Stable regulatory T cells and methods of production

Abstract

[0001] The present disclosure relates to isolated cell populations comprising stable regulatory T cells and methods for producing the same. Some embodiments provide isolated cell populations comprising regulatory T cells (also referred to as Tregs), wherein the stable regulatory T cells of the isolated cell population (a) comprise an exogenous human T cell receptor (TCR) (also referred to as TCR pMHC) that specifically binds to a target peptide complexed with a major histocompatibility complex (MHC), and (b) comprise a hypomethylated regulatory T cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus, wherein at least 80% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / lo regulatory T cells, and wherein less than 10% of the cells of the isolated cell population express FOXP3 protein from an engineered FOXP3 locus.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 397,704, filed August 12, 2022, U.S. Provisional Patent Application No. 63 / 412,032, filed September 30, 2022, and U.S. Provisional Patent Application No. 63 / 417,422, filed October 19, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] (Reference to the Electronic Sequence Listing) The contents of the electronic sequence listing (ABTH_001_03WO_SeqList_ST26.XML, size: 9,221 bytes, and creation date: August 9, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Regulatory T cells have the potential to treat diseases such as autoimmune diseases because they can target specific disease cell types. Regulatory T cells may be useful, for example, in generating local immune responses specific to specific cell types and tissues associated with a disease of interest through antigen-specific mechanisms. However, robust clinical-scale and clinical-grade production of stable, antigen-specific regulatory T cells has been difficult. Therefore, new methods for producing such regulatory T cells are needed. Summary of the Invention [Means for solving the problem]

[0004] Some embodiments provide an isolated cell population comprising regulatory T cells (also referred to as Tregs), wherein the stable regulatory T cells of the isolated cell population (a) comprise an exogenous human T cell receptor (TCR) (also referred to as TCR-pMHC) that specifically binds to a target peptide complexed with a major histocompatibility complex (MHC), and (b) comprise a hypomethylated regulatory T cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus, wherein at least 80% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / lo regulatory T cells, and wherein less than 10% of the cells of the isolated cell population express FOXP3 protein from an engineered FOXP3 locus. The target peptide is typically a specific antigenic peptide (agonist) in that it triggers intracellular signaling pathways that induce expression of genes required for T cell-mediated functions, such as cytokine secretion and suppressive activity.

[0005] In some embodiments, the present disclosure provides an isolated population of cells comprising stable CD4+ regulatory T cells (Tregs) derived from a subject with an autoimmune disease, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus, and the cells comprise an exogenous human T cell receptor (TCR) that specifically binds to a target peptide complexed with a major histocompatibility complex (MHC).

[0006] In some embodiments, the stable CD4+ Tregs do not express FOXP3 protein from the engineered FOXP3 locus. In some embodiments, the TSDR is the CNS2 region of FOXP3. In some embodiments, the MHC is MHC class I or MHC class II.

[0007] In some embodiments, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells are stable CD4+ Tregs that comprise a hypomethylated TSDR at the endogenous FOXP3 locus.

[0008] In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the cells are CD4+CD25+CD127- / lo. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the cells are CD4+CD25+CD127- / loFOXP3+.

[0009] In some embodiments, the population comprises at least 4×10 7 stable CD4+ Tregs. In some embodiments, the population comprises between 4×10 7 and 1×10 10 stable CD4+ Tregs.

[0010] In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated population are conventional CD4+ T cells. In some embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the conventional T cells comprise an exogenous human TCR. In some embodiments, the ratio of stable CD4+ Tregs to conventional T cells in the isolated population is at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, at least 500:1, at least 1000:1, or at least 10000:1. In some embodiments, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated population are CD8+ T cells. In some embodiments, the isolated population does not contain a percentage of CD8+ T cells detectable by fluorescence-activated cell sorting (FACS).

[0011] In some embodiments, at least 10% of the cells express an exogenous human TCR, hi some embodiments, at least 15%, 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% of the cells express an exogenous human TCR.

[0012] In some embodiments, the percentage of stable CD4+ Tregs containing a hypomethylated TSDR at the FOXP3 locus is reduced by no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% after a cryopreservation freeze-thaw cycle.

[0013] In some embodiments, stable CD4+ Tregs exhibit one or more functions selected from regulatory cytokine secretion activity, expression of activation markers associated with regulatory T cells, and / or suppressive activity.

[0014] In some embodiments, the exogenous human TCR comprises one or more amino acid substitutions to cysteine residues in the TCR α chain constant region and the TCR β chain constant region, wherein the cysteine residues are capable of forming one or more disulfide bonds. In some embodiments, the TCR α chain constant region comprises a T48C amino acid substitution relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR β chain constant region comprises a S57C amino acid substitution relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3.

[0015] In some embodiments, the disclosure provides a method of producing a population of cells comprising stable CD4+ regulatory T cells (Tregs), the method comprising: (a) removing CD8+ cells, CD19+ cells, and optionally CD14+ cells from a biological sample obtained from a subject with an autoimmune disease to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+ cells to produce an enriched population; (c) isolating CD4+CD25+CD127− / lo cells from the enriched population; (d) expanding the enriched population to produce an enriched population of expanded cells; and (e) quantifying the methylation status of a T-cell-specific demethylated region (TSDR) at the FOXP3 locus in the cell population, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus, thereby producing a cell population comprising stable CD4+ Tregs.

[0016] In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4+ Tregs that contain a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated population is selected for therapeutic use when the percentage of cells that contain a hypomethylated TSDR at the FOXP3 locus is 80% or greater.

[0017] In some embodiments, step (c) is performed at least twice. In some embodiments, the method further comprises activating the population of cells of step (c).

[0018] In some embodiments, activating comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the second activation step is performed 4 to 8 days after the first activation step. In some embodiments, the cells are expanded in a culture medium containing IL-2 and TNFα. In some embodiments, activating and expanding comprises culturing the population of cells for at least 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, activating and expanding comprises culturing the population of cells for no more than 15, 14, 13, or 12 days.

[0019] In some embodiments, the disclosure provides methods for producing a population of cells comprising engineered stable CD4+ Tregs, the method comprising: (a) removing CD8+ cells, CD19+ cells, and optionally CD14+ cells from a biological sample obtained from a subject with an autoimmune disease to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+ cells to produce an enriched population; (c) isolating CD4+CD25+CD127− / lo cells from the enriched population; (d) delivering a vector comprising a nucleic acid encoding an exogenous human T cell receptor (TCR) to the isolated population of (c) to produce a population of engineered cells; (e) expanding the population of engineered cells to produce an expanded population of engineered cells; and (f) quantifying the methylation status of a T-cell-specific demethylated region (TSDR) at the FOXP3 locus in the expanded population of engineered cells, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus.

[0020] In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4+ Tregs that contain a hypomethylated TSDR at the FOXP3 locus. In some embodiments, step (c) is performed at least twice. In some embodiments, the isolated population is selected for therapeutic use when the percentage of cells that contain a hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95%, or more.

[0021] In some embodiments, the method further comprises activating the population of engineered cells of step (c). In some embodiments, the activating comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the activation step is performed at least twice. In some embodiments, the second activation step is performed 4-8 days after the first activation step. In some embodiments, the cells are grown in a culture medium comprising IL-2 and TNFα.

[0022] In some embodiments, the expanded population comprises at least 1 x 10 7 The patient is a patient with a stable CD4+ Treg cell line.

[0023] In some embodiments, the methylation status of the TSDR in step (f) is assessed at least 24 hours after the cryopreservation freeze-thaw cycle. + CD25 + / high CD127 - / lo The percentage of regulatory T cells is assessed at least 24 hours after the cryopreservation freeze-thaw cycle.

[0024] In some embodiments, less than 5%, less than 4%, or less than 3% of the cells of the depleted biological sample comprise CD8+ cells, CD19+ cells, and / or CD14+ cells. In some embodiments, 1% or less of the population of cells are CD8+ cells. In some embodiments, 20% or less of the CD4+ Treg population are conventional T cells.

[0025] In some embodiments, the population of cells in step (c) is CD25 high CD45RA cells and CD25 + CD45RA + cells, including CD25 + CD45RA - In some embodiments, the isolating step (c) comprises (i) identifying a first subpopulation of CD4+ cells from the enriched population of step (b), (ii) isolating CD25+ cells from the first subpopulation, and + / high CD127 - / lo (iii) identifying a second subpopulation of cells; and (iv) isolating CD25 from the second subpopulation. high CD45RA and CD25 + / high CD45RA + Select cells and CD25 + CD45RA - This involves excluding CD4+ Tregs, thereby isolating a population of CD4+ Tregs.

[0026] In some embodiments, the TSDR is the CNS2 region of FOXP3. In some embodiments, the human subject is a male subject. In some embodiments, the human subject is a female subject.

[0027] In some embodiments, delivering a vector comprising a nucleic acid encoding an exogenous human TCR comprises transducing the vector into a cell population. In some embodiments, the exogenous human TCR is encoded as a single polypeptide comprising a TCR alpha chain and a TCR beta chain. In some embodiments, the polypeptide comprises an N-terminal TCR beta chain and a C-terminal TCR alpha chain. In some embodiments, the polypeptide comprises a self-cleaving peptide sequence located between the TCR alpha chain and the TCR beta chain. In some embodiments, the self-cleaving peptide sequence is a 2A peptide sequence. In some embodiments, the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence.

[0028] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is a VSVg-pseudotyped, self-inactivating, third-generation lentiviral vector. In some embodiments, the nucleic acid comprises a promoter operably linked to a coding sequence encoding an exogenous human TCR, and optionally the promoter is an EF-1 alpha promoter or an MND promoter. In some embodiments, the nucleic acid further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally a WPRE-mut6. In some embodiments, the coding sequence is codon-optimized.

[0029] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding an exogenous human TCR.

[0030] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the isolated population described herein and a pharmaceutically acceptable excipient. In some embodiments, the present disclosure provides a pharmaceutical composition comprising the isolated population described herein and a cryopreservation agent.

[0031] In some embodiments, the present disclosure provides a method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an isolated population or pharmaceutical composition thereof described herein.

[0032] In some embodiments, the pharmaceutical composition is administered in an amount effective to alleviate one or more symptoms of an autoimmune disease. In some embodiments, administering comprises intravenous administration. In some embodiments, administering comprises one or more injections. In some embodiments, the cells of the isolated population are autologous to the subject. [Brief explanation of the drawings]

[0033] [Figure 1A]

[0013] An exemplary method for producing a cell population containing stable regulatory T cells is provided. Figure 1A shows a schematic diagram of the overall process for isolating regulatory T cells with a CD4+CD45RA+CD25+ / highCD127lo / - phenotype. [Figure 1B] An exemplary method for producing a cell population containing stable regulatory T cells is provided. Figure 1B shows data illustrating a fluorescence-activated cell sorting (FACS) strategy for enriching a cell population for cells with a CD45RA+CD4+CD25+CD127lo / - phenotype from a CD25-enriched sample to produce an isolated cell population containing stable regulatory T cells. As shown in Figure 1A, this sorting strategy is used in two consecutive steps to obtain a high level of purity. [Figure 2] 1 shows a graph depicting the recovery of regulatory T cells after depletion and CD25 enrichment steps in an exemplary method for producing an isolated cell population comprising stable regulatory T cells. [Figure 3A] 1 shows data showing the relative abundance of various cell phenotypes after depletion and CD25 enrichment steps in an exemplary method for producing an isolated cell population comprising stable regulatory T cells. [Figure 3B] 1 shows data showing the relative abundance of various cell phenotypes after depletion and CD25 enrichment steps in an exemplary method for producing an isolated cell population comprising stable regulatory T cells. [Figure 4] Graph showing proliferation of cells after transduction with exogenous TCR. Non-transduced cells ("non-transduced") are included as a control. [Figure 5-1] 5-1 and 5-2 show graphs depicting regulatory T cell phenotype and TCR expression in cell populations produced by exemplary methods of the present disclosure. [Figure 5-2] 5-1 and 5-2 show graphs depicting regulatory T cell phenotype and TCR expression in cell populations produced by exemplary methods of the present disclosure. [Figure 6]

[0023] Figure 1 shows data demonstrating that regulatory T cells in cell populations produced by exemplary methods of the present disclosure maintain hypomethylated T cell-specific demethylated regions (TSDRs). [Figure 7] 1 shows a graph illustrating activation of lentiviral-transduced regulatory T cells of the present disclosure after cryopreservation freeze-thaw cycles. [Figure 8] Graph of the gating strategy for the purification of the CD4+ / CD45RA+CD127- / lowCD25+ cell population is shown. [Figure 9] Figure 10 shows the percent TSDR hypomethylation of different runs refined by the gating strategy shown in Figure 8 . [Figure 10A] Expression of CTLA-4 (FIG. 10A) after incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 10B] Expression of CD69 (FIG. 10B) after incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 10C] Expression of TGFβ-1 (FIG. 10C) following incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 10D] Expression of IL-10 (FIG. 10D) after incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 11] Shows the change in percent TSDR hypomethylation over time for growth of three different MS donors. [Figure 12A] Population doubling levels (FIGS. 12A and 12B) and percent TSDR hypomethylation for CD45RA+ and CD45RA- cells over 8 days of expansion are shown. The spike-in demonstrates that CD45RA+ can expand within the CD45RA- cell population. [Figure 12B]Population doubling levels (FIGS. 12A and 12B) and percent TSDR hypomethylation for CD45RA+ and CD45RA- cells over 8 days of expansion are shown. The spike-in demonstrates that CD45RA+ can expand within the CD45RA- cell population. [Figure 12C] Population doubling levels (FIGS. 12A and 12B) and percent TSDR hypomethylation for CD45RA+ and CD45RA- cells over 8 days of expansion are shown. The spike-in demonstrates that CD45RA+ can expand within the CD45RA- cell population. [Figure 13A] The gating strategy for CD25+CD127- / loCD45- and CD25+ / highCD127-loCD45- selection is shown. [Figure 13B] The gating strategy for CD25+CD127- / loCD45- and CD25+ / highCD127-loCD45- selection is shown. [Figure 13C] The gating strategy for CD25+CD127- / loCD45- and CD25+ / highCD127-loCD45- selection is shown. [Figure 14A] The histogram approach shown in Figures 13A-13C is shown. [Figure 14B] The histogram approach shown in Figures 13A-13C is shown. [Figure 14C] The histogram approach shown in Figures 13A-13C is shown. [Figure 14D] The histogram approach shown in Figures 13A-13C is shown. [Figure 14E] The histogram approach shown in Figures 13A-13C is shown. [Figure 15] Percent TSDR hypomethylation over the course of expansion for the same donor based on different selection strategies is shown. [Figure 16] Cell proliferation after restimulation on days 6, 8, or 10 is shown. [Figure 17] The effect of fold proliferation by TNF-α, a second stimulus, or both is shown. [Figure 18A] The viability and purity of cells expanded by both TNF-α and a second stimulus are shown. [Figure 18B] The viability and purity of cells expanded by both TNF-α and a second stimulus are shown. [Figure 19A] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19B] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19C] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19D] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19E] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 20] Percent TSDR hypomethylation over the course of expansion for the same donor based on different selection strategies is shown. [Figure 21] Percent TSDR hypomethylation for three different donors based on the gating strategies shown in Figures 13A-13C and 19A-19E is shown. [Figure 22A] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 22B] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 22C] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 22D] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 23-1] Figures 23-1 and 23-2 show the percentage of FOXP3+ and FOXP3- cells that expressed IL-2 and IFN-γ after activation with PMA and ionomycin. [Figure 23-2] Figures 23-1 and 23-2 show the percentage of FOXP3+ and FOXP3- cells that expressed IL-2 and IFN-γ after activation with PMA and ionomycin. DETAILED DESCRIPTION OF THE INVENTION

[0034] overview The present disclosure provides methods and compositions for treating autoimmune diseases through the generation and use of stable regulatory T cells. The present disclosure also provides methods and compositions for treating autoimmune diseases through the generation and use of engineered stable regulatory T cells comprising antigen-specific T cell receptors (TCRs). Such engineered regulatory T cells can specifically target distinct cell types and tissues associated with autoimmune diseases to prevent immune-mediated destruction, restore homeostasis, and promote repair in affected tissues. In addition, the engineered regulatory T cells of the present disclosure suppress local inflammation in the target and do not cause systemic immunosuppression. Furthermore, the engineered regulatory T cells described herein are stable (e.g., committed to a Treg phenotype) thymic-derived regulatory T cells that can persist in vivo for extended periods and provide therapeutic benefit for months or years after a single administration. These stable regulatory T cells are also, in some embodiments, resistant to proinflammatory triggers (e.g., proinflammatory cytokines). In some embodiments, the stable regulatory T cells of the present disclosure are autologous, meaning that they are obtained directly from the patient's own cells, engineered to express an exogenous antigen-specific TCR, and then administered back to the patient. The use of autologous cells minimizes the risk of rejection by the patient (e.g., graft-versus-host disease).

