ALS treatment using induced regulatory T (iTREG) cells

Abstract

To provide a method for treating amyotrophic lateral sclerosis.SOLUTION: The present disclosure provides methods for treating ALS using pentostatin and cyclophosphamide treatment followed by TREG and / or TREG / Th2 hybrid cells from de-differentiated T cells. The present disclosure further provides: methods for producing TREG and / or TREG / Th2 hybrid cells from de-differentiated T cells; the TREG and TREG / Th2 hybrid cells; populations thereof; and compositions thereof. Methods for producing de-differentiated T cells, the de-differentiated T cells, populations thereof and compositions thereof are also provided.SELECTED DRAWING: Figure 42

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 768,176, filed November 16, 2018, and U.S. Provisional Application No. 62 / 927,075, filed October 28, 2019, both of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Adoptive T-cell therapy is a novel intervention for the effective treatment of cancer and infectious diseases, autoimmune diseases, and neurodegenerative diseases. It is becoming increasingly clear that the transfer of T cells with a more primitive differentiated state, which are converted to higher growth potentials and other important attributes, is associated with improved in vivo effects after adoptive transfer. However, most forms of adoptive T-cell therapy typically require an ex vivo manufacturing process that leads to further cell differentiation. This is problematic because T cells from adult humans are already in a predominantly advanced differentiated state (referred to as effector memory cells) and often exist in an aging state under the control of checkpoint inhibitor molecules. While approaches can be taken to mitigate this limitation, including the isolation (purification) of a more naive T cell subset at the start of culture, this approach is partially limited by the small number of naive T cells present in adult human peripheral blood. Therefore, isolated T cells in a primitive differentiated state are highly desirable.

[0003] It is well known that even highly differentiated cells possess an inherent ability to dedifferentiate toward a more primitive state. In fact, in the most extreme examples, differentiated cells can be manipulated to achieve an induced pluripotent stem cell (iPSC) state, thereby allowing such iPS cells to share key characteristics with embryonic stem cells and then be further modulated toward redifferentiation toward different tissue fates. Cell therapies using such iPSC methodologies have many potential clinical applications. The generation of iPS cells from differentiated somatic cells was initially demonstrated by the transfer of major transcription factors, including Sox2, Oct3 / 4, KLF4, and c-myc, or Sox2, Oct3 / 4, Nanog, and Lin28, via viral or nonviral-mediated approaches.

[0004] However, the ability to convert somatic cells into iPS cells is inefficient and partially dependent on the degree of somatic differentiation. For example, the ability to convert mature mouse immune T cells into iPS cells is 300 times less efficient than the ability to convert mouse hematopoietic stem cells into iPS cells. Nevertheless, using gene transfer methods, it has been demonstrated that mature human peripheral blood T cells retain the ability to convert to an iPS cell state. Over the past decade, researchers have also characterized transcription factors associated with the early stages of T cell differentiation. However, the redifferentiation of T cells from various types of stem cell precursors is a relatively inefficient process, typically taking 1-2 months.

[0005] While the biology of dedifferentiation is becoming increasingly characterized, much remains unknown regarding the specific transcription factors and transcription factor dynamics associated with dedifferentiation. It is also important to recognize that gene transfer methods achieving dedifferentiation are cumbersome and associated with complications such as teratoma formation, which must be addressed through additional genetic interventions, including cell fate suicide gene programming. As a potential alternative, various pharmacological interventions can be used to achieve a degree of dedifferentiation. For example, calcineurin inhibition in cell culture using the immunosuppressant cyclosporine has resulted in molecular changes that replace the need for gene delivery of the Sox2 transcription factor to promote mouse iPS cells. In addition, rapamycin (mTOR), an immunosuppressant that inhibits its mammalian target, can induce dedifferentiation in terminal effector T cells via starvation-induced upregulation of the transcription factor KLF2, thereby increasing the T-center memory molecules CD62L and CCR7. Furthermore, the inhibition resulting from rapamycin and mTOR signaling is crucial for maintaining cell quiescence in naive T cells with reduced differentiation. It is important to note that the mTOR pathway consists of both the mTORC1 complex (containing the Raptor subunit) and the mTORC2 complex (containing the Rictor subunit). Inhibition of both mTORC1 and mTORC2 is associated with increased promotion and maintenance of memory T cells. Notably, while rapamycin can directly inhibit only mTORC1, long-term rapamycin-mediated inhibition of mTORC1 can result in downstream inhibition of mTORC2. Reduction of T cell growth factor signaling via mTOR inhibition or inhibition of other pathways is also known to upregulate another key molecule associated with T cells in a more primitive differentiated state, namely the IL-7 receptor α (CD127). Further studies have shown that inhibition of the T cell mTOR pathway via the pharmacological agent rapamycin or the Wnt-β-catenin signaling activator TWS11 promoted the dedifferentiation of human naive T cells toward a less differentiated T stem cell memory population, which has been previously identified and characterized in mouse and human T cells.Further experimental model studies have shown that pharmacological inhibition of the AKT signaling pathway, or combined inhibition of the PI3 kinase and vascular-active intestinal peptide signaling pathway, resulted in the generation of T cells with reduced differentiation and increased T cell function during adoptive transplantation.

[0006] Blocking mTOR via ex vivo culture of human T cells in rapamycin has been shown to reduce T cell expression of molecules associated with effector differentiation, such as cytokine secretion molecules and cytolytic effector molecules.

[0007] In addition, the 1,25-hydroxylated form of vitamin D (as used herein, "vitamin D") can inhibit human T cell effector function. The inhibitory effect of vitamin D on human T cell proliferation may be synergistic with immunosuppressive drug exposure using agents such as cyclosporine A or rapamycin. However, previous studies have shown that the inhibitory effect of vitamin D on T cell effectors is relatively specific to Th1 molecules rather than Th2 molecules. Furthermore, vitamin D inhibits immunosuppressive regulatory T(T) cells. REG It was shown to promote cell populations.

[0008] In some conflicting findings, human CD8 + It was determined that T cells express high levels of vitamin D receptors, and individuals with the highest levels tended to have high levels of T cell effector function and immunosenescence.

[0009] More recent studies using a mouse model of Mycobacterium tuberculosis infection have demonstrated that vitamin D is important for macrophage elimination of intracellular pathogens through mechanisms involved in IFN-γ production and autophagy. In addition, in human non-small cell cancer cell lines, vitamin D signaling can promote the cytotoxic form of autophagy, which contributes to antitumor effects when combined with radiation. Finally, vitamin D receptor signaling promotes autophagy in normal human breast tissue. Loss of such vitamin D receptor signaling has been associated with an increased risk of developing breast cancer. Despite this evidence linking vitamin D to autophagy in innate immunity (in the context of macrophages), data on the effects of vitamin D on autophagy in T cells during adaptive immunity, and, in this context, whether the potential effects of vitamin D and rapamycin on T cell autophagy are redundant, remain scarce.

[0010] These somewhat contradictory results regarding the potential role of vitamin D in T cell biology are likely related to the recently discovered broad-spectrum effects of vitamin D across the genome, both at the mRNA and microRNA levels. Therefore, the effects of vitamin D on immunity need to be evaluated within a context-dependent framework.

[0011] To maintain immune tolerance, regulatory T(T) REG ) cells are essential. REG A decrease in the quantity or quality of cells is the underlying cause of numerous primary autoimmune diseases, including, to name a few, type 1 diabetes mellitus (T1DM), multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. In addition, T REG Deficiency is associated with the acceleration of primary neurodegenerative diseases in their natural history. Finally, T REG Deficiencies are associated with serious complications in solid organ and hematopoietic stem cell transplant settings, most notably increased graft rejection rates and graft-versus-host disease (GVHD) rates. T is also associated with the maintenance of immune homeostasis. REGConsidering this important role of cells, many experimental approaches have been developed to promote T REG cells. One such promising approach is the adoptive transfer of T REG cells, which exists in two major subtypes: (1) natural (n) T REG cells derived from the thymus that are involved with age and thereby reduce the number of nT REG cells available for adoptive transfer (the "nT REG " or "natural T REG "), and (2) induced (i) T REG cells that are converted peripherally from a larger pool of effector T cells. Due to the limited number of nT<所提供的原文中此标签内容缺失,无法准确翻译,保留原文标签 REG cells, attempts to use nT REG cells in adoptive T cell therapy rely on ex vivo manufacturing methods for the isolation and subsequent expansion of nT REG cells. Clinical trials of nT REG cells for adoptive cell therapy are in the early stages of implementation and are mainly in Phase I / II clinical trials for the prevention of GVHD and the treatment of T1DM. In contrast, with regard to the potential use of iT REG cells (the ex vivo-produced T REG and T REG / Th2 cells-containing T<0000......(原文此处标签内容不完整,推测可能是 REG ,按要求保留原文标签)cells) in adoptive cell therapy, there are other issues, namely, (1) peripheral effector T cells are relatively abundant, but they mainly exist in an effector memory maturation state with limited replication and therapeutic potential, and (2) such peripheral effector T cells have a high degree of existing effector differentiation towards T cell subsets that contribute to disease onset, namely, the Th1 and Th17 subsets. Therefore, for iT REG cell therapy to be highly feasible, the following are required: (1) causing the dedifferentiation of effector T cells into a less differentiated memory phenotype with an increased proliferative potential and a clear improvement in T REG cell therapeutic potential, and (2) T REGIt will be necessary to develop ex vivo manufacturing methods that involve both promoting T cell differentiation toward the desired phenotype and eliminating pathogenic Th1 and Th17 pathways.

[0012] IT REG Cell production is initiated by the collection of lymphocyte-containing peripheral blood mononuclear cells from the subject to be treated (in the case of autologous therapy) or a normal donor (in the case of allogeneic gene therapy). Typically, this collection is performed in a steady state, i.e., without the administration of any growth factors. However, in the context of allogeneic therapy, collection may also be performed in the context of administering molecules such as granulocyte colony-stimulating factor (G-CSF) or plerixafor, 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 this disclosure, the inventors describe anti-TNF-α therapeutic agents as T REG iT REG It is stated that it can be administered before the collection of lymphocytes for production. Specifically, the inventors demonstrate that etanercept, an anti-TNF-α agent that is a recombinant receptor that preferentially inhibits serum-free, cell-free TNF-α while relatively preserving membrane-bound TNF-α on the cell surface, induces a significant change in the T cell receptor (TCR) repertoire as measured by RNA sequencing. Membrane-bound TNF-α is transmitted via the TNFR2 receptor to the T REG To provide a positive signal to cells, our method uses iT REG Before cell manufacturing T REGThis provides a robust intervention for enriching cells. Other therapeutic agents that preferentially inhibit serum-free, cell-free TNF-α, including but not limited to adalimumab, an anti-TNF-α monoclonal antibody, can also be used in this intervention.

[0013] Amyotrophic lateral sclerosis (ALS) is a primary neurodegenerative disease affecting the cerebral cortex, brainstem, and spinal cord, leading to progressive disability and typically death due to respiratory failure. ALS is familial in 10% of patients, due to various genetic events; the remaining patients have sporadic ALS, the etiology of which is unknown, but may be associated with environmental factors. Recent registry data (2013) indicates an ALS prevalence of approximately 16,000 cases in the United States, and these data also suggest that ALS disproportionately affects individuals in the 60-69 age group who are white. Veterans and potentially professional American football players appear to have an increased risk of developing ALS, thereby suggesting that chemical exposure or traumatic brain injury may increase the risk of developing the disease. ALS is a heterogeneous disease with a wide range of clinical presentations and rates of progression. The average survival time for ALS patients is 2-4 years from diagnosis, but survival can be as short as a few months or even more than 10 years. Disease scoring systems such as the patient-reported ALSFRS-R score (ALS Functional Assessment Scale, revised) do not account for the linear and nonlinear aspects of disease progression, making it difficult to estimate prognosis in ALS patients. This difficulty in estimating the rate of disease progression represents a limitation of clinical trials in ALS and indicates that potential disease biomarkers, including immunological monitoring developed by the inventors, should be emphasized as components of protocol therapy. The clinical onset of ALS is insidious, with most patients presenting with weakness in the upper or lower extremities, or difficulty speaking or swallowing (medulla oblongata onset). ALS remains a diagnosis of exclusion because blood, cerebrospinal fluid, or radiological examinations are not conclusive. Consequently, ALS is usually a diagnosis of exclusion, after other diseases have been ruled out. This process of ruling out other diseases can typically take up to a year, thereby delaying the occurrence of therapeutic attempts and clinical trials. This delay in reference is likely to be a natural consequence, as up to 50% of motor neurons may have ceased functioning by the time ALS is finally diagnosed.Given this situation, it is generally recommended to use ALS patients in clinical trials relatively soon after diagnosis.

[0014] ALS is a primary neurodegenerative disease, and neuroinflammation acts as a secondary growth factor. Evidence for this conclusion is partly derived from the observation that dysfunction of TAR DNA-binding protein 43 (TDP-43) occurs in the majority of familial and sporadic ALS patients. TDP-43 is a nuclear-confined, easily aggregated RNA and DNA-binding protein in healthy states, thereby occupying cytoplasmic inclusions found in neurons of ALS patients. While the exact mechanisms leading to alterations in the TDP-43 pathway are not yet fully understood, various cellular stress events or amplification of genetic elements that replicate themselves via RNA intermediates (posteriorly transplantable elements, RTEs) appear to be involved. Ultimately, such events result in multifaceted programmed cell death in neurons, including programmed necrosis. Notably, the necrotic cell death patterns occurring in ALS patients have been shown to be particularly immunogenic compared to more orderly apoptotic cell death. Indeed, TNF-α, a known molecular mediator of motor neuron death in ALS, can produce necrotic forms of cell death. Necrotic cell death can lead to the release of autoantigens that can be presented to the adaptive immune system for autoimmune induction. In addition, because protein aggregates themselves can be immunogenic, protein aggregates produced in ALS patients (including, but not limited to, TDP-43, SOD-1, and p62) may be targets for autoimmune responses that emerge after neurodegeneration. In fact, it has recently been shown that monocytes from ALS patients develop an inflammatory phenotype when pulsed with TDP-43-containing exosomes.

[0015] Extensive evidence exists that, in response to primary neurodegeneration, the congenital inflammasome and adaptive peripheral immune system bind, leading to further abnormal progression of ALS disease. In a superoxide dismutase-1 (SOD1) transgenic mouse model of ALS, CD3 in the spinal cord... +T cell infiltration and microglia activation were recognized as pro-inflammatory factors contributing to disease progression. Furthermore, transplantation of wild-type microglia cells with reduced inflammatory tendencies compared to host microglia cells in a PU.1 knockout mouse model of ALS reduced neurodegeneration and improved survival. In addition, for the first time in a SOD1 mouse model of ALS, CD4 + The protective role of T cells has been described, thereby demonstrating the double-edged sword nature of the peripheral immune T cell pool in ALS (acting as either a growth factor or a protective factor). In ALS patients, direct evidence of the harmful role of peripheral adaptive immune system T cells can be confirmed by demonstrating that T cells infiltrating the spinal cord express an oligoclonal T cell receptor (TCR) repertoire. Furthermore, professional antigen-presenting cells (dendritic cells) emerging from the peripheral immune system can be isolated in the spinal cord tissue of ALS patients, closely associated with inflammatory peripheral-derived monocytes and resident CNS microglia cells. 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, followed by CD4 + T-Helper-1 (Th1), CD8 + T-cell toxicity-1 (Tc1) and CD4 + It can drive the IL-23 pathway, which promotes Th17-mediated neurodegenerative immunity. Subsequent studies have shown protective CD4 in a mouse model of ALS with SOD1. + The phenotype of the T cell subset is regulatory T(T) that reduces inflammation through a mechanism partially mediated by anti-regulatory Th2 cytokines IL-4 and IL-10. REG They were characterized as a group of cells.

[0016] This biology states that microglia are important cellular components in the brain that drive neurodegeneration, and that microglia and CNS infiltration of peripheral CD4 + This is consistent with the abundant data in neuroinflammation research showing that T cells interact and influence the pathogenesis of disease. This is consistent with mouse modeling results, and FoxP3 + T REGPatients with a peripheral immune system enriched with cells and Th2 T cells showed a reduced rate of ALS progression compared to patients with a predominantly pro-inflammatory Th1 immune profile. Furthermore, T cells in ALS patients REG It has recently been found that cellular dysfunction correlates with disease progression and severity. Current clinical trials are underway for the treatment of nT for ALS. REG The use of multiple infusions of cells + low-dose IL-2 is being evaluated (ClinicalTrials.gov;NCT03241784); IL-2 is a cytokine that stimulates the STAT5 signaling pathway, thereby activating nT REG It can promote in vivo proliferation of cells.

[0017] Induced (i)T REG Cells are nT REG Unlike cell populations, it does not originate from the thymus, rather, iT REG nT is a population that is converted from other pathogenic postthymic T cell subsets, such as Th1 cells. REG and iT REG Both play important and non-redundant roles in suppressing the inflammatory response, but iT REG The development of therapies is relatively advantageous in terms of the efficacy and ease of production of regulatory T cells. Furthermore, adoptive iT cells for ALS REG The therapy is particularly effective when used in combination with the immunomonitoring techniques and host treatment regimens (pentostatin, cyclophosphamide, lamivudine) described by the inventors in this disclosure.

[0018] Riluzole (Rilutek®), the first drug approved for the treatment of ALS in 1995, is only slightly effective in reducing the morbidity and mortality of ALS. Despite significant clinical research investigating over 60 molecules for ALS therapy, only two additional molecules, the antioxidant edaravone and the tyrosine kinase inhibitor macitinib, have shown modest clinical success. Edaravone (Radicava®), recently approved by the FDA for the treatment of ALS, offers minimal clinical benefit, is expensive, and requires daily infusion therapy with two weeks on and two weeks off, while macitinib is not FDA approved. A Phase II trial of rapamycin for the treatment of ALS is currently underway (ClinicalTrials.gov identifier: NCT03359538). Rapamycin is also being investigated for its potential to treat T REG Due to its tendency to promote cell remodeling, it may be a preferred drug for use in ALS. However, long-term therapy with rapamycin has substantial toxicity, requires pharmacological monitoring, and may have contradictory effects in that it actually worsens ALS in some models, potentially limiting the expansion of the adopted T cell population.

[0019] Therefore, given the current situation with very limited treatment options, it is essential to evaluate novel strategies for the treatment of ALS. In this application, the inventors propose an inductive (i) controllable T(T) REG This paper describes novel therapeutic approaches for ALS, primarily focusing on cell therapy. [Overview of the Initiative]

[0020] This disclosure relates to the dedifferentiation of T cells and the T cells of such cells. REG or T REG This focuses on methods for differentiation into Th2 cells.

[0021] In some embodiments, the initial dedifferentiation method may include initiating culture with an input cell population (without drug administration) collected under steady state.

[0022] In some embodiments, the method involves initiating a dedifferentiation culture using an input cell population that has been treated or is being treated with an anti-TNF-α therapeutic agent that is preferentially selective for inhibiting serum-free, cell-free morphology of TNF-α, either from the subject (in an autologous context) or from a normal donor (in an allogeneic context) that has been treated with an anti-TNF-α therapeutic agent that is preferentially selective for inhibiting serum-free, cell-free morphology of TNF-α, with relative preservation of membrane-bound TNF-α. Such therapeutic agents include, but are not limited to, recombinant receptor etanercept, which can be administered by subcutaneous injection at conventional doses of 25–50 mg per week, or monoclonal antibody adalimumab, which can be administered by intravenous injection at conventional doses of 40 mg per week or 40 mg every other week. In all of these cases, the dosage of the anti-TNF-α therapeutic agent can be adjusted according to desired biomarker changes, which may include, but are not limited to, changes in the TCR repertoire as measured by RNA sequencing, as well as shifts toward type 2 TNF receptor (TNFR2) and away from type 1 TNF receptor (TNFR1) as measured by flow cytometry.

[0023] In some embodiments, the method includes: planting a cell culture input population, including T cells from a target, at cell density in a culture medium containing vitamin D, temsirolimus, and an IL-2 signaling inhibitor; stimulating the T cells by adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium in a bead:T cell ratio of 1:1 or less; or, in the most extreme example, without adding anti-CD3 / anti-CD28 co-stimulation; and incubating the cell culture input population and culture medium for a certain period to obtain dedifferentiated T cells. It is also possible to perform this dedifferentiation procedure in the absence of any bead co-stimulation.

[0024] In any of the embodiments described above, the method may further include collecting the dedifferentiated T cells described above.

[0025] In any of the embodiments described above, the method may further include, after collecting 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.

[0026] In any of the embodiments described above, the method may further include collecting the above-mentioned culture input population of cells from the subject before inoculating the above-mentioned culture input population of cells into the above-mentioned culture medium.

[0027] In any of the embodiments described above, the method may further include measuring the expression level of RAPTOR or RICTOR in the cell culture input population, wherein the period continues until the expression level of RAPTOR or RICTOR in the cell culture input population is reduced by at least 50%, more preferably 90%, compared to a T cell control population, the T cell control population being prepared under the same conditions as the cell culture input population, which is free of temsirolimus, IL-2 signaling inhibitors, and vitamin D.

[0028] In any of the embodiments described above, the method may further include measuring the expression levels of RAPTOR or RICTOR and housekeeping proteins in the culture input population of cells, the period of which continues until the expression level of RAPTOR or RICTOR normalized by the housekeeping proteins in the produced T cells is reduced by at least 50%, more preferably 90%, compared to the expression level of RAPTOR or RICTOR normalized by the housekeeping proteins in a control population of T cells produced under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D.

[0029] This disclosure also relates to dedifferentiated T cells produced by any of the embodiments described above.

[0030] The disclosure also relates to compositions comprising a population of dedifferentiated cells, wherein at least a portion of the population of dedifferentiated cells expresses RAPTOR or RICTOR at a rate of at least 50%, more preferably 90%, lower than a control population of T cells produced under the same conditions as a culture input population of cells that do not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D.

[0031] In any of the embodiments described above, the method may further include measuring at least a portion of the population of dedifferentiated cells described above, thereby expressing at least 10%, more preferably 50%, changes in the RNA expression of the following molecules compared to a control population of T cells: namely, reductions in T cell effector molecules including, but not limited to, granzyme B, IL-10, and IFN-γ; increases in transcription factors associated with dedifferentiated cells including, but not limited to, Nanog, KLF4, and KLF10; increases in the expression of molecules preferentially expressed on naive T cell subsets including, but not limited to, CD127 and IL-7 receptor α chain; reductions in transcription factors associated with TH1-type differentiation including, but not limited to, T-BET and STAT1; and relative conservation of cell survival-promoting transcription factors including, but not limited to, HIF-1α.

[0032] In any of the embodiments described above, the method may further include measuring at least a portion of the above population of dedifferentiated cells, thereby determining that they express molecules indicating cells that have experienced autophagy with at least a 10%, more preferably 50%, change in expression. For example, the above dedifferentiated cells have increased p62 expression as determined by Western blot analysis compared to control T cells. Other methods for measuring autophagy may also be applied, such as the method described in Yoshii SR. Mizushima N. Monitoring and Measuring Autophagy. International Journal of Molecular Sciences. 2017;18(9):1865.

[0033] This disclosure also relates to dedifferentiated T cells produced by any of the embodiments described above.

[0034] The disclosure also relates to compositions comprising a population of dedifferentiated cells in which at least a portion of the above population of dedifferentiated cells expresses at least 10%, more preferably 50%, a change in the RNA expression of the following molecules compared to a control population of T cells: namely, a reduction in T cell effector molecules including, but not limited to, granzyme B, IL-10, and IFN-γ; an increase in transcription factors associated with dedifferentiated cells including, but not limited to, Nanog, KLF4, and KLF10; an increase in the expression of molecules preferentially expressed on naive T cell subsets including, but not limited to, CD127 and IL-7 receptor α chain; a reduction in transcription factors associated with TH1-type differentiation including, but not limited to, T-BET and STAT1; and a relative conservation of cell survival-promoting transcription factors including, but not limited to, HIF-1α.

[0035] This disclosure also relates to compositions comprising a population of dedifferentiated cells as defined above, wherein the expression of molecules indicating cells that have experienced autophagy is altered by at least 10%, more preferably 50%. For example, the above dedifferentiated cells have increased p62 expression as determined by Western blot analysis compared to control T cells. Other methods for measuring autophagy, such as the method described in Yoshii SR, Mizushima N. Monitoring and Measuring Autophagy. International Journal of Molecular Sciences. 2017;18(9):1865, may also be applied.

[0036] This disclosure also relates to compositions comprising a population of dedifferentiated cells, wherein at least a portion of the above population of dedifferentiated cells express less than 50% of both RAPTOR and RICTOR compared to a control population of T cells.

[0037] This disclosure describes how to dedifferentiate T cells. REG or T REG This study focuses on methods for differentiating cells into Th2 cells.

[0038] In some embodiments, the method involves culturing dedifferentiated T cells of the disclosure, or T cells otherwise dedifferentiated, in a culture medium containing IL-2, IL-4, and TGF-β; adding anti-CD3 / anti-CD28 coated magnetic beads in a ratio of 3:1 (beads:T cells); adding beads in the above ratio (beads:T cells); and incubating the dedifferentiated T cells for a certain period of time. REG This includes obtaining Th2 cells.

[0039] In some embodiments, the method involves culturing dedifferentiated T cells having at least 50% less RAPTOR or RICTOR expression compared to a control population of T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, in a culture medium containing IL-2, IL-4, and TGF-β; adding anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 ratio (beads:T cells); and incubating the above dedifferentiated T cells for a certain period of time. REG This includes obtaining Th2 cells.

[0040] In some embodiments, the method involves culturing dedifferentiated T cells in a culture medium containing IL-2 and TGF-β, having at least 90% less RAPTOR or RICTOR expression compared to a control population of T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D; adding anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 ratio (beads:T cells); and incubating the above dedifferentiated T cells for a certain period of time. REG This includes obtaining cells.

[0041] In any of the above embodiments, the culture medium may further contain pemetrexed.

[0042] This disclosure also relates to T produced by any of the above methods. REG or T REG This targets Th2 cells.

[0043] This disclosure also relates to methods for treating amyotrophic lateral sclerosis in patients requiring treatment for amyotrophic lateral sclerosis.

[0044] In some embodiments, the method comprises subjecting the subject to one or more primary treatment cycles, each of which involves administering pentostatin to the subject and / or cyclophosphamide to the subject and administering a therapeutically effective amount of manufactured T REG The method includes subjecting a composition containing cells to one or more immunotherapy treatment cycles, which involves administering the composition to the subject described above.

[0045] In some embodiments, the 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 manufactured T REG This includes administering cells to the above-mentioned subjects.

[0046] In some embodiments, the method involves applying a therapeutically effective amount of manufactured T to the above-mentioned subject. REG The treatment includes one or more treatment cycles, including the administration of cells.

