Stable regulatory t cells and methods of production thereof

Abstract

The present disclosure relates to isolated populations of cells comprising stable regulatory T cells and methods of producing the stable regulatory T cells. Some aspects provide an isolated population of cells comprising regulatory T cells (also referred to as Tregs) wherein: the regulatory T cells of the isolated population of cells comprise stable regulatory T cells (a) comprising an exogenous human T cell receptor (TCR) (also referred to as TCR-pMHC) that specifically binds to a target peptide complexed with the major histocompatibility complex (MHC), and (b) comprising a hypomethylated regulatory T cell specific demethylation region (TSDR) at the endogenous FOXP3 locus, at least 80% of the cells of the isolated population of cells being CD25 + / high CD4 + CD127- / lo regulatory T cells, and less than 10% of the cells of the isolated population of cells expressing FOXP3 protein from the engineered FOXP3 locus. The target peptide is typically a specific antigen peptide (agonist) in that it triggers intracellular signaling pathways that induce T cell mediated functions such as cytokine secretion and expression of genes required for inhibitory activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 397,704 filed on August 12, 2022, U.S. Provisional Application No. 63 / 412,032 filed on September 30, 2022, and U.S. Provisional Application No. 63 / 417,422 filed on October 19, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] 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 Art

[0005] Regulatory T cells have the potential to treat diseases such as autoimmune diseases because they can target specific diseased cell types. Regulatory T cells can be used, for example, to generate local immune responses specific to certain cell types and tissues associated with the disease of interest through antigen-specific mechanisms. However, robust clinical scale and clinical-grade manufacturing of stable antigen-specific regulatory T cells has been challenging. Therefore, new methods for manufacturing such regulatory T cells are needed. Summary of the invention

[0006] Some aspects provide a cell population comprising regulatory T cells (also referred to as Tregs) separated, wherein: the regulatory T cells of the separated cell population comprise stable regulatory T cells, which (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 low-methylated regulatory T cell-specific demethylation region (TSDR) at an endogenous FOXP3 locus, at least 80% of the cells of the separated cell population are CD25+ / high CD4+CD127- / lo regulatory T cells, and less than 10% of the cells of the separated cell population express FOXP3 proteins from an engineered FOXP3 locus. The target peptide is typically a specific antigenic peptide (agonist) because it triggers intracellular signaling pathways that induce T cell-mediated functions such as cytokine secretion and expression of genes required for suppressive activity.

[0007] In some embodiments, the present disclosure provides an isolated cell population comprising stable CD4+ T regulatory cells (T regs) derived from a subject with an autoimmune disease, wherein at least 80% of the cells are stable CD4+ T regs comprising a hypomethylated T cell-specific demethylation region (TSDR) at the FOXP3 locus, and wherein the cells comprise an exogenous human T cell receptor (TCR) that specifically binds a target peptide complexed with a major histocompatibility complex (MHC).

[0008] In some embodiments, the stable CD4+ T regs do not express FOXP3 protein from an 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.

[0009] 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+ T regs comprising hypomethylated TSDR at the endogenous FOXP3 locus.

[0010] 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+.

[0011] In some embodiments, the population comprises at least 4×107 stable CD4+ T regs. In some embodiments, the population comprises 4×107 to 1×1010 stable CD4+ T regs.

[0012] 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 conventional T cells contain exogenous human TCR. In some embodiments, the ratio of stable CD4+ Treg 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 comprise a detectable percentage of CD8+ T cells by fluorescence activated cell sorting (FACS).

[0013] In some embodiments, at least 10% of the cells express the exogenous human TCR. In 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 the exogenous human TCR.

[0014] In some embodiments, after cryopreservation freeze-thaw cycles, the percentage of stable CD4+ T regs comprising hypomethylated TSDR at the FOXP3 locus decreases by 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0015] In some embodiments, the 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.

[0016] In some embodiments, the exogenous human TCR comprises one or more amino acid substitutions of cysteine ​​residues in the TCR α chain constant region and the TCR β chain constant region, and wherein the cysteine ​​residues are capable of forming one or more disulfide bonds. In some embodiments, relative to a TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises a T48C amino acid substitution, and wherein relative to a TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an S57C amino acid substitution.