[0035] Isolating a highly stable and pure population of engineered regulatory T cells produced in the thymus and obtaining a sufficiently large and pure population of the engineered regulatory T cells to treat patients has proven difficult because the population of stable thymic regulatory T cells is very small relative to the total population of lymphocytes and because there are no cell surface markers associated exclusively with regulatory T cells. Although isolated polyclonal regulatory T cells have proven effective in clinical treatment, when engineering regulatory T cells with exogenous TCRs, a higher level of purity and stability is required because sufficient numbers of exogenous TCR-engineered conventional T cells and unstable regulatory T cells (i.e., peripheral Tregs) can have deleterious effects by triggering rather than suppressing immune responses at the site of autoimmune disease.

[0036] These stably engineered regulatory T cells and methods of production offer advantages over some current methods of producing regulatory T cells for cell therapy. Alternative approaches have been used to generate populations of engineered regulatory T cells or regulatory-like T cells, but these cells have drawbacks compared to the populations of cells described herein.

[0037] One possible approach is to increase the proportion of regulatory T cells by driving FOXP3 expression through manipulation of conventional T cells or induced stem cells, either through manipulation of the FOXP3 promoter or through the introduction of an ectopic FOXP3 gene into the cells. FOXP3 expression confers a regulatory-like phenotype. However, FOXP3 is only one of several genes with upregulated expression in stable regulatory T cells, and these regulatory-like T cells have been shown to differ from natural Tregs. Indeed, when CD4+ T cells were edited to exhibit a Treg-like phenotype by introducing an exogenous promoter upstream of FOXP3, the engineered cells were shown to have reduced suppressive capacity compared to natural Tregs (Buckner, Science Translational Medicine, 2022), suggesting that these cells would not exhibit the same level of efficacy as true stable regulatory T cells when administered in a clinical setting.

[0038] Conversion of conventional CD4+ T cells has also been attempted, for example, by stimulation with TGF-β and IL-2, retinoic acid, short-chain fatty acids, and TGF-β, or rapamycin. However, these induced regulatory T cells lack the epigenetic changes associated with stable regulatory T cells, particularly regulatory T cell-specific demethylation of the conserved noncoding sequence 2 (CNS2) of the FOXP3 gene, and are therefore functionally unstable and do not retain a stable phenotype when administered in a clinical setting. Furthermore, proteomic analysis has shown that these induced Treg-like cells show little overlap in protein expression profiles with stable thymic Tregs and instead share signaling and metabolic proteins with conventional T cells (Mensink et al., Sci Rep. 2022).

[0039] Contemplated herein are populations of cells and strategies for isolating highly purified and stable thymic regulatory T cells engineered with an exogenous TCR, of sufficient purity and in sufficient numbers to be administered to a subject in therapeutic amounts. In some embodiments, the optimal population of regulatory T cells is one that expresses CD25 T cells after isolation and prior to introduction of an exogenous TCR. + / hig hCD4 + CD127 - / lo CD45RA + , as this phenotype is associated with thymic naive regulatory T cells. In some embodiments, the optimal population of regulatory T cells is CD25 T cells after isolation and prior to introduction of an exogenous TCR. high CD4 + CD127 - / lo CD45RA - as this phenotype is associated with thymic antigen-experienced regulatory T cells. These cells further exhibit a stable TSDR phenotype after introduction of an exogenous TCR, indicating no expansion of non-regulatory T cell subpopulations or loss of regulatory T cell stability (e.g., no change in cell fate - cells remain terminally differentiated).

[0040] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document.

[0041] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0042] Also, unless expressly indicated to the contrary, it is to be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0043] In the claims and the above specification, all transitional phrases such as "comprise," "including," "carry," "have," "include," "involving," "hold," "comprise," and the like, are to be understood to be open-ended, i.e., to mean including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" must be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedure.

[0044] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0045] Where a range of values is provided, each value between and including the upper and lower endpoints of the range is specifically contemplated and described herein.

[0046] regulatory T cells In some embodiments, the present disclosure provides isolated populations of cells comprising stable CD4+ regulatory T cells derived from a subject with an autoimmune disease, as well as compositions thereof. These isolated populations may be used, for example, to treat the autoimmune disease in a subject in need thereof.

[0047] The terms "regulatory T cells," "T regulatory cells," and "Tregs" are used interchangeably herein and refer to T cells that suppress the effector functions of other cell populations of the immune system (e.g., conventional CD4+ T cells, effector CD8+ T cells, antigen-presenting cells, and / or granulocytes). Regulatory T cells are defined by the expression of the cell surface markers CD4 and CD25, as well as the expression of the transcription factor FOXP3. Additionally, regulatory T cells do not express, or express at low levels, the cell surface marker CD127. Thus, regulatory T cells have the following protein expression profile: CD4 + CD25 high / + CD127lo / - FOXP3 + It is characterized by:

[0048] Throughout this disclosure, expression of the indicated proteins by a cell or population of cells is indicated by a "+" (e.g., CD4 + ), "-" (e.g., CD127 - ), "high" (e.g., CD25 high ), "int" or "intermediate" (e.g., CD25 int ), "low" (e.g., CD127 low ), "High / +" (e.g., CD25 High / + ), or "low / -" (e.g., CD127 low / - ) As used herein, various expression indices refer to the presence or absence (e.g., "+" or "-", respectively) of the indicated protein, or the relative level of protein expression as measured by conventional protein expression assays (e.g., flow cytometry, fluorescence activated cell sorting (FACS), or Western blot). Unless otherwise indicated, protein expression described throughout this application is determined by FACS. (a) Positive ( + ) indicator refers to the detectable level of expression of the indicated protein by FACS. Positive ( + ) may be further divided into populations of "low" and / or "high" subpopulations. (b) A subpopulation of "low" cells expresses the indicated protein, but at a lower level (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower) than other cells in the population, or at a lower level than expression in a control cell population (e.g., compared to CD4+ conventional T cells or CD8+ effector T cells). (c) A subpopulation of "high" cells expresses the indicated protein at a higher level than other cells in the population (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher), or at a level higher than expression in a control cell population (e.g., compared to CD4+ conventional T cells or CD8+ effector T cells). (d) Negative ( - ) indication refers to the absence of expression of the indicated protein, or an expression level of the indicated protein that is below the limit of detection for a particular detection assay (e.g., below the limit of detection for a particular fluorescent antibody and / or flow cytometer). (e) " - / Low The designation " refers to a cell population that includes cells that are (-) for the indicated protein and cells that express "low" levels of the indicated protein. (f) + / High The designation " refers to a cell population containing cells that are (+) for the indicated protein, including cells that express high levels of the indicated protein.

[0049] In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25+. In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + / high In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD4+. In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD45RA+. In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are FOXP3+.

[0050] In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + / high CD4 + CD127 - / lowRegulatory T cells are cells that express CD25 or express it at high levels, express CD4, and do not express CD127 or express it at low levels. In some embodiments, regulatory T cells do not express CD127. In some embodiments, regulatory T cells (e.g., stable regulatory T cells) express CD25 + / high CD4 + CD127 - / low CD45RA + Regulatory T cells are cells. Thus, regulatory T cells express CD25 or express it at high levels, express CD4 and CD45RA, and do not express or express it at low levels of CD127. In some embodiments, regulatory T cells express FOXP3. In some embodiments, regulatory T cells are CD25+ / highCD4+CD127- / loFOXP3+ cells. Thus, regulatory T cells express CD25 or express it at high levels, express CD4 and FOXP3, and do not express or express it at low levels of CD127. In some embodiments, regulatory T cells are CD25+ / highCD4+CD45RA+CD127- / loFOXP3+ cells. Thus, regulatory T cells express CD25 or express it at high levels, express CD4, CD45RA, and FOXP3, and do not express or express it at low levels of CD127.

[0051] During thymic regulatory T cell development, the genome organizer SATB1 (special AT-rich sequence-binding protein) binds to specific genomic sites from the CD4+CD8+ thymic stage to open chromatin and activation super-enhancers associated with many regulatory T cell signature genes, such as FOXP3, IL2RA, (CD25), CTLA4, IKZF2 (HELIOS), and IFZF4 (EOS). SATB1 and MLL4 (myeloid / lymphoid or mixed lineage leukemia 4), enzymes involved in enhancer priming, generally occupy a newly identified conserved enhancer region, termed conserved noncoding sequence 0 (CNS0), in the FOXP3 locus, followed by activation of enhancers and then promoters in CNS3 and CNS2. This results in stable hypomethylation and expression of FOXP3 and other regulatory T cell-associated genes, thereby resulting in a stable regulatory T cell phenotype (Piotrowska, et al.; Int J Mol Sci. 2021).

[0052] After hematopoietic progression, populations of T cells can transition between one phenotype and another under various conditions (see, e.g., Kitagawa et al., Nat Immunol. 2017 Feb;18(2):173-183). As used herein, "stable regulatory T cells" are regulatory T cells that contain a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus. In some embodiments, stable regulatory T cells are further defined by expression of CD4, CD25, and / or FOXP3. Tregs derived from the thymus (e.g., thymic Tregs) are stable, and regulatory T cells that can no longer transition between T cell phenotypes due to changes in the methylation status of one or more loci in the FOXP3 locus are also considered stable. In contrast, peripheral Tregs, which are not stable and can be induced in response to pro-inflammatory conditions, do not exhibit stable hypomethylation of the TSDR region of the FOXP3 locus. Hypomethylation of the TSDR, an evolutionarily conserved CpG-rich regulatory element of the FOXP3 gene, is associated with FOXP3 expression. The TSDR of the endogenous FOXP3 locus is hypomethylated when one or more methylated cytosines in the TSDR are substituted with cytosine by removing the methyl group. Therefore, the stability of regulatory T cells is determined by the presence of a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus.

[0053] In some embodiments, measuring the methylation status of the TSDR of the FOXP3 locus is as described in Kressler et al., "Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Regulatory T Phenotype," Frontiers in Immunology, January 7, 2021; or Schreiber, et al., "The Regulatory T-Specific Demethylated Region Stabilizes Foxp3 Expression Independent of NF-κB Signaling," PLOS One, February 5, 2014. In some embodiments, the TSDR of the FOXP3 locus is selected from conserved non-coding sequence 0 (CNS0), CNS3, and CNS2. In some embodiments, the TSDR of the FOXP3 locus is CNS2.

[0054] In some embodiments, stable regulatory T cells maintain a hypomethylated T-regulatory cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus in the presence of pro-inflammatory conditions (e.g., in the presence of one or more pro-inflammatory cytokines). In some embodiments, stable regulatory T cells comprise a hypomethylated T-regulatory cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus in the presence of pro-inflammatory conditions for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, or 25 days.

[0055] In some embodiments, the stable regulatory T cells further exhibit one or more of the following functions: (i) regulatory cytokine secretion activity (e.g., secretion of IL-10, TGFβ, and IL-35); (ii) expression or activation markers associated with regulatory T cells (e.g., expression of CD69, 4-1BB, CD25, CD71, and / or CTLA-4); and / or (iii) suppressive activity (e.g., the ability of the stable regulatory T cells to suppress the activation and / or proliferation of other effector cells of the immune system).

[0056] Thus, in some embodiments, the isolated cell populations provided herein comprise stable regulatory T cells that comprise expression of a hypomethylated TSDR of the FOXP3 locus, CD4, CD25, and FOXP3, and / or exhibit cytokine secretion, activation, and / or suppressive activity.

[0057] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after isolation from a biological sample. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after isolation from a biological sample. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for 1 to 20 days, 1 to 10 days, 1 to 5 days, 5 to 30 days, 5 to 20 days, 10 to 40 days, or 25 to 50 days after isolation from the biological sample.

[0058] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after transduction with a nucleic acid encoding an exogenous TCR. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with a nucleic acid encoding an exogenous TCR. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after transduction with a nucleic acid encoding an exogenous TCR. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the locus for 1 to 20 days, 1 to 10 days, 1 to 5 days, 5 to 30 days, 5 to 20 days, 10 to 40 days, or 25 to 50 days after transduction with a nucleic acid encoding an exogenous TCR.

[0059] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain hypomethylated TSDR at the FOXP3 locus over time after cryopreservation. For example, in some embodiments, the isolated cell populations described herein are cryopreserved and then thawed for use and / or analysis, which is referred to herein as cryopreservation freeze-thaw cycling. In such embodiments, the stable regulatory T cells of the isolated cell population maintain hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after cryopreservation freeze-thaw cycling. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after cryopreservation freeze-thaw cycling. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the locus for 1-20 days, 1-10 days, 1-5 days, 5-30 days, 5-20 days, 10-40 days, or 25-50 days after a cryopreservation freeze-thaw cycle.

[0060] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after administration to a subject. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after administration to a subject. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after administration to a subject. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for 1 to 20 days, 1 to 10 days, 1 to 5 days, 5 to 30 days, 5 to 20 days, 10 to 40 days, or 25 to 50 days after administration to a subject.

[0061] In some embodiments, the isolated population described herein is an autologous cell population.In this context, the term autologous refers to cells obtained from the same subject to which they are subsequently administered.For example, a cell population can be obtained from a subject, subjected to the method described herein, and then administered to the same subject (the subject from which the cell population was originally obtained) to treat autoimmune disease.In such embodiments, the cell population administered to a subject comprises autologous regulatory T cells.

[0062] In some embodiments, the isolated population described herein is an allogeneic cell population.In this context, the term allogeneic refers to the cells obtained from one subject and then administered to another subject.For example, a cell population can be obtained from a subject, subjected to the method described herein, and then administered to another subject to treat autoimmune disease.

[0063] In some embodiments, the isolated population of cells described herein (e.g., an isolated population comprising stable CD4+ regulatory T cells) is isolated from a biological sample obtained from a subject diagnosed with or suspected of having an autoimmune disease. The autoimmune disease may be selected from multiple sclerosis (e.g., progressive multiple sclerosis or relapsing-remitting multiple sclerosis), type 1 diabetes, and inclusion body myositis. In some embodiments, the autoimmune disease is multiple sclerosis (e.g., progressive multiple sclerosis). In some embodiments, the regulatory T cells are obtained from a subject diagnosed with or suspected of having multiple sclerosis (e.g., progressive multiple sclerosis). In other embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the regulatory T cells are obtained from a subject diagnosed with or suspected of having type 1 diabetes. In yet other embodiments, the autoimmune disease is inclusion body myositis. In some embodiments, the regulatory T cells are obtained from a subject diagnosed with or suspected of having inclusion body myositis.

[0064] Manipulative regulatory T cells In some embodiments, the present disclosure provides an isolated population of cells comprising regulatory T cells (e.g., stable regulatory T cells) that comprise an exogenous human T cell receptor (TCR) that binds to a target peptide. This binding occurs when the target peptide is complexed with a major histocompatibility complex (MHC) (e.g., MHC class I or MHC class II). Such populations are referred to herein as "engineered regulatory T cells" or "stable engineered regulatory T cells."

[0065] T cell receptors (TCRs) are transmembrane heterodimers containing α and β chains linked by disulfide bonds. Within these chains are complementarity-determining regions (CDRs), which determine the target peptide to which the TCR binds. TCRs activate T cells that contain them, resulting in an exaggerated immune response. Antigen-presenting cells digest specific proteins (antigens) and present their fragments (peptides) on major histocompatibility complexes (MHC). This peptide-MHC (pMHC) complex binds to the TCR, while other costimulatory molecules are activated, leading to T cell activation, proliferation, differentiation, apoptosis, or cytokine release.

[0066] An exogenous TCR can be any TCR introduced into a regulatory T cell, where the TCR is not endogenous to the regulatory T cell (i.e., naturally occurring in the regulatory T cell). For example, in some embodiments, the exogenous TCR is encoded by a nucleic acid that is not endogenous to the regulatory T cell (i.e., not naturally occurring in the genome of the regulatory T cell). In some embodiments, the nucleic acid is an engineered nucleic acid, e.g., a recombinant or synthetic nucleic acid.

[0067] A TCR specifically binds to a target peptide complexed with MHC. A TCR is considered to "specifically" bind to a target peptide complexed with MHC if the TCR has a higher binding affinity for a non-target peptide complexed with MHC compared to the target peptide complexed with MHC. A TCR may bind to a target peptide complexed with MHC with a binding affinity of at least 10 M, 10 M, 10 M, 10 M, 10 M, 10 M, 10 M, 10 M, or 10 M (e.g., 10 M to 10 M). In some embodiments, a TCR is considered to "specifically" bind to a target peptide complexed with MHC when T cells expressing the TCR are activated upon contact with the target peptide complexed with MHC (e.g., as assessed by increased CD69 expression) or are more highly activated compared to a non-target peptide complexed with MHC. In some embodiments, the peptide is presented by a cell expressing MHC.

[0068] The exogenous TCR may be a human TCR. In some embodiments, the TCR is from a monkey, a mouse, a rat, or any other animal.