[0047] In some embodiments, the method involves producing a therapeutically effective amount of T REG This may include administering cells to the subjects mentioned above. [Brief explanation of the drawing]

[0048] [Figure 1A] This shows normalized GAPDH mRNA expression in control cells and cells treated under various conditions. [Figure 1B] This shows the normalized granzyme B mRNA expression in control cells and cells treated under various conditions. [Figure 1C] This shows normalized IL-10 mRNA expression in control cells and cells treated under various conditions. [Figure 1D] Figures 1A–1D illustrate the normalized IFN-γ mRNA expression of control cells and cells treated under various conditions. They demonstrate that the combination of vitamin D and temsirolimus reduces the expression of effector molecules in human CD4+ and CD8+ cells. [Figure 2A] This shows normalized NANOG mRNA expression in control cells and cells treated under various conditions. [Figure 2B] This shows normalized KLF4 mRNA expression in control cells and cells treated under various conditions. [Figure 2C] This shows normalized KLF10 mRNA expression in control cells and cells treated under various conditions. [Figure 2D] Figures 2A–2D illustrate the normalized IL-7 receptor mRNA expression in control cells and cells treated under various conditions. The combination of vitamin D and temsirolimus increases the expression of stem cell-related transcription factors and the atomic T cell molecule IL-7 receptor-α in human CD4+ and CD8+ T cells. [Figure 3A] This shows the normalized T-BET mRNA expression in control cells and cells treated under various conditions. [Figure 3B] This shows normalized STAT1 mRNA expression in control cells and cells treated under various conditions. [Figure 3C] Figures 3A–3C show normalized HIF-1-α mRNA expression in control cells and cells treated under various conditions. These figures demonstrate that the combination of vitamin D and temsirolimus reduces the expression of effector Th1 / Tc1 cell-related transcription factors without reducing the expression of HIF-1-α, a transcription factor associated with T cell survival. [Figure 4] We demonstrate actin-normalized p62 expression in cells treated under various conditions, showing that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade induces the expression of the autophagy-related molecule p62. [Figure 5] We demonstrate actin-normalized RAPTOR expression in cells treated under various conditions, showing that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces the expression of the mTORC1-related molecule RAPTOR. [Figure 6] Western blots of GAPDH, p70S6K, SGK1, Raptor, and Rictor expression in cells treated under various conditions are shown, demonstrating that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces the expression of mTORC1-related molecule Raptor and mTORC2-related molecule Rictor. [Figure 7] The study shows normalized BIM expression by actin expression in cells treated under various conditions, illustrating that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces the expression of the pro-apoptotic molecule BIM. Figure 8 illustrates the effect of culture components on subsequent T cell yield during the dedifferentiation interval (at day 13 of culture). [Figure 8]This illustrates the effect of culture components during the dedifferentiation interval on subsequent T cell yield (on day 13 of culture). [Figure 9A] This shows the percentage of CD4 cells that are CD45RA+ after being treated under various conditions. [Figure 9B] This shows the percentage of CD4 cells that are CD62L+ and CCR7+ for cells treated under various conditions. [Figure 9C] Figures 9A–9C illustrate the effects of culture components during the dedifferentiation interval on the expression of memory markers in CD4+ T cells (at day 13 of culture). [Figure 10A] This shows the percentage of CD8 cells that are CD62L+ and CCR7+ for cells treated under various conditions. [Figure 10B] Figures 10A and 10B illustrate the effects of culture components during the dedifferentiation interval on the expression of memory markers in CD8+ T cells. [Figure 11A] Figures 11A–11D show the analysis of inflammatory Th1 / Th17 cytokines in dedifferentiated T cells cultured in polarized neutral medium. Figure 11A: Shows IFN-γ secretion from cells treated under various conditions. [Figure 11B] This shows GM-CSF secretion from cells treated under various conditions. [Figure 11C] This shows TNF-α secretion from cells treated under various conditions. [Figure 11D] This shows IL-17 secretion from cells treated under various conditions. [Figure 12A] Figures 12A–12D show the analysis of IL-2 and Th2-type cytokines in dedifferentiated T cells cultured in polarized neutral medium. Figure 12A: Shows IL-2 secretion from cells treated under various conditions. [Figure 12B]This shows IL-4 secretion from cells treated under various conditions. [Figure 12C] This shows IL-5 secretion from cells treated under various conditions. [Figure 12D] This shows IL-13 secretion from cells treated under various conditions. [Figure 13] Under hybrid Th2 / TREG polarization conditions, a favorable expansion of dedifferentiated T cells is observed compared to Th1 polarization conditions. [Figure 14A] This shows the percentage of CD4+CD45RA+ cells out of all CD4+ cells treated under various conditions. [Figure 14B] This shows the percentage of CD4+CD62L+CCR7+ cells out of all CD4+ cells treated under various conditions. [Figure 14C] Figures 14A–14C show the percentage of CD4+CD62L+CCR7+CD127+ cells out of total CD4+ cells treated under various conditions. These figures illustrate how culturing dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of naive and triple-positive T central memory CD4+ T cells. [Figure 15A] This shows the percentage of CD8+CD62L+CCR7+ cells out of all CD8 cells treated under various conditions. [Figure 15B] Figures 15A and 15B illustrate how culturing dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of triple-positive T central memory CD8+ T cells. [Figure 16A] This shows IL-2 secretion from cells treated under various conditions. [Figure 16B] This shows IL-4 secretion from cells treated under various conditions. [Figure 16C]Figures 16A–16C illustrate how culturing dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of T cells with atomic Th2 cell cytokine phenotypes: IL-2, IL-4, and IL-5 secretion. [Figure 17A] This shows IL-10 secretion from cells treated under various conditions. [Figure 17B] This shows IL-13 secretion from cells treated under various conditions. [Figure 17C] Figures 17A–17C illustrate how culturing dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of T cells with atomic Th2 cell cytokine phenotypes: IL-10, IL-13, and IL-17 secretion. [Figure 18A] This shows IFN-γ secretion from cells treated under various conditions. [Figure 18B] This shows TNF-α secretion from cells treated under various conditions. [Figure 18C] Figures 18A–18C illustrate how culturing dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of T cells with atomic Th2 cell cytokine phenotypes: IFN-γ, TNF-α, and GM-CSF secretion. [Figure 19A] This shows the percentage of CD4+ T cells in the culture, day by day and per culture inhibitor. [Figure 19B] This shows the percentage of CD4+FOXP3+ T cells in the culture, day by day and per culture inhibitor. [Figure 19C] This shows the percentage of CD4+Tbet+ T cells in the culture, day by day and per culture inhibitor. [Figure 19D]Figures 19A–19D show the percentage of CD4+GATA3+ T cells in the culture, day by day and per culture inhibitor. They illustrate that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarization conditions containing pemetrexed results in the generation of CD4+ T cells expressing FOXP3 and GATA3 transcription factors. [Figure 20A] This shows the percentage of CD8+ T cells in the culture, day by day and per culture inhibitor. [Figure 20B] This shows the percentage of CD8+FOXP3+ T cells in the culture, day by day and per culture inhibitor. [Figure 20C] This shows the percentage of CD8+ Tbet+ T cells in the culture, day by day and per culture inhibitor. [Figure 20D] Figures 20A–20D show the percentage of CD8+GATA3+ T cells in the culture, day by day and per culture inhibitor. Extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarization conditions containing pemetrexed results in the generation of CD8+ T cells expressing FOXP3 and GATA3 transcription factors. [Figure 21A] This shows IL-4 secretion from cells in the culture on a daily basis and for each culture inhibitor. [Figure 21B] This shows IL-5 secretion from cells in the culture on a daily basis and for each culture inhibitor. [Figure 21C] Figures 21A-21C illustrate how extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarity conditions results in the generation of T cells expressing a dominant Th2 cytokine phenotype: IL-4, IL-5, and IL-13 secretion. [Figure 22A] This shows IL-2 secretion from cells in the culture on a daily basis and for each culture inhibitor. [Figure 22B] IFN-γ secretion from cells in the culture is shown daily and for each culture inhibitor. [Figure 22C]Figures 22A-22C illustrate how extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarity conditions results in the generation of T cells expressing a dominant Th2 cytokine phenotype: IL-2, IFN-γ, and GM-CSF secretion. [Figure 23A] This example illustrates how anti-TNF-α therapy, etanercept therapy, can lead to significant changes in the TCR repertoire as measured by RNA sequencing, thereby representing a novel approach for target therapy prior to lymphocyte collection by apheresis. [Figure 23B] This example illustrates how anti-TNF-α therapy, etanercept therapy, can lead to significant changes in the TCR repertoire as measured by RNA sequencing, thereby representing a novel approach for target therapy prior to lymphocyte collection by apheresis. [Figure 24-1] This example illustrates that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of T cells expressing increased levels of the following molecules: CD25, CD27, 2B4, BTLA, and CTLA, compared to control Th1 / Tc1 cells. [Figure 24-2] (Continuation of Figure 24-1) [Figure 25-1] This example illustrates that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of T cells expressing increased levels of the following molecules: TIGIT, TIM3, ICOS, LAIR1, and OX40, compared to control Th1 / Tc1 cells. [Figure 25-2] (Continuation of Figure 25-1) [Figure 26A-1] This shows FOXP3 expression in CD4+ and CD8+ T cells at the start of culture and after culture, as measured by flow cytometry. [Figure 26A-2] (Continuation of Figure 26A-1) [Figure 26B-1]Shows GATA3 expression in CD4+ and CD8+ T cells at the start of culture and after culture, measured by flow cytometry. [Figure 26B-2] (Continuation of Figure 26B-1) [Figure 27A-1] Shows CD73 expression in CD4+ and CD8+ T cells at the start of culture and after culture, measured by flow cytometry. [Figure 27A-2] (Continuation of Figure 27A-1) [Figure 27B-1] Shows CD103 expression in CD4+ and CD8+ T cells at the start of culture and after culture, measured by flow cytometry. [Figure 27B-2] (Continuation of Figure 27B-1) [Figure 28A] Shows the CD150 frequency measured by flow cytometry in CD4+ and CD8+ T cells at the start of culture, after culture, and in control T cells not exposed to the mTOR inhibitor. [Figure 28B] Shows CD27 vs CD95 expression for CD4+ T cells at the start of culture and after culture, measured by flow cytometry. [Figure 29-1] Shows IL-4, IL-2, IFN-γ, TNF-α, IL-17, and GM-CSF for differently cultured cells and control cells. [Figure 29-2] (Continuation of Figure 29-1) [Figure 29-3] (Continuation of Figure 29-1) [Figure 30A-1] Shows cytokine content for the transwell assay of Th1 / Tc1 cells with or without RAPA-501 cells. [Figure 30A-2] (Continuation of Figure 30A-1) [Figure 30B-1] Shows the flow cytometry results of the assays for CD4 and PD1 in Example 24. [Figure 30B-2] (Continuation of Figure 30B-1) [Figure 31A]The secretion of IL-6, IP-10, and IFN-γ from human microglia cells, with and without exposure to RAPA-501 cells, is shown. [Figure 31B] The secretion of IL-6, IP-10, and IFN-γ from human microglia cells, with and without exposure to RAPA-501 cells, is shown. [Figure 32] The PC regimen and overall treatment approach are outlined below. [Figure 33] The lymphocyte collection regimens before and after PC are outlined below. [Figure 34] The preceding PC regimens for each repeated dose of iTREG cells are outlined below. [Figure 35] A schematic overview of monitoring patients treated with iTREG cells is provided. [Figure 36A] This study demonstrates that anti-TNF-α therapy, etanercept therapy, leads to significant changes in the TCR repertoire when measured by RNA sequencing, thereby representing a novel approach for target therapy prior to lymphocyte collection by apheresis. [Figure 36B] This study demonstrates that anti-TNF-α therapy, etanercept therapy, leads to significant changes in the TCR repertoire when measured by RNA sequencing, thereby representing a novel approach for target therapy prior to lymphocyte collection by apheresis. [Figure 37-1] This example illustrates that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of T cells expressing increased levels of the following molecules: CD25, CD27, 2B4, BTLA, and CTLA, compared to control Th1 / Tc1 cells. [Figure 37-2] (Continuation of Figure 37-1) [Figure 38-1] This example illustrates that extended culture of dedifferentiated T cells under hybrid Th2 / TREG polarization conditions results in the generation of T cells expressing increased levels of the following molecules: TIGIT, TIM3, ICOS, LAIR1, and OX40, compared to control Th1 / Tc1 cells. [Figure 38-2] (Continuation of FIG. 38-1) [Figure 39] Shows an alternative protocol design. [Figure 40A] Shows RAPA-501 GATA3 and FOXP3 measured by flow cytometry for CD4+ cells. [Figure 40B] Shows RAPA-501 GATA3 and FOXP3 measured by flow cytometry for CD8+ cells. [Figure 41-1] Shows an exemplary workflow of the dedifferentiation method of the present disclosure. [Figure 41-2] (Continuation of FIG. 41-1) [Figure 41-3] (Continuation of FIG. 41-1) [Figure 42] Shows the putative mechanism of action of the hybrid TREG / Th2 cells of the present disclosure.

Mode for Carrying Out the Invention

[0049] The present disclosure provides methods for T cell dedifferentiation and for the resulting cells, methods for producing human hybrid regulatory T / Th2 cells (hybrid T REG / Th2 cells) from dedifferentiated T cells, and methods for the treatment of ALS using induced regulatory T (iT REG ) cells.

[0050] Definitions As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0051] The use of the term "or" in the claims and the present disclosure is used to mean "and / or" unless explicitly indicated to refer to only alternatives or if the alternatives are mutually exclusive.

[0052] The use of the term "approximately" when used with a number is intended to include a + / - 10% range. For example, if a number of amino acids is identified as approximately 200, this includes 180 to 220 (plus or minus 10%).

[0053] The terms “patient,” “individual,” and “subject” are used interchangeably herein, with human patients preferred and referring to the mammalian subject being treated. In some cases, the methods of the present invention may be found to be used in laboratory animals, veterinary applications, and in the development of animal models for diseases, including but not limited to rodents such as mice, rats, and hamsters, as well as primates.

[0054] "Sample" is used in its broadest sense herein. Samples, including cells, polynucleotides, polypeptides, peptides, antibodies, etc., may include body fluids, soluble fractions of cell preparations, or culture media in which cells were 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; and cells, tissues, tissue prints, fingerprints, skin, or hair.

[0055] "Treatment" is an intervention undertaken with the intention of preventing or altering the onset of pathology or symptoms of a disorder. Therefore, "treatment" can refer to both therapeutic treatment and preventive or protective measures. Those who require treatment include those who already have a disorder and those whose disorder should be prevented. In the treatment of tumors (e.g., cancer), therapeutic agents can either directly reduce the pathology of tumor cells or confer further sensitivity to other therapeutic agents, such as radiation and / or chemotherapy.

[0056] As used herein, “treatment cycle” can generally refer to a primary treatment cycle, a first treatment cycle, a second treatment cycle, or one or more additional treatment cycles.

[0057] As used herein, “immune cells” means any cells of the immune system that can be assayed, including, but not limited to, genetically modified immune cells including 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, hybridomas, and drug-modified immune cells, as well as derivatives, precursors, or predecessors of the above cell types.

[0058] T cells, or T lymphocytes, are a subset of lymphocytes originating from the thymus that possess heterodimer receptors associated with CD3 complex proteins (e.g., rearranged T cell receptors, heterodimer proteins on the surface of T cells that are responsible for the antigen / MHC specificity of cells). T cell responses can be detected by assays of their effects on other cells (e.g., target cell death, activation of other immune cells such as B cells) or the cytokines they produce.

[0059] As used herein, the term “dedifferentiated T cells” refers to T cells dedifferentiated by any of the methods of this disclosure. In certain embodiments, dedifferentiated T cells have reduced RAPTOR or RICTOR expression compared to a control population of T cells produced under the same conditions without temsirolimus, IL-2 signaling inhibitors, and vitamin D. “Dedifferentiated T cells” does not include T cells collected from patients, i.e., naturally occurring T cells.

[0060] As used herein, the term “anti-CD3 / anti-CD28” should be understood to refer to an anti-CD3 / anti-CD28 antibody. For example, “anti-CD3 / anti-CD28 magnetic beads” should be understood to refer to magnetic beads having an associated anti-CD3 / anti-CD28 antibody moiety. In cases where it is disclosed that anti-CD3 / anti-CD28 co-stimulation is not provided, it should be understood that this also excludes co-stimulation with other forms of anti-CD3 / anti-CD28, even by a specific form such as anti-CD3 / anti-CD28 magnetic beads.

[0061] In this disclosure in which co-stimulation with an anti-CD3 / anti-CD28 antibody is performed, it should be understood that this co-stimulation may be provided with any form of anti-CD3 / anti-CD28 antibody. For example, but not limited to examples, if it is indicated that co-stimulation is performed by using anti-CD3 / anti-CD28 beads, anti-CD3 / anti-CD28 nanoparticles or microparticles may be used. In cases where it is disclosed that anti-CD3 / anti-CD28 co-stimulation is not provided, it should be understood that this may also exclude co-stimulation with other forms of anti-CD3 / anti-CD28, such as anti-CD3 / anti-CD28 magnetic beads.

[0062] As used herein, the terms “human hybrid TREG / Th2 cells,” “iTREG,” and “TREG / Th2 cells” refer to cells differentiated by the methods of this disclosure, unless otherwise specified. The “human hybrid TREG / Th2 cells,” “iTREG,” and “TREG / Th2 cells” as used herein do not include T cells collected from patients, i.e., naturally occurring T cells.

[0063] As used herein, the term “manufactured TREG cells” refers to cells produced by the dedifferentiation and redifferentiation methods of this disclosure and, unless otherwise stated, may be understood to include TREG cells and human hybrid TREG / Th2 cells.

[0064] As used herein, “control Th1 / Tc1 cells” means, unless otherwise specified, cells that have not been treated with vitamin D, temsirolimus, or IL-2 signaling inhibitors, but rather have been co-stimulated with anti-CD3 / anti-CD28 magnetic coated beads in a 3:1 ratio (beads:T cells) in a medium supplemented with 20 IU / mL of IL-2 and 20,000 IU / mL of IFN-α, and otherwise cultured in the same manner as the cells being compared. When a control population of cells (or control T cells) is referred to as being treated without culture additives including temsirolimus, vitamin D, and IL-2 signaling inhibitors, or in the context of dedifferentiated cells, it should also be understood that this population (or T cells) is further co-stimulated with anti-CD3 / anti-CD28 magnetic coated beads (beads: T cells) in a 3:1 ratio in medium supplemented with 20 IU / mL IL-2 and 20,000 IU / mL IFN-α, otherwise cultured in the same way as the cells being compared, i.e., they are "control Th1 / Tc1 cells".

[0065] This disclosure provides a novel methodology for ex vivo generation of reduced-differentiation T cells, based on converting differentiated effector memory T cells into less differentiated central memory T cells using novel pharmacological combinations and defined T cell costimulation conditions.

[0066] As shown in Figure 41, the dedifferentiated T cells of this disclosure may have a quiescent phenotype in which 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) are little to no expression. Redifferentiated T cells may have a hybrid fate characterized by GATA3 and FOXP3 expression, as well as stem cell memory characterized by CD45RA and CD150 expression, and no checkpoint protein expression.

[0067] Figure 42 shows the hybrid T of the present disclosure. REGThe presumed mechanism of action of / Th2 cells is shown, which is through inflammation mediated by CD39 or CD73 receptors and hybrid TNF-α. REG This can activate Th2 cells, which in turn allows the cells to regulate pathogenic T cells and prevent their killing.

[0068] Method for T cell dedifferentiation and resulting cells The inventors provide a novel methodology for ex vivo generation of reduced-differentiation T cells, based on converting differentiated effector memory T cells into less differentiated central memory T cells using novel pharmacological combinations and defined T cell costimulation conditions.

[0069] In one embodiment, the method comprises: planting a cell culture input population, including T cells from a subject, at cell density in a culture medium containing vitamin D, temsirolimus, and an IL-2 signaling inhibitor; stimulating the T cells by adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium in a bead:T cell ratio of 1:1 or less, or without adding any co-stimulatory beads; and incubating the cell culture input population and culture medium for a certain period of time to obtain dedifferentiated T cells. In some embodiments, the subject is treated with anti-TNF-α therapy before collecting the cell culture input population. In some embodiments, the anti-TNF-α therapy is etanercept or adalimumab. In some embodiments, co-stimulation with anti-CD3 / anti-CD28 is not performed.

[0070] In any of the above embodiments, the culture medium cannot contain IL-2, and IL-2 cannot be added to the culture medium.

[0071] In any of the embodiments described above, the cell density may be approximately 1.5 × 10⁶ T cells to 18 × 10⁶ T cells per mL. Examples, but not limited to, include 6 × 10⁶ T cells to 18 × 10⁶ T cells per mL, 12 × 10⁶ T cells to 18 × 10⁶ T cells per mL, 1.5 × 10⁶ T cells to 12 × 10⁶ T cells per mL, 1.5 × 10⁶ T cells to 6 × 10⁶ T cells per mL, 6 × 10⁶ T cells 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, but not limited to examples, it is anticipated that it may be preferable to start cell culture at a higher density, such as 9 × 10⁶ T cells per mL or 18 × 10⁶ T cells per mL.

[0072] In any of the embodiments described above, the temsirolimus can be present at concentrations ranging from approximately 0.3 μM to approximately 10 μM. Examples, but not limited to, include: approximately 0.3 μM to approximately 1 μM, 0.3 μM to approximately 0.75 μM, 0.3 μM to approximately 0.5 μM, 0.5 μM to approximately 1 μM, 0.75 μM to approximately 1 μM, 0.5 μM to approximately 0.75 μM, 0.3 μM to approximately 10 μM, 0.3 μM to approximately 5 μM, 0.3 μM to approximately 3.3 μM, 1 μM to approximately 3.3 μM, 5 μM to approximately 10 μM, and 3.3 μM. It can be present in the culture medium at concentrations of μM to approximately 10 μM, 3.3 μM to approximately 5 μM, or, as an example but not limited to, approximately 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.

[0073] In any of the embodiments described above, the IL-2 signaling inhibitor may be an anti-IL-2 receptor antibody or a fragment thereof. The IL-2 signaling inhibitor may be basiliximab or daclizumab, but are not limited to examples. The IL-2 signaling inhibitor may be 5-50 μg / mL, 5-40 μg / mL, 5-30 μg / mL, 5-20 μg / mL, 5-10 μg / mL, 10-50 μg / mL, 20-50 μg / mL, 30-50 μg / mL, 40-50 μg / mL, 30-40 μg / mL, 20-40 μg / mL, 10-40 μg / mL, 5-40 μg / mL, 5-30 μg / mL g / mL, 5-20 μg / mL, 5-10 μg / mL, 10-20 μg / mL, 10-30 μg / mL, 20-30 μg / mL, or, for example, 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 are present in the culture medium at concentrations of g / mL, 5-20 μg / mL, 5-10 μg / mL, 10-20 μg / mL, 10-20 μg / mL, 10-30 μg / mL, 20-30 μg / mL, or 50 μg / mL.

[0074] In any of the embodiments described above, the above periods may be approximately 1.5 to 5 days, 1.5 to 3.5 days, 1.5 to 2.5 days, 2.5 to 3.5 days, 2.5 to 5 days, 3.5 to 5 days, or approximately 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 days. In some embodiments, the levels of mTORC1 and mTORC2 reduction may be used as a guide for determining the optimal culture interval. In some embodiments, the optimal culture interval may be determined using other molecular signatures of dedifferentiated cells, including but not limited to: RNA expression of T cell effector molecules (i.e., decreased IFN-γ), RNA expression of transcription factors (i.e., increased KLF4), evidence of autophagy signatures (i.e., increased p62), and upregulation of markers present on naive T cell subsets (i.e., increased CD127).

[0075] In any of the embodiments described above, but not limited to examples, the bead:T cell ratio may be 1:3 or co-stimulation may not occur. The bead:T cell ratio may be 1:1–1:12, 1:1–1:3, or 1:3–1:12. Further examples, but not limited to examples, the bead:T cell ratio may 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 co-stimulation may not be available; that is, in some embodiments, anti-CD3 / anti-CD28 co-stimulation may not occur during the initial dedifferentiation process.

[0076] In any of the embodiments described above, co-stimulation of the cell culture input population can be achieved using anti-CD3 / anti-CD28 nanoparticles, which may be used at concentrations lower than those recommended. Examples of such nanoparticles, though not limited to examples, may be used at approximately 0.01 to 0.1 times, 0.025 to 0.1 times, 0.05 to 0.1 times, 0.075 to 0.1 times, 0.01 to 0.075 times, 0.01 to 0.05 times, 0.01 to 0.01 times, 0.01 to 0.025 times, 0.025 to 0.075 times, 0.025 to 0.05 times, 0.05 to 0.075 times, or approximately 0.01 times, 0.025 times, 0.05 times, 0.075 times, or approximately 0.01 times, 0.025 times, 0.05 times, 0.075 times, or approximately 0.01 times. For example, reagents such as Miltenyi® T Cell TransAct® can be used at reduced doses compared to the recommended dose of 10 μL per 1 × 10⁶ T cells, for example, 1.1 μL (a 9-fold reduction) or approximately 0.11 times, but not limited to these. Finally, in the most extreme examples, anti-CD3 / anti-CD28 co-stimulation cannot be utilized, i.e., in some embodiments, anti-CD3 / anti-CD28 co-stimulation is not performed during the initial dedifferentiation process.

[0077] Alternatively, when using anti-CD3 / anti-CD28 costimulation to produce manufactured T cells, the costimulation source may be provided by soluble anti-CD3 / anti-CD28 microparticles. While not limited to examples, soluble anti-CD3 / anti-CD28 microparticles can be used at 20% of the manufacturer's recommended concentration (e.g., Cloudz®; Bio-Techne). Further examples include soluble anti-CD3-anti-CD28 microparticles being used at 5%, 10%, 15%, 20%, 25%, or 30% of the manufacturer's recommended concentration.

[0078] In any of the embodiments described above, anti-CD3 / anti-CD28 stimulation, if performed, can be carried out using a sufficient amount of anti-CD3 / anti-CD28 to achieve the desired cellular characteristics of dedifferentiation.

[0079] In any of the embodiments described above, the culture medium may further contain 5% human serum. The culture medium may include, but is not limited to, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% human serum, and any range including values ​​in between.

[0080] In any of the embodiments described above, the culture medium may include X-Vivo 20 medium. Any suitable culture medium for culturing T cells may be used.

[0081] In any of the embodiments described above, the vitamin D may be present at concentrations of approximately 0.03 nM to approximately 1 nM, 0.03 nM to approximately 0.5 nM, 0.03 nM to approximately 0.1 nM, 0.03 nM to approximately 0.05 nM, 0.05 nM to approximately 0.1 nM, 0.05 nM to approximately 0.5 nM, 0.05 nM to approximately 1 nM, 0.1 nM to approximately 1 nM, 0.1 nM to approximately 0.5 nM, or 0.5 nM to approximately 1 nM, or, in any case not limited to examples, the vitamin D may be present at concentrations of approximately 0.03 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM.

[0082] In any of the embodiments described above, the method may further include measuring the expression levels of RAPTOR or RICTOR and housekeeping proteins in the cell culture input population described above, the period of which is continued until the expression level of RAPTOR or RICTOR in the produced T cells is reduced by at least 50% compared to the T cell control population, the T cell control population being produced under the same conditions as the cell culture input population that does not contain temsirolimus, IL-2 signaling inhibitors, and vitamin D. In some embodiments, the period of which is continued until the expression level of RAPTOR or RICTOR in the produced T cells is reduced by at least 50% compared to the T cell control population produced under the same conditions as the cell culture input population that does not contain temsirolimus, IL-2 signaling inhibitors, and vitamin D. For example, but not limited to these, the period of which is continued until the expression level of RAPTOR or RICTOR is reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99%, respectively, compared to the T cell control population produced under the same conditions as the cell culture input population that does not contain temsirolimus, IL-2 signaling inhibitors, and vitamin D.

[0083] In any of the above embodiments, the housekeeping protein may be actin. In some embodiments, the housekeeping protein may be GAPDH. In any of the above embodiments, the step of measuring the expression level may be performed by Western blot analysis.

[0084] In any of the embodiments described above, this period may be continued until the expression level of RAPTOR or RICTOR in the culture input population of the cells described above is reduced by at least 50% compared to a control population of T cells produced under the same conditions as the culture input population of cells that do not contain temsirolimus, IL-2 signaling inhibitors, and vitamin D. In some embodiments, the reduction in the expression level of RAPTOR or RICTOR may be at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more compared to the control population of T cells.

[0085] In any of the embodiments described above, the initial dedifferentiation period can be continued until the RNA expression pattern differs by at least 10%, and more optimally 50%, compared to control T cells cultured under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, i.e., a reduction in T cell effector molecules including, but not limited to, granzyme B, IL-10, and IFN-γ; an increase in transcription factors associated with cells in a reduced differentiated state, including, but not limited to, Nanog, KLF4, and KLF10; an increase in the expression of molecules preferentially expressed on naive T cell subsets including, but not limited to, CD127 and IL-7 receptor α chain; a reduction in transcription factors associated with Th1 differentiation, including, but not limited to, T-BET and STAT1; and a relative conservation of cell viability-promoting transcription factors including, but not limited to, HIF-1α.

[0086] In any of the embodiments described above, the initial dedifferentiation period may be continued until the RNA expression pattern differs by at least 10%, more optimally 50%, compared to control T cells cultured under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors; that is, until there is at least 10%, more preferably 50%, change in the expression of molecules indicating autophagy-affected cells. As an example, the dedifferentiated cells described above have increased p62 expression as determined by Western blot analysis compared to control T cells, and other methods for measuring autophagy may also be applied, but are not limited to examples, such as the method described in Yoshii SR, Mizushima N. Monitoring and Measuring Autophagy. International Journal of Molecular Sciences. 2017;18(9):1865.

[0087] In any of the embodiments described above, the culture medium may not contain human serum, temsirolimus, vitamin D, IL-2 signaling inhibitor, or any combination thereof, and may not be present in the culture medium at the start of culture. In such embodiments, human serum, temsirolimus, vitamin D, or IL-2 signaling inhibitor may be added to the culture medium at approximately the same time as or after inoculation of the cell culture input population.

[0088] For example, but not limited to, intravenous formulations of 1,25-vitamin D ("calcitriol") can be used. This formulation is preferred because it is fully soluble in culture media and has a 1,25-hydroxylation that is naturally produced 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 conceivable that other vitamin D receptor (VDR) ligands, including but not limited to lithocholic acid, may be substituted for calcitriol, as described in Maestro et al; Vitamin D receptor 2016: novel ligands and structural insights; Expert Opinion on Therapeutic Patents; Volume 26, 2016; issue 11.

[0089] In some embodiments, dedifferentiated T cells obtained by any of the methods of the present disclosure are provided. In some embodiments, a composition comprising a population of dedifferentiated T cells is provided. In some embodiments, at least a portion of the dedifferentiated T cells express less than 50% of RAPTOR or RICTOR compared to a control population of T cells produced under the same conditions as a culture input population of cells that do not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D. In some embodiments, the dedifferentiated T cells express less than 50% of RAPTOR or RICTOR compared to a control population of T cells produced under the same conditions as a culture input population of cells that do not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D. Without limiting ourselves to examples, the above-mentioned dedifferentiated T cells or populations of dedifferentiated T cells may express 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less RAPTOR or RICTOR compared to a control population of T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, respectively.

[0090] In some embodiments, a dedifferentiated T cell population or dedifferentiated T cells can be characterized by reduced RNA expression with respect to cell-lysing molecules, including but not limited to granzyme B, and / or cytokine molecules, including but not limited to IFN-γ, compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, or IL-2 signaling inhibitors. Such reductions may be at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% or more, but are not limited to examples.

[0091] In some embodiments, a dedifferentiated T cell population or dedifferentiated T cells can be characterized by increased RNA expression for iPSC-related transcription factors, including but not limited to Nanog, KLF4, and KLF10, and / or naive T cell-related molecules, including but not limited to the IL-7 receptor and CD127, compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, or IL-2 signaling inhibitors. Such increases may be at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% or more, but are not limited to examples.

[0092] In some embodiments, a dedifferentiated T cell population or dedifferentiated T cells can be characterized by reduced RNA expression of Th1 effector T cell-related transcription factors, including but not limited to T-Bet and STAT1, while simultaneously maintaining nearly equivalent HIF-1-α expression compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, or IL-2 signaling inhibitors. Such reductions may be at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% or more, but are not limited to examples. HIF-1-α expression may be about 20%, 15%, 10%, or 5%, or within the control T cell population, but are not limited to examples.

[0093] In some embodiments, a population of dedifferentiated T cells or dedifferentiated T cells can be characterized by increased p62 protein expression compared to a control population of T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors. Such an increase may be at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% or more, but are not limited to examples.

[0094] Human hybrid regulatory T / Th2 cells (hybrid T REG T cells (Th2 cells) and T cells from dedifferentiated T cells REG Manufacturing method In this disclosure, the inventors provide an ex vivo manufacturing process that results in the production of iTREG cells enhanced for early differentiation state combined with depletion of Th1 and Th17 polarization. This method requires a two-step process, the first of which consists of T cell dedifferentiation, and the second of which consists of iTREG cell production. The production of human iTREG cells from this dedifferentiated T cell substrate can be carried out using a novel combination of cytokines (standard iTREG use of IL-2 and TGF-β cytokines + additional use of the cytokine IL-4, which is classically associated with Th2 differentiation), and optionally using the novel drug pemetrexed described herein. In some embodiments, iTREG cells can be produced without pemetrexed. Since such cells express both TREG and Th2 molecules, the cells produced by this method are referred to as "human hybrid TREG / Th2 cells".

[0095] In some embodiments, the method comprises culturing dedifferentiated T cells of the disclosure in a culture medium containing IL-2, IL-4, and TGF-β; adding anti-CD3 / anti-CD28 coated magnetic beads in a ratio of, for example, 3:1 (beads:T cells); adding them in the above ratio (beads:T cells); and incubating the dedifferentiated T cells for a certain period of time to obtain TREG / Th2 cells. In some embodiments, the method comprises culturing a population of dedifferentiated T cells of the disclosure. The ratio of anti-CD3 / anti-CD28 beads can be varied as long as the co-stimulation is sufficient to differentiate the cells.