[0017] In some embodiments, the present disclosure provides a method for producing a cell population comprising stable CD4+ T regulatory cells (T regs), 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 CD25+ cells in the depleted biological sample to produce an enriched population; (c) isolating CD4+CD25+CD127- / lo cells from the enriched population; (d) expanding the enriched population to produce an expanded enriched cell population; and (e) quantifying the methylation status of the T cell-specific demethylation region (TSDR) at the FOXP3 locus in the cell population, wherein at least 80% of the cells are stable CD4+ T regs comprising a low-methylated TSDR at the FOXP3 locus, thereby producing a cell population comprising stable CD4+ T regs.

[0018] In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4+ T regs comprising hypomethylated TSDR at the FOXP3 locus. In some embodiments, if the percentage of cells comprising hypomethylated TSDR at the FOXP3 locus is 80% or greater, the isolated population is selected for therapeutic use.

[0019] In some embodiments, step (c) is performed at least twice. In some embodiments, the method further comprises activating the cell population of step (c).

[0020] In some embodiments, activation includes culturing the cell colony with anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the second activation step is performed between 4 and 8 days after the first activation step. In some embodiments, the cells are expanded in a culture medium comprising IL-2 and TNFα. In some embodiments, activation and expansion include culturing the cell colony for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days. In some embodiments, activation and expansion include culturing the cell colony for no more than 15 days, 14 days, 13 days or 12 days.

[0021] In some embodiments, the present disclosure provides a method for producing a cell population comprising engineered stable CD4+ T regs, 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 CD25+ cells in the depleted biological sample 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 an engineered cell population; (e) expanding the engineered cell population to produce an expanded engineered cell population; and (f) quantifying the methylation status of a T cell-specific demethylated region (TSDR) at the FOXP3 locus in the expanded engineered cell population, wherein at least 80% of the cells are stable CD4+ T regs comprising a low-methylated TSDR at the FOXP3 locus.

[0022] In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4+ T regs comprising hypomethylated TSDR at the FOXP3 locus. In some embodiments, step (c) is performed at least twice. In some embodiments, if the percentage of cells comprising hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95% or more, the isolated population is selected for therapeutic use.

[0023] In some embodiments, the method also includes the engineered cell colony of activation step (c). In some embodiments, activation includes culturing the cell colony with anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the activation step is performed at least twice. In some embodiments, the second activation step is performed between 4 and 8 days after the first activation step. In some embodiments, the cells are expanded in a culture medium comprising IL-2 and TNF α.

[0024] In some embodiments, the expanded population comprises at least 1×10 7 Engineered stable CD4+ T reg.

[0025] In some embodiments, the methylation status of TSDR in step (f) is assessed at least 24 hours after the cryopreservation freeze-thaw cycle. In some embodiments, the methylation status of CD4 is assessed at least 24 hours after the cryopreservation freeze-thaw cycle. + CD25 + / 高 CD127 - / lo The percentage of regulatory T cells.

[0026] 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 cell population are CD8+ cells. In some embodiments, 20% or less of the CD4+ T reg population are conventional T cells.

[0027] In some embodiments, the cell population in step (c) comprises CD25 高 CD45RA - Cells and CD25 + CD45RA + cells and do not contain CD25 + CD45RA - In some embodiments, the separation of (c) comprises: (i) identifying a first subpopulation of CD4+ cells from the enriched population of step (b); (ii) identifying CD25 + / 高 CD127 - / lo a second subset of cells; and (iii) selecting CD25 高 CD45RA - and CD25 + / 高 CD45RA + Cells were isolated and CD25 + CD45RA - , thereby isolating the CD4+ T reg population.

[0028] 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.

[0029] In some embodiments, delivery of a vector comprising a nucleic acid encoding an exogenous human TCR includes transducing a cell population with the vector. 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 positioned 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.

[0030] 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 pseudotype, a 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, optionally wherein the promoter is an EF-1α 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 WPRE-mut6. In some embodiments, the coding sequence is codon-optimized.

[0031] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding an exogenous human TCR.

[0032] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated population as described herein and a pharmaceutically acceptable excipient. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated population as described herein and a cryopreservative.

[0033] 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 described herein, or a pharmaceutical composition thereof.