[0069] The target peptide may be a peptide associated with an autoimmune disease. In some embodiments, the target peptide is associated with an autoimmune disease selected from Sjögren's syndrome, Goodpasture's syndrome, SLE, Behcet's disease, multiple sclerosis, neuromyelitis optica, myasthenia gravis, primary biliary cholangitis, ulcerative colitis, Crohn's disease, celiac disease, aplastic anemia, rheumatoid arthritis, ankylosing spondylitis, autoimmune hepatitis, type 1 diabetes, polymyositis, dermatomyositis, inclusion body myositis, pemphigus, psoriasis, Hashimoto's thyroiditis, Graves' disease, GvHD, lupus nephritis, ALS, chronic inflammatory demyelinating polyneuropathy, autoimmune pancreatitis, vitiligo, and alopecia. In some embodiments, the target peptide is associated with multiple sclerosis (e.g., progressive multiple sclerosis), type 1 diabetes, or inclusion body myositis. For example, a target peptide associated with multiple sclerosis may be a peptide belonging to myelin basic protein (MBP) (e.g., MBP(83-99)). A target peptide associated with type 1 diabetes may be a peptide belonging to the glutamic acid decarboxylase enzyme (e.g., GAD65), e.g., GAD65(555-567) or GAD65(339-352), or proinsulin. In some embodiments, the target peptide is a peptide that is overexpressed in a population of cells associated with an autoimmune disease compared to a control (e.g., compared to a population of cells not associated with an autoimmune disease).

[0070] In some embodiments, the target peptide is a peptide that is likely to increase the autoimmune response in a subject with an autoimmune disease compared to a control. In some embodiments, the target peptide is a peptide that is present at a site of autoimmune disease in a subject compared to an unaffected site in the same subject. In some embodiments, the target peptide is specifically presented by an MHC allele associated with the presence of autoimmune disease in a subject.

[0071] In some embodiments, the target peptide is a peptide that is overexpressed in cells of a subject with an autoimmune disease compared to a control (e.g., compared to a healthy subject). A target peptide is considered to be overexpressed in a cell (e.g., associated with an autoimmune disease) if expression of the target peptide in the cell is 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 80%, or at least 90% higher in the cell compared to control cells (e.g., a population of cells not associated with an autoimmune disease). In some embodiments, a target peptide is overexpressed in cells of a subject with an autoimmune disease if expression of the target peptide in cells of the subject with an autoimmune disease is 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 80%, or at least 90% higher in the subject with an autoimmune disease compared to a healthy subject (e.g., a subject without an autoimmune disease).

[0072] In some embodiments, the target peptide is a peptide that is highly expressed in cells at disease sites in subjects with an autoimmune disease compared to controls (e.g., target peptide expression in unaffected / non-diseased sites in a subject). A target peptide is considered to be highly expressed in cells at disease sites (e.g., associated with an autoimmune disease) if expression of the target peptide in cells at disease sites is 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 80%, or at least 90% higher in cells at disease sites compared to control cells (e.g., cells at non-diseased sites).

[0073] In some embodiments, the target peptide is a peptide complexed with an MHC having an HLA haplotype associated with an autoimmune disease.

[0074] In some embodiments, the exogenous TCR is (or is encoded as) a single polypeptide (e.g., comprising a β chain and an α chain). In some embodiments, the TCR comprises an N-terminal β chain and a C-terminal α chain. In other embodiments, the TCR comprises an N-terminal α chain and a C-terminal β chain.

[0075] The TCR may include a linker domain located between the α chain and the β chain. In some embodiments, the linker domain includes a self-cleaving peptide sequence (e.g., a self-cleaving peptide sequence located between the α chain and the β chain). The self-cleaving peptide sequence is a peptide sequence that induces separation of a polypeptide into two peptides using a non-classical mechanism. In some embodiments, the self-cleaving peptide sequence may induce ribosomal skipping during translation of the polypeptide. In some embodiments, the self-cleaving peptide sequence is 10 to 30, 10 to 25, 15 to 30, 15 to 25, or 18 to 22 amino acids in length. In some embodiments, the self-cleaving peptide sequence may be a 2A peptide sequence. The 2A peptide sequence may include, for example, the DXEXNPGP (SEQ ID NO: 4) amino acid motif (wherein X can be any amino acid). In some embodiments, the 2A peptide sequence is a P2A (derived from porcine teschovirus-1 2A), E2A (derived from equine rhinitis A virus), F2A (derived from foot-and-mouth disease virus), or T2A (derived from Thosea asigna virus 2A) peptide sequence. The T2A peptide sequence may, for example, include the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 5). The P2A peptide sequence may, for example, include the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 6). The E2A peptide sequence may, for example, include the amino acid sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 7). The F2A peptide sequence may, for example, include the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 8).

[0076] In some embodiments, the exogenous TCR comprises two or more polypeptides, for example, in some embodiments, the exogenous TCR comprises a first polypeptide comprising an alpha chain and a second polypeptide comprising a beta chain.

[0077] In some embodiments, the exogenous TCR comprises one or more cysteine residues present in the α chain of the TCR that can form one or more disulfide bonds with one or more cysteine residues in the β chain of the TCR. In some embodiments, the exogenous TCR comprises one or more cysteine residues present in the α chain constant region of the TCR that can form one or more disulfide bonds with one or more cysteine residues in the β chain constant region of the TCR.

[0078] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3, wherein the cysteine residue at position 48 of the alpha chain can form a disulfide bond with the cysteine residue at position 57 of the beta chain.

[0079] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3, wherein the cysteine residue at position 45 of the alpha chain can form a disulfide bond with the cysteine residue at position 77 of the beta chain.

[0080] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3, wherein the cysteine residue at position 10 of the alpha chain can form a disulfide bond with the cysteine residue at position 17 of the beta chain.

[0081] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3, wherein the cysteine residue at position 45 of the alpha chain can form a disulfide bond with the cysteine residue at position 59 of the beta chain.

[0082] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3, wherein the cysteine residue at position 15 of the alpha chain can form a disulfide bond with the cysteine residue at position 15 of the beta chain.

[0083] [Table A]

[0084] The exemplary exogenous human TCR that can be introduced into the isolated cell population described herein is known in the art.For example, the TCR that is known to target MS-related antigens such as MBP or MOG includes but is not limited to Ob.1A12, Ob.2F3, Ob.1C3, Ob.3D1, Hy.2E11, Hy.1G11, Hy.2B6 and Hy.1B11 (Wucherpfennig et al., J Immunol 1994;152:5581-5592). TCRs known to target type 1 diabetes-related antigens such as proinsulin, GAD65, or IGRP include, but are not limited to, GSE.20D11, GSE.6H9, T1D#3 C8, T1D#10 C8, PM1#11, MHB10.3, SD32.5, SD52.c1, R164, 4.13, 1E6, and D222D (Yeh et al., Frontiers in Immunology, October 26, 2017). TCRs targeting lupus-associated Smith autoantigens are described, for example, in Ooi et al. (Research Square, March 16, 2023).

[0085] Nucleic acid encoding a TCR In some embodiments, the present disclosure provides a nucleic acid encoding a TCR (e.g., an exogenous TCR). The nucleic acid may be or include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., messenger RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeras.

[0086] As used herein, a nucleic acid generally refers to an engineered nucleic acid. An engineered nucleic acid is a non-naturally occurring polynucleotide (e.g., at least two nucleotides covalently linked to each other, optionally containing phosphodiester bonds, referred to as a phosphodiester backbone). Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that are amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that are chemically or otherwise modified but are still capable of base pairing (binding) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules resulting from replication of any of the above.

[0087] The engineered nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009, and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: a 5' exonuclease, a 3' extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity chews back the 5' terminal sequence, exposing complementary sequences for annealing. Polymerase activity then fills in the gaps in the annealed domains. DNA ligase then seals the nicks, covalently linking the DNA fragments to each other. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. The MegaGate molecular cloning method may also be used. MegaGate is a less toxic Gateway technology that eliminates the ccdb toxin used in Gateway recombinase cloning and instead utilizes meganuclease-mediated digestion to eliminate background vectors during cloning (see, for example, Kramme C. et al. STAR Protoc. 2021 Oct. 22;2(4):100907, incorporated herein by reference). Other methods of producing engineered polynucleotides may also be used in accordance with the present disclosure.

[0088] In some embodiments, the present disclosure provides an expression cassette comprising an open reading frame comprising a nucleic acid encoding an exogenous TCR operably linked to a promoter. A promoter is a nucleotide sequence (e.g., ATG) at which RNA polymerase binds to initially transcribe. A promoter is typically located immediately upstream (5' end) of the transcription start site. In some embodiments, the promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably linked. In some embodiments, the promoter is an inducible promoter. An inducible promoter may be regulated in vivo, for example, by chemical agents, temperature, or light.

[0089] An open reading frame is a series of consecutive codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide, e.g., a protein. An open reading frame is operably linked to a promoter if the promoter controls transcription of the open reading frame.

[0090] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding an exogenous TCR or an expression vector comprising the same. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. For example, the vector may be a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpesvirus vector, a retroviral vector, or a baculoviral vector. Viral vectors provide efficient delivery of exogenous TCRs to regulatory T cells of the present disclosure. Exemplary viral vectors can be derived from negative-stranded RNA viruses such as lentiviruses, retroviruses (e.g., Retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpesviruses), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus.Examples of retroviruses include avian leukosis-sarcoma, avian C virus, mammalian C, B, and D viruses, oncoretroviruses, HTLV-BLV complex, alpharetroviruses, gammaretroviruses, Spumaretrovirus genus, murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0091] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector of the present disclosure comprises two long terminal repeats (LTRs) flanking an expression cassette containing the lentiviral gag, pol, and rev genes, and a nucleic acid encoding an exogenous TCR. For safety, the vector does not contain any other active lentiviral genes, such as vpr, vif, vpu, nef, and tat. In some embodiments, these genes are deleted or otherwise inactivated.

[0092] In some embodiments, the lentiviral vector is a self-inactivating vector. A self-inactivating vector is a vector in which the production of full-length vector RNA in transduced cells is significantly reduced or completely eliminated. This feature greatly minimizes the risk of the emergence of replication-competent recombinants (RCRs). Furthermore, this feature reduces the risk of abnormal expression of cellular coding sequences located adjacent to the vector integration site. Furthermore, the SIN design reduces the possibility of interference between the LTR and the promoter driving the expression of the transgene.

[0093] Self-inactivation is preferably achieved by introducing a deletion in the U3 region of the 3'LTR of the vector DNA, i.e., the DNA used to produce vector RNA.Thus, during reverse transcription, this deletion is transferred to the 5'LTR of proviral DNA.However, the LTR element involved in the polyadenylation of viral RNA is not modified.Together, this reduces or eliminates the production of full-length vector RNA in transduced cells.

[0094] In some embodiments, the nucleic acid encoding the exogenous TCR is RNA (e.g., messenger RNA (mRNA)). In some embodiments, the mRNA comprises a 5' cap, a 5' untranslated region (UTR), an open reading frame (ORF), a 3' UTR, and / or a poly(A) tail.

[0095] In some embodiments, nucleic acids are codon-optimized. Codon optimization methods are known in the art. In some embodiments, codon optimization can be used to match the codon frequency in target organisms and host organisms to ensure proper folding; bias GC content to increase RNA (e.g., mRNA) stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in encoded proteins; add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust translation speed to ensure that various domains of proteins fold properly; or reduce or eliminate problematic secondary structures in polynucleotides.

[0096] In some embodiments, the nucleic acid encoding a TCR comprises a promoter operably linked to a coding sequence encoding an exogenous human TCR. The promoter may be a viral promoter or a native promoter. In some embodiments, the promoter is a constitutively active promoter or an inducible promoter. In some embodiments, the promoter is a eukaryotic translation elongation factor 1 alpha (EF-1 alpha) promoter and an MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution) (see, e.g., Gill, D.R. et al. Gene Ther. 2001;8:1539-46 and Astrakhan, A. et al. Blood 2012;119:4395-4407).

[0097] The vector may also include a stop codon and / or an expression enhancer element. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used. In some embodiments, the enhancer element is an optimized posttranscriptional regulatory element (oPRE), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The WPRE may be a wild-type WPRE or a WPRE mutant sequence (e.g., WPRE-mut6). In some embodiments, the WPRE is as described in Zanta-Boussif, MA et al., Gene Therapy volume 16, pp. 605-619 (2009).

[0098] Isolated cell populations In some embodiments, the present disclosure provides an isolated population of cells comprising stable CD4+ regulatory T cells derived from a subject with an autoimmune disease, as well as compositions thereof. In some embodiments, the present disclosure provides an isolated population of cells comprising stable CD4+ regulatory T cells derived from a subject with an autoimmune disease, engineered to express an exogenous human TCR, as well as compositions thereof. In some embodiments, at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylation region (TSDR) in the FOXP3 locus.

[0099] The methylation status of TSDR in FOXP3 locus can be evaluated by means known in the art.For example, in some embodiments, the methylation status of TSDR in FOXP3 locus is evaluated by bisulfite treatment and digital droplet PCR (ddPCR) using methylation-specific primers and probes.In some embodiments, the methylation status of TSDR in FOXP3 locus is evaluated by single-cell sequencing method.

[0100] An isolated cell population is a cell population removed from a human body or from a sample obtained from a human body. Therefore, it is considered "isolated" from a human body. A population of cells may be isolated (e.g., obtained) from a subject or from a biological sample obtained from a subject using any known cell collection method, such as apheresis. The isolated cell population of the present disclosure may be subjected to the methods described herein to produce an isolated cell population with a higher number of regulatory T cells (e.g., stable regulatory T cells) than a population of cells obtained directly from a subject or from a biological sample obtained from a subject, for example, by apheresis.

[0101] In some embodiments, the isolated cell population comprises stable CD4+ regulatory T cells, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated cell population comprises stable CD4+ regulatory T cells, wherein 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, 80%-90%, 85%-95%, 80%-85%, 85%-90%, or 90%-95% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells of the isolated cell population are stable regulatory T cells that comprise a hypomethylated TSDR at the endogenous FOXP3 locus.

[0102] In some embodiments, the isolated population is CD25 + / high CD4 + CD127 - / low In some embodiments, the isolated cell population comprises CD25 regulatory T cells. + / high CD4 + CD127 - / low FOXP3 + In some embodiments, the isolated cell population comprises CD25 regulatory T cells. + / high CD4 + CD127 - / lo FOXP3 + CD45RA+ Includes regulatory T cells.

[0103] In some embodiments, the isolated cell population is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25 + / high CD4 + CD127 - / lo In some embodiments, the isolated cell population comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25 regulatory T cells. + / high CD4 + CD127 - / lo FOXP3 + Includes regulatory T cells.

[0104] In some embodiments, at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the regulatory T cells in the isolated population are CD45RA + In some embodiments, the isolated cell population is at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25 + / high CD4 + CD127 - / lo CD45RA + Includes regulatory T cells.

[0105] As described herein, the present disclosure provides an isolated cell population comprising stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated population described herein is engineered to express an exogenous TCR, but the FOXP3 locus remains unmodified. Thus, the cells of the isolated population described herein do not comprise an engineered FOXP3 locus. An "engineered FOXP3 locus" refers to any engineered modification (e.g., human modification) intended to alter the expression of FOXP3. Such engineered modifications include, but are not limited to, the introduction of a FOXP3 transgene, the introduction of a modified promoter, and / or the use of an exogenous agent (e.g., a gene editing system, a small molecule, or a peptide) intended to activate the expression of FOXP3.

[0106] In some embodiments, the isolated cell population comprises at least 1 x 10 2 , at least 1 x 10 3 , at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , or at least 1 × 10 10 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 2 ~1×10 10 , 1×10 3 ~1×10 10 , 1×10 4 ~1×10 10 , 1×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 10 , 1×10 8 ~1×10 10 , 1×10 5 ~1×10 9 , 1×10 6 ~1×10 8 , 1×107 ~1×10 10 or 1×10 4 ~1×10 6 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 6 ~1×10 10 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9×10 7 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 7 ~1×10 10 , 2 × 10 7 ~1×10 10 , 3×10 7 ~1×10 10 , 4×10 7 ~1×10 10 , 5×10 7 ~1×10 10 , 6×10 7 ~1×10 10 , 7×10 7 ~1×10 10 , 8×10 7 ~1×10 10 , or 9×10 7 ~1×10 10 Contains stable regulatory T cells.

[0107] In some embodiments, the isolated cell population comprises stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus, wherein the stable regulatory T cells retain markers of stability in the presence of a pro-inflammatory condition (i.e., maintain a hypomethylated TSDR at the FOXP3 locus and / or express CD25 + / high CD4 + CD127 - / lo FOXP3 +(Maintaining a protein expression profile of). A pro-inflammatory state refers to cells and factors known to drive an inflammatory immune response and can include pro-inflammatory cytokines (e.g., IL-17, IL-22, IL-21, IFNγ, IL-12, TNFα, IL-1β, IL-6, IL-1, GM-CSF, and others known in the art), immune effector cells (e.g., conventional CD4+ T cells, CD8+ effector T cells, granulocytes, etc.), and other pro-inflammatory mediators (e.g., prostaglandins, thrombin, histamine, and matrix proteases). A pro-inflammatory state can be in vitro or in vivo. In some embodiments, stable regulatory T cells within the isolated cell population maintain a hypomethylated TSDR at the endogenous FOXP3 locus in the presence of pro-inflammatory conditions (for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, or 25 days).