[0096] In some embodiments, the method comprises culturing dedifferentiated T cells having reduced RAPTOR or RICTOR expression compared to a control population of T cells produced under the same conditions without temsirolimus, an IL-2 signaling inhibitor, and vitamin D, in a culture medium containing IL-2, IL-4, and TGF-β; adding anti-CD3 / anti-CD28 coated magnetic beads in a ratio of, for example, 3:1 (beads:T cells); and incubating the dedifferentiated T cells for a certain period of time to obtain TREG / Th2 cells. In some embodiments, the method comprises culturing the population of dedifferentiated T cells of the present disclosure. The ratio of anti-CD3 / anti-CD28 beads can be varied as long as the co-stimulation is sufficient to differentiate the cells. In some embodiments, RAPTOR or RICTOR expression is normalized by a housekeeping protein, such as, for example, actin or GAPDH, but not limited to these.

[0097] In any of the above embodiments, IL-2 can be present in the culture medium at concentrations of approximately 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 approximately 100 IU / ml, 1,000 IU / ml, or 10,000 IU / ml.

[0098] In any of the above embodiments, the culture medium may further contain IL-4. In any of the above embodiments, IL-4 may be present in the culture medium at concentrations of approximately 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, but not limited to examples, when it is desirable to achieve reduced Th2 polarization, lower concentrations such as 100 IU / mL may be used.

[0099] In any of the embodiments described above, TGF-β can be present in the culture medium at a concentration of approximately 10 ng / mL. Examples, however, include concentrations of approximately 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL.

[0100] In any of the above embodiments for differentiation, the bead:T cell ratio may be 3:1. In some embodiments, an equivalent amount of an alternative form of anti-CD3 / anti-CD28 having the same effect can be used. In some embodiments, the amount of co-stimulation is sufficient to saturate the cells. In any of the above embodiments, the amount of co-stimulation may be sufficient to increase the expression of GATA3 and FOXP3 in human hybrid TREG / Th2 cells.

[0101] In any of the embodiments described above, the culture medium may further contain pemetrexed. Pemetrexed may be present in concentrations of approximately 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 values ​​such as 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, or 100 nM. In some embodiments, the culture medium does not contain pemetrexed, and pemetrexed is not added to the culture medium.

[0102] In any of the embodiments described above, the incubation period for the dedifferentiated T cells may be, but are not limited to examples, 3 to 40 days, 2 to 20 days, 3 to 10 days, 3 to 6 days, 6 to 10 days, 10 to 40 days, 10 to 20 days, 10 to 15 days, 15 to 40 days, 20 to 40 days, 30 to 40 days, 20 to 30 days, or 15 to 30 days, or 15 to 20 days. In some embodiments, but are not limited to examples, shorter culture intervals, such as 3 to 10 days, may be considered for hybrid Th2 / TREG cells in a very limited differentiation state.

[0103] This disclosure also relates to methods and TREG cells produced by any of the above methods without using IL-4.

[0104] In some embodiments, TREG or TREG / Th2 cells produced by the method of the present disclosure may have increased flow cytometry expression of at least one of the following compared to control Th1 / Tc1 cells: CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, and OX40. In some embodiments, 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%, or at least 90% or more, but are not limited to examples.

[0105] In some embodiments, TREG or TREG / Th2 cells produced by the method of the present disclosure may have reduced secretion of inflammatory cytokines compared to control Th1 / Tc1 cells. Such cytokines may include, but are not limited to, IFN-γ and TNF-α. In some embodiments, but are not limited to, 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%, or at least 90% or more.

[0106] In some embodiments, TREG or TREG / Th2 cells produced by the method of the present disclosure may have reduced TBET, increased FOXP3 expression, and / or increased IL-4 secretion and increased GATA3 expression compared to control Th1 / Tc1 cells. In some embodiments, 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%, or at least 90% or more, but are not limited to examples.

[0107] In some embodiments, a population of TREG or TREG / Th2 cells may have at least 5% of CD4+ or CD8+ cells expressing GATA3. Without limiting to examples, a population of TREG or TREG / Th2 cells 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 cells express GATA3 is determined by flow cytometry. In some embodiments, T REG or T REG / Th2 cell populations are T REG or T REG Compared to the characteristics of the control T cell population of the Th2 cell population produced, CD4 cells expressing GATA3 + or CD8 + The frequency of increased T cells can be indicated. In some embodiments, the increased frequency may be greater than 50%. Examples, but not limited to, the increase may be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000%, or greater.

[0108] In some embodiments, a population of TREG or TREG / Th2 cells may have at least 5% of CD4+ or CD8+ cells expressing FoxP3. Without limiting to examples, a population of TREG or TREG / Th2 cells may have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 45% of CD4+ or CD8+ T cells expressing FoxP3. In some embodiments, whether cells express FoxP3 is determined by flow cytometry. In some embodiments, T REG or T REG / Th2 cell populations are T REG or T REG Compared to the characteristics of the control T cell population of the Th2 cell population produced, CD4 cells expressing FOXP3 + or CD8 + The frequency of increased T cells can be indicated. In some embodiments, the increased frequency may be greater than 50%. Examples, but not limited to, the increase may be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000%, or greater.

[0109] In some embodiments, a population of Tregs or Treg / Th2 cells can have at least 10% of CD4+ or CD8+ cells that express CD73. By way of example and not limitation, a population of Tregs or Treg / Th2 cells can have at least 10%, at least 15%, at least 20%, or at least 25% of CD4+ T cells that express CD73. By way of example and not limitation, a population of Tregs or Treg / Th2 cells can have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80% of CD8+ T cells that express CD73. In some embodiments, whether a cell expresses CD73 is determined by flow cytometry. In some embodiments, T REG or T REG / Th2 cell population can show an increased frequency of CD4 REG or CD8 REG T cells that express CD73 as compared to the control T cell population characteristics of T cells produced by the T + or CD8 + cell population. In some embodiments, the increased frequency 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.

[0110] In some embodiments, a population of Tregs or Treg / Th2 cells can have at least 10% of CD4+ or CD8+ cells that express CD103. By way of example and not limitation, a population of Tregs or Treg / Th2 cells can have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of CD4+ or CD8+ T cells that express CD103. In some embodiments, whether a cell expresses CD103 is determined by flow cytometry. In some embodiments, the REG or T REG / Th2 cell population can show an increased frequency of CD4 REG or T REG cells that express CD103 as compared to the control T cell population characteristics of the T cells produced by the T + or CD8 + T cell population. In some embodiments, the increased frequency 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.

[0111] In some embodiments, the T REG or T REG / Th2 cell population can have at least 5% of CD4 + or CD8 + cells that express both FOXP3 and GATA3 when measured by flow cytometry. By way of example and not limitation, the T REG or T REG / Th2 cell population can have at least 5%, 10%, 20%, 30%, 40%, or 50% of CD4 + or CD8 + cells that express both FOXP3 and GATA3.

[0112] In some embodiments, a population of TREG or TREG / Th2 cells may have at least 20% of CD4+ or CD8+ cells expressing CD150. Without limiting to examples, a population of TREG or TREG / Th2 cells 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 can obtain an increased frequency of CD150-expressing cells compared to a control population of T cells incubated without exposure to an mTOR inhibitor. In some embodiments, whether cells express CD150 is determined by flow cytometry. In some embodiments, T REG or T REG / Th2 cell populations are T REG or T REG Compared to the characteristics of the control T cell population, the population of Th2 cells produced CD4 cells expressing CD150. + or CD8 + The frequency of increased T cells can be indicated. In some embodiments, the increased frequency may be greater than 50%. Examples, but not limited to, the increase may be 50%, 100%, 200%, 300%, 500%, 1000%, 2000%, 3000%, or greater.

[0113] In some embodiments, a population of TREG or TREG / Th2 cells can express at least 5 pg / mL / 1 × 10⁶ cells / day of IL-4 after co-stimulation with anti-CD3 / anti-CD28 beads in a bead:T cell ratio of 3:1. Examples, but not limited to, a population of TREG or TREG / Th2 cells can express at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 pg / mL / 1 × 10⁶ cells / day. 6It can express IL-4 in cells.

[0114] In some embodiments, a population of TREG or TREG / Th2 cells can express at least 100 pg / mL / 1 × 10⁶ cells / day of IL-2 after co-stimulation with anti-CD3 / anti-CD28 beads in a bead:T cell ratio of 3:1.

[0115] In some embodiments, a population of TREG or TREG / Th2 cells can express at least 100 pg / mL / 1 × 10⁶ cells / day of IFN-γ or GM-CSF after co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio.

[0116] In some embodiments, populations of TREG or TREG / Th2 cells can express less than 10 pg / mL / 1 × 10⁶ cells / day of TNF-α or IL-17 after co-stimulation with anti-CD3 / anti-CD28 beads in a bead:T cell ratio of 3:1.

[0117] In some embodiments, TREG or TREG / Th2 cells can express both GATA3 and FOXP3. In some embodiments, TREG or TREG / Th2 cells can express GATA3, FOXP3, CD103, and CD73. In some embodiments, a population of TREG or TREG / Th2 cells can 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.

[0118] In any of the embodiments described above, TREG or TREG / Th2 cells, or population thereof, may have at least one or any combination of the above characteristics, insofar as the characteristics are interchangeable.

[0119] Inducible controllability T(iT REG ) A method for treating ALS using cells In this protocol, the inventors have developed the iT of ALS. REG Pentostatin plus cyclophosphamide regimens are used as immunodepletion and immunosuppression methods to enhance cell therapy. This PC regimen may have a direct beneficial effect due to its ability to deplete and suppress Th1 immune cells associated with the ALS pathogen. In addition, the PC regimen is more effective in iT REG It functions as a host modulator to increase the immune T cell space for cell therapy. Specifically, in this protocol, the inventors reduced the dosage of the PC regimen to mitigate any potential adverse effects of the regimen in a new ALS patient population. In this protocol, the inventors set the starting dose of pentostatin to 4 mg / m². 2 From 1 mg / m² 2 The number of pentostatin infusions was reduced from the previous value of four infusions per cycle to the current protocol value of one infusion per cycle, and the initial cyclophosphamide dose was reduced from 200 mg per day to 100 mg per day. Secondly, the inventors reduced the intensity of the PC regimen in terms of the stated target regarding the degree of immunodepletion conferred by the PC regimen. Because a more cautious approach is mandated in the ALS patient population, the current protocol PC regimen attempted to reduce the ALC count to a more conservative level, i.e., less than 750 cells per microliter. In general, this level of immunodepletion is not associated with severe long-term immunodeficiency in terms of a high opportunistic infection rate.

[0120] Without being constrained by theory, PC regimens are expected to deplete and suppress the Th1 / Tc1-type adaptive immune subset involved in the progression of the ALS pathogen. However, this therapy does not address the underlying primary events in ALS, namely, misfolded RNA species, insufficient autophagy for clearance of harmful RNA / DNA products, and subsequent RNA / DNA activation of congenital inflammation at the P2X7 receptor-driven NLRP3 inflammasome level. Such inflammasome activation, which has been shown to be functional in ALS models, subsequently drives IL-1-β activation, one of the earliest and most potent pro-inflammatory signals that drives adaptive T-cell inflammation, primarily mediated by the Th1 / Tc1 subset. Indeed, NLRP3 inhibition has recently been proposed to represent a novel approach to 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. Patients with ALS may, in some cases, have elevated levels of human endogenous retroviral-K (HERV-K), which drives disease development in model systems, regulated by TDP-43 accumulation, a major mechanistic component of ALS. To translate this biology clinically, a clinical trial (NCT02437110) has been initiated to evaluate an HIV antiviral cocktail of darunavir, ritonavir, raltegravir, and zidovudine. The NRTI molecule lamivudine (3TC) has also been described as inhibiting 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 three drug regimens—lamivudine, zidovudine, and abacavir—to reduce inflammation in patients with Aicardi-Goutieres syndrome (AGS), a disease that mimics the congenital inflammatory events in ALS, namely the accumulation of intracellular RNA species, activation of inflammatory pathways, and the resulting generation of systemic Th1-driven inflammation.Lamivudine is characterized as a potent inhibitor of the NLRP3 inflammasome, and since it is desirable to generate a treatment plan that is well-tolerated in the ALS patient population, the inventors chose to pursue lamivudine monotherapy as a non-invasive receptive therapy (NRTI) in this protocol.

[0121] Therefore, without being constrained by theory, a sequential strategy of depleting and suppressing the first Th1 / Tc1 response (via PC regimens) and then controlling the driving force inflammasome activation is expected to represent a novel approach to provide sustainable modulation of the complex neuroinflammatory network involved in ALS. The use of lamivudine in ALS treatment platforms is expected to be iT REG This could also be beneficial for the next step of further integrating cell therapy into the platform, namely, three treatment modes (pentostatin / cyclophosphamide, lamivudine, and iT). REG Each of these cells operates at least partially by shunting the transport of ATP away from the P2X7-driven NLRP3-mediated inflammasome toward the immunosuppressive molecule, adenosine. Firstly, pentostatin increases adenosine by inhibiting adenosine deaminase, thereby preventing the conversion of adenosine to inosine. Secondly, lamivudine is a known inhibitor of P2X7, thereby directly inhibiting the inflammasome. Thirdly, iT REG Cells provide CD39 and CD73-mediated ectonucleotidase activity that processes ATP toward adenosine. Regarding this final process, it is important to note that microglial cells essentially utilize CD39 and CD73 to suppress neuroinflammation.

[0122] In summary, these data provide evidence that the primary neurodegenerative process in ALS gives rise to a secondary inflammatory response, which on the one hand can still accelerate disease progression, but on the other hand can regulate inflammasome activation, deplete and suppress the Th1 / Tc1 subset, and T REGThis provides evidence that therapeutic interventions can involve multiple steps, including the promotion of a subset of T cells. Given this information, there is great interest in evaluating immunomodulatory therapies in ALS patients. Optimal control of neuroinflammation in ALS patients requires a three-step treatment approach addressing each of the above components: (1) control of innate inflammasome activation (by lamivudine administration, as described below); (2) reduction of pre-existing Th1 inflammatory cells (by pentostatin / cyclophosphamide regimens, as described in more detail below); and (3) iT cell transfer by adoptive T cell transplantation. REG The inventors hypothesized that cell activation is necessary. There are several reasons why such a combination approach may be necessary. First, if the underlying inflammasome activation is not addressed by maintenance therapy, host modulation therapy and T REG The immunological and therapeutic gains achieved during cell therapy are likely to be eroded by the underlying primary neurodegenerative processes. Secondly, optimized T1 therapy for hosts with uncontrolled Th1-driven inflammation. REG Even the injection of a single cell population represents a difficult immunological challenge for many reasons: existing Th1 cells are adopted into T cells. REG Cells can express differentiation plasticity, thereby providing protection. REG The population can be transformed into a pathogenic subset that can contribute to the development of disease. Thirdly, the efficacy of adoptive T cell populations is intricately linked to the degree of immune T cell space, which can be largely defined by the presence of T cell growth factors such as IL-7 and IL-15, and the creation of such an immune space is produced by host-prepared regimens including pentostatin + cyclophosphamide (PC) regimens, which have been used for decades in allogeneic hematopoietic stem cell transplantation and are now being used in the field of adoptive T cell transplantation for cancer treatment. Notably, T cells in neurodegenerative or autoimmune diseases REG Previous clinical trials involving cell therapy did not incorporate host preparation regimens such as PC regimens.

[0123] In some embodiments, the method comprises subjecting the subject to one or more primary treatment cycles, each of which involves administering pentostatin to the subject and / or cyclophosphamide to the subject and administering a therapeutically effective amount of manufactured T REG This includes subjecting a cell-containing composition to one or more immunotherapy treatment cycles, which involves administering the composition to the subject described above. Those skilled in the art can determine the therapeutically effective dose by methods known in the art and disclosed herein.

[0124] In the embodiments described above, each of the one or more immunotherapy treatment cycles further comprises administering a nucleoside reverse transcriptase inhibitor to the subject. The nucleoside reverse transcriptase inhibitor may be an NLRP3 inflammasome inhibitor, or the nucleoside reverse transcriptase inhibitor may be lamivudine. Each of the one or more immunotherapy treatment cycles may further comprise 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 days 1 and 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 days 1, 2, 3, 4, and 5 of each of the one or more immunotherapy treatment cycles. The above method may include two or more immunotherapy treatment cycles, each of which may be separated by any value between 0-4 weeks, 0-3 weeks, 0-2 weeks, 0-1 week, and, not limited to, 0 weeks, 1 week, 2 weeks, 3 weeks, or 4 weeks, as shown above. Each of the above one or more immunotherapy treatment cycles may be 18 weeks long. Each of the above one or more immunotherapy treatment cycles may further include administering an adenosine receptor modulator to the subject, as disclosed in U.S. Patent No. 9,326,983, incorporated herein by reference, lamivudine, zidovudine, stabudine, cordycepin, azidothymidine, abacavir, [ka] Other nucleoside reverse transcriptase inhibitors may be used, including but not limited to their chemical derivatives, pharmaceutically acceptable salts, and combinations thereof.

[0125] In the embodiments described above, the one or more primary treatment cycles may be 2 to 5 weeks, 2 to 4 weeks, 2 to 3 weeks, 3 to 4 weeks, or 4 to 5 weeks, and may be any value in between, such as 2 weeks, 3 weeks, 4 weeks, or 5 weeks, and may be any value in between, such as 2 weeks, 3 weeks, 4 weeks, or 5 weeks, and may be any value in between, and may be any value in between, and may be any value in between, and may be any value in between, such as 0 to 1 week or 1 to 2 weeks, and may be any value in between, such as 1 day, 1 week, or 2 weeks, and may be any value in between

[0126] In the embodiments described above, the method may further include collecting peripheral lymphocytes from the subject before one or more primary treatment cycles. After collecting peripheral lymphocytes from the subject, the method may include the prepared T REG The method may further include culturing the above peripheral lymphocytes to obtain the cells. The method may further include collecting peripheral lymphocytes from the above subject after one or more primary treatment cycles. The method may further include collecting peripheral lymphocytes from the above subject after one or more primary treatment cycles, and then producing the above T REG The method may further include culturing the peripheral lymphocytes described above to obtain the cells. The method may further include measuring the absolute lymphocyte count (ALC) in the subject after each of the one or more primary treatment cycles described above, and proceeding to one or more immunotherapy treatment cycles described above if the ALC is less than 750 per μl. In some embodiments, the target ALC value may vary and may be, for example, 0, 250, 500, 750, 1000, 1250, or 1500 cells per microliter.

[0127] In the embodiments described above, the first cycle of one or more immunotherapy treatment cycles is separated from the last cycle of one or more primary treatment cycles by 0 to 2 weeks. Each of the two or more immunotherapy treatment cycles is separated by 0 to 1 week or 1 to 2 weeks, and by any value between 0 weeks, 1 week, or 2 weeks, but not limited to examples.

[0128] In the embodiments described above, the dose of pentostatin is, but is not limited to examples, 0.5 mg / m². 2 ~4mg / m 2 , 1 mg / m² 2 ~4mg / m 2 , 2 mg / m² 2 ~4mg / m 2 The dosage, and, as an example but not limited to, 0.5 mg / m² 2 , 1 mg / m² 2 , 1.5 mg / m² 2 , 2 mg / m² 2 , 2.5 mg / m² 2 , 3 mg / m² 2 3.5 mg / m² 2 , and 4 mg / m² 2 The values ​​can be any values ​​between these. The pentostatin described above can be administered on any day of each of the one or more primary treatment cycles described above. As an example, but not limited to, pentostatin can be administered to the subject on day 1, or on days 1 and 4, of each of the one or more primary treatment cycles described above.

[0129] In the embodiments described above, cyclophosphamide can be administered to the subject in doses ranging from 50 mg to 400 mg. While not limited to examples, doses of cyclophosphamide can be between 50, 100, 150, 200, 250, 300, 350, or 400 mg and any combination of 400, 350, 300, 250, 200, 150, 100, or 50 mg, and any value between 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg. While not limited to examples, cyclophosphamide can be administered on days 1, 2, and 3, or on days 1, 2, 3, 4, and 5, for each of the one or more primary treatment cycles described above.

[0130] In the embodiments described above, the pentostatin and cyclophosphamide may be administered to the subject in a single composition. The single composition may be administered intravenously to the subject. The steps of administering the pentostatin and cyclophosphamide to the subject may include administering a first composition containing pentostatin to the subject and administering a second composition containing cyclophosphamide to the subject.

[0131] In the above embodiment, the lamivudine can be administered to the subject at a dose of 150 mg to 150 mg twice daily.

[0132] In the above embodiment, the manufactured T REG The cells, during each of the one or more immunotherapy treatment cycles described above, include, but are not limited to, 1 × 10¹⁶ cells per kg of the subject's body weight. 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 Cells, 2 × 10⁶ cells per kg of body weight of the subject 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 Cells, 3 x 10⁶ cells per kg of body weight of the subject 6 Cells ~ 5 × 10 per kg of body weight of the subject 6Cells, 4 x 10 per kg of body weight of the subject 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 Cells, 1 x 10⁶ cells per kg of body weight of the subject 6 Cells ~ 4 x 10 per kg of body weight of the subject 6 Cells, 1 x 10⁶ cells per kg of body weight of the subject 6 Cells ~ 3 x 10 per kg of body weight of the subject 6 Cells, 1 x 10⁶ cells per kg of body weight of the subject 6 Cells ~ 2 × 10⁻¹⁶ cells per kg of body weight of the subject 6 Cells, and any values ​​between them, but not limited to the example, 1 × 10 per kg of body weight of the subject. 6 Cells, 2 × 10⁶ cells per kg of body weight of the subject 6 Cells, 3 x 10⁶ cells per kg of body weight of the subject 6 Cells, 4 x 10 per kg of body weight of the subject 6 Cells, or 5 × 10⁶ cells per kg of body weight of the subject. 6 It can be administered to the above-mentioned targets in doses such as cells. Examples, though not limited to, include, per injection, manufactured T REG Approximately 1 x 10⁻¹⁶ cells 6 ~About 200×10 6 Cells can be administered. Further examples, though not limited to, include manufactured T cells. REG Approximately 1 x 10⁶ cells per injection. 6 ~About 200×10 6 , 10×10 6 ~About 200×10 6 , 50×10 6 ~About 200×10 6 , 100×10 6 ~About 200×10 6 , at least 1 × 10 6 , 10×10 6 , 50×10 6 , 100×10 6 , or 200×10 6 Cells can be administered. In some embodiments, approximately 40 × 10 6 Cells / injections may be administered. In some embodiments, approximately 120 × 10 6 Cells / injections may be administered. The above manufactured TREG The cells can include central memory versus effector memory cell ratios selected from 1:1, 3:1, 10:1, 1:3, and 1:10. REG The above composition containing cells is normal T REG It can further contain cells. The above manufactured T REG The cells can be administered to the subject on day 8 of each of the one or more immunotherapy treatment cycles described above. In some embodiments, iT REG and nT REG These can be administered in combination with the target.

[0133] In some embodiments, the 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, 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 manufactured T REG This may include administering cells to the above-mentioned subjects.

[0134] In the above embodiment, the first treatment cycle may be 14 days long. The step of administering pentostatin to the subject may be performed on day 1 of the first treatment cycle. Pentostatin may be administered at a dose of 1 mg / m² during the first treatment cycle. 2The above-mentioned subjects may be administered the above-mentioned dose. Cyclophosphamide may be administered to the above-mentioned subjects at a dose of 100 mg during the above-mentioned first treatment cycle. The above-mentioned step of administering cyclophosphamide to the above-mentioned subjects may be repeated during the above-mentioned first treatment cycle, and the above-mentioned step of administering cyclophosphamide to the above-mentioned subjects may be performed on days 1, 2, and 3 of the above-mentioned first treatment cycle, although this is not an example.

[0135] In the above embodiment, the second treatment cycle may be 14 days long. The step of administering pentostatin to the subject during the second treatment cycle may be performed on day 1 of the second treatment cycle. Pentostatin is administered at a dose of 2 mg / m² during the second treatment cycle. 2 The above dose can be administered to the above subjects. The above step of administering cyclophosphamide to the above subjects during the second treatment cycle can be repeated during the second treatment cycle. The above step of administering cyclophosphamide to the above subjects during the second treatment cycle can be performed on days 1, 2, and / or 3 of the second treatment cycle, although this is not an example. Cyclophosphamide can be administered to the above subjects at a dose of 100 mg during the second treatment cycle.

[0136] In the embodiments described above, the subject may be subjected to 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. Each of the two or more additional treatment cycles may be spaced 0 to 1 week, or 1 to 2 weeks apart, for example, any value between 0 weeks, 1 week, or 2 weeks, but not limited to examples. 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. Pentostatin may be administered at a dose of 2 mg / m² during each of the one or more additional treatment cycles. 2 The above dose may be administered to the above subjects. The above step of administering cyclophosphamide to the above subjects may be repeated between each of the above one or more additional treatment cycles. The above step of administering cyclophosphamide to the above subjects between each of the above one or more additional treatment cycles may be performed on days 1, 2, 3, 4, and / or 5 of each of the above one or more additional treatment cycles. Cyclophosphamide may be administered to the above subjects in a dose of 100 mg to 200 mg between each of the above one or more additional treatment cycles. The above additional cycle further comprises measuring the creatinine clearance (CrCl) of the above subjects before administering pentostatin to the above subjects between each of the above one or more additional treatment cycles, and adjusting the dose of pentostatin administered to the above subjects based on the above CrCl, where CrCl > 60 mL / min / 1.73 m 2 In this case, pentostatin is 2 mg / m². 2 It is administered at 60 mL / min / 1.73 m 2 >CrCl>30mL / min / 1.73m 2 In this case, pentostatin is 1 mg / m². 2 It was administered as follows: CrCl < 30 mL / min / 1.73 m 2In such a case, pentostatin is not administered. The above additional cycles may further include measuring the absolute neutrophil count (ANC) before administering cyclophosphamide to the subject during each of the above one or more additional treatment cycles, and adjusting the dose of cyclophosphamide administered to the subject based on the ANC. When ANC > 1000 per microliter, cyclophosphamide is administered at a dose of 100 mg. When ANC is 500 - 999 per microliter, cyclophosphamide is administered at a dose of 50 mg. When ALC < 50 per microliter or ANC < 500 per microliter, cyclophosphamide is not administered. The above one or more additional treatment cycles may include at least two additional treatment cycles. The final cycle of the above at least two additional treatment cycles includes measuring the absolute lymphocyte count (ALC) and the absolute neutrophil count (ANC) before administering cyclophosphamide to the subject during the above final cycle of the above at least two treatment cycles, and adjusting the dose of cyclophosphamide administered to the subject based on the ALC and ANC. Cyclophosphamide can be administered at a dose of 200 mg when ALC > 1250 / microliter. Cyclophosphamide can be administered at a dose of 100 mg when ANC > 1000 / microliter and 750 < ALC < 1250 / microliter. Cyclophosphamide can be administered at a dose of 50 mg when ANC is 500 - 999 / microliter. Cyclophosphamide may not be administered when ANC < 500 / microliter and / or ALC < 750 / microliter.

[0137] In the embodiments described above, each of the above treatment cycles, the above second treatment cycle, and the above one or more additional treatment cycles may be spaced 0 to 2 weeks apart. However, as an example, each of the above treatment cycles, the above second treatment cycle, and the above one or more additional treatment cycles may be spaced 0 to 1 week, or 1 to 2 weeks apart, or any value between 0 weeks, 1 week, or 2 weeks, but as an example, there may be spacings of 0 weeks, 1 week, or 2 weeks apart.

[0138] In the embodiments described above, the method may further include measuring the creatinine clearance (CrCl) of the subject before administering pentostatin to the subject in the first treatment cycle described above, and adjusting the dose of pentostatin administered to the subject based on the CrCl, where CrCl > 60 mL / min / 1.73 m 2 In this case, pentostatin is 1 mg / m². 2 It is administered at 60 mL / min / 1.73 m 2 >CrCl>30mL / min / 1.73m 2 In this case, pentostatin is administered at 0.5 mg / m2, and CrCl < 30 mL / min / 1.73 m 2 In this case, pentostatin is not administered.

[0139] In the embodiments described above, the method may further include measuring the absolute neutrophil count (ANC) before administering cyclophosphamide to the subject in the first treatment cycle described above, and adjusting the dose of cyclophosphamide administered to the subject based on ALC and ANC, where if ANC > 1000 per microliter, cyclophosphamide is administered at a dose of 100 mg; if ANC is between 500 and 999 per microliter, cyclophosphamide is administered at a dose of 50 mg; and if ANC < 500 per microliter, cyclophosphamide is not administered.

[0140] In the embodiments described above, the method may further include measuring the creatinine clearance (CrCl) of the subject before administering pentostatin to the subject in the second treatment cycle described above, and adjusting the dose of pentostatin administered to the subject based on the CrCl, where CrCl > 60 mL / min / 1.73 m 2 In this case, pentostatin is 2 mg / m². 2 It is administered at 60 mL / min / 1.73 m 2 >CrCl>30mL / min / 1.73m 2 In this case, pentostatin is 1 mg / m². 2 It was administered as follows: CrCl < 30 mL / min / 1.73 m 2 In this case, pentostatin is not administered.

[0141] In the embodiments described above, the method may further include measuring the absolute neutrophil count (ANC) before administering cyclophosphamide to the subject in the second treatment cycle described above, and adjusting the dose of cyclophosphamide administered to the subject based on the ANC, such that if the ANC > 1000 per microliter, cyclophosphamide may be administered at a dose of 100 mg; if the ANC is between 500 and 999 per microliter, cyclophosphamide may be administered at a dose of 50 mg; and if the ANC < 500 per microliter, cyclophosphamide is not administered.

[0142] In the embodiments described above, the method may further include measuring the absolute lymphocyte count (ALC) in the subject before each of the one or more additional treatment cycles described above, and adjusting the treatment of the subject based on the ALC, wherein if ALC < 750 per microliter, the subject is subjected to a maintenance treatment cycle, which includes subjecting the patient to one or more immunotherapy treatment cycles, and if ALC > 750 per microliter, the patient is subjected to one or more additional treatment cycles described above.

[0143] In the embodiments described above, the method may further include measuring the absolute lymphocyte count (ALC) in the subject before each of the one or more additional treatment cycles described above, and adjusting the treatment of the subject based on the ALC, wherein if ALC < 750 per microliter, the subject is given a maintenance treatment cycle including administration of a nucleoside reverse transcriptase inhibitor, without administering any further one or more additional treatment cycles before the maintenance treatment cycle, and if ALC > 750 per microliter, the subject is continued to one or more additional treatment cycles.