[0034] In some embodiments, the pharmaceutical composition is administered in an effective amount to alleviate one or more symptoms of an autoimmune disease. In some embodiments, administration comprises intravenous administration. In some embodiments, administration comprises one or more infusions. In some embodiments, the cells of the isolated colony are autologous relative to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A to Figure 1B Exemplary methods for generating cell populations comprising stable regulatory T cells are provided. Figure 1A shows the separation of CD4 + CD45RA + CD25 + / 高 CD127 lo / - Schematic representation of the overall process of phenotyping regulatory T cells. Figure 1B Data depicting a fluorescence activated cell sorting (FACS) strategy for enriching cells with CD45RA from CD25-enriched samples are shown. + CD4 + CD25 + CD127 lo / - Phenotyping a cell population of cells to generate an isolated cell population containing stable regulatory T cells. Figure 1AAs shown, this sorting strategy was employed in two consecutive steps to achieve a high level of purity.

[0036] Figure 2 Shown is a graph depicting the recovery of regulatory T cells following depletion and CD25 enrichment steps in an exemplary method of generating an isolated cell population comprising stable regulatory T cells.

[0037] FIG. 3A to FIG. 3B Shown are data depicting the relative amounts of various cell phenotypes following depletion and CD25 enrichment steps in an exemplary method of generating an isolated cell population comprising stable regulatory T cells.

[0038] Figure 4 Shown are graphs depicting the growth of cells after transduction with exogenous TCR. Untransduced cells ("untransduced") served as a control.

[0039] Figure 5 Shown are graphs depicting the phenotype and TCR expression of regulatory T cells in a cell population generated by an exemplary method of the present disclosure.

[0040] Figure 6 Data demonstrating that regulatory T cells in a cell population generated by an exemplary method of the present disclosure maintain hypomethylated T cell-specific demethylated regions (TSDRs) are shown.

[0041] Figure 7 Shown are graphs depicting activation of lentiviral-transduced regulatory T cells of the present disclosure following cryopreservation freeze-thaw cycles.

[0042] Figure 8 Shown for CD4 + / CD45RA + CD127 - / 低 CD25 + Diagram of the gating strategy for purification of cell populations.

[0043] Fig. 9 Shown by Figure 8 The gating strategy depicted in Figure 5 shows the percentage of TSDR hypomethylation in different runs of purification.

[0044] FIG. 10A to FIG. 10D Shown are the expression of CTLA-4 ( Fig. 10A )、CD69( Fig. 10B )、TGFβ-1( Fig. 10C ) and IL-10( Fig. 10D ) expression.

[0045] Fig.11Shown is the change in TSDR hypomethylation percentage over time for amplification of three different MS donors.

[0046] FIG. 12A to FIG. 12C CD45RA is shown during the eight-day expansion period + and CD45RA - The population doubling level of cells ( Fig. 12A and Fig. 12B ) and TSDR hypomethylation percentage. + CD45RA - Expansion within a population.

[0047] FIG. 13A to FIG. 13C Shown for CD25 + CD127 - / lo CD45 - and CD25 + / 高 CD127 -lo CD45 - Select the gating strategy.

[0048] FIG. 14A to FIG. 14E Shows FIG. 13A to FIG. 13C The histogram method shown in .

[0049] Fig.15 Shown are the percentages of TSDR hypomethylation during amplification of the same donor based on different sorting strategies.

[0050] Fig.16 Expansion of cells after restimulation on day 6, day 8, or day 10 is shown.

[0051] Fig.17 The effect of fold amplification of TNF-α, second stimulation, or both is shown.

[0052] FIG. 18A to FIG. 18B Viability and purity of cells expanded by both TNF-α and secondary stimulation are shown.

[0053] FIG. 19A to FIG. 19E Shown for FIG. 13A to FIG. 13C An alternative histogram method to the gating strategy shown in .

[0054] Fig. 20 Shown are the percentages of TSDR hypomethylation during amplification of the same donor based on different sorting strategies.

[0055] Fig.21 Shown based on FIG. 13A to FIG. 13C and FIG. 19A to FIG. 19E TSDR hypomethylation percentages of three different donors using the gating strategy depicted in Figure 5.

[0056] FIG. 22A to FIG. 22D The percentage of TSDR hypomethylation and FOXP3 according to the proportion of Tregs in the initial population based on two experiments are shown. + Cell percentage.