[0108] In some embodiments, the isolated cell population comprises stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus, where the stable regulatory T cells retain markers of stability over time (i.e., maintain a hypomethylated TSDR at the FOXP3 locus and / or express CD25 + / high CD4 + CD127 - / lo FOXP3 +(Maintain a protein expression profile of . For example, in some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. In some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after activation and / or expansion. In some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with an exogenous TCR. In some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after a cryopreservation freeze-thaw cycle.

[0109] In some embodiments, stable regulatory T cells are expressed ex vivo on CD25 T cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. + / high CD4 + CD127 - / lo FOXP3 + In some embodiments, stable regulatory T cells maintain a protein expression profile of CD25 ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after activation and / or expansion. + / high CD4 + CD127 - / lo FOXP3 +In some embodiments, stable regulatory T cells maintain a protein expression profile of CD25 ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with an exogenous TCR. + / high CD4 + CD127 - / lo FOXP3 + In some embodiments, stable regulatory T cells maintain a protein expression profile of CD25 ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after a cryopreservation freeze-thaw cycle. + / high CD4 + CD127 - / lo FOXP3 + Maintain the protein expression profile of

[0110] In some embodiments, at least 50% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 50% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 50% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 60% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 60% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 60% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 70% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 70% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 70% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 80% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 80% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 80% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 90% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation.In some embodiments, at least 90% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation, hi some embodiments, at least 90% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation.

[0111] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed at multiple (e.g., two or more) time points during the isolation and production process. In some embodiments, the percentage of stable regulatory T cells containing hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% between these two or more time points.

[0112] For example, in some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from the biological sample, and again within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11%, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample.

[0113] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In some embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11%, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion.

[0114] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11%, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR.

[0115] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of stable regulatory T cells containing a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0116] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after transduction with an exogenous human TCR, and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of stable regulatory T cells containing a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample, and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0117] In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percentage point(s) for at least 5, 6, 7, 8, 9, 10, or 11 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells increases by 1, 2, 3, 4, or 5 percentage point(s) for at least 5, 6, 7, 8, 9, 10, or 11 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 15 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points during at least 15 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percent during at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 15 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0118] The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed about 1, about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of the cells with an exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction of the cells with an exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed about 1-21, 1-7, 4-14, 4-7, 7-10, 7-14, 10-21, or 14-21 days after transduction of the cells with an exogenous human TCR.

[0119] In some embodiments, at least 5%, at least 10%, at least 15%, 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 95% of the regulatory T cells of the isolated cell population express the exogenous TCR (e.g., after transduction of the isolated cell population with an exogenous TCR). In some embodiments, 10% to 60% or 20% to 50% of the regulatory T cells of the isolated cell population express the exogenous TCR. In some embodiments, at least 5%, at least 10%, at least 15%, 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 95% of the stable regulatory T cells of the isolated cell population express the exogenous TCR (e.g., after transduction of the isolated cell population with an exogenous TCR). In some embodiments, 10% to 60% or 20% to 50% of the stable regulatory T cells of the isolated cell population express an exogenous TCR.

[0120] In some embodiments, the TSDR at the endogenous FOXP3 locus of stable regulatory T cells of the isolated cell population of regulatory T cells remains hypomethylated until the isolated cell population is administered to a subject. In some embodiments, the TSDR at the endogenous FOXP3 locus of stable regulatory T cells of the isolated cell population of regulatory T cells remains hypomethylated after a cryopreservation freeze-thaw cycle.

[0121] In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) of the isolated cell population exhibit one or more cellular functions associated with regulatory T cells when activated by binding to pMHC. Non-limiting examples of such cellular functions include cytokine secretion activity, expression of specific activation markers, and suppressive activity. Cytokine secretion activity simply refers to the secretion of specific anti-inflammatory cytokines (e.g., IL-10, TGFβ, and IL-35). Activation markers include, but are not limited to, CD69, 4-1BB, CD25, CD71, or CTLA-4. Regulatory T cells express one or more of these markers, for example, upon contact with a target peptide complexed with MHC.

[0122] Suppressive activity refers to the suppression of activation, proliferation, and cytokine production of non-regulatory T cells (e.g., CD8+ T cells and CD4+ conventional T cells) to partially suppress overactivity of the immune system. Regulatory T cells exhibit suppressive activity when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. In some embodiments, the target peptide is presented by a cell expressing MHC. For example, in some embodiments, regulatory T cells suppress the activation, proliferation, and cytokine production of conventional T cells that have specificity for a shared target peptide complexed with MHC, e.g., a shared target peptide presented by an antigen-presenting cell (APC). In some embodiments, regulatory T cells suppress the proliferation and growth of non-regulatory T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% compared to a control (e.g., non-regulatory T cells in the absence of regulatory T cells).

[0123] In some embodiments, regulatory T cells suppress IFN-γ production from conventional T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% compared to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress IFN-γ production from conventional T cells by 50% to 99%, 75% to 99%, or 80% to 100% compared to a control (e.g., when present in a population comprising regulatory T cells at a ratio of 1:1 to 1:8 compared to conventional T cells). In some embodiments, regulatory T cells suppress CD71 production from conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% compared to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress CD71 production by conventional T cells by 20% to 90% or 30% to 80% compared to a control (e.g., when present in a population containing regulatory T cells at a ratio of 1:1 to 1:8 compared to conventional T cells). In some embodiments, regulatory T cells suppress CD25 production from conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% compared to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress CD25 production by conventional T cells by 40% to 70% compared to a control (e.g., when present in a population containing regulatory T cells at a ratio of 1:1 to 1:8 compared to conventional T cells).

[0124] In some embodiments, regulatory T cells exhibit cytokine secretion activity (e.g., secretion of IL-10) when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells, e.g., a target peptide presented by an APC. In some embodiments, regulatory T cells exhibit expression of activation markers when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. For example, in some embodiments, regulatory T cells exhibit expression of CD69, 4-1BB, CD25, CD71, and / or CTLA-4 when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. In some embodiments, regulatory T cells exhibit suppressive activity when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. For example, in some embodiments, regulatory T cells suppress the activation of conventional T cells with specificity for a shared target peptide complexed with MHC.

[0125] The cellular function of stable regulatory T cells may be assessed about 1, about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of the cells with an exogenous human TCR. The cellular function of stable regulatory T cells may be assessed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction of the cells with an exogenous human TCR. The cellular function of stable regulatory T cells may be assessed about 1-21, 1-7, 4-14, 4-7, 7-10, 7-14, 10-21, or 14-21 days after transduction of the cells with an exogenous human TCR.

[0126] In some embodiments, transduced regulatory T cells (e.g., isolated populations of transduced regulatory T cells) retain their cellular function (e.g., the ability to be activated) after cryopreservation freeze-thaw cycling. In some embodiments, transduced regulatory T cells can be activated and / or expanded after cryopreservation freeze-thaw cycling. In some embodiments, regulatory T cells can exhibit cytokine secretion activity, expression of specific activation markers, and / or suppressive activity after cryopreservation freeze-thaw cycling.

[0127] In some embodiments, prior to activation and / or transduction with an exogenous human T cell receptor, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of cells are CD25 + / high CD4 + CD127 - / lo is.

[0128] An isolated cell population comprising stable regulatory T cells may also contain a small number of non-regulatory T cells (e.g., conventional T cells). Non-regulatory T cells may be NK T cells, B cells, CD8+ T cells, neutrophils, eosinophils, CD14+ cells, or conventional (CD4+) T cells derived from peripheral blood and lymph nodes. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising stable regulatory T cells are non-regulatory T cells. In some embodiments, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 10%, about 2% to about 5%, or about 5% to about 10% of the cells in an isolated cell population comprising regulatory T cells are non-regulatory T cells. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising regulatory T cells are non-regulatory T cells comprising an exogenous human TCR. Conventional T cells generally produce IL-2 and other interleukin factors. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising regulatory T cells are conventional T cells. In some embodiments, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 10%, about 2% to about 5%, or about 5% to about 10% of the cells in an isolated cell population comprising regulatory T cells are conventional T cells. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising regulatory T cells are conventional T cells comprising an exogenous human TCR. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of conventional T cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of conventional CD4+ T cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of B cells.In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable amount of NK T cells. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable amount of CD14+ cells. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable number of eosinophils. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable number of neutrophils. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable amount of CD8+ T cells.

[0129] The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or stable regulatory T cells) may be assessed about 1, about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of the cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or stable regulatory T cells) may be assessed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction of the cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or stable regulatory T cells) may be assessed about 1-21, 1-7, 4-14, 4-7, 7-10, 7-14, 10-21, or 14-21 days after transduction of the cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) may be assessed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after a cryopreservation freeze-thaw cycle.

[0130] In some embodiments, the ratio of regulatory T cells to conventional T cells in the isolated population of cells comprising regulatory T cells is at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 100:1.

[0131] Methods for producing stable regulatory T cells In some embodiments, the present disclosure provides a method for producing an isolated population of cells containing stable CD4+ regulatory T cells derived from a subject with an autoimmune disease. Previous methods for producing antigen-specific regulatory T cells have not resulted in clinical-scale and clinical-grade production of cell populations containing high concentrations of stable antigen-specific regulatory T cells. While polyclonal T cells have been successfully produced and administered in clinical settings, the use of stable thymic regulatory T cells engineered with exogenous TCRs poses increased safety risks and requires improved cell purity and regulatory T cell stability. Without wishing to be bound by theory, stable thymic regulatory T cells comprise a small percentage of leukocytes, and if the isolated regulatory T cell population is not highly pure, other types of leukocytes are engineered with exogenous TCRs. In sufficient numbers, these engineered non-regulatory T cells may have a deleterious effect by triggering rather than suppressing immune responses at the site of autoimmune disease. Through the integration and modification of several biomarker selection processes, including an unexpectedly effective dual selection process using CD25, and in some cases CD45RA, the methods designed herein provide the field with the tools to generate populations of antigen-specific regulatory T cells with high yields and high relative concentrations of stable TCR-transduced regulatory T cells.

[0132] In some embodiments, the methods of the disclosure involve depletion of conventional T cells and CD25 + / high CD4 + CD127 - / lo In some embodiments, the methods of the present disclosure include depletion of conventional T cells, selection for cells having a CD25 phenotype to produce a cell population comprising stable regulatory T cells. + / high CD4 + CD127 - / loand engineering the stable regulatory T cells to comprise an exogenous human TCR that specifically binds to a target peptide complexed with MHC. In some embodiments, a method for producing a cell population of stable regulatory T cells comprises isolating a biological sample comprising regulatory T cells from a human subject with an autoimmune disease.

[0133] An exemplary method of the present disclosure is provided in FIGS. 1A-1B. In the embodiment provided in FIG. 1A, a biological sample (e.g., a blood sample) is first isolated from a human subject with an autoimmune disease using apheresis. Cells of the biological sample are labeled with anti-CD8 antibodies (targeting non-CD4 conventional T cells), anti-CD19 antibodies (targeting B cells), and anti-CD14 antibodies (targeting monocytes), and the labeled cells are removed from the biological sample to produce a depleted biological sample. In some embodiments, the volume of the depleted biological sample is then reduced (e.g., by removing water / liquid) to aid in downstream processing of the sample. The sample is then labeled with an anti-CD25 antibody (e.g., a CD25-PE-biotin antibody) and a secondary molecule (e.g., an anti-biotin microbead) to produce a CD25-enriched cell population. In some embodiments, a CD25-PE-biotin antibody is used. The CD25-PE-biotin antibody comprises an anti-CD25 antibody bound to a tandem conjugate of phycoerythrin (PE) and biotin. In some embodiments, this enrichment step may be repeated two or more times (e.g., two or more times). These CD25 cultures are then further processed by sorting the cells using a TCR gating strategy utilizing fluorescence-activated cell sorting (FACS) or Tyto sorting (Miltenyi) as described in Figures 1A and 1B. First, the cells are labeled with anti-CD45RA, anti-CD4, and anti-CD127 antibodies. The cells are then selected in multiple sorting steps. In each step, in some embodiments, the labeled CD25 + / high CD4 + CD127 - / lo The cells were first identified as singlets, then as live cells, then as CD4+ cells, and then as CD25+ cells. + / high CD127- / low Identify cells and then CD25 high CD45RA and CD25 + / high CD45RA + The cells are first identified by a gating strategy that involves identifying a specific population of cells (e.g., CD4 + CD25 High CD127 - / low Cells containing the protein expression profile of CD45RA, and CD4 + CD25 + CD127 - / low CD45RA + In some embodiments, the sorting procedure is performed twice to optimize the purity of the cells.

[0134] In some embodiments, the present disclosure provides a method for producing an isolated population comprising stable CD4+ regulatory T cells, comprising: (a) removing CD8+ cells and CD19+ cells from a biological sample obtained from a subject with an autoimmune disease to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+ cells to produce an enriched population; and (c) isolating CD4+CD25+CD127+ cells from the enriched population. - / lo In some embodiments, the present disclosure provides a method for producing an isolated population comprising stable CD4+ regulatory T cells, comprising: (a) removing CD8+ cells and CD19+ cells from a biological sample obtained from a subject with an autoimmune disease to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+ cells to produce an enriched population; and (c) isolating CD4+CD25+CD127+ cells from the enriched population. - / lo and (d) engineering the population of cells of (c) to express an exogenous human T cell receptor (TCR) that specifically binds to a target peptide complexed with MHC. In some embodiments, step (a) further comprises removing CD14+ cells from the biological sample. In some embodiments, step (a) further comprises removing CD56+ cells from the biological sample.

[0135] In some embodiments, the method further comprises quantifying the methylation status of a T cell-specifically demethylated region (TSDR) at the FOXP3 locus in the population of cells, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the methylation status of the TSDR is quantified after the isolating step (c). In some embodiments, the methylation status of the TSDR is quantified after the isolating step (d).

[0136] In some embodiments, the enriched population contains CD4+CD25+CD127 - / lo The step of isolating cells comprises (i) isolating CD4 + (ii) identifying a first subpopulation of cells; and (iii) isolating CD25 from the first subpopulation. + / high CD127 - / lo (iii) identifying a second subpopulation of cells; and (iv) isolating CD25 from the second subpopulation. high CD45RA and CD25 + / high CD45RA + Select cells and CD25 + CD45RA - This involves excluding the CD4+ Tregs, thereby isolating a population of stable CD4+ Tregs.

[0137] In some embodiments, the methods provided herein further include isolating a biological sample comprising regulatory T cells from a human subject with an autoimmune disease. In some embodiments, sample isolation is performed using an apheresis technique (e.g., a leukapheresis technique for isolating white blood cells). In some embodiments, isolating a biological sample comprising regulatory T cells from a human subject with an autoimmune disease is performed by removing blood from the subject and separating the blood into plasma and cells. In some embodiments, the apheresis technique involves removing whole blood from the subject and separating the whole blood to remove desired cell types (e.g., T cells). In some embodiments, the separation step of the apheresis is performed using continuous flow centrifugation or intermittent flow centrifugation.

[0138] Cells expressing specific biomarkers (e.g., CD8+ cells, CD19+ cells, CD14+ cells, and / or CD56+ cells) may be removed from a sample (e.g., a biological sample) using FACS, antibody pull-down assay techniques, or any other method known to those of skill in the art for removing cells expressing specific biomarkers. In some embodiments, cells expressing specific biomarkers may be removed from a sample by labeling the cells with microbeads (e.g., anti-CD8, anti-CD14, anti-CD19, and / or anti-CD56 microbeads) and then subjecting the sample to FACS (to remove cells expressing the specific biomarkers).

[0139] A depleted biological sample is a biological sample (e.g., isolated from a human subject with an autoimmune disease) that has been processed to remove CD8+ cells, CD19+ cells, CD14+ cells, and / or CD56+ cells. In some embodiments, removing CD8+ cells, CD19+ cells, CD14+ cells, and / or CD56+ cells from a biological sample produces a depleted biological sample having less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, or less than 3% of its total cells comprising CD8+ cells, CD19+ cells, CD14+ cells, and / or CD56+ cells. In some embodiments, removing CD8+ cells, CD19+ cells, CD14+ cells, and / or CD56+ cells from a biological sample produces a depleted biological sample having less than 0.5% of its total cells comprising CD8+ cells.