[0144] In the embodiments described above, each of the one or more immunotherapy treatment cycles may be 18 weeks long, but is not limited to examples. In some embodiments, immunotherapy treatment cycles may be spaced 0 to 4 weeks apart. Immunotherapy treatment cycles may be repeated, including indefinitely. Repeating immunotherapy cycles may occur according to the regimen or in case of relapse. Immunotherapy cycles may occur 1 to 4 times per year, but is not limited to examples. Pentostatin is administered at a dose of 2 mg / m² on days 1 and 4 of each of the one or more immunotherapy treatment cycles described above. 2The above subjects may be administered the above dose. Cyclophosphamide may be administered to the above subjects in a dose of 100 mg on day 1, day 2, day 3, day 4, and / or day 5 of each of the above one or more immunotherapy treatment cycles. Each of the above one or more immunotherapy treatment cycles may further include administering a nucleoside reverse transcriptase inhibitor to the above subjects. The above nucleoside reverse transcriptase inhibitor may be lamivudine. The above lamivudine may be administered to the above subjects in a dose of 150 mg per day to 150 mg twice daily during each of the above one or more immunotherapy treatment cycles. Each of the above one or more immunotherapy treatment cycles may further include measuring the creatinine clearance (CrCl) of the above subjects during each of the above one or more immunotherapy treatment cycles and adjusting the dose of lamivudine administered to the above subjects based on the above CrCl, where CrCl > 50 mL / min / 1.73 m 2 In this case, lamivudine is administered twice daily at a dose of 150 mg, with a flow rate of 50 mL / min > CrCl > 30 mL / min / 1.73 m 2 In this case, lamivudine is administered at 150 mg, and CrCl < 30 mL / min / 1.73 m 2 In this case, lamivudine is not administered.

[0145] In the above embodiment, the method involves producing a therapeutically effective amount of T REG This may include administering cells to the subjects mentioned above.

[0146] In the above embodiment, the manufactured T REG The cells can be administered to the subject on day 8 of each of the above one or more immunotherapy treatment cycles. Examples, but not limited to, manufactured T cells. REG The cells can be administered to the subject on any day of the immunotherapy cycle described above, for example, on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0147] In the above embodiment, the manufactured T REGThe number of cells is 1 × 10⁶ per kg of the subject's body weight. 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 In cells, and this is not limited to the example, 1 x 10⁻¹⁶ per kg of the subject's body weight. 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 Cells, 2 × 10⁶ cells per kg of body weight of the subject 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 Cells, 3 x 10⁶ cells per kg of body weight of the subject 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 Cells, 4 x 10 per kg of body weight of the subject 6 Cells ~ 5 × 10 per kg of body weight of the subject 6 Cells, 1 x 10⁶ cells per kg of body weight of the subject 6 Cells ~ 4 x 10 per kg of body weight of the subject 6 Cells, 1 x 10⁶ cells per kg of body weight of the subject 6 cells ~3×10 6 Cells, 1 x 10⁶ cells per kg of body weight of the subject 6 Cells ~ 2 × 10⁻¹⁶ cells per kg of body weight of the subject 6 Cells, and, as an example, 1 × 10⁶ per kg of body weight of the subject. 6 Cells, 2 × 10⁶ cells per kg of body weight of the subject 6 Cells, 3 x 10⁶ cells per kg of body weight of the subject 6 Cells, 4 x 10 per kg of body weight of the subject 6 Cells, or 5 × 10⁶ cells per kg of body weight of the subject. 6 It can be administered at any value between cells, etc.

[0148] Each of the one or more treatment cycles described above may further include administering pentostatin to the subject and / or cyclophosphamide to the subject. Pentostatin is administered at a dose of 1 mg / m². 2 ~2mg / m 2 The dosage, and, as an example but not limited to, 1 mg / m² 2 , 1.5 mg / m² 2 , or 2 mg / m² 2The above subjects may be administered any value between 100 mg and 200 mg. The above dose of cyclophosphamide may be any value between 100 mg, 150 mg, or 200 mg, but is not limited to examples. The above pentostatin may be administered to the above subjects on days 1 and 4 of each of the above one or more treatment cycles. The above cyclophosphamide may be administered to the above subjects on days 1, 2, 3, 4, and / or 5 of each of the above one or more treatment cycles. The above subjects may have been previously treated with pentostatin and cyclophosphamide. Each of the above one or more treatment cycles may further include administering a nucleoside reverse transcriptase inhibitor to the above subjects. The above nucleoside reverse transcriptase inhibitor may be an NLRP3 inflammasome inhibitor. The above nucleoside reverse transcriptase inhibitor may be lamivudine. Each of the above one or more treatment cycles may be spaced 4 weeks apart.

[0149] In the above embodiment, this method is used with the above manufactured T REG Simultaneously with the cells, normal T cells were also found in the above target. REG The method may further include administering cells. In the above embodiments, the method may further include collecting peripheral lymphocytes from the subject before one or more of the above treatment cycles. In the above embodiments, after collecting peripheral lymphocytes from the subject, the method cultures the peripheral lymphocytes to produce the above-produced T REG This can further include obtaining cells. [Examples]

[0150] The following examples illustrate the methods of the Disclosure and the resulting dedifferentiation and iT REG Or provided to better illustrate redifferentiated T cells. These examples are not intended to limit or otherwise modify the scope of the methods, cells, and compositions disclosed herein.

[0151] Example 1: The combination of vitamin D and temsirolimus reduces T cell effector molecules. The inventors directly evaluated the individual effects of vitamin D, mTOR inhibition (using parenteral forms of rapamycin and temsirolimus), and the combination of vitamin D and temsirolimus on human T cell effector molecule expression (see Figure 1).

[0152] Figures 1A-1D show the relationship between the combination of vitamin D and temsirolimus and human CD4 + and CD8 + This illustrates the reduction of effector molecule expression in cells. For columns #2 to #5, T cells were subjected to a 3-day dedifferentiation interval, including low levels of anti-CD3 / anti-CD28 costimulation (beads to T cells ratio; 1:3), high doses of temsirolimus (1 μM), vitamin D (0.1 or 1.0 nM), and culture in X-Vivo 20 medium. Column 1 represents the control culture (without temsirolimus, without vitamin D, using a 3:1 beads to T cells ratio, and containing type I polarizing cytokine IFN-α (20,000 IU / mL; unless otherwise specified, this amount is used for the control culture in Examples 1 to 11 below)). The second column represents cultures with a low bead-to-T cell ratio and temsirolimus, but without vitamin D. In contrast, the third column represents cultures 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) combined with temsirolimus. At the end of the dedifferentiation interval, cells were harvested, RNA was isolated, and RNA expression analysis was performed using the Luminex Quantigene method. All results shown represent relative RNA expression, and the results are normalized to a value of 1.0 for the Th1 / Tc1 control culture.

[0153] A 3-day culture interval was used, containing low levels of T cell costimulation (a 1:3 ratio of anti-CD3 / anti-CD28 beads to T cells; the typical ratio used in the literature is the reverse, 3:1), temsirolimus (1 μM), either 0.1 or 1.0 nM of vitamin D, or a combination of temsirolimus and high-dose vitamin D. After culturing, RNA was collected and effector molecule expression levels were compared to control cultures.

[0154] As shown in Figure 1A, various cultures exhibited similar RNA expression of housekeeping control genes, including GAPDH. In contrast, compared to control Th1 / Tc1 cell cultures that did not receive vitamin D or temsirolimus, the addition of temsirolimus, vitamin D, or a combination of temsirolimus and vitamin D to the culture resulted in decreased RNA expression of T cell effector molecules, including the cytotoxic molecule granzyme B (Figure 1B) and the cytokine molecules IL-10 (Th2 cytokine, Figure 1C) and IFN-γ (Th1 cytokine, Figure 1D). Therefore, granzyme B and IFN-γ as dedifferentiation markers indicate that vitamin D is effective at concentrations of 0.1–1.0 nM. Temsirolimus at a dose of 1 μM acted beneficially as a dedifferentiation agent alone (column 2, reduction of granzyme B and IFN-γ) and did not suppress the effect of vitamin D when used in combination (column 5).

[0155] Therefore, using low levels of co-stimulation (a 1:3 ratio of anti-CD3 / anti-CD28 beads to T cells) and a short 3-day culture interval, the addition of temsirolimus, vitamin D, or a combination of temsirolimus and vitamin D can reduce both Th1 and Th2 cytokine effector mechanisms as well as cytotoxic effector mechanisms.

[0156] Example 2: The combination of vitamin D and temsirolimus alters key transcription factors associated with dedifferentiation. The inventors also evaluated the effects of vitamin D, temsirolimus, or a combination thereof on the expression of major transcription factors after low levels of co-stimulation.

[0157] Figures 2A to 2D show that the combination of vitamin D and temsirolimus is effective for human CD4 + and CD8 + This example illustrates the increased expression of stem cell-related transcription factors and the atomic T cell molecule IL-7 receptor-α in T cells. The combination of vitamin D and temsirolimus increases the expression of human CD4 + and CD8 + This illustrates the reduction of effector molecule expression in T cells. For columns #2 to #5, T cells were subjected to a 3-day dedifferentiation interval, including low levels of anti-CD3 / anti-CD28 costimulation (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 medium. The first column represents the control culture (no temsirolimus, no vitamin D, use of a 3:1 bead-to-T cell ratio, and inclusion of type I polarizing cytokine IFN-α). The second column represents the culture with a low bead-to-T cell ratio and temsirolimus but without vitamin D, and in contrast, the third column represents the culture with vitamin D (0.1 nM) but without temsirolimus. The fourth column represents cultures containing high-dose ("HD") vitamin D (1.0 nM) but without temsirolimus. The fifth column represents cultures containing both high-dose vitamin D (1.0 nM) and temsirolimus. At the end of the dedifferentiation interval, cells were collected, RNA was isolated, and RNA expression analysis was performed using the Luminex Quantigene method. All results shown represent relative RNA expression, and the results are normalized to a value of 1.0 for the Th1 / Tc1 control culture.

[0158] As shown in Figure 2A, temsirolimus or the temsirolimus + vitamin D combination results in upregulation of the Nanog transcription factor, which is recognized as one of the few essential factors necessary for the dedifferentiation of somatic cells toward 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 iPSC state.

[0159] Therefore, using low levels of co-stimulation, temsirolimus increases the iPSC transcription factor Nanog, and this temsirolimus-promoting effect is not neutralized by vitamin D at concentrations in the range of 0.1–1.0 nM.

[0160] In comparison, neither temsirolimus nor vitamin D alone increased the RNA expression of the KLF4 molecule, which is one of the classical transcription factors associated with iPSC status. However, the combination of temsirolimus and vitamin D (1.0 nM) increased KLF4 RNA expression. Therefore, it is preferable to include both temsirolimus and vitamin D in attempts to dedifferentiate T cells. As shown in Figure 2B, neither temsirolimus nor vitamin D alone acted to upregulate the beneficial dedifferentiation molecule KLF4, but the combination of temsirolimus (1 μM) and vitamin D (1.0 nM) synergistically upregulated KLF4.

[0161] The related transcription factor KLF10 was also upregulated when using a combination of temsirolimus and vitamin D (1.0 nM). As shown in Figure 2C, a 1 μM dose of temsirolimus acts alone to beneficially upregulate the dedifferentiation molecules KLF10, Nanog, and IL-7 receptor α. Vitamin D does not act alone to upregulate these molecules, but when used in combination, it does not suppress the effect of temsirolimus (column 5).

[0162] Finally, the inventors evaluated cultured cells for RNA expression of IL-7 receptor α, which is upregulated in T cells with reduced differentiation. Importantly, temsirolimus alone was able to upregulate IL-7 receptor α, but vitamin D alone could not. Nevertheless, as shown in Figure 2D, the combination of vitamin D (1.0 nM) and temsirolimus resulted in upregulation of IL-7 receptor α.

[0163] In summary, these data indicate that T cell differentiation can be forced using low levels of co-stimulation combined with temsirolimus, and that including temsirolimus and vitamin D in the culture is preferable for a more complete dedifferentiation pattern.

[0164] Example 3: The combination of vitamin D and temsirolimus reduces key transcription factors associated with Th1 differentiation while maintaining HIF-1-α expression. The inventors also evaluated the effects of vitamin D, temsirolimus, or a combination thereof, on the expression of key transcription factors associated with Th1 differentiation, namely T-BET and STAT1.

[0165] Figures 3A-3C show that the combination of vitamin D and temsirolimus reduces the expression of effector Th1 / Tc1 cell-related transcription factors without reducing the expression of HIF-1-α, a transcription factor related to T cell survival. The combination of vitamin D and temsirolimus reduces the expression of human CD4 + and CD8 +This illustrates the reduction of effector molecule expression in cells. For columns #2 to #5, T cells were subjected to a 3-day dedifferentiation interval, including low levels of anti-CD3 / anti-CD28 costimulation (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 medium. The first column represents the control culture (no temsirolimus, no vitamin D, use of a 3:1 bead-to-T cell ratio, and inclusion of type I polarizing cytokine IFN-α). The second column represents the culture with a low bead-to-T cell ratio and temsirolimus but without vitamin D, and in contrast, the third column represents the culture with vitamin D (0.1 nM) but without temsirolimus. The fourth column represents cultures containing high-dose ("HD") vitamin D (1.0 nM) but without temsirolimus. The fifth column represents cultures containing both high-dose vitamin D (1.0 nM) and temsirolimus. At the end of the dedifferentiation interval, cells were collected, RNA was isolated, and RNA expression analysis was performed using the Luminex Quantigene method. All results shown represent relative RNA expression, and the results are normalized to a value of 1.0 for the Th1 / Tc1 control culture.

[0166] Importantly, each drug or combination of drugs downregulated both T-BET RNA (Figure 3A) and STAT1 RNA (Figure 3B). As shown in Figures 3A–3C, vitamin D at doses of 0.1–1.0 nM acted alone to beneficially downregulate the differentiation molecules T-BET and STAT1. Temsirolimus at a dose of 1 μM, when combined with 1.0 nM vitamin D, did not adversely downregulate the survival-promoting transcription factor HIF-1α. However, temsirolimus at a dose of 1 μM acted alone to beneficially downregulate the differentiation molecules T-BET and STAT1, and produced similar results when combined with vitamin D (neither of these drugs are antagonists).

[0167] Quite in contrast, 1 μM temsirolimus, 0.1–1.0 nM vitamin D, or a combination thereof did not downregulate HIF-1-α, a key transcription factor important for T cell survival as a factor crucial for antitumor effects (Figure 3C).

[0168] In summary, these data demonstrate that a combination of low-level co-stimulation, temsirolimus, and vitamin D can reduce the transcription factors required for Th1 generation without inhibiting HIF-1-α, a key transcription factor necessary for overall T cell survival.

[0169] Example 4: A combination of vitamin D, temsirolimus, and an anti-IL-2 receptor monoclonal antibody increases autophagy signatures. The inventors also evaluated the effects of vitamin D, temsirolimus, or a combination thereof, on the autophagy process, which is important for promoting the stem-like dedifferentiation state. The level of autophagy can be partially determined by Western blot analysis, followed by upregulation of the autophagy substrate p62.

[0170] Figure 4 shows that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade induces the expression of the autophagy-related molecule p62. Human CD4 + and CD8 + T cells were subjected to a dedifferentiation protocol, which included a 3-day culture using low-level costimulation (beads to T cells ratio of 1:3), temsirolimus ("TEM" as shown in Figure 4, at concentrations of 1.0 or 0.3 μM), vitamin D ("D" as shown, at concentrations of 0.01, 0.03, 0.1, 0.3, or 1.0 nM), and an anti-IL-2 receptor monoclonal antibody (daclizumab, 50 μg / ml, "DAC" as shown). After the 3-day culture interval, T cells were harvested, proteins were isolated, and Western blot analysis was performed for the autophagy-related gene p62 and the housekeeping gene actin.

[0171] As shown in Figure 4, vitamin D content in T cell cultures was important for increased autophagy, as measured by upregulated p62. Vitamin D doses of 0.01–0.1 nM, in conjunction with temsirolimus at concentrations of 0.3–1.0 μM, beneficially upregulated the autophagy marker p62 during dedifferentiation. Specifically, in culture #6 (fifth column) in Figure 4, p62 expression was very low by Western blot analysis, consistent with low levels of autophagy. As indicated in the legend of the figure, this culture condition received low levels of co-stimulation, temsirolimus, and the anti-IL-2 receptor monoclonal antibody daclizumab, but not vitamin D.

[0172] In contrast, the other culture conditions, each with vitamin D supplementation, increased p62 expression (effective dose range of vitamin D, 0.01 nM to 1.0 nM). Figure 4 also demonstrates 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.

[0173] In summary, these data indicate that vitamin D with low levels of costimulation is an efficient method for inducing T cell autophagy, either alone or in combination with other T cell inhibitors, namely anti-IL-2 receptor reagents or the mTOR inhibitor temsirolimus.

[0174] Example 5: The combination of vitamin D, temsirolimus, and an anti-IL-2 receptor monoclonal antibody results in optimal disruption of the mTORC1 complex. The inventors also evaluated the effects of various T cell culture conditions on the expression of Raptor, a key component of the mTORC1 signaling complex. Importantly, it has recently been discovered that inhibition of mTORC1 is crucial for the reprogramming of somatic cells into the iPSC state.

[0175] Figure 5 shows that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces the expression of the mTORC1-related molecule, RAPTOR. Human CD4 + and CD8 + T cells were subjected to a dedifferentiation protocol, which included a 3-day culture using low-level costimulation (beads to T cells ratio of 1:3), temsirolimus ("TEM" as shown in Figure 5, at concentrations of 1.0 or 0.3 μM), vitamin D ("D" as shown, at concentrations of 0.01, 0.03, 0.1, 0.3, or 1.0 nM), and an anti-IL-2 receptor monoclonal antibody (daclizumab, 50 ng / ml, "DAC" as shown). After the 3-day culture interval, T cells were harvested, proteins were isolated, and Western blot analysis was performed for the mTORC1 complex protein Raptor and the housekeeping gene actin.

[0176] As shown in Figure 5, optimal inhibition of the mTORC1 complex was achieved when T cells were co-stimulated with a low bead-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) (shown in the first column; Culture 1).

[0177] As shown in Figure 5, the omission of daclizumab results in a slight increase in RAPTOR expression, thereby demonstrating the role of an anti-IL-2 receptor reagent for optimal mTORC1 inhibition. Therefore, the anti-IL-2 receptor monoclonal antibody daclizumab (dose, 50 μg / mL) plays a beneficial role in suppressing Raptor (column 2), an mTORC1 subunit molecule.

[0178] As shown in Figure 5, the optimal inhibition of the mTORC1 subunit molecule RAPTOR by vitamin D is at a vitamin D dose of 0.03–0.1 nM; concentrations lower or higher than this range result in suboptimal inhibition of RAPTOR. Therefore, vitamin D levels as low as 0.03 nM are sufficient for optimal inhibition of RAPTOR. However, reducing the vitamin D level to 0.01 nM results in suboptimal RAPTOR inhibition. Furthermore, increasing the vitamin D level beyond 0.1 nM can be detrimental, as shown by culture 7 (vitamin D concentration of 0.3 nM) which exhibited higher levels of RAPTOR expression.

[0179] Furthermore, as shown in Figure 5, optimal downregulation of RAPTOR requires a vitamin D and temsirolimus combination with an optimal temsirolimus dose of 1.0 μM, as culture 9 supplemented with temsirolimus at a concentration of 0.3 μM exhibited higher levels of RAPTOR expression.

[0180] Example 6: A combination of vitamin D, temsirolimus, and an anti-IL-2 receptor monoclonal antibody disrupts both the mTORC1 and mTORC2 complexes. The inventors also evaluated the mTORC2 complex, which is not directly sensitive to the inhibitory effect of rapamycin but may be affected by conditions that result in prolonged mTORC1 blockade. Importantly, inhibition of mTORC2 can promote a stem cell-like state.

[0181] Figure 6 shows that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces the expression of mTORC1-related molecule RAPTOR and mTORC2-related molecule Rictor. Human CD4 + and CD8 +T cells were subjected to a dedifferentiation protocol, which included a 3-day culture using low-level costimulation (beads to T cells ratio of 1:3), temsirolimus ("TEM" as shown in Figure 6, concentration of 1.0 μM), vitamin D ("D" as shown, concentrations of 0.03, 0.1, 0.3, or 1.0 nM), and an anti-IL-2 receptor monoclonal antibody (daclizumab, 50 ng / ml, "DAC" as shown). After the 3-day culture interval, T cells were harvested, proteins were isolated, and Western blot analysis was performed for mTORC1 complex protein Raptor; mTORC2 complex protein Rictor; post-mTORC1 protein, p70S6K; post-mTORC2 protein, SGK1, and the housekeeping gene, GAPDH.

[0182] As shown in Figure 6, T cell cultures in media containing temsirolimus, vitamin D, and the anti-IL-2 receptor antibody daclizumab showed a decrease in both the mTORC1 molecule RAPTOR and the mTORC2 molecule Rictor compared to a control culture that did not contain any of the three inhibitors. The levels of the post-mTORC1 molecule p70S6K and the post-mTORC2 molecule SGK1 were relatively maintained. Therefore, vitamin D (concentration of 0.03–1.0 nM) was effective during combination therapy differentiation for downregulation of the mTORC2 subunit Rictor. Temsirolimus at a concentration of 1 μM was also effective during combination therapy differentiation for downregulation of the mTORC2 subunit Rictor. Furthermore, the anti-IL-2 receptor monoclonal antibody daclizumab (dose, 50 μg / mL) did not discard the ability of temsirolimus and vitamin D to downregulate the mTORC2 subunit Rictor.

[0183] Therefore, T cell culture using low levels of co-stimulation and a tripartite inhibitory regimen of temsirolimus, vitamin D, and an anti-IL-2 receptor monoclonal antibody represents a novel method for reducing both the RAPTOR and Rictor subunits.

[0184] Example 7: A combination of vitamin D, temsirolimus, and an anti-IL-2 receptor monoclonal antibody reduces the expression of the pro-apoptotic Bcl2 family member gene BIM. Autophagy at the mitochondrial level (mitophagy) can alter the quality of mitochondrial proteins, and in particular, mitophages may exhibit favorable shifts from apoptotic precursor family members such as BIM to anti-apoptotic precursor family member genes, particularly towards bcl2 family member genes. Furthermore, culture methods that reduce apoptotic tendencies are associated with increased dedifferentiation capacity.

[0185] Figure 7 shows that a combination of vitamin D, temsirolimus, and anti-IL-2 receptor blockade reduces the expression of the pro-apoptotic molecule, BIM. Human CD4 + and CD8 + T cells were subjected to a dedifferentiation protocol, which included a 3-day culture using low-level costimulation (beads to T cells ratio of 1:3), temsirolimus ("TEM" as shown in Figure 7, at concentrations of 1.0 or 0.3 μM), vitamin D ("D" as shown, at concentrations of 0.01, 0.03, 0.1, 0.3, or 1.0 nM), and an anti-IL-2 receptor monoclonal antibody (daclizumab, 50 ng / ml, "DAC" as shown). After the 3-day culture interval, T cells were harvested, proteins were isolated, and Western blot analysis was performed for the pro-apoptosis-related gene BIM and the housekeeping gene actin.

[0186] To evaluate this, the inventors measured BIM levels in T cells cultured with low-intensity co-stimulation (a 1:3 bead-to-T cell ratio) and in the presence of various inhibitors. As shown in Figure 7, T cell cultures containing a combination of temsirolimus, vitamin D (0.1 nM), and the anti-IL-2 receptor monoclonal antibody daclizumab exhibited the lowest levels of BIM expression. However, Figure 7 also shows that the anti-IL-2 receptor monoclonal antibody daclizumab (dose, 50 μg / ml) plays a beneficial role in suppressing BIM, a pro-apoptotic molecule (column 2). Each of the three inhibitors appeared to play a role in BIM inhibition, as the absence of any single inhibitor increased BIM levels.

[0187] Therefore, we conclude that the combination inhibitor regime represents a method for inducing a favorable shift in mitochondrial regulation of apoptotic tendencies.

[0188] Example 8: A three-inhibitor dedifferentiation regimen yields subsequent proliferative T cells after the removal of the inhibitors. To demonstrate that a 3-day regimen consisting of low levels of co-stimulation, temsirolimus, vitamin D, and an anti-IL-2 receptor monoclonal antibody resulted in a dedifferentiated state capable of redifferentiation, we performed an experiment in which, after removing the inhibitor from the culture, we re-stimulated the cells on day 3 of culture using high levels of co-stimulation (a bead-to-T cell ratio of 3:1). After 10 days (13 days of total culture), T cells were harvested, counted, and evaluated by flow cytometry.

[0189] Figure 8 illustrates the effect of culture components during the dedifferentiation interval on subsequent T cell yield (at day 13 of culture). Human CD4 + and CD8 +T cells were subjected to a 3-day dedifferentiation interval, including culture in X-Vivo 20 medium supplemented with low levels of anti-CD3 / anti-CD28 costimulation (bead-to-T cell ratio, 1:3 or 1:1 as shown), temsirolimus (1 μM, or low dose ["Lo"], 0.1 μM), vitamin D (0.1 nM, or high dose of 1.0 nM ["HD"], or low dose of 0.01 nM), anti-IL-2 receptor monoclonal antibody (daclizumab, 50 μg / ml), and 5% human AB serum. The first column represents the control culture (without temsirolimus, vitamin D, or anti-IL-2R antibody). The second column represents cultures without anti-IL-2R antibody, the fourth column represents cultures without serum supplementation, the fifth column represents low-dose vitamin D, the sixth column represents results using high-dose vitamin D, the seventh column represents results using low-dose temsirolimus, the eighth column represents cultures without temsirolimus, and the ninth column represents results using a higher bead ratio. After a 3-day interval, the medium was replaced with fresh X-Vivo 20 without inhibitors, providing high-level costimulation (3:1 bead-to-T cell ratio), and T cell growth cytokines IL-2 (100 IU / ml) and IL-7 (10 ng / ml) were added. On day 13 of culture, viable T cells were enumerated, and the overall yield is shown compared to the input number on day 0.

[0190] Figure 8 shows the number of T cells after the redifferentiation stage. As these data show (column #3), T cells maintained over the first 3-day dedifferentiation interval using low levels of co-stimulation, temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody had a satisfactory T cell yield (over 250% of the culture input).

[0191] Quite in contrast, very low yields were observed in the culture medium shown in column #4, which did not receive serum supplementation during the first three-day culture interval. Therefore, this data demonstrates that the first three-day culture interval should include X-Vivo 20 culture medium supplemented with 5% AB serum.

[0192] Furthermore, lowering the vitamin D concentration to 0.01 nM or increasing it to 1.0 nM resulted in very low yields (data are shown in columns #5 and #6, respectively). Therefore, the preferred concentration of vitamin D is 0.1 nM.

[0193] Furthermore, when the temsirolimus concentration was reduced to 0.1 μM, the resulting T cell yield decreased (column #7). Therefore, the preferred concentration of temsirolimus is 1.0 μM.

[0194] Finally, when the co-stimulation level increased during the dedifferentiation interval (the bead-to-T cell ratio changed from 1:3 to 1:1, and the results are shown in the last column #9), the number of T cells obtained was very low. As shown in Figure 8, a low level of co-stimulation should be used during dedifferentiation (a 1:3 co-stimulated bead-to-T cell ratio), because increasing the ratio to 1:1 significantly reduces the ability to produce T cells from the dedifferentiated state (last column). Therefore, the preferred bead-to-T cell ratio during the dedifferentiation phase of culture is 1:3.

[0195] Example 9: The culture section of the first three components expressed CD4, a cell surface molecule consistent with reduced differentiation. + This leads to the generation of T cells. CD4 obtained at various points during the redifferentiation stage of the culture + T cells were evaluated for the expression of memory markers using flow cytometry.

[0196] Figures 9A-9C show the memory marker CD4 (on day 13 of culture). +The effects of culture components during the dedifferentiation interval on T cell expression are illustrated. Human CD4+ and CD8+ T cells were subjected to a 3-day dedifferentiation interval including culture in X-Vivo 20 medium supplemented with low levels of anti-CD3 / anti-CD28 costimulation (beads to T cells 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 (daclizumab, 50 μg / ml), and 5% human AB serum (as shown in Figures 9A-9C above). After the 3-day interval, the medium was replaced with fresh X-Vivo 20 without inhibitors, providing high-level costimulation (beads to T cells ratio, 3:1), and T cell growth cytokines IL-2 (100 IU / ml) and IL-7 (10 ng / ml) were added. + and CD45RA + Co-expression of markers (results shown in the panel above, evaluated on day 13 of culture); CD4 + CD62L + , and CCR7 + Co-expression of markers (bottom left panel; evaluated on day 3 of culture); and CD4 + CD62L + , CCR7 + , and CD127 + Flow cytometry was used to evaluate the co-expression of markers (bottom right panel; evaluated on day 10 of culture). All results are based on CD4 at the start of culture. + This is shown in comparison to the T cell values ​​(the last column in Figures 9A-9C, "Day 0 Input Value").

[0197] As shown in Figure 9A, compared to values ​​from input T cells on day 0, T cells initially proliferated with a combination of temsirolimus, vitamin D, and an anti-IL-2 receptor monoclonal antibody exhibited relatively conserved expression of the CD45RA marker expressed on naive T cells (column #3). Quite in contrast, the absence of these three molecules during the initial culture interval resulted in depletion of the naive T cell population (culture #1). Furthermore, the exclusion of temsirolimus during the initial culture interval resulted in depletion of the naive T cell population (culture #6).

[0198] As shown in Figure 9B, each of the T cell cultures that first proliferated during the 3-day interval incorporating low levels of co-stimulation exhibited increased T cell expression of the central memory molecules CD62L and CCR7.

[0199] Finally, as shown in Figure 9C, T cells initially proliferated with a combination of temsirolimus, vitamin D, and an anti-IL-2 receptor monoclonal antibody showed a significant increase in the expression of T cells that were triple-positive for CD62L, CCR7, and IL-7 receptor α (CD127) (compared to input cells on day 0). Elimination of the three inhibitors during the initial 3-day culture (column #1) neutralized the initial culture section's ability to promote the expansion of this triple-positive population. In addition, reducing or eliminating temsirolimus alone from the initial culture section also significantly reduced the frequency of triple-positive T cells (columns #5 and 6).

[0200] In summary, these data indicate that the initial culture intervals of the three drugs result in the conversion of CD4+ T cells from a primarily terminal effector memory population to a poorly differentiated T cell population, including the co-expression of CD62L, CCR7, and CD127, which represent a very limited state of T cell differentiation.

[0201] Example 10: The culture section of the first three components expresses CD8, a cell surface molecule consistent with reduced differentiation. + This leads to the generation of T cells. CD8 obtained at various points during the redifferentiation stage of the culture + T cells were evaluated for the expression of memory markers using flow cytometry.