[0057] Fig.23 FOXP3 expressing IL-2 and IFN-γ after activation with PMA and ionomycin is shown + and FOXP3 - proportion. DETAILED DESCRIPTION

[0058] Overview

[0059] The present disclosure provides methods and compositions related to treating autoimmune diseases by producing and utilizing stable regulatory T cells. The present disclosure also provides methods and compositions related to treating autoimmune diseases by producing and utilizing engineered stable regulatory T cells comprising antigen-specific T cell receptors (TCR). This engineered regulatory T cell can specifically target discrete cell types and tissues associated with autoimmune diseases, so as 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 at the target and do not cause systemic immunosuppression. In addition, the engineered regulatory T cells described herein are stable (e.g., directed to Treg phenotype) thymus-derived regulatory T cells, which can exist in vivo for a long time and can provide months or years of therapeutic benefits after a single dose. In some embodiments, these stable regulatory T cells are also resistant to proinflammatory triggers (e.g., proinflammatory cytokines). In some embodiments, the stable regulatory T cells of the present disclosure are autologous cells, which means that they are obtained directly from the patient's own cells, engineered to express foreign antigen-specific TCRs, and then administered back into the patient. The use of autologous cells minimizes the risk of patient rejection (e.g., graft-versus-host disease).

[0060] Isolating highly stable and pure engineered regulatory T cell populations produced in the thymus and obtaining sufficiently large and pure engineered regulatory T cell populations to treat patients has proven to be challenging, both because stable thymic regulatory T cell populations are very small relative to the total population of lymphocytes and because there are no cell surface markers associated only with regulatory T cells. Although isolated polyclonal regulatory T cells have been shown to be effective in clinical therapy, when regulatory T cells are engineered with exogenous TCRs, higher levels of purity and stability are required because conventional T cells and unstable regulatory T cells (i.e., peripheral Tregs) engineered with exogenous TCRs, if present in sufficient numbers, may have deleterious effects by causing rather than suppressing immune responses at sites of autoimmune disease.

[0061] These stable engineered regulatory T cells and production methods provide advantages over several existing methods of producing regulatory T cells for cell therapy.Alternative methods have been used to generate populations of engineered regulatory T cells or regulatory-like T cells, but these cells have disadvantages relative to the cell populations described herein.

[0062] One possible approach is to increase the proportion of regulatory-like T cells by engineering conventional T cells or induced stem cells (by engineering the FOXP3 promoter or introducing an ectopic FOXP3 gene into cells). FOXP3 expression confers a regulatory-like phenotype. However, FOXP3 is only one of several genes that are upregulated in stable regulatory T cells, and studies have shown that these regulatory-like T cells are different from natural Tregs. In fact, when CD4+ T cells were edited to display a Treg-like phenotype by introducing an exogenous promoter upstream of FOXP3, engineered cells were shown to have reduced suppressive capacity relative to natural Tregs (Buckner, Science Translational Medicine, 2022), suggesting that when administered in a clinical setting, these cells will not exhibit the same level of efficacy as true stable regulatory T cells.

[0063] The conversion of conventional CD4+ T cells has also been attempted, for example by stimulation with TGF-β and IL-2, stimulation with retinoic acid, short-chain fatty acids and TGF-β, or stimulation with rapamycin. However, these induced regulatory T cells lack epigenomic changes associated with stable regulatory T cells, especially regulatory T cell-specific demethylation at the conserved noncoding sequence 2 (CNS2) of the FOXP3 gene, and are therefore functionally unstable, and will not retain a stable phenotype when applied in a clinical setting. In addition, proteomic analysis shows that these induced Treg-like cells have very little overlap with stable thymic Tregs in protein expression profiles, and on the contrary, share signaling and metabolic proteins with conventional T cells (Mensink et al., Sci Rep. 2022).