[0140] Cell selection techniques may be used to select cells with a particular phenotype (e.g., cells expressing a particular biomarker, such as CD4, CD25, or CD45RA, or cells with no or low expression of a specific biomarker, such as CD127). The cell selection technique may be FACS technology, a Tyto device, an antibody pull-down assay technique, a magnetic cell separation technique, or any other method of selecting cells expressing a particular biomarker known to those skilled in the art. In some embodiments, the cell selection technique involves labeling cells expressing a particular biomarker (e.g., CD25) with a biotinylated antibody targeting that biomarker, and then performing a pull-down assay (e.g., using anti-biotin microbeads or streptavidin conjugates). In some embodiments, the cell selection technique involves labeling cells expressing a particular biomarker (e.g., CD25, CD4, and / or CD45RA) with an antibody targeting that biomarker. Cells labeled with the antibody targeting the particular biomarker can then be selected from a sample (e.g., a CD25-enriched population) using FACS.

[0141] Then, after the CD25 enrichment step, + / high CD4 +CD127 - / lo A multi-step selection process is followed to select regulatory T cells. In some embodiments, the selection process is carried out on a Tyto device (Miltenyi). Those skilled in the art will understand that software for visualizing selected cell populations can display data in various ways, including dot plots and histograms. When a population is identified based on the presence or absence of a single protein, the identification can be carried out using dot plots or histograms. When a population needs to be identified based on the presence or absence of two proteins (for example, CD25 and CD45RA), the identification should be carried out using dot plots.

[0142] In some embodiments, prior to the first sorting step, the CD25 enriched cell population is separated into multiple subpopulations of cells, and the first sorting step is performed simultaneously and / or sequentially on the multiple subpopulations. + / high CD4 + CD127 - / lo This provides rapid processing of large numbers of cells that need to be processed to isolate a sufficient number of cells that are a small percentage of the total. In some embodiments, the CD25-enriched cell population is separated into two, three, four, five, six, or more subpopulations, each of which is sorted in a first sorting step. In some embodiments, after the first sorting step, the sorted subpopulations are combined before a second sorting step.

[0143] In some embodiments, the present disclosure provides a method for producing an isolated population comprising stable CD4+ regulatory T cells, comprising: (a) removing CD8+ cells and CD19+ cells from a biological sample obtained from a subject with an autoimmune disease to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+ cells to produce an enriched population; and (c) isolating CD4 regulatory T cells from the enriched population. + CD25 + CD127 - / lo and isolating cells, the isolating comprising (i) isolating CD4 +(ii) identifying a first subpopulation of cells; and (iii) isolating CD25 from the first subpopulation. + / high CD127 - / lo (iii) identifying a second subpopulation of cells; and (iv) isolating CD25 from the second subpopulation. high CD45RA - and CD25 + / high CD45RA + Select cells and CD25 + CD45RA - and thereby isolating a population of stable CD4+ Tregs.

[0144] In some embodiments, CD25 is isolated from the CD25 enriched population. + / high CD4 + CD127 - / lo Selecting the cells includes identifying a first subpopulation of CD4+ cells from the CD25 enriched population. + / high CD127 - / lo A second subpopulation of cells is identified from the first subpopulation. high CD45RA and CD25 + / high CD45RA + A population of cells is selected from the second subpopulation for isolation.

[0145] In some embodiments, the first subpopulation comprises at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD4+ cells.

[0146] In some embodiments, the second subpopulation is at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD25 + / high CD4 + CD127 - / lo In some embodiments, the second subpopulation comprises at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD25 + / high CD4 + CD127 - / lo FoxP3 + Includes regulatory T cells.

[0147] In some embodiments, the population selected for isolation is CD25 high CD4 + CD127 - / low CD45RA(CD25 high CD45RA - ) and CD25 + / high CD4 + CD127 - / low CD45RA + (CD25+ / high CD45RA + In some embodiments, the population selected for isolation comprises at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% CD25 regulatory T cells. high CD45RA - In some embodiments, the population selected for isolation comprises at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% CD25 cells. + / high CD45RA + CD25 in the final population high CD45RA and CD25 + / high CD45RA + It is understood that the percentages of cells total approximately equal 80%, 90%, or 100% of the total cell population.

[0148] In some embodiments, the population selected for isolation has less than 10% CD25 + CD4 + CD127 - / low In some embodiments, the population selected for isolation comprises less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% CD25 + CD4 + CD127 - / low CD45RA - Contains cells.

[0149] In some embodiments, the methods described herein result in an isolated population of cells in which at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated cell population comprises stable CD4+ regulatory T cells, wherein 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, 80%-90%, 85%-95%, 80%-85%, 85%-90%, or 90%-95% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus.

[0150] In some embodiments, an isolated population of cells produced by the methods described herein is selected for therapeutic use if at least 80% of the cells are stable CD4+ Tregs that comprise a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, an isolated population of cells is selected for therapeutic use if at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+ Tregs that comprise a hypomethylated TSDR at the FOXP3 locus. In some embodiments, an isolated population of cells is selected for therapeutic use if 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, an isolated population of cells is selected for therapeutic use if 80%-90%, 85%-95%, 80%-85%, 85%-90%, or 90%-95% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus.

[0151] In some embodiments, the methods provided herein further comprise introducing an exogenous TCR that specifically binds to a target peptide complexed with MHC into a population of cells comprising stable CD4+ T regulatory cells. In some embodiments, introducing the exogenous TCR comprises transfecting the cell population with a nucleic acid encoding the exogenous TCR. In some embodiments, introducing the exogenous TCR comprises transducing the cell population with a nucleic acid encoding the exogenous TCR. The nucleic acid encoding the exogenous TCR may be an RNA (e.g., mRNA) molecule or a DNA molecule. In some embodiments, the nucleic acid is a vector or a plasmid. In some embodiments, the nucleic acid is delivered to the population of cells using a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, or a baculoviral vector.

[0152] In some embodiments, the method or process for producing a cell population of regulatory T cells further comprises activating and / or expanding the population of regulatory T cells (e.g., after the depletion, enrichment, and manipulation steps described herein). In some embodiments, the regulatory T cells are activated prior to transfection or transduction of the regulatory T cells with an exogenous TCR. In some embodiments, the regulatory T cells are activated with an anti-CD28 antibody and / or an anti-CD8 antibody. In some embodiments, activating the regulatory T cells comprises contacting the regulatory T cells with an antigen that specifically binds to an exogenous TCR belonging to the isolated cell population. In some embodiments, the antibody (e.g., anti-CD3, anti-CD28, and / or a TCR-specific antibody) is conjugated or complexed to beads, e.g., magnetic beads or polymer beads. In some embodiments, the antibody is covalently bound to a polymer matrix.

[0153] In some embodiments, the methods provided herein further comprise a second activation and / or expansion step, hi some embodiments, the second activation and / or expansion step is performed 5, 6, or 7 days after the first activation and / or expansion step.

[0154] In some embodiments, activating and / or expanding regulatory T cells comprises culturing the isolated cell population in a cell culture medium. In some embodiments, the medium comprises IL-2. In some embodiments, the medium comprises approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, 1,400, 1,600, 1,800, 2,000, 3,000, or 4,000 IU / mL of IL-2. In some embodiments, the medium comprises 500-1,500 or 800-1,200 IU / mL of IL-2. In some embodiments, the medium comprises approximately 1,000 IU / mL of IL-2. In some embodiments, the medium comprises 1,000 IU / mL of IL-2. In some embodiments, the medium further comprises TNFα. In some embodiments, the medium contains about 500, 750, 1000, 1500, 1750, 2000, 2250, 2500, or about 3000 IU / mL of TNFα. In some embodiments, the medium contains 2000-3000 or 2250-2750 IU / mL of TNFα. In some embodiments, the medium contains about 2500 IU / mL of TNFα. In some embodiments, the medium contains 2500 IU / mL of TNFα.

[0155] In some embodiments, activating and expanding the regulatory T cells comprises culturing the cells of the population for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, activating and expanding the regulatory T cells comprises culturing the cells of the population for 2-14, 2-10, 2-5, 5-10, or 5-14 days. In some embodiments, activating and expanding the regulatory T cells comprises culturing the cells of the population for 15, 14, 13, or 12 days or less. In some embodiments, after activation and / or expansion, the isolated cell population comprises at least 1 x 10 regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus. 2 , at least 1 x 10 3 , at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , or at least 1 × 10 10 Stable CD25 + / high CD4 + CD127 - / lo Includes regulatory T cells.

[0156] In some embodiments, the method or process for producing a cell population of regulatory T cells may further comprise the step of cryopreserving the cell population. In some embodiments, the method or process for producing a cell population of regulatory T cells may further comprise the step of thawing the cryopreserved cell population. A cryopreservation freeze-thaw cycle refers to the process of cryopreserving a cell population and subsequently thawing the cryopreserved cell population.

[0157] Pharmaceutical Composition In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated cell population of regulatory T cells (e.g., stable regulatory T cells) described herein. In some embodiments, the pharmaceutical composition comprises a cell population of regulatory T cells (e.g., stable regulatory T cells) described herein and a pharmaceutically acceptable excipient.

[0158] As used herein, a pharmaceutically acceptable excipient may also be referred to as a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, or a pharmaceutically acceptable adjuvant. The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of appropriate dosage and treatment regimens for using the particular compositions described herein in various treatment regimens.

[0159] Pharmaceutical compositions should typically be sterile and stable under the conditions of manufacture and storage. Sterile injectable formulations may be prepared using non-toxic parenterally acceptable diluents or solvents. Pharmaceutical compositions for use according to the present invention may contain pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers, which are non-toxic to subjects at the dosages and concentrations used. In some embodiments, the pharmaceutical composition may contain an organic solvent, such as, but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide (including mixtures or combinations thereof).

[0160] The pharmaceutical composition may comprise an effective amount of stable regulatory T cells sufficient to induce a desired biological response. For example, in some embodiments, the effective amount of stable regulatory T cells described herein may refer to a number of cells sufficient to ameliorate symptoms associated with an autoimmune disease (e.g., progressive multiple sclerosis). As will be understood by those skilled in the art, the effective amount of the solution or preparation provided herein may vary depending on various factors, such as the desired biological response, e.g., the specific disease to be treated, the specific symptoms to be alleviated, the cells or tissues to be targeted, and the age, sex, and general health of the subject.

[0161] In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus) is at least 1 x 10 2 , at least 1 x 10 3 , at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , or at least 1 × 10 10 In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus) comprises 1 x 10 2 ~1×10 10 , 1×10 3 ~1×10 10 , 1×10 4 ~1×10 10 , 1×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 10 , 1×10 8 ~1×10 10 , 1×10 9 ~1×10 10In some embodiments, the effective amount of stable regulatory T cells is 1 x 10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9×10 7 In some embodiments, the effective amount of stable regulatory T cells comprises 1 x 10 7 ~1×10 10 , 2 × 10 7 ~1×10 10 , 3×10 7 ~1×10 10 , 4×10 7 ~1×10 10 , 5×10 7 ~1×10 10 , 6×10 7 ~1×10 10 , 7×10 7 ~1×10 10 , 8×10 7 ~1×10 10 , or 9×10 7 ~1×10 10 Contains stable regulatory T cells.

[0162] In some embodiments, isolated cell populations (e.g., intended for use in pharmaceutical compositions) are cryopreserved (e.g., subjected to one or more cryopreservation freeze-thaw cycles). That is, the isolated cell populations produced herein may be combined with a cryoprotectant that forms chemical bonds with water and lowers the melting temperature by increasing the total concentration of solutes in the system. Non-limiting examples of cryoprotectants include glycerol, dimethyl sulfoxide (DMSO), ethanediol, and propanediol. While conventional methods, often involving the use of serum and DMSO, may be used, the present disclosure also contemplates the use of freezing media manufactured under cGMP conditions and formulated to be serum-free and non-animal in origin (e.g., using <10% DMSO in the freezing cocktail). Other cryopreservation techniques are also provided herein, including more advanced cooling techniques that vitrify cells without the use of cryoprotectants, for example. See, for example, Shinshu University, "A new way to 'freeze' cells promises to transform the common cell-freezing practice." ScienceDaily.com, April 2019. This process of ultra-rapid cooling utilizes inkjet cell printing to cool cells at a rate of 10,000°C / second, causing near-vitrification of the cells.

[0163] Treatment method In some embodiments, the present disclosure provides a method of treating an autoimmune disease in a subject in need thereof, comprising administering an isolated population of cells comprising stable CD4+ regulatory T cells or a composition thereof. In some embodiments, the autoimmune disease is selected from multiple sclerosis (e.g., progressive multiple sclerosis), type 1 diabetes, and inclusion body myositis. In some embodiments, the present disclosure provides a method of administering an isolated cell population (and related pharmaceutical compositions) comprising regulatory T cells described herein to a subject (e.g., a subject with an autoimmune disease). In some embodiments, the present disclosure provides a method of administering to a subject a cell population or pharmaceutical composition comprising regulatory T cells described herein in an amount effective to alleviate one or more symptoms of the autoimmune disease. In some embodiments, the present disclosure provides a method of treating an autoimmune disease in a subject, comprising administering to the subject a cell population or pharmaceutical composition comprising regulatory T cells described herein. In some embodiments, at least a portion of the population of cells are autologous cells (i.e., obtained from the same subject to which they are subsequently administered). In some embodiments, a population of cells is isolated from a subject, subjected to a method of producing a cell population described herein (e.g., to enrich the relative concentration of regulatory T cells within the population), engineered to express an exogenous TCR, and then administered to the same subject to treat a disease.

[0164] In some embodiments, the autoimmune disease is selected from Sjögren's syndrome, Goodpasture's syndrome, SLE, Behcet's disease, multiple sclerosis, neuromyelitis optica, myasthenia gravis, primary biliary cholangitis, ulcerative colitis, Crohn's disease, celiac disease, aplastic anemia, rheumatoid arthritis, ankylosing spondylitis, autoimmune hepatitis, type 1 diabetes, polymyositis, dermatomyositis, inclusion body myositis, pemphigus, psoriasis, Hashimoto's thyroiditis, Graves' disease, GvHD, lupus nephritis, ALS, chronic inflammatory demyelinating polyneuropathy, autoimmune pancreatitis, vitiligo, and alopecia. In some embodiments, the autoimmune disease is selected from multiple sclerosis, type 1 diabetes, and inclusion body myositis.

[0165] In some embodiments, treating a disease (or treatment thereof) refers to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of an autoimmune disease or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have occurred and / or after the disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or inhibit the onset or progression of the disease. For example, treatment may be administered to a susceptible individual before the onset of symptoms (e.g., to identify genetic factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0166] A subject refers to an individual organism, e.g., an individual human. In some embodiments, the subject is a human subject, such as a male or female subject. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be male or female.

[0167] Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intravenous, subcutaneous, intravenous, intrathecal administration, direct delivery to a selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intramuscular, intradermal, intratumoral, and other parenteral routes of administration. If desired, administration routes may be combined. In some embodiments, the isolated populations or compositions thereof described herein are administered intravenously.

[0168] The cell population or pharmaceutical composition comprising regulatory T cells may be administered as a bolus dose. In some embodiments, administration of the cell population or pharmaceutical composition comprising regulatory T cells comprises one or more infusions of the isolated cell population or pharmaceutical composition into the subject (e.g., the cells are infused through a central line, similar to a blood transfusion).

[0169] Further Numbered Embodiments Further numbered embodiments of the present disclosure are provided as follows:

[0170] Embodiment 1. An isolated cell population comprising regulatory T cells, wherein the regulatory T cells of the isolated cell population (a) comprise an exogenous human T cell receptor (TCR) that specifically binds to a target peptide complexed with a major histocompatibility complex (MHC), and (b) comprise stable regulatory T cells that comprise a hypomethylated regulatory T cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus, wherein at least 80% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / lo regulatory T cells, and wherein less than 10% of the cells of the isolated cell population express FOXP3 protein from an engineered FOXP3 locus.

[0171] Embodiment 2. The isolated cell population of embodiment 1, wherein the MHC is MHC class I or MHC class II.

[0172] Embodiment 3. The isolated cell population of any one of the preceding embodiments, wherein at least 70% of the cells of the isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

[0173] Embodiment 4. The isolated cell population of embodiment 3, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells of the isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

[0174] Embodiment 5. The isolated cell population of any one of the preceding embodiments, wherein at least 85%, at least 90%, or at least 95% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / lo regulatory T cells.

[0175] Embodiment 6. The isolated cell population of any one of the preceding embodiments, wherein at least 70% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / loFOXP3+ regulatory T cells.

[0176] Embodiment 7. The isolated cell population of embodiment 6, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the isolated cell population are CD25+ / highCD4+CD127- / loFOXP3+ regulatory T cells.

[0177] Embodiment 8. The isolated cell population of any one of the preceding embodiments, wherein the percentage of cells of the isolated cell population comprising stable regulatory T cells does not decrease by more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percentage point(s) during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0178] Embodiment 9. The isolated cell population of embodiment 8, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0179] Embodiment 10. The isolated cell population of embodiment 8, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0180] Embodiment 11. The isolated cell population of embodiment 8, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0181] Embodiment 12. The isolated cell population of any one of the preceding embodiments, wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion following transduction of the stable regulatory T cells with an exogenous human TCR.