[0202] Figures 10A and 10B show the memory marker CD8 +The effects of culture components during the dedifferentiation interval on T cell expression are illustrated. Human CD4+ and CD8+ T cells were subjected to a 3-day dedifferentiation interval including culture in X-Vivo 20 medium supplemented with low levels of anti-CD3 / anti-CD28 costimulation (beads to T cells 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 (daclizumab, 50 μg / ml), and 5% human AB serum (as shown in Figures 10A-10B above). After the 3-day interval, the medium was replaced with fresh X-Vivo 20 without inhibitors, providing high-level costimulation (beads to T cells ratio, 3:1), and T cell growth cytokines IL-2 (100 IU / ml) and IL-7 (10 ng / ml) were added. + CD62L + , and CCR7 + Co-expression of markers (left panel; evaluated on day 10 of culture); and CD8 + CD62L + , CCR7 + , and CD127 + Flow cytometry was used to evaluate co-expression of markers (right panel; evaluated on day 10 of culture). All results were based on CD8 at the start of culture. + This is shown in comparison to the T cell values ​​(the last column in Figures 10A-10B, "Day 0 Input Value").

[0203] As shown in Figure 10A, each of the T cell cultures that first proliferated during the 3-day interval incorporating low levels of co-stimulation showed central memory molecules CD62L and CD8 of CCR7. + It exhibited increased T cell expression.

[0204] Finally, as shown in Figure 10B, T cells initially proliferated with a combination of temsirolimus, vitamin D, and an anti-IL-2 receptor monoclonal antibody were triple-positive for CD62L, CCR7, and IL-7 receptor α (CD127) CD8 +T cell expression was significantly increased (compared to input cells on day 0). Elimination of the three inhibitors during the initial 3-day culture period (column #1) neutralized the initial culture section's ability to promote the expansion of this triple-positive population. In addition, reducing or eliminating temsirolimus alone from the initial culture section also significantly reduced the frequency of triple-positive T cells (columns #5 and #6).

[0205] In summary, these data show that the initial culture intervals of the three drugs include co-expression of CD62L, CCR7, and CD127, which represent a very limited state of T cell differentiation, primarily from a terminal effector memory population to a poorly differentiated T cell population. + This demonstrates that it can induce T cell conversion.

[0206] Example 11: Dedifferentiated T cells have an inherent bias toward low cytokine potentials. Figures 11A–11D highlight the components of the dedifferentiation process, including low levels of co-stimulation (anti-CD3 / anti-CD28 beads versus T cells ratio of 1:3, which is reduced compared to the conventional method described in Kalamasz D, Long SA, Taniguchi R, Buckner JH, Berenson RJ, Bonyhadi M. Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and Anti-CD28 antibodies) Journal of immunotherapy (Hagerstown, Md:1997).2004;27(5):405–418), the mTOR inhibitor temsirolimus; vitamin D; and the use of anti-IL-2 receptor monoclonal antibodies.

[0207] Figures 11A-11D show the analysis of inflammatory Th1 / Th17 cytokines in dedifferentiated T cells cultured in polarized neutral medium. Human CD4 + and CD8 +T cells were subjected to a 3-day dedifferentiation procedure, including co-stimulation with anti-CD3 / anti-CD28 (3 / 28) coated magnetic beads at a low ratio (beads to T cells ratio, 1:3), and supplementation with 5% human serum, as shown, containing the following culture components: temsirolimus (Y indicates a concentration of 1 μM, Y and Lo indicate concentrations of 0.1 μM), vitamin D (Y indicates a concentration of 0.1 nM, Y and Lo indicate concentrations of 0.01 nM), anti-IL-2 receptor monoclonal antibody (daclizumab, 50 μg / ml), and supplementation with 5% human serum. After 3 days, dedifferentiated T cells were co-stimulated in a medium supplemented with the T cell proliferation cytokines rhu IL-2 (100 IU / ml) and rhu IL-7 (10 ng / ml), which are not potent in inducing T cell polarization (typical bead-to-T cell ratio of 3:1). After 10 days of culture (total, 13 days of culture), T cells were harvested, washed, re-stimulated with 3 / 28 beads (3:1 ratio) for 24 hours, and the supernatant was collected and tested for cytokine content by Luminex multi-analyte analysis. All results shown are 1 × 10⁶ 6 It is expressed as a cytokine at the pg / ml level per cell / ml / 24 hours.

[0208] To evaluate whether the dedifferentiated T cell state exhibited a unique bias toward a specific cytokine secretion pattern, the inventors cultured dedifferentiated T cells using high levels of co-stimulation (bead-to-T cell ratio, 3:1) and maintenance in a medium containing only T cell growth cytokines IL-2 and IL-7, without any inhibitors.

[0209] As detailed in Figures 11A–11D, T cells resulting from redifferentiation from each of the dedifferentiation precursor states exhibited very low levels of secretion of inflammatory cytokines, including IFN-γ (most values ​​less than 1000 pg / ml), TNF-α (most values ​​less than 100 pg / ml), and IL-17 (all values ​​less than 10 pg / ml). Notably, GM-CSF was secreted at much higher levels, sometimes exceeding 10,000 pg / ml, under several conditions. GM-CSF levels were regulated by dedifferentiation states consisting of higher doses of temsirolimus (1.0 μM) and higher doses of vitamin D (0.1 nM). Therefore, it is desirable to expand T cells from dedifferentiation methods incorporating these higher concentrations of temsirolimus and vitamin D to regulate the resulting GM-CSF T cell cytokine secretion.

[0210] In particular, including a low concentration of vitamin D (0.01 nM) during the dedifferentiation interval resulted in slightly higher levels of IFN-γ and TNF-α compared to the use of a higher concentration of vitamin D (0.1 nM). Therefore, it is preferable to use a concentration of vitamin D of approximately 0.1 nM for the regulation of T cell secretion resulting from the inflammatory cytokine IFN-γ.

[0211] Furthermore, as shown in Figures 12A–12D, the T cells resulting from the redifferentiation of each of the dedifferentiated progenitor T cells secreted very low levels of IL-2, but again, the levels were lower when higher concentrations of temsirolimus and vitamin D were incorporated compared to conditions using lower concentrations of these drugs.

[0212] Figures 12A-12D show the analysis of IL-2 and Th2-type cytokines in dedifferentiated T cells cultured in polarized neutral medium. Human CD4 + and CD8 +T cells were subjected to a 3-day dedifferentiation procedure, including co-stimulation with anti-CD3 / anti-CD28 (3 / 28) coated magnetic beads at a low ratio (beads to T cells ratio, 1:3), and supplementation with 5% human serum, as shown, containing the following culture components: temsirolimus (Y indicates a concentration of 1 μM, Y and Lo indicate concentrations of 0.1 μM), vitamin D (Y indicates a concentration of 0.1 nM, Y and Lo indicate concentrations of 0.01 nM), anti-IL-2 receptor monoclonal antibody (daclizumab, 50 μg / ml), and supplementation with 5% human serum. After 3 days, dedifferentiated T cells were co-stimulated in a medium supplemented with the T cell proliferation cytokines rhu IL-2 (100 IU / ml) and rhu IL-7 (10 ng / ml), which are not potent in inducing T cell polarization (typical bead-to-T cell ratio of 3:1). After 10 days of culture (total, 13 days of culture), T cells were harvested, washed, re-stimulated with 3 / 28 beads (3:1 ratio) for 24 hours, and the supernatant was collected and tested for cytokine content by Luminex multi-analyte analysis. All results shown are 1 × 10⁶ 6 It is expressed as a cytokine at the pg / ml level per cell / ml / 24 hours.

[0213] The resulting T cells also exhibited very low levels of secretion of Th2 cytokines IL-4 (<20 pg / ml) and IL-5 (<60 pg / ml). However, IL-13 levels were elevated under several T cell culture conditions, and lower cytokine secretion was detected in conditions incorporating higher concentrations of temsirolimus and vitamin D compared to conditions using lower concentrations of these agents.

[0214] In summary, these data demonstrate that T cell redifferentiation in a medium containing T cell growth cytokines (IL-2 and IL-7) without strong polarization signals (without the addition of IFN-α, IL-4, or TGF-β) after the Step 1 dedifferentiation process exhibits an inherent bias toward T cells with low cytokine potentials, and in particular, we demonstrated low levels of harmful cytokines IFN-γ, TNF-α, and IL-17. This observation is particularly strong when the dedifferentiation process incorporates low levels of co-stimulation and proliferation in a medium containing 1.0 μM temsirolimus, 0.1 nM vitamin D, and an anti-IL-2 receptor monoclonal antibody.

[0215] Example 12: Hybrid T REG Favorable expansion of dedifferentiated T cells under Th2 polarization conditions and in the presence of the novel drug pemetrexed. The inventors of this invention have shown that T cell redifferentiation is T REG The effects of dedifferentiation components were evaluated when polarizing cytokines IL-2 and TGF-β, or Th1 polarizing cytokine IFN-α, were incorporated.

[0216] In fact, either TBET or GATA3 is necessary for maintaining immune resistance. REG It has been shown to maintain cellular capacity. Nevertheless, T REG Despite this evidence regarding the role of either TBET or GATA3 in cellular function, due to the very strong binding of TBET and the resulting Th1-type pathway in autoimmunity, we have fabricated T REG We chose to prioritize GATA3 expression in cells. Therefore, we have adopted the dedifferentiation process of step 1, and the subsequent, T REG Either of the redifferentiation processes in step 2, which include both polarization signals (IL-2, TGF-β) and the main Th2 polarization signal (IL-4), is used to create human "hybrid" T REG - We evaluated whether it could generate Th2 cells. REG The intentional addition of IL-4 to cell ex vivo cultures is T REGSince it has been shown to either promote or suppress the phenotype, the results from the literature from experimental mouse models are mixed in this respect. REG In addition to this conflicting mouse literature regarding the role of exogenous IL-4 in cell production, human T REG Information on the ex vivo role of IL-4 in cells is scarce, but in this study, IL-4 is found to be beneficial in human T cells. REG It was found that the cells retained their function.

[0217] iT REG Cells are iT REG Human iT cells with a hybrid Th2 component are characterized as having a tendency for in vivo differentiation plasticity, which allows them to be converted into pathogenic Th1 or Th17 subsets. REG Cells may be favored for adoptive T cell therapy. On the other hand, differentiation into the Th2 phenotype is iT REG When coded in manufacturing, the Th2 bias predictably limits plasticity to the Th1 / Th17 phenotype.

[0218] Furthermore, the inventors have found that the drug pemetrexed is iT REG We evaluated whether it could be beneficial in promoting the cellular phenotype. Preferred iT REG There are precedents for the use of drugs for cell generation, most notably the mTOR inhibitor rapamycin. REG It is associated with a shift toward cells. However, pemetrexed is iT REG It is not characterized as having a promoting effect. Pemetrexed has a complex mechanism of action as a folate antimetabolite.

[0219] Figure 13 shows the hybrid Th2 / REGUnder polarization conditions, dedifferentiated T cells show a favorable expansion compared to Th1 polarization conditions. Human CD4+ and CD8+ T cells were subjected to a 3-day dedifferentiation procedure ("Step 1"). As shown in Figure 13, the dedifferentiation intervention in Step 1 variedly to include 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 various combinations of inhibitors (T, D, or T, D, B). After 3 days, the dedifferentiated T cells were variedly subjected to Th1 polarization 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 T cells were co-stimulated in culture medium supplemented with polarization conditions (IL-2, TGF-β, and rhu IL-4 [1000 IU / ml]) (typical bead-to-T cell ratio, 3:1). In addition, T cells were cultured in the presence of variable polarization conditions, either without the novel inhibitory molecule pemetrexed ("0") or in the presence of variable concentrations of pemetrexed as shown (10 nM ["10"], 33 nM ["33"], or 100 nM ["100"]). After a total of 10 days of culture, n=24 cultures were collected, viable cells were listed, and the results were graphed above (y-axis = cell number × 10). 6 The number of input cells is 1.5 × 10⁶. 6 (It was a single individual.)

[0220] As shown in Figure 13, the redifferentiation ability of T cells after dedifferentiation in step 1 depended on the specific components added during dedifferentiation, the specific cytokines added during redifferentiation, and the presence of pemetrexed during redifferentiation.

[0221] Notably, attempts to redifferentiate a sufficient number of T cells after dedifferentiation in Step 1 were unsuccessful under Th1 polarization conditions (see Figure 13, cultures #9–#16; the yield of all T cells was less than the T cell input). A very limited ability to redifferentiate along the Th1 pathway was observed when the dedifferentiation conditions included temsirolimus and vitamin D alone or in combination with an anti-IL-2 receptor reagent, and this was also observed when pemetrexed was not added during Step 2 culture or at a concentration in the range of 10–100 nM.

[0222] Quite in contrast, attempts to redifferentiate a sufficient number of T cells after dedifferentiation in step 1 resulted in hybrid T REG -Success under Th2 polarization conditions (see Figure 13, cultures #2 and #5). Notably, under the most severe dedifferentiation conditions (containing temsirolimus, vitamin D, and anti-IL-2 receptor monoclonal antibody), T REG -Sufficient T cells under Th2 polarization conditions were observed only when pemetrexed was added to the step 2 culture at a concentration of 10 nM.

[0223] Using these most stringent dedifferentiation step 1 conditions, pure T in step 2 REG Attempts to redifferentiate a sufficient number of T cells under polarized conditions (IL-2 + TGF-β, IL-4-free) were unsuccessful, even in the presence of pemetrexed (cultures #20 and #21).

[0224] In summary, from a numerical perspective, T REG - Use Th2 hybrid polarization conditions (IL-2, TGF-β, and IL-4) and pemetrexed at a concentration of 10 nM to optimize the success of T cell redifferentiation.

[0225] Example 13: Hybrid T REG Culture of dedifferentiated T cells under Th2 conditions is limited to the differentiated state of CD4. + and CD8 + This leads to T cell generation. The inventors also evaluated the effects of the dedifferentiation process in Step 1, followed by the redifferentiation in Step 2, on the T cell memory state under various cytokine polarization conditions / various pemetrexed conditions. Specifically, the study showed that T cells in a limited differentiated state improved the therapeutic usefulness for adoptive cell therapy. Therefore, limited T cell differentiation is likely to be an advantageous feature of the Step 1 / Step 2 T cell production method.

[0226] Figures 14A to 14C show the hybrid Th2 / T REG Culture of dedifferentiated T cells under polarization conditions, naive and triple-positive T cells, central memory CD4 + This exemplifies how it leads to the generation of T cells. Human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure and subsequent culture in a medium containing variable polarization conditions and variable presence of pemetrexed, as shown in Figure 13. Of the total N=24 culture conditions, only those cultures with favorable cell yields were further evaluated, and it was shown that all cultures containing pemetrexed ("+") had a concentration of 10 nM. REG Unless otherwise specified ("TReg, IL4 absent"), all samples contained IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of incubation, cultures were collected and evaluated by flow cytometry for the following content: naive CD4 + T cells (total CD4 co-expressing CD45RA) + Expressed as a percentage of T cells; Figure 14A), Central Memory CD4 + T cells (total CD4 co-expressing both CD62L and CCR7) + T cells expressed a percentage of the cells; Figure 14B), as well as triple-positive central memory CD4 + T cells (total CD4 co-expressing CD62L, CCR7, and CD127) + % of T cells expressed; Figure 14C).

[0227] As shown in Figure 14A, Hybrid T REG- Redifferentiation in step 2 in the Th2 state (with or without 10 nM pemetrexed) is preferred in the experimental mouse model of adoptive T cell therapy, CD4 + CD45RA + This resulted in a high frequency of naive T cell subsets.

[0228] In addition, as shown in Figure 14C, Hybrid T REG - Redifferentiation in the Th2 state in step 2 (with or without 10 nM pemetrexed) is associated with CD4 having triple-positive co-expression of memory markers CD62L, CCR7, and CD127. + This resulted in a high frequency of T cell subsets. This triple-positive memory phenotype is a marker for T cells in a very primitive differentiation state.

[0229] As shown in Figure 14C, CD4 was triple positive for CD62L, CCR7, and CD127. + The frequency of T cells is, REG Compared to polarization conditions, Hybrid T REG - The value was higher under TH2 polarization conditions.

[0230] Furthermore, as shown in Figure 14C, the use of more stringent Step 1 differentiation conditions, including not only temsirolimus and vitamin D but also anti-IL-2 receptor monoclonal antibodies, results in hybrid T REG -Using Th2 polarization conditions yielded the highest frequency of CD4 cells that were triple-positive for CD62L, CCR7, and CD127.

[0231] Figures 15A and 15B show the hybrid Th2 / T REG Culture of dedifferentiated T cells under polarization conditions leads to triple-positive T cells with central memory CD8 + This exemplifies how it leads to the generation of T cells. Human CD4 + and CD8 +T cells were subjected to a 3-day dedifferentiation procedure and subsequent culture in a medium containing variable polarization conditions and variable presence of pemetrexed, as shown in Figure 13. Of the total N=24 culture conditions, only those cultures with favorable cell yields were further evaluated, and it was shown that all cultures containing pemetrexed ("+") had a concentration of 10 nM. REG Unless otherwise specified ("TReg, IL4 absent"), all samples contained IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of incubation, cultures were collected and evaluated for the following content by flow cytometry: Central Memory CD8 + T cells (total CD8 co-expressing both CD62L and CCR7) + T cells expressed a percentage of the data (Figure 15A), as well as triple-positive central memory CD8 + T cells (total CD8 co-expressing CD62L, CCR7, and CD127) + A percentage of T cells expressed this value; (Figure 15B).

[0232] Furthermore, as shown in Figure 15B, the frequency of CD8 cells that were triple-positive for CD62L, CCR7, and CD127 was, pure T REG Compared to polarization conditions, Hybrid T REG - It was higher under TH2 polarization conditions. Furthermore, as shown in Figures 15A-15B, the use of more stringent Step 1 differentiation conditions, including not only temsirolimus and vitamin D but also anti-IL-2 receptor monoclonal antibodies, resulted in hybrid T REG - Using Th2 polarization conditions, CD8 was triple positive for CD62L, CCR7, and CD127. + It produced T cells at the highest frequency.

[0233] In summary, these data show that hybrid T REG -T cell redifferentiation using Th2 cytokine polarization (IL-2, TGF-β, and IL-4), and pemetrexed (10 nM) after dedifferentiation in step 1, is preferably limited to a restricted differentiation state of CD4 cells. + and CD8+ This demonstrates the generation of T cells.

[0234] Example 14: Hybrid T REG Culture of dedifferentiated T cells under Th2 polarization conditions yields T cells with a primitive Th2 cell cytokine phenotype. The cytokine secretion patterns of T cells redifferentiated under the culture conditions of Step 2 after dedifferentiation in Step 1 were also evaluated. Cytokine secretion is an indicator of T cell effector function, and therefore, T REG It is generally desirable that cells have the potential for reduced cytokine secretion, particularly with respect to major inflammatory cytokines such as IL-17, IFN-γ, and TNF-α. The proposed hybrid T REG -In the case of a Th2 cell population, such cells are also expected to secrete several classifications of Th2 cytokines.

[0235] Figures 16A–16C demonstrate that culturing dedifferentiated T cells under hybrid Th2 / TReg polarization conditions results in the production of T cells with an atomic Th2 cell cytokine phenotype, as shown by high levels of IL-2 and IL-4 secretion and low levels of IL-5 secretion. Human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure and subsequent culture in a medium containing variable polarization conditions and variable presence of pemetrexed, as shown in Figure 13. Of the total N=24 culture conditions, only those cultures with favorable cell yields were further evaluated, and it was shown that all cultures containing pemetrexed ("+") had a concentration of 10 nM. REG Unless otherwise indicated ("TReg, IL4 absent"), all conditions included IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of culture, T cells were harvested, washed, re-stimulated with 3 / 28 beads (3:1 ratio) for 24 hours, and the resulting supernatant was collected and tested for cytokine content by Luminex multi-analyte analysis. All results shown are 1 × 10⁶ 6It is expressed as a cytokine at the pg / ml level per cell / ml / 24 hours.

[0236] As shown in Figure 16A, regardless of whether pemetrexed was added to the culture, hybrid T REG T cells redifferentiated under TH2 cytokine polarization conditions (IL-2, TGF-β, and IL-4) showed the highest levels of IL-2 secretion. This result is consistent with prior understanding that IL-2 secretion in T cells is a characteristic of early-differentiated T cells, as seen in T cells redifferentiated under hybrid culture conditions.

[0237] Figures 17A–17C show hybrid Th2 / T as indicated by low levels of IL-10, IL-13, and IL-17 secretion. REG This study demonstrates that culturing dedifferentiated T cells under polarized conditions leads to the production of T cells with an atomic Th2 cell cytokine phenotype. (Human CD4) + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure and subsequent culture in a medium containing variable polarization conditions and variable presence of pemetrexed, as shown in Figure 13. Of the total N=24 culture conditions, only those cultures with favorable cell yields were further evaluated, and it was shown that all cultures containing pemetrexed ("+") had a concentration of 10 nM. REG Unless otherwise indicated ("TReg, IL4 absent"), all conditions included IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of culture, T cells were harvested, washed, re-stimulated with 3 / 28 beads (3:1 ratio) for 24 hours, and the resulting supernatant was collected and tested for cytokine content by Luminex multi-analyte analysis. All results shown are 1 × 10⁶ 6 It is expressed as a cytokine at the pg / ml level per cell / ml / 24 hours.

[0238] Figures 18A–18C show hybrid Th2 / T REGThis study demonstrates that culturing dedifferentiated T cells under polarized conditions leads to the production of T cells with an atomic Th2 cell cytokine phenotype. Human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure and subsequent culture in a medium containing variable polarization conditions and variable presence of pemetrexed, as shown in Figure 13. Of the total N=24 culture conditions, only those cultures with favorable cell yields were further evaluated, and it was shown that all cultures containing pemetrexed ("+") had a concentration of 10 nM. REG Unless otherwise indicated ("TReg, IL4 absent"), all conditions included IL-2, TGF-β, and IL-4 ("TReg"). After a total of 10 days of culture, T cells were harvested, washed, re-stimulated with 3 / 28 beads (3:1 ratio) for 24 hours, and the resulting supernatant was collected and tested for cytokine content by Luminex multi-analyte analysis. All results shown are 1 × 10⁶ 6 It is expressed as a cytokine at the pg / ml level per cell / ml / 24 hours.

[0239] Furthermore, as shown in Figure 16B, regardless of whether or not pemetrexed was added to the culture, hybrid T REG -T cells redifferentiated under TH2 conditions (IL-2, TGF-β, and IL-4) showed the highest levels of IL-4 secretion. Since IL-4 is the major cytokine that directs Th2 polarization, T cells produced under hybrid conditions are indeed 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 Figure 16C; IL-10, see Figure 17A; IL-13, see Figure 17B) or effector Th1 / Th17 cytokines (IFN-γ, see Figure 18A; TNF-α, see Figure 18B; GM-CSF, see Figure 18C; IL-17, see Figure 17C).

[0240] Importantly, the lack of IL-4 inclusion in the T cell redifferentiation process resulted in higher levels of inflammatory cytokines IFN-γ, TNF-α, and GM-CSF (see Figures 18A–18C).

[0241] In summary, these data show that T from dedifferentiated cells in step 1 REG The redifferentiation of T cells toward the phenotype occurs because the T cells that emerge from these conditions have a significantly reduced ability to secrete cytokines associated with inflammatory diseases, resulting in hybrid T cells. REG - This demonstrates that the Th2 polarization conditions are being utilized optimally.

[0242] Example 15: Hybrid T REG Culture of dedifferentiated T cells under Th2 conditions is enhanced hybrid T REG / Results in T cells with a Th2 transcription factor profile. The T cell cytokine phenotype is determined by major transcription factors. The association between transcription factors and T cell subsets is as follows: FOXP3 is associated with T REG Cell development is directed; TBET directs Th1 cell development, and GATA3 directs Th2 cell development.

[0243] To evaluate these transcription factors using our manufacturing method, T cells are first subjected to the dedifferentiation procedure in step 1, and then hybrid T cells are used. REG -The cells were redifferentiated under TH2 culture conditions (IL-2, TGF-β, and IL-4). In addition, the inventors compared the effects of pemetrexed with those of classical mTOR inhibitors. In their experiments, instead of using an oral formulation of the mTOR inhibitor (rapamycin; Sirolimus®), they used temsirolimus (Toracel®), the water-soluble parent form of the drug.

[0244] iT REG The phenotype is considered unstable, and therefore, the inventors have found that hybrid T is stable at delayed points including days 20 and 32 of culture. REG-The stability of redifferentiated T cells was evaluated using Th2 culture conditions. In addition, to rigorously test phenotypic stability, T cells were subjected to high levels of co-stimulation (a 3:1 bead-to-T cell ratio) between days 24 and 32 of culture and grown in a culture medium free of cytokines or pharmacological agents.

[0245] Figures 19A to 19D show hybrid Th2 / T REG Extended culture of dedifferentiated T cells under polarized conditions results in CD4 cells expressing FOXP3 and GATA3 transcription factors. + This exemplifies how it leads to the generation of T cells. Human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure, followed by co-stimulation (3:1 bead-to-T cell ratio) with or without the pharmacological inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM) to create hybrid Th2 / T cells. REG The cells were grown in a medium containing polarization conditions (IL-2; TGF-β; IL-4). The cultures were re-stimulated with 3 / 28 beads on both day 14 and day 24 of culture. On day 24 of culture, the culture medium did not contain exogenous cytokines or pharmacological inhibitors to assess the stability of transcription factor expression. T cells were harvested on days 12, 20, and 32 of culture and subjected to surface flow cytometry (CD4 marker) and intracellular staining for the following transcription factors: FOXP3, Tbet, and GATA3. The above data represent the percentage of CD4 cells in the entire culture population (Figure 19A), T REG Figure 19B shows the percentage of CD4 cells expressing the transcription factor FOXP3, Figure 19C shows the percentage of CD4 cells expressing the Th1 transcription factor Tbet, and Figure 19D shows the percentage of CD4 cells expressing the Th2 transcription factor GATA3.

[0246] For details of Figures 19A to 19D, T REG-T cells redifferentiated under Th2 conditions gradually shifted towards CD4 cell dominance in the culture over time (Figure 19A). As shown in Figure 19B, CD4 cells expressed FOXP3 at high frequency and stably from day 12 to day 32 of culture, regardless of the presence of temsirolimus or pemetrexed in the culture.

[0247] As shown in Figure 19C, there was a very low frequency of contamination by the Th1 transcription factor TBET even in the absence of pharmacological inhibitors. However, the most consistently reduced TBET levels were observed under hybrid polarization conditions including pemetrexed. Finally, as shown in Figure 19D, hybrid T1 polarization was supplemented with pemetrexed. REG - In T cells produced under TH2 conditions, the highest expression of Th2-related GATA3 at the end of culture was observed.

[0248] Figures 20A to 20D show hybrid Th2 / T REG Extended culture of dedifferentiated T cells under polarized conditions results in CD8 cells expressing FOXP3 and GATA3 transcription factors. + This exemplifies how it leads to the generation of T cells. Human CD4 + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure, followed by co-stimulation (3:1 bead-to-T cell ratio) with or without the pharmacological inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM) to create hybrid Th2 / T cells. REG The cells were grown in a medium containing polarization conditions (IL-2; TGF-β; IL-4). The cultures were re-stimulated with 3 / 28 beads on both day 14 and day 24 of culture. On day 24 of culture, the culture medium did not contain exogenous cytokines or pharmacological inhibitors to assess the stability of transcription factor expression. T cells were harvested on days 12, 20, and 32 of culture and subjected to surface flow cytometry (CD8 marker) and intracellular staining for the following transcription factors: FOXP3, Tbet, and GATA3. The above data represent the percentage of CD8 cells in the entire culture population (Figure 20A), T REGFigure 20B shows the percentage of CD8 cells expressing the transcription factor FOXP3, Figure 20C shows the percentage of CD8 cells expressing the Th1 transcription factor Tbet, and Figure 20D shows the percentage of CD8 cells expressing the Th2 transcription factor GATA3.

[0249] As shown in Figure 20A, the CD8 cell content decreased gradually and modestly over time in the culture. REG Cellular function is generally attributed to the CD4 cell subset, but CD8 + T REG The cells are also well described, and CD4 + and CD8 + T cells containing both T cell subsets REG It should be noted that the use of populations may be advantageous for diversifying antigen specificity. Therefore, the method described by the inventors is CD4- and CD8-T REG Since it generates both, it may be partially advantageous.

[0250] As shown in Figure 20B (upper right panel), CD8 manufactured using this method. + The T cells were indeed stable over time in culture and enriched for stable FOXP3 expression, independently of the presence of pharmacological inhibitors.

[0251] As shown in Figure 20C (lower left panel), T REG - Redifferentiation under Th2 polarization conditions is generally associated with low levels of the Th1 transcription factor TBET and CD8 + T cell expression was induced, but the lowest levels were consistently observed in the presence of pemetrexed.

[0252] Finally, as shown in Figure 20D (bottom right panel), T REG - Under TH2 conditions, redifferentiation is actually shifted to Th2 type differentiation, as indicated by increased expression of the GATA3 transcription factor CD8. + This resulted in T cells.

[0253] Figures 40A-40B also show CD4 + and CD8+ Flow cytometry of GATA3 and FOXP3 in redifferentiated TREG-Th2 cells in both subsets is shown.

[0254] In summary, these transcription factor analyses show that hybrid T REG - Redifferentiation under Th2 culture conditions + pemetrexed addition is CD4, which expresses both FOXP3 and GATA3, while limiting TBET expression. + and CD8 + This suggests that it may be optimal because it allows for the preservation of both T cells.

[0255] Example 16: Hybrid T REG Culture of dedifferentiated T cells under Th2 conditions yields T cells with an enhanced Th2 cytokine secretion profile. In addition to transcription factor measurement, the inventors of this invention have also measured cytokine secretion capacity using hybrid T REG -T cells redifferentiated under Th2 polarization conditions were also evaluated. As shown in Figures 21A-21D, REG -All redifferentiated cultures grown under Th2 polarization conditions yielded IL-4-capable T cells, thereby demonstrating the inherent ability of this method to achieve Th2 polarity even in the absence of pharmacological inhibitors.

[0256] Figures 21A-21D show the hybrid Th2 / T REG This example illustrates how extended culture of dedifferentiated T cells under polar conditions leads to the generation of T cells expressing a dominant Th2 cytokine phenotype: IL-4, IL-5, and IL-13 secretion. (Human CD4) + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure, followed by co-stimulation (3:1 bead-to-T cell ratio) with or without the pharmacological inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM) to create hybrid Th2 / T cells. REGT cells were grown in a culture medium containing polarization conditions (IL-2; TGF-β; IL-4). The cultures were re-stimulated with 3 / 28 beads on both day 14 and day 24 of culture. On day 24 of culture, the culture medium was free of exogenous cytokines or pharmacological inhibitors to assess the stability of transcription factor expression. T cells were harvested, washed, and re-stimulated with 3 / 28 beads (3:1 ratio) for 24 hours on days 12, 20, and 32 of culture. The resulting supernatant was collected and tested for cytokine content by Luminex multi-analyte analysis. All results shown are 1 × 10⁻⁶. 6 Cytokine levels are expressed in pg / ml units per cell / ml / 24 hours. Th2 cytokine IL-10 was also evaluated: all values ​​are 1 × 10⁻⁶. 6 The level was less than 20 pg / ml per cell / mL / 24 hours.