[0064] Contemplated herein is a cell population and a strategy for isolating the cell population, which cell population comprises highly pure and stable thymic regulatory T cells engineered with exogenous TCRs, which are of sufficient purity and sufficient number to be administered to a subject in therapeutic amounts. In some embodiments, after isolation and before introduction of the exogenous TCRs, the optimal population of regulatory T cells is CD25 + / 高 CD4 + CD127 - / lo CD45RA + , because this phenotype is associated with thymic naive regulatory T cells. In some embodiments, after isolation and before the introduction of exogenous TCRs, the optimal population of regulatory T cells also comprises CD25 高 CD4 + CD127 - / lo CD45RA - , as this phenotype is associated with thymic antigen-experienced regulatory T cells. These cells also displayed a stable TSDR phenotype after introduction of exogenous TCRs, indicating that there was no overgrowth of non-regulatory T cell subsets or loss of regulatory T cell stability (e.g., cell fate was not altered – cells remained terminally differentiated).

[0065] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter in which they are individually cited, which in some cases may be incorporated in their entirety.

[0066] The indefinite articles "a" and "an" as used in the specification and claims should be understood to mean "at least one" unless explicitly stated to the contrary.

[0067] It should also 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 unless explicitly stated to the contrary.

[0068] In the claims and the foregoing specification, all transitional phrases such as "includes," "comprising," "carrying," "having," "containing," "involving," "holding," "consisting of," etc. shall be understood as open-ended, i.e., meaning including but not limited to. As provided in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0069] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0070] Where a range of values ​​is provided, every value between and including the upper and lower ends of that range is specifically contemplated and described herein.

[0071] Regulatory T cells

[0072] In some embodiments, the present disclosure provides isolated cell populations and compositions thereof, which contain stable CD4+ T regulatory cells derived from subjects with autoimmune diseases. These isolated populations can be used, for example, to treat autoimmune diseases in subjects in need thereof.

[0073] 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 and the expression of the transcription factor FOXP3. Further, regulatory T cells do not express or express the cell surface marker CD127 at low levels. Thus, regulatory T cells are characterized by the following protein expression profiles: CD4 + CD25 高 / + CD127 lo / - FOXP3 + .

[0074] Throughout this disclosure, expression of a indicated protein by a cell or cell population may include reference to various expression indicators, such as "+" (e.g., CD4 + ), “-” (e.g., CD127 - ), “high” (e.g., CD25 高), "int", or "medium" (e.g., CD25 int ), “low” (e.g., CD127 低 ), "high / +" (e.g., CD25 高 / + ) or “low / -” (e.g., CD127 低 / - ). In this article, various expression indicators refer to the presence or absence of the indicated protein (e.g., "+" or "-", respectively) or the relative level of protein expression 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.

[0075] (a) Positive ( + ) indicators refer to the expression levels of the indicated proteins detectable by FACS. + ) are further divided into "low" and / or "high" subpopulations.

[0076] (b) A "low" cell subpopulation expresses the indicated protein, but its expression level is lower than the expression level of other cells in the population (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the expression level of other cells in the population) or lower than the expression level in a control cell population (e.g., relative to CD4+ conventional T cells or CD8+ effector T cells).

[0077] (c) A "high" cell subpopulation expresses the indicated protein at a level that is higher than the expression level of other cells in the population (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than the expression level of other cells in the population) or higher than the expression level in a control cell population (e.g., relative to CD4+ conventional T cells or CD8+ effector T cells).

[0078] (d) Negative - ) indicator means that expression of the indicated protein is absent, or that the expression level of the indicated protein is below the detection limit of a particular detection assay (e.g., below the detection limit of a particular fluorescent antibody and / or flow cytometer).

[0079] (e) Indicator “ - / 低 ” refers to a cell population comprising cells that are (-) for the indicated protein and cells that express “low” levels of the indicated protein.

[0080] (f) Indicator “ + / 高 ” refers to a cell population comprising cells that are (+) for the indicated protein, including cells expressing high levels of the indicated protein.

[0081] 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+. + / 高 In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) are CD4+. In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) are CD45RA+. In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) are FOXP3+.

[0082] In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) are CD25 + / 高 CD4 + CD127 - / 低 That is, the regulatory T cells express or express high levels of CD25, express CD4, and do not express or express low levels of CD127. In some embodiments, the regulatory T cells do not express CD127. In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) are CD25 + / 高 CD4 + CD127 - / 低 CD45RA + Cells. Therefore, regulatory T cells express or express high levels of CD25, express CD4 and CD45RA, and do not express or express low levels of CD127. In some embodiments, regulatory T cells express FOXP3. In some embodiments, regulatory T cells are CD25+ / high CD4+CD127- / loFOXP3+ cells. Therefore, regulatory T cells express or express high levels of CD25, express CD4 and FOXP3, and do not express or express low levels of CD127. In some embodiments, regulatory T cells are CD25+ / high CD4+CD45RA+CD127- / loFOXP3+ cells. Therefore, regulatory T cells express or express high levels of CD25, express CD4, CD45RA and FOXP3, and do not express or express low levels of CD127.