[0182] Embodiment 13. The isolated cell population of embodiment 12, wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 10% for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 10% for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0183] Embodiment 14. The isolated cell population of embodiment 12, wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 5% for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 5% for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0184] Embodiment 15. The isolated cell population of embodiment 12, wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 1% for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 1% for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0185] Embodiment 16 The isolated cell population of any one of the preceding embodiments, comprising at least 1 x 10 stable regulatory T cells.

[0186] Embodiment 17. The isolated cell population of embodiment 16, comprising 1 x 10 to 1 x 10 stable regulatory T cells.

[0187] Embodiment 18 The isolated cell population of any one of the preceding embodiments, wherein less than 5%, less than 2%, or less than 1% of the cells of the isolated cell population express FOXP3 protein from the engineered FOXP3 locus.

[0188] Embodiment 19. The isolated cell population of any one of the preceding embodiments, wherein less than 10% of the cells of the isolated cell population express FOXP3 protein from (a) an endogenous open reading frame operably linked to an engineered FOXP3 promoter, or (b) an exogenous FOXP3 transgene.

[0189] Embodiment 20 The isolated cell population of any one of the preceding embodiments, wherein at least 90%, at least 95%, or 100% of the cells of the isolated cell population comprise an unmodified endogenous FOXP3 locus.

[0190] Embodiment 21 The isolated cell population of any one of the preceding embodiments, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% of the cells of the isolated cell population comprise an engineered FOXP3 gene or engineered FOXP3 protein.

[0191] Embodiment 22 The isolated cell population of any one of the preceding embodiments, wherein at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the cells of the isolated cell population do not contain an exogenous FOXP3 gene or exogenous FOXP3 protein.

[0192] Embodiment 23 The isolated cell population of any one of the preceding embodiments, wherein at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the cells of the isolated cell population do not exhibit ectopic FOXP3 expression.

[0193] Embodiment 24 The isolated cell population of any one of the preceding embodiments, wherein at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the cells of the isolated cell population express physiological levels of functional FOXP3 protein.

[0194] Embodiment 25 The isolated cell population of any one of the preceding embodiments, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% of the cells of the isolated cell population overexpress functional FOXP3 protein.

[0195] Embodiment 26 The isolated cell population of any one of the preceding embodiments, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells of the isolated cell population are conventional T cells.

[0196] Embodiment 27. The isolated cell population of any one of the preceding embodiments, wherein about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 5%, or about 5% to about 10% of the cells in the isolated cell population are conventional T cells.

[0197] Embodiment 28. The isolated cell population of embodiment 26 or 27, wherein the conventional T cells of the isolated population comprise an exogenous human TCR.

[0198] Embodiment 29. The isolated cell population of embodiment 28, wherein about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, or about 60% of the conventional T cells comprise an exogenous TCR.

[0199] Embodiment 30 The isolated cell population of any one of the preceding embodiments, wherein the ratio of regulatory T cells to conventional T cells in the isolated cell population is at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 100:1.

[0200] Embodiment 31 The isolated cell population of any one of the preceding embodiments, wherein less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells of the isolated cell population are CD8+ T cells.

[0201] Embodiment 32. The isolated cell population of embodiment 31, wherein CD8+ T cells are undetectable, optionally undetectable by fluorescence-activated cell sorting (FACS).

[0202] Embodiment 33 The isolated cell population of any one of the preceding embodiments, wherein at least 10% of the regulatory T cells of the isolated cell population express an exogenous human TCR.

[0203] Embodiment 34. The isolated cell population of embodiment 33, wherein at least 15%, 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% of the regulatory T cells of the isolated cell population express an exogenous human TCR.

[0204] Embodiment 35 The isolated cell population of any one of the preceding embodiments, wherein the percentage of CD25+ / highCD4+CD127- / lo regulatory T cells is assessed 1 hour to 120 days after transduction.

[0205] Embodiment 36. The isolated cell population of any one of embodiments 1 to 34, wherein the percentage of CD25+ / highCD4+CD127- / lo regulatory T cells is assessed at least 1, 12, 24, 48, 72, 96, or 120 hours after transduction.

[0206] Embodiment 37. The isolated cell population of any one of embodiments 1 to 34, wherein the percentage of CD25+ / highCD4+CD127- / lo regulatory T cells is assessed 7 to 14 days after transduction.

[0207] Embodiment 38. The isolated cell population of any one of embodiments 1 to 34, wherein the percentage of CD25+ / highCD4+CD127- / lo regulatory T cells is assessed at least 10, 15, 30, 60, or 120 days after transduction.

[0208] Embodiment 39 The isolated cell population of any one of the preceding embodiments, wherein the TSDR at the endogenous FOXP3 locus remains hypomethylated after cryopreservation freeze-thaw cycles, at least until the cells of the isolated cell population are administered to a subject.

[0209] Embodiment 40. The isolated cell population of any one of the preceding embodiments, wherein the stable regulatory T cells of the isolated cell population exhibit one or more functions selected from: (a) cytokine secretion activity, (b) expression of activation markers associated with regulatory T cells, and / or (c) suppressive activity.

[0210] Embodiment 41. The isolated cell population of embodiment 40, wherein one or more functions are assessed 1 hour to 14 days after transduction.

[0211] Embodiment 42. The isolated cell population of embodiment 40, wherein the one or more functions are assessed at least 12, 24, 48, 72, 96, or 120 hours after transduction.

[0212] Embodiment 43. The isolated cell population of embodiment 40, wherein the one or more functions are assessed at least 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction.

[0213] Embodiment 44. The isolated cell population of any one of embodiments 40 to 43, wherein the one or more functions are assessed after cryopreservation of the isolated cell population, optionally at least 24 hours after cryopreservation.

[0214] Embodiment 45 The isolated cell population of any one of the preceding embodiments, wherein at least 10% of the stable regulatory T cells are CD45RA+.

[0215] Embodiment 46. The isolated cell population of embodiment 45, wherein at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the stable regulatory T cells are CD45RA+.

[0216] Embodiment 47. The isolated cell population of any one of the preceding embodiments, wherein at least 10% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / loCD45RA+ prior to activation and transduction with an exogenous human TCR.

[0217] Embodiment 48. The isolated cell population of any one of the preceding embodiments, wherein at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / loCD45RA+ prior to activation and transduction with an exogenous human TCR.

[0218] Embodiment 49 The isolated cell population of any one of the preceding embodiments, wherein the exogenous human TCR is encoded as a single polypeptide.

[0219] Embodiment 50. The isolated cell population of embodiment 49, wherein the exogenous human TCR comprises an N-terminal beta chain and a C-terminal alpha chain.

[0220] Embodiment 51. The isolated cell population of embodiment 49 or 50, wherein the polypeptide comprises an alpha chain and a beta chain, and wherein the polypeptide comprises a self-cleaving peptide sequence located between the alpha chain and the beta chain.

[0221] Embodiment 52 The isolated cell population of embodiment 51, wherein the self-cleaving peptide sequence is a 2A peptide sequence.

[0222] Embodiment 53 The isolated cell population of embodiment 52, wherein the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence.

[0223] Embodiment 54 The isolated cell population of any one of the preceding embodiments, wherein the exogenous human TCR comprises one or more amino acid substitutions to cysteine residues in the TCR alpha chain constant region and the TCR beta chain constant region, wherein the cysteine residues are capable of forming one or more disulfide bonds.

[0224] Embodiment 55. The isolated cell population of embodiment 54, wherein the TCR alpha chain constant region comprises a T48C amino acid substitution relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR beta chain constant region comprises a S57C amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 3.

[0225] Embodiment 56. A method of producing the isolated cell population of any one of the preceding embodiments, the method comprising: (a) isolating a biological sample comprising regulatory T cells from a human subject having an autoimmune disease; (b) removing CD8+ cells, CD19+ cells, and optionally CD14+ cells from the biological sample to produce a depleted biological sample; (c) selecting CD25+ cells from the depleted biological sample to produce a CD25-enriched cell population; (d) selecting CD25+ / highCD4+CD127- / loCD45RA+ cells from the CD25-enriched cell population to produce one or more positive fractions; (e) selecting CD25+ / highCD4+CD127- / loCD45RA+ cells from the one or more positive fractions to produce a cell population comprising stable regulatory T cells; and (f) engineering the cell population comprising stable regulatory T cells such that the stable regulatory T cells comprise an exogenous human TCR, thereby producing the isolated population of cells.

[0226] Embodiment 57. The method of embodiment 56, wherein less than 5%, less than 4%, or less than 3% of the cells of the depleted sample comprise CD8+ cells, CD19+ cells, and / or CD14+ cells.

[0227] Embodiment 58 The method of embodiment 56 or 57, wherein less than 0.5% of the cells in the depleted sample comprise CD8+ cells.

[0228] Embodiment 59. The method of any one of embodiments 56 to 58, wherein the selecting in (d) comprises identifying CD4+CD45RA+ cells, then identifying CD25+ / highCD127- / lo cells from the identified CD4+CD45RA+ cells to identify a first population of CD25+ / highCD4+CD127- / loCD45RA+ cells, and then selecting the first population of CD25+ / highCD4+CD127- / loCD45RA+ cells from the first subpopulation to produce one or more positive fractions.

[0229] Embodiment 60. The method of embodiment 59, wherein the selecting of (e) comprises selecting and identifying CD4+CD45RA+ cells, then identifying CD25+ / highCD127- / lo cells from the identified CD4+CD45RA+ cells to identify a second population of CD25+ / highCD4+CD127- / loCD45RA+ cells, and then selecting the second population of CD25+ / highCD4+CD127- / loCD45RA+ cells from the second subpopulation to produce a cell population comprising stable regulatory T cells.

[0230] Embodiment 61 The method of any one of embodiments 56-60, wherein the autoimmune disease is multiple sclerosis, optionally progressive multiple sclerosis, type 1 diabetes, or inclusion body myositis.

[0231] Embodiment 62 The method of embodiment 61, wherein the human subject has the genetic HLA haplotype DR2a / b, DR3, or DR4.

[0232] Embodiment 63 The method of any one of embodiments 56-62, wherein the human subject is a male subject.

[0233] Embodiment 64 The method of any one of embodiments 56-62, wherein the human subject is a female subject.

[0234] Embodiment 65. The method of any one of embodiments 56-64, wherein manipulating the isolated cell population comprises transducing the cell population with a nucleic acid encoding an exogenous human TCR.

[0235] Embodiment 66. The method of any one of embodiments 56-65, further comprising activating and expanding cells of the isolated cell population to produce a cell population comprising at least 1 x 10 stable CD25+ / highCD4+CD127- / lo regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

[0236] Embodiment 67. The method of embodiment 66, wherein activating and expanding comprises culturing the cells of the isolated cell population for at least 5, 6, 7, 8, 9, or 10 days.

[0237] Embodiment 68. The method of embodiment 67, wherein activating and expanding comprises culturing the cells of the isolated cell population for no more than 15, 14, 13, or 12 days.

[0238] Embodiment 69. The method of any one of embodiments 65 to 68, wherein the nucleic acid is a vector.

[0239] Embodiment 70. The method of embodiment 69, wherein the vector is a viral vector.

[0240] Embodiment 71. The method of embodiment 70, wherein the viral vector is a lentiviral vector.

[0241] Embodiment 72. The method of any one of embodiments 65 to 71, wherein the nucleic acid comprises a promoter operably linked to a coding sequence encoding an exogenous human TCR, and optionally the promoter is an EF-1α promoter or an MND promoter.

[0242] Embodiment 73. The method of any one of embodiments 65 to 72, wherein the nucleic acid further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally a WPRE-mut6.

[0243] Embodiment 74. The method of embodiment 72 or 73, wherein the coding sequence is codon-optimized.

[0244] Embodiment 75. A vector described in any one of embodiments 69 to 74.

[0245] Embodiment 76 A pharmaceutical composition comprising the isolated cell population of any one of the preceding embodiments and a pharmaceutically acceptable excipient.

[0246] Embodiment 77. A composition comprising the isolated cell population of any one of the preceding embodiments and a cryopreservative.

[0247] Embodiment 78. A method comprising administering to a subject the pharmaceutical composition of embodiment 76, wherein the subject has an autoimmune disease.

[0248] Embodiment 79. The method of embodiment 78, wherein the autoimmune disease is multiple sclerosis, optionally progressive multiple sclerosis, type 1 diabetes, or inclusion body myositis.

[0249] Embodiment 80 The method of embodiment 78 or 79, wherein the pharmaceutical composition is administered in an amount effective to alleviate one or more symptoms of the autoimmune disease.

[0250] Embodiment 81 A method comprising administering to a subject the isolated cell population of any one of the preceding embodiments.

[0251] Embodiment 82 The method of embodiment 81, wherein the subject has an autoimmune disease.

[0252] Embodiment 83 The method of embodiment 82, wherein the autoimmune disease is multiple sclerosis, optionally progressive multiple sclerosis, type 1 diabetes, or inclusion body myositis.

[0253] Embodiment 84. The method of any one of embodiments 81-83, wherein administering comprises intravenous administration.

[0254] Embodiment 85. The method of any one of embodiments 81-84, wherein administering comprises one or more injections.

[0255] Embodiment 86 The method of any one of embodiments 81-85, wherein the cells of the isolated cell population are autologous to the subject. [Example]

[0256] Example 1. Manufacturing method for the production of autologous stable regulatory T cells expressing exogenous TCRs The generation of a cell population containing stable thymus-derived regulatory T cells expressing an exogenous TCR is demonstrated in this example. The overall enrichment and sorting strategy is shown in Figures 1A-1B.

[0257] Leukapheresis, depletion, and enrichment First, leukapheresis transfusions from human subjects were collected, transported to a central processing facility, and held at 4°C until processing approximately 24 hours after collection. The incoming leukapheresis product was washed in phosphate-buffered saline containing 0.5% human serum albumin (HSA) to reduce platelet counts and then labeled with a cocktail of magnetic microbeads (CD8, CD14, and CD19 microbeads; Miltenyi) using an automated cell processing system.

[0258] After depletion of cytotoxic T cells (CD8+), B cells (CD19+), and monocytes (CD14+) using magnetic cell separation technology, CD25+ cells were enriched. The volume of the depleted biosample was reduced by centrifugation. The depleted biosample was then labeled with CD25-PE-biotin antibody (Miltenyi) according to the manufacturer's recommendations. The cells were then washed with phosphate-buffered saline containing 0.5% HSA and labeled with anti-biotin microbeads (Miltenyi). CD25+ cells were then isolated using magnetic cell separation technology to produce a CD25-enriched sample.

[0259] Figure 2 shows the viable cells (VC) of all cells after apheresis, removal of CD8+, CD19+, and CD14+ cells (depleted sample), and selection for CD25+ (CD25-enriched sample) for Run 1. Approximately 98% of the total cells in the population are CD3+ cells after the CD25 selection step to produce the CD25-enriched sample, and approximately 95% of the cells are CD4+ T cells.

[0260] Table 1 shows the CD4+ and naive Treg cell composition of the apheresis cell population after the depletion and enrichment steps for five additional manufacturing runs.

[0261] [Table 1]

[0262] Figure 3A shows the viable count (VC) of naive regulatory T cells (specifically, CD4+CD25+ / highCD127- / loCD45RA+ cells) after apheresis, removal of CD8+, CD19+, and CD14+ cells (depleted sample), and selection for CD25+ (CD25-enriched sample) for Run 1. Figure 3B shows the recovery rate of naive regulatory T cells (CD4+CD25+ / highCD127- / loCD45RA+ cells) after each step, indicating that approximately 80% of CD4+CD25+ / highCD127- / loCD45RA+ cells were recovered after the depletion step and 75% of CD4+CD25+ / highCD127- / loCD45RA+ cells were recovered after the CD25-enrichment step.

[0263] Table 2 shows the VC of naive regulatory T cells after apheresis, depletion, and enrichment, as well as the recovery rate from the previous step of naive regulatory T cells after depletion and enrichment.

[0264] [Table 2]

[0265] Gating Strategy The CD25-enriched samples were then stained with fluorescently labeled anti-CD45RA, anti-CD4, and anti-CD127 antibodies to detect CD4 + CD25 + / high CD127 - / lo CD45RA +Phenotypes were sorted by fluorescence-activated cell sorting (FACS) to generate cell populations containing stable regulatory T cells. FACS was performed in two rounds: a first debulk fraction sort and a second fractional purity sort. For debulk sorting, the CD25-enriched sample was divided into multiple fractions that were sorted across multiple cell sorters. After debulk sorting, the fractions were combined for the second round of purity sorting. The purpose of differentially sorting cells in debulk and / or purity sorting is to expedite the sorting process by distributing the sorting across multiple machines and ensure that cells remain viable. The gating strategies used for debulk and purity sorting were identical. For the gating strategy, CD45RA + CD4 + Cells were first selected and then CD25 + / high CD127 - / lo CD45RA cells + CD4 + We used a dot plot-based approach to select from a cell population to optimize the purity of stable regulatory T cells in the population as determined by their TSDR phenotype. As shown in Figure 1B, this FACS gating strategy begins with first selecting CD45RA on a dot plot. + vs. CD4 + The cells were plotted and selected based on their population distribution using a box gate. From the selected population of cells, CD25 + / high Cells vs. CD127 - / lo Plot the cells and gate using a polygon gate to identify CD4 + CD25 + / high CD127 - / lo CD45RA + A population of cells was selected. This FACS strategy was developed to optimize the selection of stable naive regulatory T cells based on the TSDR hypomethylation phenotype. CD4 for run 1 + CD25 + / high CD127 - / lo CD45RA + The composition of the cell population before sorting, after debulking, and after production of the cell population is provided in Table 3.