[0257] Notably, temsirolimus secretes the effector Th2 cytokines IL-5 (Figure 21B) and IL-13 (Figure 21C). REG -While it blunted the redifferentiation ability of T cells under Th2 conditions, the use of pemetrexed completely preserved the ability of T cells to secrete IL-5 and IL-13. Therefore, these data suggest that pemetrexed is effective in T REG -The use of pemetrexed is more compatible with the manufacture of Th2 hybrid subsets than conventional mTOR inhibitors such as temsirolimus. REG This provides further evidence that it is preferable compared to the use of accelerators.

[0258] Furthermore, T REG - All T cells redifferentiated under Th2 polarization conditions exhibited relatively low levels of expression of IL-2 (Figure 22A), IFN-γ (Figure 22B), IL-17 (all below 20 pg / ml), and TNF-α (all below 20 pg / ml).

[0259] Figures 22A-22D show the hybrid Th2 / T REGThis example illustrates how extended culture of dedifferentiated T cells under polar conditions leads to the generation of T cells expressing a dominant Th2 cytokine phenotype: IL-2, IFN-γ, and GM-CSF secretion. (Human CD4) + and CD8 + T cells were subjected to a 3-day dedifferentiation procedure, followed by co-stimulation (3:1 bead-to-T cell ratio) with or without the pharmacological inhibitor temsirolimus (1.0 μM) or pemetrexed (10 nM) to create hybrid Th2 / T cells. REG T cells were grown in a culture medium containing polarization conditions (IL-2; TGF-β; IL-4). The cultures were re-stimulated with 3 / 28 beads on both day 14 and day 24 of culture. On day 24 of culture, the culture medium was free of exogenous cytokines or pharmacological inhibitors to assess the stability of transcription factor expression. T cells were harvested, washed, and re-stimulated with 3 / 28 beads (3:1 ratio) for 24 hours on days 12, 20, and 32 of culture. The resulting supernatant was collected and tested for cytokine content by Luminex multi-analyte analysis. All results shown are 1 × 10⁻⁶. 6 Cytokine levels are expressed in pg / ml units per cell / ml / 24 hours. Inflammatory cytokines IL-17 and TNF-α were also evaluated: all values ​​are 1 × 10⁻⁶. 6 The level was less than 20 pg / mL per cell / mL / 24 hours.

[0260] Notably, GM-CSF secretion increased with further supplementation of pemetrexed. REG -This was observed to a higher degree in redifferentiated T cells under TH2 hybrid culture conditions (10 nM, Figure 22C). Inferred from experimental studies in the literature, T REG - It is unclear whether the enhanced GM-CSF capacity within the Th2 hybrid population is necessarily harmful or beneficial.

[0261] In summary, these data are T REG-The redifferentiation of T cells in a Th2 polarized state is favorable because it results in T cells with a low ability to secrete Th1 and Th17 cytokines associated with inflammatory diseases. Pemetrexed is used in hybrid T REG -Inclusion in Th2 polarization conditions is advantageous for increasing Th2 cytokine production capacity, which would further provide protection against differentiation plasticity toward Th1 and Th17 subsets.

[0262] Example 17: Use of selective anti-TNF-α reagents before lymphocyte collection by apheresis to beneficially modify the input T cell TCR repertoire. Figures 23A and 23B illustrate the use of RNA-based T-cell receptor sequencing to detect broad upregulation and downregulation of T-cell TCR specificity after treatment with the TNF-α inhibitor etanercept. In Figure 23, RNA was isolated from peripheral blood mononuclear cells from ALS patients before and after treatment with etanercept therapy. The RNA was subjected to TCR repertoire profiling as previously described by Rosati E, Dowds CM, Liaskou E, Henriksen EKK, Karlsen TH, Franke A. Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol. 2017;17(1):61. In Figure 23A, approximately 25% of TCR specificity was upregulated in post-treatment samples (as shown in red); in stark contrast, approximately 25% of TCR specificity was downregulated in post-treatment samples (as shown in blue). As shown in Figure (B) in the upper right, several T cell clones increased in frequency from 0.01 before etanercept (near the detection limit of the assay) to post-treatment values ​​ranging from 247 to 486, thus etanercept therapy resulted in significant T cell clonal expansion, which is consistent with T cell expansion exceeding 4-log. As shown in Figure 23B in the lower right, several T cell clones decreased in frequency from 259 to 598 before etanercept (near the detection limit of the assay) to post-treatment values ​​of 0.01, thus etanercept therapy resulted in significant T cell clonal contraction, which is consistent with T cell contraction exceeding 4-log.

[0263] Figures 23A-B show that anti-TNF-α therapy with etanercept, which preferentially inhibits the serum cell-free morphology of TNF-α that promotes TNFR1-expressing Th1 cells, is associated with extensive changes in the upregulation and downregulation of T cell receptors. These observations suggest that by pre-treating subjects with etanercept or any other anti-TNF-α therapeutic agent that preferentially inhibits the serum cell-free morphology of TNF-α (monoclonal antibody, adalimumab, etc.), the T cell receptor repertoire can be shifted away from Th1-phenotypic T cells based on antigen specificity, thereby enriching TREG-phenotypic T cells based on antigen specificity.

[0264] Example 18: Characterization of TREG-Th2 hybrid populations as cell products enriched for the expression of CD25, CD27, 2B4, BTLA, and CTLA4. Figure 24 shows that the produced iTREG / Th2 hybrid population exhibited increased expression of CD25, CD27, 2B4, BTLA, and CTLA4 compared to the control Th1 / Tc1 culture. In Figure 24, the iTREG / Th2 hybrid population was generated by the previously detailed method using the early phase of T cell differentiation, followed by redifferentiation in a medium containing IL-2, TGF-β, and IL-4. On day 11 of iTREG / Th2 production, cells were harvested and subjected to flow cytometry to evaluate relevant molecules of CD4+ and CD8+ T cell expression, namely CD25, CD27, 2B4, BTLA, and CTLA4, compared to three separate control conditions to evaluate Th1 / Tc1 polarization.

[0265] Figure 24 shows that hybrid TREG-Th2 cells produced according to the described conditions exhibit increased expression of the following cell surface molecules by flow cytometry compared to control Th1 / Tc1 cells: CD25, CD27, 2B4, BTLA, and CTLA4.

[0266] As illustrated in Figure 24, the iTREG / Th2 hybrid cell product has CD4+ and CD8+ T cells expressing CD25, CD27, 2B4, BTLA, and CTLA4 at levels at least 10%, more preferably 50%, higher than control Th1 / Tc1 cells.

[0267] The IL-2 receptor CD25 is crucial for the ability of TREG cells to manage autoimmunity, particularly CD8+ T cell-driven responses. Therefore, CD25 expression on iTREG / Th2-producing cell products is a desirable feature.

[0268] CD27, a co-stimulatory molecule whose expression is increased on TREG cells, has been shown to contribute to the inhibitory function of TREG. Therefore, CD27 expression on iTREG / Th2-producing cell products is a desirable feature.

[0269] Recently, 2B4 (CD244) has been shown to inhibit the CD8+ T cell response by reducing glycolysis and cell division. Therefore, 2B4 expression on iTREG / Th2-producing cell products is a desirable feature.

[0270] BTLA (CD272) is a co-inhibitory receptor, and ligation of BTLA with the herpesvirus entry vector HVEM promotes TREG cell induction and inhibition of effector immune responses. Therefore, BTLA expression on iTREG / Th2-producing cell products is a desirable feature.

[0271] CTLA4 is a key effector molecule in TREG cells, as recently demonstrated by its ability to limit immunity to malaria infection. Therefore, CTLA4 expression on iTREG / Th2-producing cell products is a desirable feature.

[0272] Example 19: Characterization of TREG-Th2 hybrid populations as cell products enriched for the expression of TGIT, TIM 3, ICOS, LAIR1, and OX40. Figure 25 shows that the produced iTREG / Th2 hybrid population had increased expression of TIGIT, TIM3, ICOS, LAIR1, and OX40 compared to the control Th1 / Tc1 culture. In Figure 25, the iTREG / Th2 hybrid population was generated by the previously detailed method using the early phase of T cell differentiation, followed by redifferentiation in a medium containing IL-2, TGF-β, and IL-4. On day 11 of iTREG / Th2 production, cells were harvested and subjected to flow cytometry to evaluate relevant molecules of CD4+ and CD8+ T cell expression, namely TIGIT, TIM3, ICOS, LAIR1, and OX40, compared to three distinct control conditions to evaluate Th1 / Tc1 polarization. Figure 25 shows that hybrid TREG-Th2 cells produced according to the described conditions result in the generation of T cells expressing increased levels of the following cell surface molecules by flow cytometry: TIGIT, TIM3, ICOS, LAIR1, and OX40, compared to control Th1 / Tc1 cells.

[0273] As illustrated in Figure 25, the iTREG / Th2 hybrid cell product has CD4+ and CD8+ T cells expressing TIGIT, TIM3, ICOS, LAIR1, and OX40 at levels at least 10%, more preferably 50%, higher than control Th1 / Tc1 cells.

[0274] TIGIT is a cell surface co-inhibitory receptor molecule associated with regulatory T cell function. Therefore, TIGIT expression on iTREG / Th2-producing cell products is a desirable feature.

[0275] TIM3 is a co-inhibitory receptor that mediates the inhibitory effect on TREG cells. Therefore, TIM3 expression on iTREG / Th2-producing cell products is a desirable feature.

[0276] ICOS is a recently determined co-stimulatory molecule that helps maintain regulatory T cell-mediated immunosuppression for the control of immune responses in the central nervous system. Therefore, ICOS expression on iTREG / Th2-producing cell products is a desirable feature.

[0277] LAIR1 (CD305) is a multifaceted inhibitory molecule capable of blocking inflammation through multiple processes, including the suppression of activated effector memory T cells. Therefore, LAIR1 expression on iTREG / Th2-producing cell products is a desirable feature.

[0278] OX40 is a co-stimulatory molecule. Therefore, OX40 expression on iTREG / Th2-producing cell products is a desirable feature.

[0279] Example 20: Characterization of GATA3 and FOXP3 expression in TREG / Th2 hybrid populations Steady-state apheresis samples were obtained, enriched for lymphocytes using a Ficoll gradient, and then seeded in G-Rex culture vessels. The cells were incubated in complete medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM), and basiliximab (30 μg / mL). After an initial dedifferentiation period, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 bead-to-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 (CD4 and CD8) and intracellular molecular expression (GATA3 and FOXP3), and evaluated by flow cytometry. The results in Figures 26A-B show FOXP3 and GATA3 expression in CD4+ and CD8+ T cells at the start of culture and after culture (Th2 / TREG), as measured by flow cytometry. The percentages provided indicate the number of cells considered positive for CD4+ or CD8+ and intracellular markers (indicated by boxes).

[0280] The results shown in Figures 26A-B illustrate the phenotypes of the produced Th2 / TREG cell products. Type II cytokine-phenotypic T cells can be partially characterized by their expression of the transcription factor GATA3, while regulatory T cell populations can be partially identified by their expression of the transcription factor FoxP3. At the start of culture, very low frequencies of T cells expressed either GATA3 or FoxP3. Quite in contrast, T cell products produced under Th2 / TREG culture conditions expressed high frequencies of T cells that were either single-positive for GATA3, single-positive for FOXP3, or double-positive (not shown) for both GATA3 and FOXP3. Importantly, as shown, this transcription factor profile was expressed in both the produced CD4+ (upper panel) and CD8+ (lower panel) T cells. IL-4-free control cultures significantly reduced the frequency of GATA3-positive T cells, thereby demonstrating the crucial role of IL-4 in the production of the Th2 / TREG hybrid population (not shown).

[0281] Most of the phenotypic characterization of T cell products produced according to the TREG / Th2 methods detailed in this disclosure can be confirmed at the end of culture. However, it is important to note that T cell products can be cryopreserved, and therefore the phenotypic characterization of T cells in the thawed state reflects the actual product adopted into the subject. TREG / Th2 cells in the thawed state can be characterized compared to control Th1 / Tc1 cells by: (a) increased expression of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, and OX40 by flow cytometry; (b) decreased IFN-γ and TNF-α and increased IL-4 secretion by Luminex cytokine secretion analysis; and (c) altered expression of T cell fate transcription factors, namely decreased TBET and increased FOXP3 and GATA3.

[0282] Example 21: Characterization of CD73 and CD103 expression in TREG / Th2 hybrid populations Steady-state apheresis samples were obtained, enriched for lymphocytes using a Ficoll gradient, and then seeded in G-Rex culture vessels. The cells were incubated in complete medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM), and basiliximab (30 μg / mL). After an initial dedifferentiation period, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 bead-to-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 (CD4 and CD8), and for the ectonucleotidase molecule CD73 or the integrin molecule CD103, and evaluated by flow cytometry. The results in Figures 27A-B show CD73 and CD103 expression in CD4+ and CD8+ T cells at the start of culture and after culture (Th2 / TREG), as measured by flow cytometry. The percentages provided indicate the number of cells considered positive for CD4+ or CD8+, and for either the ectonucleotidase molecule or the integrin molecule, respectively (indicated by boxes).

[0283] Regulatory T cell populations can suppress pathogenic effector T cell populations through several defined mechanisms, including the expression of CD39 and CD73 ectonucleotidase molecules, which act to hydrolyze pro-inflammatory ATP against immunosuppressive adenosine substrates. Indeed, TREG cells expressing CD39 have increased suppressive function and have been associated with the resolution of inflammatory bowel disease. Furthermore, the suppressive function of human TREG cells is partially mediated by CD73. As shown below in Figure 27A, T cells produced under Th2 / TREG culture conditions can have increased expression of the TREG-associated effector molecule CD73, and CD39 was also highly expressed on TREG / Th2-produced T cells (not shown). In addition to CD39 / CD73 ectonucleotidases, TREG cell function also correlates with the expression of CD103, an integrin that directs epithelial lymphocyte localization. In fact, CD103 and IL-2 receptor signaling cooperate to maintain immune resistance in the intestinal mucosa, and furthermore, CD103-expressing TREG cells are important for improving experimental chronic GVHD. As shown below in Figure 27B, T cells produced under Th2 / TREG culture conditions may have increased expression of the TREG-related effector molecule CD103.

[0284] Example 22: Characterization of CD150 and CD27 / CD95 expression in TREG / Th2 hybrid populations Steady-state apheresis samples were enriched for lymphocytes using a Ficoll gradient, seeded in G-Rex culture vessels, and incubated in complete medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM), and basiliximab (30 μg / mL). After this initial dedifferentiation period, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 bead-to-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 cytometry analysis for CD4, CD8, CD150, CD27, CD95, CD45RA, CD62L, and CCR7. The results are shown in Figures 28A-B.

[0285] T cells cultured under TREG (RAPA-501) conditions were compared to culture input T cells ("Day 0") and to control cultured T cells grown without mTOR inhibitors ("control"). As shown in Figure 28A, both the CD4+ and CD8+ T cell subsets contained within the RAPA-501 cell product showed significantly increased expression of the stem cell marker CD150 compared to both culture input T cells and control cultured T cells. As shown in Figure 28B, the RAPA-501 cell product was also enriched with the T stem cell memory (TSCM) phenotype compared to culture input cells. Since the T cells obtained under these conditions were effector memory CD45RO+, the control culture lacked this population (not shown). The left panel (culture input T cells) and the right panel (RAPA-501 cells) show the expression of TSCM markers CD45RA, CD62L, and TSCM markers CD95 and CD27 after gated on CCR7, and similar differences in the expression of these TSCM markers were observed for CD8+ T cells (not shown).

[0286] In experimental models, the efficacy of adoptive T cell therapy depends on the success of T cell population transplantation and in vivo persistence. Importantly, T cell differentiation status helps to direct in vivo persistence, with less differentiated cells increasing persistence. In our initial research, mouse rapamycin-resistant T cells expressing the T central memory (TCM) phenotype showed increased in vivo engraftment compared to control T cells. Furthermore, human rapamycin-resistant T cells also showed increased engraftment in human-to-mouse models of xenograft-versus-host disease. Other researchers have determined that T cells with reduced differentiation compared to the T effector memory (TEM) population, including TCM subsets, naive T cell subsets, and more recently T stem cell memory (TSCM) subsets, increase in vivo persistence and mediate the increased in vivo effect. This relationship between T cell differentiation status and in vivo T cell function can be applied to TREG cells as follows: (1) TCM-phenotypic TREG cells were more effective in reducing experimental GVHD compared to TEM-phenotypic TREG cells; and (2) TREG cells expressing the stem cell marker CD150 were highly effective in preventing stem cell graft rejection. As shown below in Figure 28A, T cells produced under Th2 / TREG culture conditions were enriched for cells in a reduced differentiated state consistent with a T stem cell subset including expression of the CD150 marker.

[0287] Example 23: Characterization of cytokine secretion in a TREG / Th2 hybrid population Steady-state apheresis samples were enriched for lymphocytes using a Ficoll gradient, seeded in G-Rex culture vessels, and incubated in complete medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM), and basiliximab (30 μg / mL). After an initial dedifferentiation period, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 bead-to-T cell ratio, and cytokines (IL-4 (1000 IU / mL), IL-2 (10,000 IU / mL), and TGF-β (100 ng / mL)) were added. This culture is referred to as condition "A". Condition "B" was the same culture condition but without IL-4 addition. Condition "C" reflects standard TREG culture conditions with rapamycin (1 μM), IL-2 (100 IU / mL), and TGF-β (10 ng / mL). Condition "D" reflects a Th1-type control culture prepared in the presence of IFN-α without an mTOR inhibitor. After culturing, T cells were harvested, stimulated with anti-CD3 / anti-CD28 beads, and the resulting supernatant was tested for cytokine content using the Luminex assay.

[0288] It may be important to evaluate the cytokine secretion of the produced Th2 / TREG cells. First, it is important that the cell product can secrete IL-4, a driver cytokine for subsequent Th2 differentiation. Second, it is desirable that the adoptive transplanted T cell population is able to secrete IL-2, as this ability exhibits precursor function that allows T cells to proliferate more readily in vivo without requiring exogenous IL-2. Finally, it is important that the Th2 / TREG cell population reduces the secretion of Th1 or Th17 cytokines IFN-α, TNF-α, IL-17, and GM-CSF. As shown in Figure 29, the produced Th2 / TREG cell product secreted IL-4 and IL-2 while minimizing the secretion of Th1 or Th17 cytokines.

[0289] Example 24: Characterization of Th1 / Tc1 suppression by TREG / Th2 hybrid population Steady-state apheresis samples were enriched for lymphocytes using a Ficoll gradient, seeded in G-Rex culture vessels, and incubated in complete medium containing vitamin D (0.3 nM), temsirolimus (3.0 μM), and basiliximab (30 μg / mL). After an initial dedifferentiation period, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 bead-to-T cell ratio for ex vivo production of Th2 / TREG cells, and cytokines (IL-4 (1000 IU / mL), IL-2 (10,000 IU / mL), and TGF-β (100 ng / mL)) were added. In parallel, T cells were cultured in the presence of the type I polarizing cytokine IFN-α to generate effector Th1 / Tc1 cells. Th1 / Tc1 cultures were generated from the same donor (autologous, "AUTO") or unrelated donor (allogeneic, "ALLO") as the RAPA-501 cell cultures. After ex vivo culture, Th1 / Tc1 effector T cells were seeded in the bottom chamber of a Transwell plate and co-stimulated with anti-CD3 / anti-CD28 coated beads in a 3:1 bead-to-T cell ratio. 24 hours after Th1 / Tc1 cell co-stimulation, RAPA-501 cells were added to the top chamber of the Transwell plate in a 1:1 Th1 / Tc1 to RAPA501 ratio. (A) RAPA-501 regulation of cytokine content. Culture supernatants were collected at 24 hours (before adding RAPA-501 cells to the top chamber) and 48 hours (with or without RAPA-501 cell addition) and tested for cytokine content by Luminex assay. The results for IL-2, IFN-γ, GM-CSF, and TNF-α content are expressed in units of pg / ml / 24 hours / 1 × 10⁶ cells / ml. (B) RAPA-501 cell regulation of PD1 Th1 / Tc1 cell expression is performed in an antigen-independent manner. Autologous or allogeneic Th1 / Tc1 cells were collected at 48 hours, either after or without the addition of RAPA-501 cells at 24 hours, and then subjected to flow cytometry to evaluate PD1 expression.

[0290] During the development of the RAPA-501 cell product, the inventors conducted experiments to characterize the molecular mechanism of observed T cell suppression. One method for evaluating potential mechanisms is the Transwell assay, in which effector T cells and RAPA-501 cells are separated by a filter that prevents cell-to-cell contact but allows cell communication via small soluble media such as cytokines. Figures 30A-B show that RAPA-501 cells regulate effector T cells in a contact-independent manner (experiments performed in a Transwell container). RAPA-501 cells suppressed the cytokine secretion capacity of effector T cells by acting in a T cell receptor-independent manner. Specifically, because no co-stimulatory beads were added to the Transwell chamber containing RAPA-501 cells, RAPA-501 cells did not require co-stimulation to regulate inflammatory cytokine levels, including IL-2, IFN-γ, GM-CSF, and TNF-α (Figure 30A). The ability of TREG cells to consume IL-2 is a generally described phenomenon, but previous studies have identified the requirement of intercellular contact for IL-2 consumption. Therefore, RAPA-501 cells appear to be able to regulate the levels of multiple inflammatory cytokines to some extent independently in a contact-independent manner. These results suggest that RAPA-501 cells are a favorable candidate for cytokine neutralization. Secondly, we found that RAPA-501 cells regulated an additional aspect of effector T cell biology, namely the promotion of programmed death-1 (PD-1) checkpoint molecule expression on effector T cells, in a contact-independent manner (using Transwell experiments). Importantly, as shown in Figure 30B, RAPA-501 cells upregulated PD1 expression in both autologous and allogeneic Th1 / Tc1 cells, thereby further revealing that one mechanism of RAPA-501 cell repressive function occurs TCR-independently via a soluble mediator.

[0291] Example 25: Characterization of cytokine secretion suppression by CNS microglia cells using a TREG / Th2 hybrid population. Human microglia cells (HMC3 cell line) were first activated with IFN-γ (10 ng / ml; 24 hours), then with LPS (10 ng / ml; 3 hours), and subsequently seeded into the lower chamber of a transwell with or without the addition of RAPA-501 cells prepared as described above (left panel), or with the addition of RAPA-501 cells (right panel) (RAPA-501 to HMC3 ratio, 1:40). RAPA-501 cells were generated using the method described in the patent application to produce hybrid Th2 / TREG phenotypic T cells. After 24 hours, cell-free supernatant was collected and evaluated for IL-6, IFN-γ, and IP-10 content by Luminex assay (cytokine secretion measured at pg / ml / 1 × 10⁶ cells / ml / 24 hours). The results are shown in Figure 31.

[0292] Microglia are CNS-resident antigen-presenting cells that can develop into pro-inflammatory factors in ALS. The ability of manufactured human Th2 / TREG cells to suppress human microglia inflammation was previously unknown. To address this, we sequentially cultured the human microglia cell line HMC3 in IFN-γ and subsequently induced a pro-inflammatory state with LPS endotoxin. As shown in Figures 31A-31B, the addition of RAPA-501 Th2 / TREG cell products to the pro-inflammatory microglia reduced the culture supernatant content of pro-inflammatory cytokines IL-6, IP-10, and IFN-γ. In this experiment, the observed immunosuppressive effect occurred in a Transwell vessel with a highly diluted TREG to inflammatory microglia ratio of 1:40, thereby demonstrating 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 TREG to microglia ratio).

[0293] Example 26: iT of ALS using pentostatin, cyclophosphamide, and lamivudine host modulating platform REG Cell therapy. Figure 32 details the PC regimen and the overall treatment approach. The PC regimen is administered in 2-week cycles, with doses of pentostatin or cyclophosphamide escalating over cycles 1–4 (total duration of the PC regimen is 8 weeks). Pentostatin is administered on either day 1 or days 1 and 4 of the 14-day cycle, and cyclophosphamide (Cy) is administered on either days 1, 2, and 3 or days 1, 2, 3, 4, and 5 of the 14-day cycle. For cycle 4, if ALC is less than 1250 cells per microliter, the Cy dose is increased to 200 mg per day. After immunodepletion and immunosuppression are achieved by the administration of the PC regimen, the first iT REG Cell injection is performed in week 8 of treatment. The inflammasome inhibitor lamivudine is administered continuously at a dose of 150 mg BID from week 8 to week 26 of the protocol.

[0294] Figure 33 shows iT REG This provides further details regarding cell production and shows lymphocyte collection by apheresis regimens before and after the PC regimen. Lymphocytes from ALS patients are collected by steady-state apheresis (10-15 liters collected) performed immediately before or after the PC regimen. Collection before the PC regimen is preferable because more T cells are found and immunosuppression does not occur. REG Manufacturing may be more advantageous. In comparison, collection after the PC regime is iT REG This could be advantageous because inflammatory Th1 / Tc1 cells that contaminate the culture are depleted in vivo before production. REG After manufacturing, the product is treated as indicated by injections #2, #3, and #4. REG To enable repeated administration of the cells, they are cryopreserved in therapeutic doses.

[0295] Figure 34 shows multiple iT REG The cell injection strategy will be explained in more detail, iT REG The sequence of PC regimens preceding each repeated dose of cells is shown. REGAdminister the following before infusion: (1) to deplete and suppress inflammatory Th1 / Tc1 cells that contribute to the onset of the disease, and (2) to adoptively transferred iT REG To increase in vivo levels of homeostatic cytokines such as IL-7 and IL-15, enabling in vivo expansion of the population. The PC regimen involves 2 mg / m² on days 1 and 4. 2 It consists of a combination of pentostatin at a dose and a constant dose of cyclophosphamide at 100 mg per day from days 1 to 5. After a 2-day rest period, iT REG The cells are administered (day 8 of the regimen). The inflammasome inhibitor lamivudine is administered to iT REG It is administered continuously from week 8 onwards to limit inflammation during cell therapy.

[0296] Figure 35 shows iT REG Further details regarding the monitoring of patients treated with cells are provided, showing that ALS monitoring is performed approximately monthly by both patient-reported ALSFRS-R and clinician-reported Appel scores, as indicated. Immunology labs to monitor the inflammatory status of ALS patients are assessed approximately monthly, as indicated.

[0297] Example 27: Use of selective anti-TNF-α reagents before lymphocyte collection by apheresis to beneficially modify the input T cell TCR repertoire. Figures 36A–36B show that anti-TNF-α therapy with etanercept, which preferentially inhibits the serum cell-free morphology of TNF-α that promotes TNFR1-expressing Th1 cells, is associated with extensive changes in the upregulation and downregulation of T cell receptors. Figures 36–36B illustrate the use of RNA-based T cell receptor sequencing to detect extensive upregulation and downregulation of T cell TCR specificity after treatment with the TNF-α inhibitor, etanercept. In Figures 36–36B, RNA was isolated from peripheral blood mononuclear cells from ALS patients before and after treatment with etanercept therapy. RNA was subjected to TCR repertoire profiling as previously described by Rosati E, Dowds CM, Liaskou E, Henriksen EKK, Karlsen TH, Franke A. Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol. 2017;17(1):61. As shown in Figure 36A, approximately 25% of TCR specificity was upregulated in the post-treatment samples (shown in red); in striking contrast, approximately 25% of TCR specificity was downregulated in the post-treatment samples (shown in blue). As shown in Figure 36B, etanercept therapy resulted in significant T cell clonal expansion, thereby consistent with T cell expansion exceeding 4-log, as several T cell clones increased from a frequency of 0.01 pre-etanercept (near the detection limit of the assay) to a post-treatment value ranging from 247 to 486. As shown in Figure 36B, etanercept therapy resulted in significant T cell clonal contraction, as several T cell clones decreased from a frequency of 259 to 598 pre-etanercept to a post-treatment value of 0.01, thereby consistent with T cell clonal contraction exceeding 4-log. These observations demonstrate that by pre-treating subjects with etanercept or any other anti-TNF-α therapeutic agent that preferentially inhibits the serum noncellular form of TNF-α (monoclonal antibody, adalimumab, etc.), the T cell receptor repertoire can be shifted to detach from T cells with an antigen-specific Th1 phenotype, thereby enabling T REG型This demonstrates the enrichment of phenotypic T cells.

[0298] Example 28: T as a cell product enriched for the expression of CD25, CD27, 2B4, BTLA, and CTLA4 REG - Characterization of the Th2 hybrid population. Figure 37 shows a hybrid T manufactured according to the conditions described. REG -Th2 cells show increased expression of the following cell surface molecules: CD25, CD27, 2B4, BTLA, and CTLA4, compared to control Th1 / Tc1 cells, as measured by flow cytometry. Figure 37 shows hybrid T cells containing IL-2, TGF-β, and IL-4. REG In Th2 medium, using the initial phase of T cell differentiation followed by redifferentiation, iT cells are differentiated according to the previously detailed method. REG A Th2 hybrid population was generated. Cells were harvested and the relevant molecules, namely CD4, CD25, CD27, 2B4, BTLA, and CTLA4, were extracted. + and CD8 + T cell expression was evaluated using flow cytometry, and the results were compared to three separate control conditions to assess Th1 / Tc1 polarization.

[0299] As illustrated in Figure 37, iT REG The / Th2 hybrid cell product has CD4+ and CD8+ T cells expressing CD25, CD27, 2B4, BTLA, and CTLA4 at levels at least 10%, more preferably 50%, higher than control Th1 / Tc1 cells.

[0300] The IL-2 receptor CD25 is responsible for managing autoimmune responses, particularly CD8+ T cell-driven responses. REG It is important for the function of cells. Therefore, iT REG CD25 expression on / Th2-producing cell products is a desirable feature.

[0301] T REG CD27, a co-stimulatory molecule whose expression on cells has increased, REGIt has been shown to contribute to the inhibitory function of iT. REG CD27 expression on / Th2-producing cell products is a desirable feature.

[0302] 2B4 (CD244) has recently been affected by the decline of glycolysis and cell division, leading to CD8 + It has been shown to inhibit the T cell response. Therefore, iT REG Expression of 2B4 on / Th2-producing cell products is a desirable feature.

[0303] BTLA (CD272) is a co-inhibitory receptor, and ligation between BTLA and the herpesvirus entry vector HVEM is T REG It promotes cell induction and inhibition of effector immune responses. Therefore, iT REG BTLA expression on / Th2-producing cell products is a desirable feature.

[0304] CTLA4 has recently demonstrated its ability to limit immunity to malaria infection, REG It is an important effector molecule in cells. Therefore, iT REG CTLA4 expression on / Th2-producing cell products is a desirable feature.