[0083] In the development of thymic regulatory T cells, the genomic organizer SATB1 (special AT-rich sequence binding protein) binds to specific genomic sites from the CD4+CD8+ thymocyte stage to open chromatin and activate super enhancers associated with many regulatory T cell signature genes, such as FOXP3, IL2RA, (CD25), CTLA4, IKZF2 (HELIOS) and IFZF4 (EOS). The enzymes SATB1 and MLL4 (myeloid / lymphoid or mixed lineage leukemia 4) involved in enhancer initiation usually occupy the newly identified conservative enhancer region at the FOXP3 locus, named as conservative non-coding sequence 0 (CNS0), and then activate the enhancer at CNS3 and CNS2, and then activate the promoter. This leads to stable hypomethylation and expression of FOXP3 and other regulatory T cell-related genes, thereby leading to a stable regulatory T cell phenotype (Piotrowska et al.; Int J Mol Sci.2021).

[0084] After hematopoietic development, T cell populations are able to switch between one phenotype to another under various conditions (see, e.g., Kitagawa et al., Nat Immunol. 2017 February; 18 (2): 173–183). As used herein, "stable regulatory T cells" are regulatory T cells comprising a low methylated T cell-specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, stable regulatory T cells are also defined by the expression of CD4, CD25 and / or FOXP3. Tregs derived from the thymus (e.g., thymic T regs) are stable, and regulatory T cells that are no longer able to switch between T cell phenotypes due to changes in the methylation state of one or more loci in the FOXP3 locus are also considered stable. In contrast, unstable and peripheral Tregs that can be induced in response to a proinflammatory state do not exhibit stable hypomethylation of the TSDR region of the FOXP3 locus. Hypomethylation of TSDR, an evolutionarily conserved CpG-rich regulatory element of the FOXP3 gene, is associated with the expression of FOXP3. Hypomethylation of the TSDR of the endogenous FOXP3 locus occurs when the methyl group from one or more methylated cytosines in the TSDR has been removed to replace the methylated cytosine with cytosine. Therefore, the stability of T regulatory cells is determined by the presence of a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus.

[0085] In some embodiments, the methylation status of the TSDR of the FOXP3 locus is measured as described in the following documents: Kressler et al. "Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Regulatory TPhenotype" Frontiers in Immunology, January 7, 2021; or Schreiber et al. "The Regulatory T-Specific Demethylated Region Stabilizes Foxp3 Expression Independently of NF-κB Signaling" PLOS One, February 5, 2014. In some embodiments, the TSDR of the FOXP3 locus is selected from conserved noncoding sequence 0 (CNS0), CNS3 and CNS2. In some embodiments, the TSDR of the FOXP3 locus is CNS2.

[0086] In some embodiments, stable regulatory T cells maintain low methylation regulatory T cell-specific demethylation regions (TSDRs) at endogenous FOXP3 loci in the presence of proinflammatory conditions (e.g., in the presence of one or more proinflammatory cytokines). In some embodiments, stable regulatory T cells comprise low methylation regulatory T cell-specific demethylation regions (TSDRs) at endogenous FOXP3 loci in the presence of proinflammatory conditions for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days.

[0087] In some embodiments, the stable regulatory T cells also 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 stable regulatory T cells to suppress the activation and / or proliferation of other effector cells of the immune system).

[0088] Thus, in some embodiments, the isolated cell populations provided herein comprise stable T regulatory cells that comprise hypomethylated TSDR of the FOXP3 locus, expression of CD4, CD25 and FOXP3, and / or exhibit cytokine secretion, activation and / or suppression activity.

[0089] In some embodiments, the stable regulatory T cells of the isolated cell population provided herein maintain the low methylation TSDR at the FOXP3 locus over time after separation from the biological sample. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain the low methylation TSDR at the FOXP3 locus after separation from the biological sample, and continue for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days. In some embodiments, the stable regulatory T cells maintain the low methylation TSDR at the FOXP3 locus after separation from the biological sample, and continue for more than 5 days, more than 10 days, more than 15 days or more than 20 days. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 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 isolation from a biological sample.