[0266] [Table 3]

[0267] The purity and recovery of naive Tregs during the selection process for five additional manufacturing runs are shown in Table 4.

[0268] [Table 4]

[0269] Regulatory T cells were activated using T cell TransAct Activation Reagent (Miltenyi) for approximately 48 hours after FACS. The activated regulatory T cells were then transduced with a third-generation VSV-pseudotyped, self-inactivating lentiviral vector encoding an exogenous TCR that specifically binds to a target peptide associated with multiple sclerosis (e.g., progressive multiple sclerosis), designated "TCR-A." The vector construct contains an N-terminal TCR β chain and a C-terminal TCR α chain, with a linker domain containing a GSG amino acid sequence followed by a P2A self-cleaving peptide. The TCR β-GSG-P2A-TCR α fusion construct was expressed under the EF1α promoter. The percentage of transduced cells, measured by FACS analysis using an anti-Vβ antibody, is shown in Figure 5.

[0270] The transduced regulatory T cells were then expanded in T cell expansion medium supplemented with 1,000 IU / mL of IL-2 until harvest (day 9, 10, or 11). The cell number, size, population doubling, and viability of non-transduced and transduced regulatory T cells were assessed and are shown for run 1 (Figure 4). Notably, the transduced regulatory T cells shared a similar growth profile to the non-transduced regulatory T cells, suggesting that the exogenous TCR did not impair cell growth and proliferation.

[0271] The population doubling levels and % viability for five additional manufacturing runs are shown in Table 5. The % target dose achieved for the entire process for all six manufacturing runs is also shown. The % target dose was calculated by determining the final total number of naive Tregs based on the initial number and population doubling level of naive Tregs before expansion after the selection process, assuming a transduction efficiency of 25%. The % target dose was calculated based on a target dose of 100 million transduced cells. As shown, the manufacturing process in this example achieves a Treg product with high purity and sufficient cells to dose a patient from a single leukopack. As used herein, PDL is the population doubling level used throughout. TDN refers to transduced cells.

[0272] [Table 5]

[0273] Transduced cells were collected on days 0–11 of the activation and expansion process, and TSDR hypomethylation at the FOXP3 CNS2 locus was measured by ddPCR assay. Genomic DNA (gDNA) was obtained from enriched regulatory T cells and bisulfite-treated. A digital droplet PCR (ddPCR) assay using methylation-specific primers and probes was then employed to quantify unmethylated and methylated sequences. The TSDR hypomethylation status of cells collected for all six transduced manufacturing runs is shown in Figure 6. During expansion, TSDR hypomethylation levels did not decrease by more than 10% for any manufacturing run and remained above 80% for all manufacturing runs. Table 6 below shows the TSDR hypomethylation status for all six donor samples processed using the method described in this example.

[0274] These data indicate that the resulting regulatory T cells are stable, thymus-derived regulatory T cells.

[0275] [Table 6]

[0276] Treg activity after cryopreservation freeze-thaw cycles Regulatory T cells produced by the methods of this example and either untransduced or transduced with lentiviral vectors encoding the TCRs described herein were cryopreserved for storage. Cells were frozen and stored in a vapor-phase nitrogen storage freezer.

[0277] After a period of refrigeration, the frozen cell population was thawed. The cell population was divided into two experimental groups. The first experimental group was activated with anti-CD3 and anti-CD28 antibodies (transduced (TDN)), whereas the second experimental group was not activated (untransduced (UNT)). Both groups were grown in medium containing IL-2. After incubation, IL-10 levels were measured after 1, 2, and 3 days.

[0278] The increase in IL-10 levels for each of the three-day runs for cells activated with anti-CD3 and anti-CD28 antibodies is shown in Figure 7. The cells showed increased secretion of IL-10 cytokine over the three days, demonstrating that the cell populations were able to be activated and retain their cellular function after cryopreservation freeze-thaw cycles (Figure 7). Cells that were not activated did not show an increase in IL-10 levels. This data further demonstrates that regulatory T cells retained function.

[0279] Example 2. Gating strategy optimized for GMP conditions A second gating strategy was developed using a 1D histogram-based approach rather than a 2D dot-based approach to reduce user error in a GMP manufacturing setting. Cells were prepared as in Example 1 up until the FACS sorting step. In the FACS sorting step, the gating scheme used, shown in Figure 8, was such that CD25 enriched cells were enriched in the histogram (CD4 + >CD45RA + (tissue)>CD127 - / lo (tissue) 40%>CD25 Hi(tissue) 70%) using CD4 + , CD45RA + , CD127 - / lo and CD25 + / high The approach was based on one-dimensional histogram plots sequentially gated on TCR-A and TCR-E. For runs with the same run number as in Example 1, both gating strategies were performed in parallel on the same donor sample. Cells were transduced with TCR-A or a disulfide-modified, codon-optimized version called "TCR-E v2."

[0280] The characteristics of the cells before sorting are shown in Table 7, and the viability of the cells after each step of the manufacturing process is shown in Table 8.

[0281] [Table 7]

[0282] [Table 8]

[0283] The purity and recovery of naive Tregs during the sorting process for each run is shown in Table 9.

[0284] [Table 9]

[0285] The characteristics of the produced cells are shown in Table 10 below.

[0286] [Table 10]

[0287] The TSDR status of the cells is shown in Table 11 below and Figure 9. One run with a TSDR level above 100% is due to the donor being female. Because the TSDR assay measures the FoxP3 locus on the X chromosome, and females have one silenced (methylated) copy of the X chromosome, the TSDR demethylation level is doubled for female subjects to obtain the percentage of cells with a hypomethylated FoxP3 locus.

[0288] [Table 11]

[0289] Treg activity after cryopreservation freeze-thaw cycles Regulatory T cells produced by the method in this example for runs 5-7, either untransduced or transduced with the indicated lentiviral vectors, were cryopreserved for storage. Cells were frozen and stored in a vapor-phase nitrogen storage freezer.

[0290] After a period of refrigeration, the frozen cell population was thawed. The cell population was divided into two experimental groups. The first experimental group was activated with anti-CD3 and anti-CD28 antibodies, while the second experimental group was not. Both groups were grown in medium containing IL-2. After incubation, regulatory T cell activation markers and cytokine levels were measured at 1, 2, and 3 days (IL-10, CTLA-4, TGF-β-1, and CD69).

[0291] Figures 10A-10B show the increased levels of IL-10, CTLA-4, TGF-β-1, and CD69 for all runs over three days for cells activated with anti-CD3 and anti-CD28 beads. Activated cells showed increased levels of IL-10 (Figure 10D), CTLA-4 (Figure 10A), TGF-β-1 (Figure 10C), and CD69 (Figure 10B) over the period, demonstrating that the cell populations were activated and could retain their cellular function after cryopreservation freeze-thaw cycles (Figures 10A-10D). Non-activated cells did not show increased levels. This data further demonstrates that regulatory T cells retained function.

[0292] Example 3: High-stringency manufacturing strategies for stable Treg selection Challenges in generating stable Tregs from patient populations Following the establishment of the manufacturing process using healthy donor samples, the manufacturing process was evaluated using samples from MS donors. For certain MS donors, lower initial TSDR hypomethylation levels and / or less stable TSDR hypomethylation levels over the course of expansion (from day 0 to harvest) were observed compared to results using healthy donor cells. Figure 11 shows the change in TSDR hypomethylation over expansion for three MS donors. For two of the three MS donors, the initial TSDR was less than 90%, and the TSDR decreased by more than 10% over the course of expansion. This is likely due to differences in Treg populations between healthy donors and autoimmune donors, including MS donors, making the boundary between Treg cells and conventional T cells less clear during cell sorting. These results suggested that for some donors, increasing the stringency of the gating strategy likely improved the purity of the isolated Treg population and, therefore, the proportion of stable Tregs obtained in the process. However, increasing stringency likely resulted in a decrease in yield. This example describes a more stringent gating approach coupled with a modified expansion protocol that enhances Treg expansion, which is required to overcome the reduced yields resulting from the increased stringency of stable Treg selection.

[0293] Assessment of Treg subpopulations for inclusion in the process The selection strategy described in the above examples focuses on the selection of naive Tregs. Considering the need for a more stringent selection strategy for a particular donor and the possibility that this strategy may eliminate the increase in the proportion of naive Tregs, the present inventors have used CD45RA to select naive Tregs. + We evaluated whether a selection step is necessary in the context of a more stringent sorting strategy or whether more stringently selected antigen-experienced Tregs could be used. Naive Tregs are CD4 + CD25 + CD127 - / lo CD45RA + Antigen-experienced Tregs are CD4+ CD25 high CD127 - / lo CD45 - Both of these populations are stable Tregs with a stable TSDR hypomethylation phenotype. To assess whether CD45RA-based selection is necessary, we performed antigen-experienced (CD45RA - ) and naive (CD45RA + ) Tregs were isolated and compared for proliferation capacity and stability.

[0294] Cells were prepared prior to the FACS sorting step as described in Example 1. Cells were then subjected to two rounds of FACS sorting for debulking and purity. - and CD45RA + Both populations were sorted on CD4, CD25, and CD127 by debulk sorting. Cells were first sorted on CD4 + and then using a polygon gate set at 70% of the standard polygon gate for Treg identification and selection, CD25 + / high CD127 - / lo Tregs were identified. The selection was repeated and CD45RA cells were identified as CD25 + / high CD127 - / lo After identification of the Treg population, CD45RA cells were identified and selected. + For cells, CD25 + / high CD127 - / lo Following identification of the Treg population, CD45RA + Cells were identified and selected. Naive (CD45RA + ) Treg percentages are shown in Table 12 below.

[0295] At the end of selection, three groups were established. (a) CD45RA + Treg-enriched CD45RA + Treg fraction (b) CD45RA - Treg-enriched CD45RA - Treg fraction (c) “Spike-in” Treg-80% enriched CD45RA - Treg fraction + 20% enriched CD45RA + Treg fraction

[0296] [Table 12]

[0297] The cells were then expanded in culture for 8 days as described in Example 1. Figures 12A-12C show the population doubling level (PDL) and TSDR hypomethylation after cell expansion. As shown in Figures 12A and 12C, CD45RA + The CD45RA population showed increased TSDR hypomethylation along with a dramatic increase in proliferation compared to the CD45RA population. - CD45RA compared to Tregs + Due to the relative proliferation capacity of naive Tregs, CD45RA + These findings suggest that naive Tregs are required for the expansion process. Importantly, the "spike-in" samples express CD45RA + Naive Tregs express a large excess of CD45RA - We show that they can still proliferate effectively in the presence of antigen-experienced Tregs.

[0298] Development of a more stringent gating strategy for the isolation of stable Tregs In light of the above findings, CD45RA + To increase the stringency of selection while still capturing Tregs, we developed an improved gating strategy. In particular, this strategy targets antigen-experienced (CD4 + CD25 high CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + ) Tregs, while CD4 + CD25 + CD127- / lo Specifically, CD45RA non-Tregs are excluded. By separately identifying both antigen-experienced and naive Tregs, sufficient numbers of Tregs can be obtained from a single leukopack to produce a cell therapy product. Antigen-experienced Tregs have high suppressive capacity and can suppress any non-Tregs both ex vivo and in vivo. This approach allows for the elimination of CD45RA non-Tregs. + Identify cells and then (either sequentially or simultaneously) use a CD127 gating scheme in combination with a critical "not" or L-shaped gate as the final step in the gating scheme. - / lo and CD25 + Identify antigen-experienced (CD4 + CD25 highい CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + ) Tregs are selected, while unwanted CD4 + CD25 + CD127 - / lo CD45RA - This gating approach is illustrated in Figures 13A-13C and 14A-14E, where Figures 13A-13C show a dot plot approach of the CD25 / CD127 selection step and Figures 14A-14E show a dot plot approach of the CD127 selection step. - / lo , followed by CD25 + We present a histogram approach to selecting

[0299] To further illustrate the gating strategy, CD25-enriched cells were stained with fluorescently labeled antibodies that recognize human CD4, CD127, and CD45RA cell surface markers. The cells had already been labeled with CD25 antibodies in the enrichment step of the process. The cells were then sorted twice using a FACS instrument (using the same strategy in each step) using the gating scheme described below. In the dot plot-based approach shown in Figures 13A-13C, the first step of the gating process was to select CD4 + T cells were first identified from all live cell events (Figure 13A). In a second step, CD25 T cells were identified based on two-parameter flow plots. + CD127 - / lo Treg cells were identified, thereby allowing the spatial context of the Treg cell population to be visualized relative to another cell population (i.e., Tconv cells), and CD25 and CD127 levels were visualized by drawing a polygon gate in the upper left quadrant to capture CD25 + CD127 - / lo The cells were captured. The polygonal gate was then shifted upward to reduce the selected cell population to 70% of the original population, thereby increasing the stringency of selection (Figure 13B). In a final step, a "not" or L-shaped gate was applied to visualize CD45RA and CD25 levels and identify naive Tregs (CD4 + CD25 + CD127 - / lo CD45RA + ) and antigen-experienced Tregs (CD4 + CD25 high CD127 - / low CD45RA - ) cell populations were analyzed by CD25 + The CD45RA cell population was selected for expansion by excluding it from the sorting gate (Figure 13C). Both antigen-experienced and non-stable Treg populations expressed CD45RA - However, they can be distinguished by CD25 levels, and antigen-experienced Tregs express CD25 high and the non-stable Treg population is CD25 +Naive Tregs express only CD45RA + and CD25 + Because CD45RA cells are CD25 + or CD25 high The threshold for determining whether or not a Treg is present is determined based on the CD25 level in the naive Treg population, and high is defined as a CD25 level above that expressed in the naive Treg population. The L-shaped gate is important because it allows the selection of two important populations of cells: CD25 high CD45RA antigen-experienced Tregs and CD25 + CD45RA + Select naive Tregs and CD25 + Exclude CD45RA contaminating cells.

[0300] The histogram-based approach was implemented as an operational alternative for certain manufacturing situations and yielded similar results as the dot plot strategy (Figure 14A-E). Using continuous histogram flow plots, we identified the lowest expression of CD127 (i.e., 40 ± 2% CD127 - / lo ), followed by the highest expression of CD25 (i.e., 70±2% CD25 + ) with CD4 + T cells were selected (Figures 14A-14C). The cell population was further refined by selecting 70±2% of Treg cells in a two-parameter flow plot of CD25 vs. CD127 (Figure 14D). Naive Tregs (CD4 + CD25 + CD127 - / lo CD45RA + ) and antigen-experienced Tregs (CD4 + CD25 high CD127 - / lo CD45RA - ) were identified using the same "not" or L-shaped gate as in the dot plot-based approach (Figure 14E).

[0301] The MS donor used in run 10 provided a second leukopack (run 12), which was processed using a new sorting strategy (histogram + L-shaped gate). With the exception of the FACS sorting step, all other elements of the manufacturing process were as described in Example 1. Table 13 shows the characteristics of the cells after enrichment and before sorting, demonstrating that the cell characteristics were very similar between the two runs before the sorting step. In the context of Table 13 and all tables herein, N / A means that the data was not collected and therefore not available.

[0302] [Table 13]

[0303] Table 14 shows the characteristics of the cells after isolation and before expansion. As expected, the purity of Tregs after sorting remains high with the stringent sorting approach, but the proportion of naive Tregs is reduced by including antigen-experienced Tregs in the sorting strategy compared to the original process described in Example 1. The recovery of naive Tregs was also reduced due to the increased stringency of the gating protocol.

[0304] [Table 14]

[0305] Population doubling levels and % survival are shown in Table 15. The % target dose achieved for the entire process is also shown and was calculated similarly to Example 1, except that for the new selection strategy, both populations are selected, so the target dose calculation was based on the total number of Tregs rather than the total number of naive Tregs.

[0306] [Table 15]

[0307] Figure 15 and Table 16 below show the % TSDR hypomethylation after sorting and over the course of expansion. The L-shaped gate approach achieved an initial TSDR hypomethylation of over 95% (compared to 87.79% with the sorting approach from Example 1). Furthermore, with the L-gate-based approach, TSDR hypomethylation levels decreased by less than 10% over the course of expansion.