[0305] Example 29: T as a cell product enriched for the expression of TIGIT, TIM3, ICOS, LAIR1, and OX40 REG - Characterization of the Th2 hybrid population. Figure 38 shows a hybrid T manufactured according to the conditions described. REG - This shows that Th2 cells result in the generation of T cells expressing increased levels of the following cell surface molecules by flow cytometry compared to control Th1 / Tc1 cells: TIGIT, TIM3, ICOS, LAIR1, and OX40. Figure 38 shows iT REG / Th2 hybrid populations were generated by the previously detailed method using the early phase of T cell differentiation, followed by redifferentiation in a medium containing IL-2, TGF-β, and IL-4. REGOn the 11th day of / Th2 production, cells were collected and CD4 + and CD8 + Flow cytometry was performed to evaluate molecules associated with T cell expression, namely TIGIT, TIM3, ICOS, LAIR1, and OX40, and compared to three separate control conditions for assessing Th1 / Tc1 polarization.

[0306] As illustrated in Figure 38, iT REG The / Th2 hybrid cell product has CD4+ and CD8+ T cells expressing TIGIT, TIM3, ICOS, LAIR1, and OX40 at levels at least 10%, more preferably 50%, higher than control Th1 / Tc1 cells.

[0307] TIGIT is a cell surface co-inhibitory receptor molecule associated with regulatory T cell function, including, for example, its contribution to the immunosuppressive environment in B-cell non-Hodgkin lymphoma. Therefore, iT REG TIGIT expression on / Th2-producing cell products is a desirable feature.

[0308] TIM3 includes, for example, the suppression of T cells invading head and neck squamous cell carcinoma. REG It is a co-inhibitory receptor that mediates the inhibitory effect on cells. Therefore, iT REG TIM3 expression on / Th2-producing cell products is a desirable feature.

[0309] ICOS is a co-stimulatory molecule that has recently been determined to help maintain immunosuppression by regulatory T cells for the control of immune responses in the central nervous system. REG ICOS expression on / Th2-producing cell products is a desirable feature.

[0310] LAIR1 (CD305) is a multifaceted inhibitory molecule that can block inflammation through multiple steps, including the suppression of activated effector memory T cells. Therefore, iT REG LAIR1 expression on / Th2-producing cell products is a desirable feature.

[0311] OX40 is a co-stimulatory molecule. Therefore, iT REG OX40 expression on / Th2-producing cell products is a desirable feature.

[0312] T as detailed in this disclosure REG Most of the phenotypic characterization of T cell products produced according to the / Th2 method can be confirmed at the end of culture. However, it is important to note that T cell products can be cryopreserved, and therefore the phenotypic characterization of T cells in the thawed state reflects the actual product adopted into the subject. REG Th2 cells can be characterized compared to control Th1 / Tc1 cells by: (a) increased expression of CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, and OX40 by flow cytometry; (b) decreased IFN-γ and TNF-α and increased IL-4 secretion by Luminex cytokine secretion analysis; and (c) altered expression of T cell fate transcription factors, namely decreased TBET and increased FOXP3 and GATA3.

[0313] iT REG Apheresis for production. Prior to treatment with the pentostatin / cyclophosphamide regimen, subjects undergo a lymphocyte apheresis procedure. The purpose of this peripheral lymphocyte collection is to provide iT cells for adoptive T cell therapy. REG It is the process of manufacturing cells.

[0314] Apheresis consists of collecting 10-15 liters on a CS-3000 or equivalent machine. The apheresis product is sent to the protocol sponsor, Rapa Therapeutics, and iT REG The cells are produced by ex vivo culture using special culture conditions.

[0315] Research purpose The first objective was to investigate the relationship between PC regimens and maintenance lamivudine therapy in an inflammatory subset of ALS patients. REG Determine the safety of cell injection.

[0316] Second objective: IT REG The ability of therapy to inhibit inflammatory markers in ALS patients will be determined by a preliminary method. iT REG Determines the effectiveness of the therapy.

[0317] Eligibility Criteria Individuals with sporadic or familial ALS who have been diagnosed with suspected, possible, or confirmed sporadic or familial ALS, supported by a clinical laboratory, according to the World Federation of Neurology El Escorial Criteria. Eligibility requirements include: age 18 to 75 years; Karnovsky Performance Status ≥ 70%; ejection fraction (MUGA or 2D echocardiography) within the normal limits of the healthcare institution; serum creatinine ≤ 2.0 mg / dl; AST and ALT ≤ 3 times the upper limit of normal; bilirubin ≤ 1.5 (except in cases due to Gilbert's disease); and corrected DLCO ≥ 50% on pulmonary function tests.

[0318] To evaluate secondary endpoints, subjects must have evidence of inflammatory markers in peripheral blood cell populations after evaluation of at least two separate blood samples during the screening interval. Assays used to assess the potential patient inflammatory state include flow cytometry, cytokine secretion analysis, and cellular signaling events by Western blot analysis. Other tests may include testing or in vitro sensitization of the T cell receptor repertoire to potential autoantigens such as motor neuron proteins or ALS-related protein aggregates. Cytokine secretion can be measured without stimulation (autonomous cytokine secretion), and with anti-CD3 / anti-CD28 costimulation; LPS endotoxin exposure; CD40 ligand exposure; and adenosine A2 AThe evaluation will be conducted using a variety of modalities, including, but not limited to, T cell receptor agonism and antagonistism; T cell (PD1, TIM-3) and monocyte (CD47, CD200) checkpoint inhibition; and assessment of T cell receptor clonality by RNA sequencing. The decision of whether a potential subject is considered to have sufficient inflammation to be included in the study will be made based on a total matrix analysis of these studies, and this decision will be made by the study PI or lead associate researcher in consultation with the medical director of Rapa Therapeutics Lab where the immunoassays will be performed.

[0319] Exclusion criteria include patients actively receiving riluzole (Rilutek®) or edaravone (Radicava®) therapy (except in cases where a stable dose has been taken for more than one month). The following also represent exclusion criteria: any clinical trial intervention within 30 days of the protocol; pulmonary vital capacity measurement less than 60% of the predicted level; active uncontrolled infection; hypertension not adequately controlled by three or fewer medications; history of cerebrovascular event within 6 months of enrollment; history of pulmonary embolism within 6 months of enrollment; or clinically significant cardiac pathology (class III or IV heart failure according to NYHY, uncontrolled angina, severe uncontrolled ventricular arrhythmia, or electrocardiographic evidence of acute ischemia or active conduction abnormality, as defined by myocardial infarction within 6 months prior to enrollment). Patients with a history of coronary artery bypass grafting or angioplasty will undergo cardiac evaluation and be considered on a case-by-case basis. Patients seropositive for HIV, hepatitis B, or hepatitis C will be excluded. Patients who are known to be pregnant or who have recently been diagnosed with pregnancy are excluded, as are patients of childbearing age who do not wish to use contraception. Patients may be excluded at the discretion of the PI, or if their participation is deemed to pose an unacceptable medical or psychological risk.

[0320] Treatment of ALS patients with PC regimens The goal of the 8-week PC regimen is to partially deplete and suppress Th1 / Tc1 cells, which contribute to the development of ALS disease. In addition, iT immediately after the PC regimen REG From week 8 onward, when cell injection continues, the PC regimen is also intended to acutely induce the creation of T cell homeostatic cytokines, particularly IL-7 and IL-15.

[0321] The PC regimen is administered in 14-day cycles, but an additional delay of up to two weeks between cycles may be permitted if logistical problems occur or if additional time is needed to evaluate and / or treat any adverse events. For cycle #1, pentostatin (1 mg / m² on day 1) 2 iv) is administered in combination with cyclophosphamide (100 mg po daily on days 1, 2, and 3). Cycle #2, unless dose-limiting toxicity occurs, is administered with an increased dose of pentostatin (2 mg / m² on day 1). 2 iv) consists of the combination of the same dose of cyclophosphamide (100 mg po per day on days 1, 2, and 3). Cycles #3 and #4, administered as long as dose-limiting toxicity does not occur and the absolute lymphocyte count exceeds 750 cells per microliter, consist of two doses of pentostatin (2 mg / m² on days 1 and 4) combined with cyclophosphamide (100 mg po per day on days 1, 2, 3, 4, and 5) for 5 days. 2 It consists of (iv).

[0322] If the ALC count is 750 cells per microliter or less before cycle #3 or cycle #4, no further cycles are administered, and the patient proceeds to maintenance therapy with lamivudine. If the absolute lymphocyte count exceeds 1250 cells per microliter before cycle #4, the dose of cyclophosphamide is doubled (200 mg po per day on days 1, 2, 3, 4, and 5).

[0323] Specific details regarding pentostatin administration: (a) Preparation: Pentostatin is reconstituted by the dispensing department to a concentration of 2 mg / ml according to the vial instructions. The appropriate patient-specific dose is then added to 0.9% sodium chloride to make a total volume of 50 mL; (b) Dosage and administration: The dosage of pentostatin is adjusted for renal impairment (see below); each dose of pentostatin is administered intravenously over 30-60 minutes; (c) Premedication and antiemetic therapy: Before infusion, infusion 1 liter of 0.9% sodium chloride over 30-60 minutes. Pentostatin may be emetogenic. The guidelines for anti-vomiting regimens are as follows (variations are permitted at the discretion of the PI): (1) 12 mg of dexamethasone by intravenous infusion 60 minutes prior to each dose of pentostatin; (2) In addition, oral dexamethasone may be administered for the first 5 days of each cycle if necessary for vomiting control; (3) Ondansetron may be administered at a dose of 8 mg by intravenous infusion 60 minutes prior to each dose of pentostatin; (4) For the remainder of the treatment, ondansetron may be administered orally at a dose of 8 mg (tablet) every 12 hours from day 1 to day 14 of the cycle if necessary; and (5) aprepitant may be added to the anti-vomiting regimen as needed in patients with uncontrolled nausea and vomiting.

[0324] Specifications related to pentostatin dose reduction: Serum creatinine levels are obtained before calculating the planned doses of pentostatin and CrCl. CrCl is obtained from 24-hour urine or calculated using the Cockcroft-Gault formula. If the subject experiences an increase in creatinine levels during pentostatin and cyclophosphamide therapy, subsequent administration is modified as follows: CrCl ≥ 60 (mL / min / 1.73m²). 2 Regarding: Administer 100% of the intended pentostatin dose (for cycles #1 and #2, pentostatin 1 mg / m²). 2 For cycles #3 and #4, pentostatin 2 mg / m² 2For 30 ≤ CrCl < 60: Administer 50% of the intended pentostatin dose (for cycles #1 and #2, 0.5 mg / m² of pentostatin). 2 For cycles #3 and #4, pentostatin 1 mg / m² 2 Regarding CrCl<30: Pentostatin is retained.

[0325] Pentostatin is rarely associated with neurological toxicity (seizures, coma), so particular attention should be paid to assessing CNS toxicity. If the PC regimen is associated with any new neurological toxicity of grade 2 or higher severity, or with an exacerbation of any existing neurological toxicity, the institutional PI should be contacted to discuss whether further pentostatin therapy and further protocol therapy are permitted.

[0326] Specific aspects of cyclophosphamide administration: hydration. Because cyclophosphamide can cause cystitis, it is important for patients to maintain adequate hydration. At a minimum, patients should drink at least 2-4 liters of fluids per day to maintain clear urine. It is also especially important to empty the bladder before sleep. Oral cyclophosphamide is administered at a fixed dose of 100 mg per day on days 1, 2, and 3 (for cycles #1 and #2), or on days 1, 2, 3, 4, and 5 (for cycles #3 and #4). However, for patients who do not have a substantial reduction in ALC (as defined by ALC exceeding 1250 cells per microliter) before cycle #4, the cyclophosphamide dose for cycle #4 is increased to 200 mg per day over days 1, 2, 3, 4, and 5. If a patient cannot tolerate oral therapy, IV infusion of cyclophosphamide is permitted. The IV dose is the same as the intended oral dose. For IV infusion, cyclophosphamide is reconstituted to a concentration of 20 mg / ml by the HUMC dispensing department according to the vial instructions. The appropriate dose (100 mg or 200 mg) is then diluted in 100 ml of D5W or 0.9% sodium chloride and administered intravenously over 30 minutes.

[0327] A PC cycle is not expected to result in a significant decrease in the absolute number of neutrophils. However, if the ANC falls below a certain value at the time of the decision immediately preceding the next cycle, the dose of cyclophosphamide will be adjusted as follows: (1) 100% of the intended dose will be administered for ANC values ​​of 1000 cells or more per microliter; (2) 50% of the intended dose will be administered for ANC values ​​of 500 to 999 cells per microliter; and (3) if the ANC value is 500 cells per microliter, no cyclophosphamide will be administered. In addition, for ANC values ​​of less than 500 cells per microliter, the decision to initiate G-CSF therapy may be considered by the PI.

[0328] The quantitative goal of the 8-week PC regimen is to reduce the ALC level to approximately 750 cells per microliter. This degree of depletion and suppression of T cells, which contribute to the pathogenesis of the disease, is necessary to control the neuroinflammatory process. REG The inventors hypothesize that this will enable successful cell transplantation and biological activity. However, potentially, a more severe reduction in host Th1 / Tc1 cells may be necessary. REG It may be necessary to allow cells to exert their full inhibitory function. In such cases, the PC regimen is iT REG Prior to cell therapy, it may be enhanced or extended to target lower ALC values, such as 500, 250, or 0 ALC per microliter. On the other hand, potentially, iT REG Cell therapy is highly effective, and even an ALC value of 750 cells per microliter may be considered too restrictive. In such cases, the PC regimen can be decentralized or shortened in duration to target higher ALC values ​​such as 1000, 1250, or 1500 cells per microliter.

[0329] Implementation of lamivudine maintenance therapy Once the PC regimen is complete, the patient will proceed to maintenance therapy with lamivudine, which will continue until the end of the 6-month trial period of the protocol. Lamivudine (oral tablets) will be administered at a dose of 150 mg po BID. If the estimated creatinine clearance decreases to less than 50 ml / min, lamivudine will be reduced to a dose of 150 mg po once daily, and lamivudine will be discontinued if the estimated creatinine clearance is less than 30 ml / min.

[0330] As previously stated, the stated purpose of lamivudine is to downregulate the NLRP3 inflammasome, which represents the proximal event in the ALS pathogen. Therefore, we anticipate that other inflammasome inhibitors may be suitable, or perhaps possibly suitable, for use in our protocol platform. For example, inflammasome inhibitors with potentially improved risk-benefit ratios have been developed.

[0331] Lamivudine, because its mechanisms of action are actually complementary, iT REG It is important to note that this is not expected to antagonize cell therapy. Complementary controls include rapamycin (which can inhibit a wide variety of T cell responses) and IL-2 (in vivo T REG T cells (which have a narrow therapeutic window in terms of promoting proliferation and can promote inflammatory T cell populations) REG This is in contrast to other interventions proposed for cell therapy.

[0332] supportive care In cases of neutropenia, systemic antibiotic prophylactics are not necessary. The decision to initiate antibiotics depends on the protocol PI.

[0333] All patients may receive oral antiviral prophylaxis for HSV or VZV using acyclovir (or its prodrug valacyclovir) from the start of protocol therapy until the end of the study.

[0334] All patients may receive oral antifungal prophylaxis (first-line drug: fluconazole) from the start of protocol therapy until the end of the trial. Alternative drugs are permitted in accordance with the approval of protocol PI.

[0335] All patients will begin prophylaxis against Pneumocystis PJP at the time of participation in the clinical trial (and continue until their final visit at the end of the trial). Patients will receive oral cotrimoxazole (trimethoprim 160 mg / sulfamethoxazole 800 mg) one tablet orally on Mondays, Wednesdays, and Fridays. Alternative schedules or alternative medications are permitted in accordance with the approval of the protocol PI.

[0336] IT REG Cell-based treatment for ALS patients: Product manufacturing and phenotype As previously explained in detail, iT REG The cell product is produced from autologous T cells collected by apheresis either at the time of trial entry prior to the PC regimen or at the time of completion of the 8-week PC regimen. Each apheresis collectible may have its own advantages: early collectibles have a higher T cell yield, while post-PC collectibles consist of a T cell population that is relatively depleted of Th1 / Tc1 cells.

[0337] iT REG Cells transition from an inflammatory phenotype to an anti-inflammatory T cell. REG Because it is manufactured based on the principle of effector T cell conversion to a phenotype, CD4 + CD25 + , and relatively rare nT cells typically characterized by low expression of CD127. REG Obtaining a population of natural T15 is expensive and time-consuming, requiring either monoclonal antibody / column selection methods or flow cytometry. REG A purification process would likely be unnecessary. In addition, CD8 + T REG Cells have been shown to mediate immunosuppression, iT REG iT may be beneficial in that it provides increased diversity to cell therapies. REG CD8 from a cell population + There will likely be no need to remove T cells.

[0338] T15, a limited number of differentiated states, can be defined as central memory type based on the expression of cell surface markers such as CD62L and CCR7. REG It has been shown that the cells increased their in vivo regulatory function. On the other hand, T cells in a more differentiated effector memory state REG Cell acquisition is also known to enable upcontrol of inhibitory-mediating molecules such as IL-10 and CTLA-4, ectonucleotidase molecules CD39 and CD73, and cell-lysing molecules such as perforin and fas ligand. These data include populations of both central and effector memory subsets. REG We have shown that injecting cell products is beneficial, and therefore, the iT that we utilize reg This indicates that the cell product has representations from both subsets.

[0339] In addition, iT REG Cells need to express FoxP3, a transcription factor that directs the regulatory T cell differentiation program. Furthermore, it has been shown that FoxP3 expression and the resulting regulatory function can deteriorate over time, therefore iT REG The cell product needs to exhibit stable FoxP3 expression over long periods in the culture medium.

[0340] Furthermore, because FoxP3 can be transiently expressed by genuine inflammatory T cell subsets, it has been shown in humans that FoxP3 alone is insufficient for identifying regulatory T cell phenotypes. Therefore, iT cells that express FoxP3 but also relatively lack co-expression of Th1 / Tc1 transcription factor TBET or Th1 cytokines such as IL-2 or IFN-γ, which are associated with inflammatory T cell subsets, are not suitable for identification. REG It is important to produce cell products.

[0341] Finally, iT REG It is important that cell products reduce the ability of differentiation plasticity from the regulatory phenotype to the inflammatory phenotype.REG It has been well documented that cells can be relatively unstable in their repressive phenotype, which can lead to transformation into inflammatory T cell subsets that can actually contribute to the mediation of neurodegenerative diseases. REG The cell product should stably express FoxP3 and also show a reduced tendency towards conversion to the Th1 / Tc1 subset. REG As a further protective measure against cell differentiation plasticity, the inventors intentionally directed IL-4 to iT so that any such differentiation is directed towards the Th2 type line. REG It is incorporated into the cell manufacturing process. Here, the Th2 type lineage is T REG Cell maintenance and T REG The importance of cell suppression function is thought to be T REG It has been described as the default pathway of cells and can mediate anti-inflammatory effects in the setting of ALS. Despite this evidence of the potentially beneficial role of the Th2-like state of regulatory T cells, REG The method for manufacturing cell therapy does not involve the intentional addition of exogenous IL-4 during culture. REG (As illustrated by recent examples of manufacturing).

[0342] At the end of manufacturing, iT REG The cell product is used to determine the therapeutic cell dose (1-5 × 10⁻¹⁰ 6 Frozen storage in at least four single-use aliquots per cell / kg.

[0343] IT REG Cell-based treatment for ALS patients: T REG combination of cell populations IT REG The cell population is divided into 1-5 × 10⁶ cells per kg of recipient body weight. 6 It is injected in cellular doses. This dose has a relatively low T compared to previous studies. REG Cell therapy is facilitated by several factors: PC regimens, iT REG Provides sufficient immunological space for cell engraftment; iT REGCells express memory profiles associated with cell persistence after adoptive transplantation; and iT REG The cell products are cryopreserved in at least four clinically relevant therapeutic doses, thereby enabling multiple treatment cycles.

[0344] As previously explained in detail, iT REG The cell products contain diverse memory differentiation states (central memory [CM] plus effector memory [EM]), thereby enabling both long-term and immediate control of neuroinflammation. The ratio of such central memory populations to effector memory populations can be controlled for optimal results according to the clinical situation, i.e., iT of CM:EM cells based on clinical parameters. REG The distribution can be 1:1, 3:1, 10:1, 1:3, or 1:10.

[0345] Similarly, depending on the clinical situation, CD4 + iT REG Cell vs. CD8 + iT REG It may be possible to improve therapeutic effects by controlling the cell ratio.

[0346] Finally, iT REG and nT REG Since cells express different T cell receptor repertoires and can therefore be complementary in mediating immunosuppression, we believe that iT REG The optimal therapy using cells is nT REG This is assumed to be achievable through the co-administration of cells.

[0347] IT REG Cell therapy for ALS patients: in combination with medication IT REG Cell therapy, when combined with a platform that includes the immunomodulatory effects of PC regimens and the inflammasome inhibitory effects of lamivudine, may be sufficient to control neuroinflammation.

[0348] However, the inventors believe that by changing the platform, iTREG It is assumed that cell therapy may be optimized. Examples, though not limited to, include: modifying the intensity of the PC regimen, substituting cyclophosphamide with another drug to synergize with pentostatin, or adding a third component to the PC regimen, such as adding low-dose IL-2 therapy after anti-TNF therapy, which, according to our reasoning, could be optimized in vivo. REG It predictably increases the number of cells. Low-dose IL-2 therapy is described, but is not limited to, as an example: 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.

[0349] Furthermore, the inventors anticipate that lamivudine may be replaced with more potent or more specific inflammasome inhibitors similar to those recently synthesized.

[0350] Ultimately, the driving force for inflammation in ALS is initiated by more proximal events, such as the accumulation of misfolded RNA elements and insufficient autophagy. In this respect, there is a rationale for using drugs that can promote autophagy, particularly rapamycin, in ALS therapy. However, clinical trials of rapamycin for the treatment of ALS have only just begun (ClinicalTrials.gov identifier: NCT03359538), and furthermore, this protocol evaluates sequential therapy of rapamycin (which may be associated with substantial toxicity), constant doses of rapamycin (which may result in large inter-patient drug variability), and relatively low doses of rapamycin (which do not guarantee the high drug levels necessary for potent inhibition of the mTOR pathway and consequently promotion of autophagy). To circumvent these limitations, we have developed iT REGCell therapy is combined with rapamycin to promote autophagy using the following parameters: limit drug toxicity and iT REG The use of intermittent rapamycin therapy to limit the potential for cellular rapamycin inhibition (e.g., adding a 1-week recovery with mTOR inhibitor therapy followed by a 3-week recovery with mTOR therapy); the use of variable doses of rapamycin, including a loading dose of rapamycin, combined with serological testing of rapamycin levels to ensure homogeneous drug levels for more consistent inhibition of the mTOR pathway; and the use of high-dose rapamycin therapy to achieve serum rapamycin levels of 30 ng / ml in preference to typical target levels of approximately 5–12 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.

[0351] Furthermore, rapamycin therapy may not be optimal for promoting autophagy in neurodegenerative diseases due to insufficient drug penetration into the central nervous system. In this regard, even intravenous therapy with the rapamycin analog temsirolimus did not result in significant drug levels in the cerebrospinal fluid. To overcome this limitation, the inventors envision administering temsirolimus through an indwelling onmaya reservoir in a manner similar to that used in the treatment of lysosomal storage diseases, in order to achieve consistent CSF drug levels of an mTOR inhibitor to optimally promote autophagy in the context of neurodegenerative diseases.

[0352] IT REG Cell-based treatment for ALS patients: Immunological monitoring ALS iT REGIn the context of cell therapy, it will be important to quantify the success of cell therapy in terms of its ability to modulate disease-related neuroinflammatory pathways. In other words, monitoring the clinical course of neurodegenerative diseases is insufficient considering the large variability in disease progression across patient cohorts. The ability to optimally treat neuroinflammation, including in combination with various pharmacological agents, is crucial. REG Multiple cell injections will likely be required. Therefore, it will be important to use immunobiological markers to support guidance in treatment decision-making.

[0353] IT REG Therapeutic decisions regarding cell injection and repeated administration of related pharmacological agents are based on specialized testing of peripheral blood mononuclear cells developed by the inventors. These tests address several key issues related to inflammatory monitoring, including spontaneous cytokine measurement; coordination between T cells and monocytes in cytokine measurement; the roles of recombinant human CD40 ligand, T cell checkpoint inhibitor pathways, and monocyte checkpoint pathways in demasking cytokine secretion; evaluation of inflammasome activation by various techniques such as protein quantification by Western blotting; evaluation of adenosine receptor biology of peripheral T cells as an indicator of inflammatory events; use of flow cytometry to evaluate FoxP3 transcription factor co-expression with Th1-related molecules TBET, IL-2, or IFN-γ; characterization of the T cell receptor repertoire by RNA sequencing; and detection of antigen-specific T cell responses to potential neuronal autoantigens, such as protein aggregates that occur during disease onset.

[0354] Protocol evaluation A clinical evaluation by a physician or intermediate provider is performed on day 1 of each cycle of the PC regimen, which is intended to be a 14-day cycle. The patient is examined once during the 14-day PC cycle (ideally around day 8 of the cycle) by a local provider. At these visits, a differential and complete metabolic panel (CBC) is obtained (a complete metabolic panel typically includes about 14 tests, including electrolytes, creatinine, liver transaminases, and bilirubin; the specific panel used is not mandatory for the protocol), and the laboratory results are sent to the protocol researchers.

[0355] Upon completion of the interval including the PC regimen (approximately 2 months), patients will be examined monthly at 3, 4, 5, and 6 months, with the 6-month visit representing the end-of-trial visit. The trials conducted at these clinical evaluation points will include: (1) preliminary medical history and physical examination; (2) CBC with differential and platelet counts; (3) complete metabolic panel; and (4) immunosubset enumeration (TBNK panel).

[0356] To monitor the immunological parameters of the study, and to enable centralized monitoring and deeper analysis, peripheral blood samples are sent to Rapa Therapeutics. The blood sample consists of 30 ml in a top green heparinized tube (for cell assays) and 5 ml in a top red tube (for serum assays), both sent to Rapa Therapeutics.

[0357] Using the same samples sent to Rapa Therapeutics, the inventors investigated the effects of PC regimens and lamivudine maintenance therapy on pro-inflammatory or anti-inflammatory cytokines or cell subsets as measured by cellular signaling events, RNA expression, supernatant / Luminex assay, flow cytometry, and phosphorylation analysis by Western blotting.

[0358] Serum is evaluated for potential biomarkers of ALS. Examples include, but are not limited to, the biomarkers 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.

[0359] The inventors characterize the patient's TCR repertoire and evaluate whether therapeutic interventions affect the repertoire.

[0360] In vitro studies fall under the general category of “immune characterization studies.” These focus on the isolation of different cell subsets by multi-parameter FACS analysis, or subsequent characterization by magnetic beads. Specifically, peripheral blood mononuclear cells (PBMCs) are analyzed by flow cytometry for hematopoietic lineage, immune function subsets, cytokine production, and the expression of markers indicating activation status. Cell subsets are analyzed for T cell receptor repertoire diversity. Cells can be activated in vitro with a number of different stimuli, including specific antigens and mitogens known to activate different pathways of T lymphocyte or monocyte function. Assays may include T cell proliferation, cytokine production, and gene expression. Specific assays used in ongoing data analysis may be modified, deleted, or replaced as field technology and knowledge evolve during the research process, without constituting a change in the research objectives.

[0361] Response criteria Patient-reported ALSFS-R scores and clinician-reported Appel scores are measured at various points in time, as described above.

[0362] Toxicity standards Toxicity is graded according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) (available from http: / / ctep.info.nih.gov). A copy of CTCAE version 4.0 can be downloaded from the CTEP homepage. All therapeutic areas and personnel involved in the trial must have access to a copy of CTCAE version 4.0.

[0363] Any Grade 4 or 5 toxicity (CTCAE version 4.0) with attributes likely or certainly associated with the study drug (pentostatin, cyclophosphamide, lamivudine) is considered a dose-limiting toxicity (DLT). The following toxicities are not considered DLTs: biochemical Grade 4 toxicity (excluding renal and hepatic values), Grade 4 vomiting, Grade 4 fever, and Grade 4 toxicity associated with infection that resolves within 7 days.

[0364] Statistical considerations This study design incorporates a standard 3+3 methodology for evaluating the safety of the pentostatin and cyclophosphamide regimen and the maintenance of lamivudine therapy (platform). If no patients develop DLT in the first three patients through completion of the pentostatin / cyclophosphamide regimen, the regimen is deemed safe to expand the cohort to a total of n=10 patients. On the other hand, if one of the first three patients develops DLT, the increase is to a total of n=6 patients. In such a case, if one or fewer of the first six patients develop DLT, the cohort can proceed to an increase to n=10.

[0365] Once this platform is successfully developed, the inventors will use iT REG To evaluate the safety and potential efficacy of cell injection. Initially, a pilot study will be conducted to investigate the inhibition of disease-related neuroinflammatory pathways. REG Evaluate the ability of cells to be injected multiple times.

[0366] Once the ability to effectively modulate biomarkers associated with neurodegeneration is documented, the inventors will use iT REG A Phase II clinical trial will be conducted (using either historical control data or a randomized cohort design) to evaluate whether cell therapy can improve clinical outcomes for ALS patients.

[0367] Risk / Benefit Analysis The estimated survival time for patients involved in this study is predicted to be approximately 2 to 4 years from the start of the study.

[0368] The first protocol component consists of a four-cycle immunodepletion and immunosuppression regimen (PC regimen) comprising pentostatin and cyclophosphamide. The inventors hypothesize that the PC regimen eliminates and suppresses pathogenic immune cells that contribute to ALS progression, and therefore patients may benefit from this effect in the form of improved quality of life or ultimately reduced disease progression. However, there may be unexpected toxicity of the PC regimen with respect to the central nervous system. Although drug modifications have been made to help ensure that the PC regimen is relatively safe in this new ALS patient population, the PC regimen may have contradictory effects and may actually increase the rate of ALS progression or cause some other neurological toxicity. Rarely, pentostatin may also cause toxicity to other organs such as the heart or kidneys. The most common toxicity expected from the PC regimen is lymphocyte depletion, although this effect is part of the rationale for the treatment. On the other hand, the PC regimen may eliminate myeloid cells, thereby increasing the likelihood of bacterial or fungal infections. PC regimens are expected to be associated with T-cell immunosuppression and therefore may lead to opportunistic viral infections.

[0369] The second protocol component consists of maintenance therapy with the antiviral drug lamivudine. If the drug works as hypothesized to reduce inflammation originating from the central nervous system, the patient may benefit from this therapy. Lamivudine is generally a very well-tolerated drug, with the exception of gastrointestinal side effects and pancreatitis.