[0308] [Table 16]

[0309] New Reproduction Process Because the newly developed gating strategy selects fewer naive Tregs than the original process, the expansion process was modified to increase Treg expansion.

[0310] The expansion protocol was modified to include the addition of TNF-α to the expansion medium and a second anti-CD28 / anti-CD3 stimulation. First, the effect of restimulation was evaluated at various time points after transduction. For this experiment, cells were prepared according to the method described in Example 1, except that cells were gated only on CD4, CD25, and CD127, and not on CD45RA. Cells were then activated and transduced as in Example 1, and a second activation step using anti-CD3 and anti-CD28 was performed on days 6, 8, and 10 after the first activation step. This restimulation on day 6 resulted in similar proliferation as restimulation on days 7 or 8. However, advantageously, restimulation on day 6 of the expansion process was found to result in the greatest increase in proliferation when cells were harvested on day 10, crucially not requiring an extended expansion process (Figure 16). Next, the effect of adding TNF-α to the expansion protocol was evaluated. Cells prepared by the method described in this Example (including the L-shaped gate) were expanded as described in Example 1, with or without a second stimulus on day 6, and with or without 2500 IU / mL of TNF-α, to evaluate the effect of TNF-α on Treg proliferation, alone or in combination with a second stimulus. As shown in Figure 17, the addition of TNF-α throughout the expansion process, combined with a second stimulus with anti-CD3 and anti-CD28 on day 6 of the expansion process, provided the greatest increase in proliferation. However, each individual change to the process also increased proliferation. As shown in Figure 18, cells expanded in TNF-α in combination with a second stimulus exhibited the highest viability and purity at harvest compared to either the process change alone or the original expansion protocol.

[0311] Tregs generated through a novel gating and expansion process The manufacturing process described in this example was then applied to a second replicate MS donor. The MS donor from Run 11 provided an additional leukopack. This leukopack was processed using both dot plot and histogram-based stringent sorting strategies and with improvements to the growth conditions described above. In the CD127 / CD25 selection step (FIG. 19C), minor modifications were made to the histogram-based L-shaped gate approach. The lowest expression of CD127 (i.e., 30±2% CD127 - / lo ), followed by the highest expression of CD25 (i.e., 85±2% CD25 + ) with CD4 + T cells were selected. The cell population was further refined by selecting 85±2% Treg cells in a two-parameter flow plot of CD25 vs. CD127 (FIG. 19D). The L-shaped gate itself was unchanged (FIG. 19E). Prior to the sorting step, cells were treated as in Example 1 (FIGS. 19A-19E). Table 17 shows the characteristics of the cells after enrichment and before sorting, demonstrating that the characteristics of the cells from each run were very similar prior to the sorting step.

[0312] [Table 17]

[0313] The properties of the cells after sorting are shown in Table 18 below.

[0314] [Table 18]

[0315] Table 19 below shows the characteristics of the production cells. The % target dose was calculated as above.

[0316] [Table 19]

[0317] Figure 20 and Table 20 below show TSDR hypomethylation across expansion for this donor using the process described in Example 2 and both the dot plot and histogram-based L-shaped gating strategies described in this example.

[0318] [Table 20]

[0319] The comparative data presented in this example demonstrate that a strict gating strategy combined with an updated expansion process results in a highly stable Treg population and generates sufficient transduced Tregs to achieve the target dose.

[0320] This process was repeated with an additional MS donor and two additional healthy donors, with both L-shaped gating approaches, and similar results were achieved. Table 21 shows the characteristics of the cells after enrichment and before sorting.

[0321] [Table 21]

[0322] The properties of the cells after sorting are shown in Table 22 below.

[0323] [Table 22]

[0324] The characteristics of the production cells are shown below in Table 23. The target dose was calculated as described earlier in this example.

[0325] [Table 23]

[0326] The TSDR status of the cells is shown in Table 24 below and in FIG.

[0327] [Table 24]

[0328] For all donors processed using the selection and expansion strategy described in this example, a post-sorting TSDR hypomethylation status of over 90% was achieved, which did not decrease by more than 10% over the expansion process. This demonstrates that this process generates highly pure stable Treg populations from various donors. In addition, the process was also able to achieve sufficient numbers of transduced Tregs to achieve the target dose.

[0329] Example 4: Evaluation of TSDR threshold levels We investigated the threshold level of TSDR hypomethylation, which represents the level of stable Tregs required for stable Treg cell production. To maintain a stable level of Treg cells throughout the expansion process, we first examined the percentage of stable Tregs on day 0, indicating low or no growth of contaminating conventional T cells or other cellular impurities. To do this, we spiked various percentages of conventional T cells into Tregs isolated from the same healthy donor on day 0 and tracked TSDR levels throughout the expansion process. TSDR hypomethylation levels and FOXP3 + Levels correlated with initial Treg purity and remained stable throughout expansion with a 10% or less decline in TSDR hypomethylation when Treg purity was >60% (Figures 22A-22D). The Treg purity of the expanded cell product was then assessed for the presence of conventional T cells and the production of pro-inflammatory cytokines upon activation with PMA and ionomycin. Cryopreserved cells from harvest were thawed into fresh medium and then stimulated with PMA and ionomycin for 4 hours at 37°C and 5% CO2. Intracellular cytokine (ICC) and IL-2 production in response to IFN-γ were measured by flow cytometry. Figure 23 shows FoxP3 expressing IL-2 and IFN-γ after activation with PMA and ionomycin. + and FoxP3 -Figure 1 shows the percentage of T cells secreting IFN-γ and IL-2. In the absence of conventional T cells, 100% Treg products showed little or no secretion of IFN-γ or IL-2, indicating that only a small number of unstable Treg cells had expanded from the initial culture. As the initial Treg purity decreased, the percentage of T cells secreting IFN-γ and IL-2 cytokines increased, with the greatest increase observed when the starting Treg purity was less than 90%. This increase was likely due to an increased Tcon-to-Treg ratio. Upon activation, some conventional T cells upregulate FoxP3, which suggests that IFN-γ and IL-2 upregulate some FoxP3. + This suggests that high TSDR levels are important for maintaining a stable population of cells that can suppress contaminating conventional T cells.

Claims

1. 1. An isolated population of cells comprising stable CD4+ regulatory T cells (Tregs) derived from a subject with an autoimmune disease, comprising: at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus; An isolated population, wherein the cells comprise an exogenous human T cell receptor (TCR) that specifically binds to a target peptide complexed with a major histocompatibility complex (MHC).

2. 2. The isolated population of claim 1, wherein said stable CD4+ Tregs do not express FOXP3 protein from an engineered FOXP3 locus.

3. 3. The isolated population of claim 1 or 2, wherein the TSDR is the CNS2 region of FOXP3.

4. The isolated population of any one of claims 1 to 3, wherein the MHC is MHC class I or MHC class II.

5. 5. The isolated population of any one of claims 1 to 4, wherein at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

6. At least 80%, at least 85%, at least 90%, or at least 95% of the cells are CD4 ＋ CD25 ＋ CD127 －／ｌｏ 10. The isolated population of any one of the preceding claims, wherein

7. At least 80%, at least 85%, at least 90%, or at least 95% of the cells are CD4 ＋ CD25 ＋ CD127 －／ｌｏ FOXP3 ＋ 10. The isolated population of any one of the preceding claims, wherein

8. At least 4 x 10 ７ 10. The isolated population of any one of the preceding claims, comprising stable CD4+ Tregs.

9. 4 x 10 ７ ~1 x 10 １０ 9. The isolated population of claim 8, comprising stable CD4+ Tregs.

10. 10. The isolated population of any one of the preceding claims, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated population are conventional CD4+ T cells.

11. 11. The isolated population of claim 10, wherein less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the conventional T cells comprise the exogenous human TCR.

12. 10. The isolated population of any one of the preceding claims, wherein the ratio of stable CD4+ Tregs to conventional T cells in said isolated population is at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, at least 500:1, at least 1000:1, or at least 10000:

1.

13. Less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated population are CD8 ＋ 10. The isolated population of any one of the preceding claims, which is a T cell.

14. 14. The isolated population of claim 13, wherein the isolated population does not contain a percentage of CD8+ T cells detectable by fluorescence-activated cell sorting (FACS).

15. 10. The isolated population of any one of the preceding claims, wherein at least 10% of the cells express the exogenous human TCR.

16. 16. The isolated population of claim 15, wherein at least 15%, 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% of the cells express the exogenous human TCR.

17. 10. The isolated population of any one of the preceding claims, wherein the percentage of stable CD4+ Tregs comprising hypomethylated TSDR at the FOXP3 locus is decreased by no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% after a cryopreservation freeze-thaw cycle.

18. the stable CD4+ Tregs a. Regulatory cytokine secretion activity, b. Expression of activation markers associated with regulatory T cells, and / or c. suppressive activity.

19. 10. The isolated population of any one of the preceding claims, wherein the exogenous human TCR comprises one or more amino acid substitutions to cysteine residues in the TCR alpha chain constant region and the TCR beta chain constant region, wherein the cysteine residues are capable of forming one or more disulfide bonds.

20. The isolated population of claim 19, wherein the TCR alpha chain constant region comprises a T48C amino acid substitution relative to the TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 1, and the TCR beta chain constant region comprises a S57C amino acid substitution relative to the TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO:

3.

21. 1. A method for producing a population of cells comprising stable CD4+ regulatory T cells (Tregs), comprising: a. CD8 from a biological sample obtained from a subject with an autoimmune disease ＋ cells, CD19 ＋ cells, and optionally CD14 ＋ removing cells to produce a depleted biological sample; b. Enriching the depleted biological sample for CD25+ cells to produce an enriched population; c. CD4+CD25+CD127 from the enriched population －／ｌｏ isolating the cells; d. Expanding the enriched population to produce an enriched population of expanded cells; and e. quantitating the methylation status of a T cell-specific demethylated region (TSDR) at the FOXP3 locus in the population of cells, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus; thereby producing a population of cells containing stable CD4+ Tregs.

22. 22. The method of claim 21, wherein at least 85%, at least 90%, or at least 95% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus.

23. 23. The method of claim 21 or 22, wherein the isolated population is selected for therapeutic use if the percentage of cells containing hypomethylated TSDR in the FOXP3 locus is 80% or greater.

24. The method of any one of claims 21 to 23, wherein step (c) is performed at least twice.

25. 25. The method of any one of claims 21 to 24, further comprising activating the population of cells of step (c).

26. 26. The method of claim 25, wherein said activating comprises culturing said population of cells with an anti-CD3 antibody and an anti-CD28 antibody.

27. 27. The method of claim 25 or 26, wherein the second activation step is carried out 4 to 8 days after the first activation step.

28. 28. The method of claim 27, wherein the cells are grown in a culture medium containing IL-2 and TNFα.

29. 29. The method of any one of claims 21 to 28, wherein said activating and expanding comprises culturing said population of cells for at least 5, 6, 7, 8, 9, 10, 11, or 12 days.

30. 30. The method of any one of claims 21 to 29, wherein said activating and expanding comprises culturing said population of cells for no more than 15, 14, 13, or 12 days.

31. 1. A method for producing a population of cells comprising engineered stable CD4+ Tregs, comprising: a. CD8 from a biological sample obtained from a subject with an autoimmune disease ＋ cells, CD19 ＋ cells, and optionally CD14 ＋ removing cells to produce a depleted biological sample; b. Enriching the depleted biological sample for CD25+ cells to produce an enriched population; c. CD4+CD25+CD127 from the enriched population －／ｌｏ isolating the cells; d. delivering a vector comprising a nucleic acid encoding an exogenous human T cell receptor (TCR) to the isolated population of (c) to produce a population of engineered cells; e. Expanding the population of engineered cells to produce an expanded population of engineered cells; f. Quantifying the methylation status of a T cell-specific demethylated region (TSDR) at the FOXP3 locus in an expanded population of the engineered cells, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus.

32. 32. The method of claim 31, wherein at least 85%, at least 90%, or at least 95% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus.

33. 33. The method of claim 31 or 32, wherein step (c) is performed at least twice.

34. 34. The method of any one of claims 31 to 33, wherein the isolated population is selected for therapeutic use if the percentage of cells containing hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95% or more.

35. 35. The method of any one of claims 31 to 34, further comprising activating the population of engineered cells of step (c).

36. 36. The method of claim 35, wherein said activating comprises culturing said population of cells with an anti-CD3 antibody and an anti-CD28 antibody.

37. 37. The method of claim 35 or 36, wherein the activation step is performed at least twice.

38. 38. The method of claim 37, wherein the second activation step is performed 4 to 8 days after the first activation step.

39. The method of any one of claims 31 to 38, wherein the cells are grown in a culture medium comprising IL-2 and TNFα.

40. the expanded population is at least 1 x 10 ７ 40. The method of any one of claims 31 to 39, comprising engineered stable CD4+ Tregs.

41. 41. The method of any one of claims 31 to 40, wherein the methylation status of the TSDR in step (f) is assessed at least 24 hours after a cryopreservation freeze-thaw cycle.

42. CD4 ＋ CD25 ＋／ｈｉｇｈ CD127 －／ｌｏ 42. The method of any one of claims 31 to 41, wherein the percentage of regulatory T cells is assessed at least 24 hours after a cryopreservation freeze-thaw cycle.

43. Less than 5%, less than 4%, or less than 3% of the cells in the depleted biological sample are CD8 ＋ cells, CD19 ＋ cells, and / or CD14 ＋ 43. The method of any one of claims 21 to 42, comprising cells.

44. 44. The method of any one of claims 21 to 43, wherein no more than 1% of the population of cells are CD8+ cells.

45. 45. The method of any one of claims 21 to 44, wherein no more than 20% of the CD4+ Treg population are conventional T cells.

46. The population of cells in step (c) is ｈｉｇｈ CD45RA cells and CD25+CD45RA+ cells, ＋ CD45RA － 46. The method of any one of claims 21 to 45, which is cell-free.

47. (c) isolating comprises (i) isolating CD4 ＋ (ii) identifying a first subpopulation of cells; and (iii) isolating CD25 from said first subpopulation. ＋／ｈｉｇｈ CD127 －／ｌｏ (iii) identifying a second subpopulation of cells; and (iii) isolating CD25 from said second subpopulation. ｈｉｇｈ CD45RA and CD25 ＋／ｈｉｇｈ CD45RA ＋ Select cells and CD25 ＋ CD45RA － 47. The method of claims 21-46, comprising excluding a population of CD4+ Tregs, thereby isolating the population of CD4+ Tregs.

48. 48. The method of any one of claims 21 to 47, wherein the TSDR is the CNS2 region of FOXP3.

49. 49. The method of any one of claims 21 to 48, wherein the human subject is a male subject.

50. 49. The method of any one of claims 21 to 48, wherein the human subject is a female subject.

51. 51. The method of any one of claims 31-50, wherein said delivering said vector comprising nucleic acid encoding said exogenous human TCR comprises transducing said cell population with said vector.

52. 52. The method of any one of claims 31 to 51, wherein the exogenous human TCR is encoded as a single polypeptide comprising a TCR alpha chain and a TCR beta chain.

53. 53. The method of claim 52, wherein the polypeptide comprises an N-terminal TCR beta chain and a C-terminal TCR alpha chain.

54. 54. The method of claim 52 or 53, wherein the polypeptide comprises a self-cleaving peptide sequence located between the TCR alpha chain and the TCR beta chain.

55. 55. The method of claim 54, wherein the self-cleaving peptide sequence is a 2A peptide sequence.

56. 56. The method of claim 55, wherein the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence.

57. The method of any one of claims 31 to 56, wherein the vector is a viral vector.

58. 58. The method of claim 57, wherein the viral vector is a lentiviral vector.

59. 59. The method of claim 58, wherein the lentiviral vector is a VSVg-pseudotyped self-inactivating third-generation lentiviral vector.

60. 60. The method of any one of claims 31 to 59, wherein the nucleic acid comprises a promoter operably linked to a coding sequence encoding the exogenous human TCR, optionally wherein the promoter is an EF-1α promoter or an MND promoter.

61. 61. The method of any one of claims 31 to 60, wherein the nucleic acid further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally a WPRE-mut6.

62. 62. The method of claim 60 or 61, wherein the coding sequence is codon-optimized.

63. The vector according to any one of claims 57 to 62.

64. 10. A pharmaceutical composition comprising the isolated population of any one of the preceding claims and a pharmaceutically acceptable excipient.

65. 10. A pharmaceutical composition comprising the isolated population of any one of the preceding claims and a cryopreservative.

66. 65. A method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 64 or the isolated population of any one of claims 1 to 20.

67. 67. The method of claim 66, wherein the pharmaceutical composition is administered in an amount effective to alleviate one or more symptoms of the autoimmune disease.

68. 68. The method of claim 66 or 67, wherein the administering comprises intravenous administration.

69. 69. The method of any one of claims 66-68, wherein said administering comprises one or more injections.

70. 70. The method of any one of claims 66 to 69, wherein the cells of the isolated population are autologous to the subject.

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