[0370] The third protocol component is iT REG It consists of multiple injections of cells. Patients can benefit from this therapy because the cell therapy, which controls inflammation, occurs directly within the microenvironment where inflammation is initiated; because the cell therapy operates through multiple molecular mechanisms that cannot be easily replicated through drug therapy; and because the effects of the cell therapy can be prolonged by the memory cell effect.

[0371] Alternative protocol design Figure 39 provides an alternative protocol design. Lymphocytes are collected by steady-state apheresis, and the apheresis products are shipped to Rapa Therapeutics (Rockville, MD). After RAPA-501 cell production, n=4 doses of RAPA-501 cells are cryopreserved in single-use infusion bags at clinically indicated cell doses. The treatment interval is 6 months, followed by a 6-month observation interval. Cohort #1 consists of 40 × 10⁶ cells. 6 Patients receive RAPA-501 cells administered over four cycles at a cell / infusion dose. Cohort #1 represents the safety cohort and utilizes a standard 3+3 design. Progression to Cohort #2 occurs if 0 / 3 or 1 / 6 or fewer patients experience dose-limiting toxicity (DLT). Cohort #2 involves a T-cell dose of 120 × 10⁶ 6 The same 4 cycles of RAPA-501 cells as in the above trial protocol were administered, except for an increase in the number of cells / injections. In cohort #3, monotherapy (40 or 120 × 10¹⁶ cells, respectively, according to cohort #1 or #2) was administered before each of the four RAPA-501 cell injections. 6 We will evaluate the highest dose of RAPA-501 cells that can be safely administered as either cell therapy (or injection) + host conditioning with a PC regimen. The PC regimen includes pentostatin (2 mg / m² on days 1 and 4). 2 The treatment consists of cyclophosphamide (100 mg daily, days 1-5), no treatment on days 6 and 7, and RAPA-501 cell infusion on day 8.

[0372] Dedifferentiation Embodiments: 1. A method for dedifferentiating T cells, The process involves planting a culture input population of cells, including target-derived T cells, at cell density in a culture medium containing vitamin D, temsirolimus, and an IL-2 signaling inhibitor, and Add anti-CD3 / anti-CD28 coated magnetic beads to the above T cells and culture medium in a bead:T cell ratio of 1:1 to 1:12, A method comprising incubating the above-mentioned cell culture input population and culture medium for a certain period of time to obtain dedifferentiated T cells. 2. The method according to Embodiment 1, further comprising collecting the dedifferentiated T cells described above. 3. After collecting the dedifferentiated T cells as described above, The above involves packaging at least a portion of the dedifferentiated T cells into a package, The method according to Embodiment 2, further comprising freezing the above package containing the above portion of dedifferentiated T cells. 4. Before inoculating the above cell culture input population into the above culture medium, The method according to any one of Embodiments 1 to 3, further comprising collecting a culture input population of the above cells from the above subject. 5. The method according to any one of Embodiments 1 to 4, wherein the culture medium described above does not contain IL-2, and IL-2 is not added to the culture medium described above. 6. The method according to any one of Embodiments 1 to 5, wherein the cell density is at least 1.5 × 10⁶ T cells per 1 mL. 7. The method according to any one of Embodiments 1 to 6, wherein the above-mentioned temsirolimus is present in the culture medium at a concentration of approximately 0.3 μM to approximately 1 μM. 8. The method according to any one of Embodiments 1 to 6, wherein the above-mentioned temsirolimus is present in the culture medium at a concentration of approximately 1 μM. 9. The method according to any one of Embodiments 1 to 8, wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody or a fragment thereof. 10. The method according to Embodiment 9, wherein the IL-2 signaling inhibitor is basiliximab or daclizumab. 11. The method according to any one of Embodiments 1 to 10, wherein the above-mentioned IL-2 signaling inhibitor is present in the culture medium at a concentration of 5 to 50 μg / mL. 12. The method according to any one of Embodiments 1 to 11, wherein the above period is approximately 3 days. 13. The method according to any one of Embodiments 1 to 12, wherein the beads:T cell ratio is 1:3. 14. The method according to any one of Embodiments 1 to 13, wherein the culture medium further comprises 5% human serum. 15. The method according to any one of Embodiments 1 to 14, wherein the culture medium described above includes X-Vivo 20 medium. 16. The method according to any one of Embodiments 1 to 15, wherein the above vitamin D is present in the above culture medium at a concentration of approximately 0.03 nM to approximately 1 nM. 17. The method according to any one of Embodiments 1 to 15, wherein the above vitamin D is present in the above culture medium at a concentration of approximately 0.1 nM. 18. Further comprising measuring the expression levels of RAPTOR or RICTOR in the culture input population of the above cells, The method according to any one of embodiments 1-11 and 13-17, wherein the above period continues until the expression level of RAPTOR or RICTOR in the cell culture input population is reduced by at least 50%, more preferably 90%, compared to a control population of T cells produced under the same conditions as the cell culture input population that does not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D. 19. Further comprising measuring the expression levels of RAPTOR or RICTOR and housekeeping proteins in the culture input population of the above cells, The method according to any one of embodiments 1-11 and 13-17, wherein the above period continues until, after normalization for housekeeping protein expression levels, the expression levels of RAPTOR or RICTOR, respectively, in the culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D are reduced by at least 50% compared to a control population of T cells produced under the same conditions. 20. The method according to Embodiment 19, wherein the housekeeping protein is actin or GAPDH. 21. The method according to any one of embodiments 18 to 20, wherein the above step of measuring the expression level is performed by Western blot analysis. 22. Further comprising measuring the expression levels of RAPTOR or RICTOR in the culture input population of the above cells, The method according to any one of embodiments 1-11 and 13-17, wherein the above period continues until the expression level of RAPTOR or RICTOR in the cell culture input population is reduced by at least 50%, more preferably 90%, compared to a control population of T cells produced under the same conditions as the cell culture input population that does not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D. 23. Dedifferentiated T cells produced by the method described in any one of Embodiments 1 to 22. 24. A composition comprising a population of dedifferentiated T cells, At least a portion of the above population of dedifferentiated T cells express less than 50% of both RAPTOR or RICTOR compared to the T cell control population, and the T cell control population is prepared under the same conditions as the cell culture input population, which does not contain temsirolimus, IL-2 signaling inhibitors, and vitamin D. 25. A method for dedifferentiating T cells, The process involves planting a culture input population of cells, including T cells derived from the target, at cell density in a culture medium containing vitamin D and temsirolimus, The above T cells are stimulated by adding anti-CD3 / anti-CD28 coated magnetic beads to the above T cells and culture medium in a bead:T cell ratio of 1:1 or less. A method comprising incubating the above-mentioned cell culture input population and culture medium for a certain period of time to obtain dedifferentiated T cells. 26. The method according to Embodiment 25, further comprising collecting the dedifferentiated T cells described above. 27. After collecting the dedifferentiated T cells described above, The above involves packaging at least a portion of the dedifferentiated T cells into a package, The method according to Embodiment 26, further comprising freezing the above package containing the above portion of dedifferentiated T cells. 28. Before inoculating the above cell culture input population into the above culture medium, The method according to any one of embodiments 25 to 27, further comprising collecting a culture input population of the above cells from the above subject. 29. The method according to any one of Embodiments 25 to 28, wherein the culture medium described above does not contain IL-2, and IL-2 is not added to the culture medium described above. 30. The method according to any one of Embodiments 25 to 29, wherein the cell density is 1.5 × 10⁶ T cells per 1 mL. 31. The method according to any one of Embodiments 25 to 30, wherein the above-mentioned temsirolimus is present in the culture medium at a concentration of approximately 0.3 μM to approximately 1 μM. 32. The method according to any one of Embodiments 25 to 30, wherein the above-mentioned temsirolimus is present in the culture medium at a concentration of approximately 1 μM. 33. The method according to any one of embodiments 25 to 32, wherein the above period is approximately 3 days. 34. The method according to any one of embodiments 25 to 33, wherein the beads:T cell ratio is 1:3. 35. The method according to any one of Embodiments 25 to 34, wherein the culture medium further comprises 5% human serum. 36. The method according to any one of Embodiments 25 to 35, wherein the culture medium described above includes X-Vivo 20 medium. 37. The method according to any one of Embodiments 25 to 36, wherein the above vitamin D is present in the above culture medium at a concentration of approximately 0.03 nM to approximately 1 nM. 38. The method according to any one of Embodiments 25 to 37, wherein the above vitamin D is present in the above culture medium at a concentration of approximately 0.1 nM. 39. Further comprising measuring the expression levels of RAPTOR or RICTOR in the culture input population of the above cells, The method according to any one of embodiments 25-32 and 34-38, wherein the above period continues until the expression level of RAPTOR or RICTOR in the above cell culture input population is reduced by at least 50% compared to a control population of T cells produced under the same conditions as the cell culture input population that does not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D. 40. Further includes measuring the expression levels of RAPTOR, RICTOR, and housekeeping proteins in the culture input population of the above cells, The method according to any one of embodiments 25-32 and 34-38, wherein the above period continues until, after normalization for housekeeping protein expression, the expression level of RAPTOR or RICTOR in the cell culture input population is reduced to 50%, or more preferably 90%, compared to a control population of T cells produced under the same conditions as the cell culture input population that does not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D. 41. The method according to Embodiment 40, wherein the housekeeping protein is actin or GAPDH. 42. The method according to any one of embodiments 39 to 41, wherein the above step of measuring the expression level is performed by Western blot analysis. 43. Further comprising measuring the expression levels of RAPTOR or RICTOR in the culture input population of the above cells, The method according to any one of embodiments 25-32 and 34-38, wherein the above period continues until the expression level of RAPTOR or RICTOR in the cell culture input population is reduced by at least 50%, more preferably 90%, compared to a control population of T cells produced under the same conditions as the cell culture input population that does not contain temsirolimus, an IL-2 signaling inhibitor, and vitamin D. 44. Dedifferentiated T cells produced by the method described in any one of embodiments 25 to 43. 45. A dedifferentiated T cell population characterized by at least 10% and more preferably 50% reduction in RNA expression for the following T cell differentiation molecules: cell lysis molecules including but not limited to granzyme B, and cytokine molecules including but not limited to IFN-γ, compared to a control population of T cells cultured without the culture additives identified by these methods. 46. ​​A dedifferentiated T cell population characterized by at least a 10% increase, more preferably a 50% increase, in RNA expression for the following T cell differentiation molecules: transcription factors associated with induced pluripotent stem cells, including but not limited to Nanog, KLF4, and KLF10, and molecules associated with naive T cells, including but not limited to the IL-7 receptor and CD127, compared to a control population of T cells cultured without the culture additives identified by these methods. 47. A dedifferentiated T cell population characterized by at least a 10% decrease, more preferably a 50% decrease, in RNA expression of the following T cell differentiation molecules: T-Bet and STAT1, but not limited to these, and transcription factors associated with Th1 effector T cells, compared to a control population of T cells cultured without the culture additives identified by these methods, but incidentally having equivalent expression of cell survival-related transcription factors, including but not limited to HIF-1-α. 48. A dedifferentiated T cell population characterized by an at least 10% and more preferably 50% increase in the expression of autophagy molecular markers, including but not limited to an increase in the protein level of the autophagy-related molecule p62 as determined by Western blot analysis, compared to a control population of T cells cultured without the culture additives identified by these methods. 49. The method according to any one of Embodiments 1 to 22, wherein the above step of adding anti-CD3 / anti-CD28 coated magnetic beads to the T cells and culture medium in a bead:T cell ratio of 1:1 to 1:12 is not performed. 50. The method according to any one of Embodiments 25 to 43, wherein the above step of adding anti-CD3 / anti-CD28 coated magnetic beads to the above T cells and culture medium in a bead:T cell ratio of 1:1 or less is not performed in order to stimulate the above T cells. 51. The following characteristics: Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of granzyme B, IL-10, and IFN-γ was reduced by at least 10%, more preferably by 50%. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs among Nanog, KLF4, KLF10, and CD127 was increased by at least 10%, more preferably by 50%. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of T-Bet and STAT1 was reduced by at least 10%, more preferably by 50%. HIF-1-α expression was within approximately 20% of a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, p62 expression was increased by at least 10%, more preferably by 50%. Compared to a control population of T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, the expression levels of RAPTOR or RICTOR are reduced by at least 50%, more preferably 90%. The expression levels of RAPTOR or RICTOR, normalized by housekeeping proteins, are reduced by at least 50%, more preferably 90%, compared to a control population of T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, and A population of dedifferentiated T cells characterized by one or more of those combinations. 52. The following characteristics: Compared to control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of granzyme B, IL-10, and IFN-γ was reduced by at least 10%, more preferably by 50%. Compared to control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs among Nanog, KLF4, KLF10, and CD127 was increased by at least 10%, more preferably by 50%. Compared to control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of T-Bet and STAT1 was reduced by at least 10%, more preferably by 50%. HIF-1-α expression in control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors was within approximately 20%. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, p62 expression was increased by at least 10%, more preferably by 50%. Compared to control T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, the expression levels of RAPTOR or RICTOR are reduced by at least 50%, more preferably 90%. The expression levels of RAPTOR or RICTOR, normalized by housekeeping proteins, are reduced by at least 50%, more preferably 90%, compared to control T cells produced under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, and Dedifferentiated T cells characterized by one or more of those combinations.

[0373] Redifferentiation Embodiment: 1. A method for differentiating dedifferentiated T cells into TREG / Th2 cells, Dedifferentiated T cells are cultured in a culture medium containing IL-2, IL-4, and TGF-β, and anti-CD3 / anti-CD28 coated magnetic beads are added in a 3:1 ratio (beads:T cell ratio). A method comprising incubating the above-mentioned dedifferentiated T cells for a certain period of time to obtain TREG / Th2 cells. 2. The method according to Embodiment 1, wherein the culture medium further comprises pemetrexed. 3. The method according to any one of Embodiments 1 to 2, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 4. The method according to any one of Embodiments 1 to 3, wherein the above IL-4 is present in the culture medium at a concentration of approximately 1000 IU / mL. 5. The method according to any one of Embodiments 1 to 4, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 6. The method according to Embodiment 2, wherein the pemetrexed described above is present in the culture medium at a maximum concentration of 100 nM. 7. The method according to Embodiment 2, wherein the above pemetrexed is present in the culture medium at a concentration of approximately 10 nM. 8. The method according to any one of Embodiments 6 to 7, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 9. The method according to any one of Embodiments 6 to 8, wherein the above IL-4 is present in the culture medium at a concentration of approximately 1000 IU / mL. 10. The method according to any one of Embodiments 6 to 9, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 11. A method for differentiating dedifferentiated T cells into TREG / Th2 cells, The method involves culturing dedifferentiated T cells, wherein the dedifferentiated T cells express RAPTOR and RICTOR at levels at least 10% lower than control T cells in a culture medium containing IL-2, IL-4, and TGF-β. Add anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 ratio (beads:T cell ratio). A method comprising incubating the dedifferentiated T cells for a certain period of time to obtain TREG / Th2 cells. 12. The method according to Embodiment 11, wherein the culture medium further comprises pemetrexed. 13. The method according to any one of Embodiments 11 to 12, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 14. The method according to any one of Embodiments 11 to 13, wherein the above IL-4 is present in the culture medium at a concentration of approximately 1000 IU / mL. 15. The method according to any one of Embodiments 11 to 14, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 16. The method according to Embodiment 12, wherein the pemetrexed described above is present in the culture medium at a maximum concentration of 100 nM. 17. The method according to Embodiment 12, wherein the above pemetrexed is present in the culture medium at a concentration of approximately 10 nM. 18. The method according to any one of Embodiments 16 to 17, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 19. The method according to any one of Embodiments 16 to 18, wherein the above IL-4 is present in the culture medium at a concentration of approximately 1000 IU / mL. 20. The method according to any one of Embodiments 16 to 19, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 21. The method according to any one of Embodiments 1 to 20, wherein the culture medium is X-Vivo 20 supplemented with 5% human AB serum. 22. The method according to any one of Embodiments 1 to 21, wherein the above period is 3 to 40 days. 23. TREG / Th2 cells produced by the method described in any one of Embodiments 1 to 22. 24. A method for differentiating dedifferentiated T cells into TREG cells, The process involves culturing dedifferentiated T cells in which RAPTOR and RICTOR expression is reduced compared to a control population of T cells, in a culture medium containing IL-2 and TGF-β. Adding anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 ratio (beads:T cell ratio), A method comprising incubating the above-mentioned dedifferentiated T cells for a certain period of time to obtain TREG cells. 25. The method according to Embodiment 24, wherein the culture medium further comprises pemetrexed. 26. The method according to any one of Embodiments 24 to 25, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 27. The method according to any one of Embodiments 24 to 26, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 28. The method according to Embodiment 25, wherein the above pemetrexed is present in the culture medium at a maximum concentration of 100 nM. 29. The method according to Embodiment 25, wherein the above pemetrexed is present in the culture medium at a concentration of approximately 10 nM. 30. The method according to any one of Embodiments 28 to 29, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 31. The method according to any one of Embodiments 28 to 30, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 32. A method for differentiating dedifferentiated T cells into TREG cells, The method involves culturing dedifferentiated T cells, wherein the dedifferentiated T cells express RAPTOR and RICTOR at levels at least 10% lower than control T cells in a culture medium containing IL-2 and TGF-β. Add anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 ratio (beads:T cell ratio). A method comprising incubating the above-mentioned dedifferentiated T cells for a certain period of time to obtain TREG cells. 33. The method according to Embodiment 32, wherein the culture medium further comprises pemetrexed. 34. The method according to any one of Embodiments 32 to 33, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 35. The method according to any one of Embodiments 32 to 34, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 36. The method according to Embodiment 33, wherein the pemetrexed described above is present in the culture medium at a maximum concentration of 100 nM. 37. The method according to Embodiment 33, wherein the above pemetrexed is present in the culture medium at a concentration of approximately 10 nM. 38. The method according to any one of embodiments 36 to 37, wherein the above IL-2 is present in the culture medium at a concentration of approximately 100 IU / mL. 39. The method according to any one of Embodiments 36 to 38, wherein the above TGF-β is present in the culture medium at a concentration of approximately 10 ng / mL. 40. The method according to any one of Embodiments 24 to 39, wherein the culture medium is X-Vivo 20 medium supplemented with 5% AB serum. 41. The method according to any one of embodiments 24 to 40, wherein the above period is 3 to 40 days. 42. The method according to any one of Embodiments 1 to 41, wherein the dedifferentiated T cells described above have reduced expression of RAPTOR and RICTOR compared to a control population of T cells. 43. TREG cells produced by the method described in any one of embodiments 1 to 22 and 24 to 42. 44. Lymphocytes collected by apheresis for subsequent T cell culture are obtained in a steady state or after treatment of the target with an anti-TNF-α therapeutic agent, a cell-free form of TNF-α, most notably the recombinant receptor molecule etanercept, or the monoclonal antibody adalimumab, produced by the method of any one of embodiments 1-22 and 24-42, which is relatively selective in terms of neutralizing serum, and are TREG cells or hybrid TREG / Th2 cells. 45. TREG cells or hybrid TREG / Th2 cells or populations thereof that have increased expression by flow cytometry of at least one of the following molecules: CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OX40, and combinations thereof, compared to control Th1 / Tc1 cells, as described by the method of any one of embodiments 1-22 and 24-42. 46. ​​TREG cells or hybrid TREG / Th2 cells or populations thereof that exhibit reduced secretion of inflammatory cytokines, including IFN-γ and TNF-α, compared to control Th1 / Tc1 cells, as produced by the method of any one of embodiments 1-22 and 24-42. 47. TREG cells or hybrid TREG / Th2 cells produced by the method of any one of embodiments 1-22 and 24-42, wherein the above TREG cells or hybrid TREG / Th2 cells or populations thereof have modified expression of T cell fate transcription factors, most notably a decrease in TBET and an increase in FOXP3, compared to control Th1 / Tc1 cells. 48. TREG cells or hybrid TREG / Th2 cells produced by the method of any one of embodiments 1-22 and 24-42, wherein the above-described TREG cells or hybrid TREG / Th2 cells or populations thereof have additional phenotypic traits, including increased secretion of the Th2 cytokine IL-4 and increased expression of the Th2 transcription factor GATA3, compared to control Th1 / Tc1 cells. 49. A population of TREG or TREG / Th2 cells having at least 5% of CD4+ or CD8+ T cells expressing GATA3. 50. Population of TREG or TREG / Th2 cells having at least 5% of CD4+ or CD8+ T cells expressing FoxP3. 51. A population of TREG or TREG / Th2 cells having at least 10% of CD4+ or CD8+ T cells expressing CD73. 52. A population of TREG or TREG / Th2 cells having at least 10% of CD4+ or CD8+ T cells expressing CD103. 53. A population of TREG or TREG / Th2 cells having at least 20% CD4+ or CD8+ T cells expressing CD150. A population of TREG or TREG / Th2 cells expressing at least 5 pg / mL / 1 × 10⁶ cells / day of IL-4 after co-stimulation with anti-CD3 / anti-CD28 beads in a bead:T cell ratio of 54.3:1. A population of TREG or TREG / Th2 cells expressing at least 100 pg / mL / 1 × 10⁶ cells / day of IL-2 after co-stimulation with anti-CD3 / anti-CD28 beads in a bead:T cell ratio of 55.3:1. A population of TREG or TREG / Th2 cells expressing at least 100 pg / mL / 1 × 10⁶ cells / day of IFN-γ or GM-CSF after co-stimulation with anti-CD3 / anti-CD28 beads in a bead:T cell ratio of 56.3:1. A population of TREG or TREG / Th2 cells expressing less than 100 pg / mL / 1×10⁶ cells / day after co-stimulation with anti-CD3 / anti-CD28 beads at a bead:T cell ratio of 57.3:1. 58. The following characteristics: Compared to control Th1 / Tc1 cells, the expression of one or more of the following, as measured by flow cytometry, is increased by at least 10%: CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OXO40, and combinations thereof. IFN-γ secretion was reduced by at least 10% compared to control Th1 / Tc1 cells. TNF-α secretion was reduced by at least 10% compared to control Th1 / Tc1 cells. TBET expression was reduced by at least 10% compared to control Th1 / Tc1 cells. FOXP3 expression increased by at least 10% compared to control Th1 / Tc1 cells. When measured by flow cytometry, CD4 expressing GATA3 + or CD8 + T cells make up at least 5%, When measured by flow cytometry, CD4 expressing FOXP3 + or CD8 + T cells make up at least 5%, When measured by flow cytometry, CD4 expressing CD73 + or CD8 + T cells make up at least 5%, When measured by flow cytometry, CD4 expressing CD103 + or CD8 + T cells make up at least 5%, CD4 expressing both FOXP3 and GATA3 as measured by flow cytometry + or CD8 + Cells make up at least 5%, When measured by flow cytometry, CD4 expressing CD150 + or CD8 + T cells make up at least 20%, T REG or T REG Compared to the T cell population specific to the T cells produced, the expression of one or more of the following GATA3, FoxP3, CD73, CD103, and CD150 is increased by at least 50%. After co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, IL-4 levels of at least 5 pg / mL / 1×10⁻¹⁰ 6 Cells / day secretion, After co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, IL-2 levels of at least 100 pg / mL / 1×10⁻¹⁰ 6 Cells / day secretion, Co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, followed by IFN-γ at 100 pg / mL / 1×10⁶ 6 Secretion of less than cells / day, Co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, followed by GM-CSF at 100 pg / mL / 1×10⁶ 6 Secretion of less than cells / day, TNF-α levels of 10 pg / mL / 1×10⁻¹⁰ after co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio. 6 Secretion of less than cells / day, IL-17 levels of 10 pg / mL / 1×10⁻¹⁰ after co-stimulation with anti-CD3 / anti-CD28 beads at a 3:1 bead:T cell ratio. 6 Secretion of less than cells / day, and A population of TREG or TREG / Th2 cells having one or more of those combinations. 59. The following characteristics: Compared to control Th1 / Tc1 cells, the expression of one or more of the following, as measured by flow cytometry, is increased by at least 10%: CD25, CD27, 2B4, BTLA, CTLA4, TIGIT, TIM3, ICOS, LAIR1, OXO40, and combinations thereof. IFN-γ secretion was reduced by at least 10% compared to control Th1 / Tc1 cells. TNF-α secretion was reduced by at least 10% compared to control Th1 / Tc1 cells. TBET expression was reduced by at least 10% compared to control Th1 / Tc1 cells. FOXP3 expression increased by at least 10% compared to control Th1 / Tc1 cells. After co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, IL-4 levels of at least 5 pg / mL / 1×10⁻¹⁰ 6 Cells / day secretion, After co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, IL-2 levels of at least 100 pg / mL / 1×10⁻¹⁰ 6 Cells / day secretion, Co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, followed by IFN-γ at 100 pg / mL / 1×10⁶ 6 Secretion of less than cells / day, Co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio, followed by GM-CSF at 100 pg / mL / 1×10⁶ 6 Secretion of less than cells / day, TNF-α levels of 10 pg / mL / 1×10⁻¹⁰ after co-stimulation with anti-CD3 / anti-CD28 beads in a 3:1 bead:T cell ratio. 6 Secretion of less than cells / day, IL-17 levels of 10 pg / mL / 1×10⁻¹⁰ after co-stimulation with anti-CD3 / anti-CD28 beads at a 3:1 bead:T cell ratio. 6 Secretion of less than cells / day, Expression of GATA3, FOXP3, CD73, and CD103, and TREG or TREG / Th2 cells having one or more of those combinations. 60. Dedifferentiated T cells have the following characteristics: Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of granzyme B, IL-10, and IFN-γ was reduced by at least 10%, more preferably by 50%. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs among Nanog, KLF4, KLF10, and CD127 was increased by at least 10%, more preferably by 50%. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of T-Bet and STAT1 was reduced by at least 10%, more preferably by 50%. HIF-1-α expression was within approximately 20% of a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, p62 expression was increased by at least 10%, more preferably by 50%. Compared to a control population of T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, the expression levels of RAPTOR or RICTOR are reduced by at least 50%, more preferably 90%. The expression levels of RAPTOR or RICTOR, normalized by housekeeping proteins, are reduced by at least 50%, more preferably 90%, compared to a control population of T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, and The method according to any one of claims 1 to 41, comprising one or more combinations thereof. 61. Dedifferentiated T cells have the following characteristics: Compared to control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of granzyme B, IL-10, and IFN-γ was reduced by at least 10%, more preferably by 50%. Compared to control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs among Nanog, KLF4, KLF10, and CD127 was increased by at least 10%, more preferably by 50%. Compared to control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, the expression of one or more mRNAs of T-Bet and STAT1 was reduced by at least 10%, more preferably by 50%. HIF-1-α expression in control T cells incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors was within approximately 20%. Compared to a control T cell population incubated under the same conditions without temsirolimus, vitamin D, and IL-2 signaling inhibitors, p62 expression was increased by at least 10%, more preferably by 50%. Compared to control T cells produced under the same conditions as a culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, the expression levels of RAPTOR or RICTOR are reduced by at least 50%, more preferably 90%. The expression levels of RAPTOR or RICTOR, normalized by housekeeping proteins, are reduced by at least 50%, more preferably 90%, compared to control T cells produced under the same conditions as the culture input population of cells without temsirolimus, IL-2 signaling inhibitors, and vitamin D, and The method according to any one of claims 1 to 41, comprising one or more combinations thereof.

Claims

1. Induced hybrid T ＲＥＧ A method for producing Th2 cells, Dedifferentiated T cells were cultured in a culture medium containing IL-2, IL-4, and TGF-β to induce hybrid T cells. ＲＥＧ / This includes culturing for a period of time sufficient to produce Th2 cells, A method wherein the dedifferentiated T cells are prepared by culturing T cells in the presence of vitamin D, temsirolimus, and an anti-IL-2 receptor antibody or its antigen-binding fragment.

2. The method according to claim 1, wherein the culture medium further comprises anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 ratio (beads:T cell ratio).

3. The method according to claim 1, wherein IL-2 is present in the culture medium at a concentration of 100 IU / mL to 10,000 IU / mL.

4. The method according to claim 1, wherein the TGF-β is present in the culture medium at a concentration of 5 ng / mL to 10 ng / mL.

5. The method according to claim 1, wherein the aforementioned period is 3 to 40 days.

6. The method according to claim 1, wherein the concentration of IL-4 is 100 IU / mL to 1000 IU / mL.

7. The method according to claim 1, wherein the dedifferentiated T cells express RAPTOR and RICTOR at a level reduced by at least 50% compared to a control population of T cells.

8. Induced hybrid T ＲＥＧ A method for producing Th2 cells, The process involves culturing T cells in a first culture medium containing vitamin D, temsirolimus, and an IL-2 signaling inhibitor for a first period sufficient to produce dedifferentiated T cells, The dedifferentiated T cells are isolated from the first culture medium, Dedifferentiated T cells are cultured in a second culture medium containing IL-2, IL-4, and TGF-β. To the second culture medium, add anti-CD3 / anti-CD28 coated magnetic beads in a 3:1 ratio (beads:T cell ratio), The dedifferentiated T cells are induced in a second culture medium as hybrid T cells. ＲＥＧ This includes incubating for a second period sufficient to produce Th2 cells, A method wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody or its antigen-binding fragment.

9. The method according to claim 8, wherein IL-2 is present in the second culture medium at a concentration of 100 IU / mL to 10,000 IU / mL.

10. The method according to claim 8, wherein the TGF-β is present in the second culture medium at a concentration of 5 ng / mL to 10 ng / mL.

11. The method according to claim 8, wherein the second period is 3 to 40 days.

12. The method according to claim 8, wherein IL-4 is present in the second culture medium at a concentration of 100 IU / mL to 1000 IU / mL.

13. The method according to claim 8, wherein the temsirolimus is present in the first culture medium at a concentration of at least 1 μM.

14. The method according to claim 8, wherein the IL-2 signaling inhibitor is basiliximab or daclizumab.

15. The method according to claim 8, wherein the IL-2 signaling inhibitor is present in the first culture medium at a concentration of 5 μg / mL to 50 μg / mL.

16. The method according to claim 8, wherein the first period is approximately three days.

17. The method according to claim 8, further comprising adding anti-CD3 / anti-CD28 coated magnetic beads to the first culture medium in a bead:T cell ratio of 1:1 to 1:

12.

18. The method according to claim 8, wherein the vitamin D is present in the first culture medium at a concentration of about 0.03 nM to about 1 nM.

19. The method further includes measuring the expression level of RAPTOR or RICTOR in the T cells, The method according to claim 8, wherein the first period continues until the expression level of RAPTOR or RICTOR in the T cells is reduced by at least 50% compared to a control population of T cells cultured under the same conditions without temsirolimus, an IL-2 signaling inhibitor, and vitamin D.

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