[0090] In some embodiments, the stable regulatory T cells of the cell colonies separated provided herein maintain the hypomethylated TSDR at the FOXP3 locus over time after transduction with nucleic acid encoding exogenous TCR. For example, in some embodiments, the stable regulatory T cells of the cell colonies separated maintain the hypomethylated TSDR at the FOXP3 locus after transduction with nucleic acid encoding exogenous TCR, and continue for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days. In some embodiments, the stable regulatory T cells maintain the hypomethylated TSDR at the FOXP3 locus after transduction with nucleic acid encoding exogenous TCR, and continue for more than 5 days, more than 10 days, more than 15 days or more than 20 days. In some embodiments, 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 transduction with a nucleic acid encoding an exogenous TCR.

[0091] In some embodiments, the stable regulatory T cells of the cell colonies of the separation provided herein maintain the hypomethylated TSDR at the FOXP3 locus over time after cryopreservation. For example, in some embodiments, the cell colonies of the separation described herein are cryopreserved, thawed afterwards for use and / or analysis, which is referred to herein as cryopreservation freeze-thaw cycles. In such embodiments, the stable regulatory T cells of the cell colonies of the separation maintain the hypomethylated TSDR at the FOXP3 locus after the cryopreservation freeze-thaw cycles, for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days. In some embodiments, the stable regulatory T cells maintain the hypomethylated TSDR at the FOXP3 locus after the cryopreservation freeze-thaw cycles, for more than 5 days, more than 10 days, more than 15 days or more than 20 days. 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 cryopreservation freeze-thaw cycles.

[0092] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain the low methylation TSDR at the FOXP3 locus over time after being applied to a subject. For example, in some embodiments, the stable regulatory T cells of the isolated cell populations maintain the low methylation TSDR at the FOXP3 locus after being applied to a subject, for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days. In some embodiments, the stable regulatory T cells maintain the low methylation TSDR at the FOXP3 locus after being applied to a subject, for more than 5 days, more than 10 days, more than 15 days or more than 20 days. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 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 administration to a subject.

[0093] 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 subsequently administered. For example, a cell population can be obtained from a subject, subjected to the methods described herein, and then administered to the same subject (the cell population was initially obtained from the subject) to treat an autoimmune disease. In such embodiments, the cell population administered to the subject comprises autologous regulatory T cells.

[0094] In some embodiments, the isolated population described herein is an allogeneic cell population. In this context, the term allogeneic refers to 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 methods described herein, and then administered to another subject in order to treat an autoimmune disease.

[0095] In some embodiments, the cell colonies of separation described herein (e.g., a colony of separation comprising stable CD4+ T regulatory cells) are separated from biological samples obtained from subjects diagnosed with or suspected of having an autoimmune disease. The autoimmune disease can 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, regulatory T cells are obtained from subjects 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, regulatory T cells are obtained from subjects diagnosed with or suspected of having type 1 diabetes. In other embodiments, the autoimmune disease is inclusion body myositis. In some embodiments, regulatory T cells are obtained from subjects diagnosed with or suspected of having inclusion body myositis.

[0096] Engineered regulatory T cells

[0097] In some embodiments, the disclosure provides a kind of cell colony of separation, it comprises regulatory T cells (for example, stable regulatory T cells), and these regulatory T cells comprise exogenous human T cell receptor (TCR) that binds target peptide.When target peptide is compounded with major histocompatibility complex (MHC) (for example, MHC class I or MHC class II), this combination occurs.Such colony is referred to as "engineered regulatory T cells" or "stable engineered regulatory T cells" in this article.

[0098] The T cell receptor (TCR) is a transmembrane heterodimer comprising an alpha chain and a beta chain connected by a disulfide bond. Within these chains is a complementary determining region (CDR), which determines the target peptide that the TCR will bind to. The TCR activates the T cell in which it is located, resulting in excessive immune responses. Antigen presenting cells digest certain proteins (antigens) and display their fragments (peptides) on the major histocompatibility complex (MHC). This peptide-MHC (pMHC) complex binds to the TCR, and other costimulatory molecules are activated, resulting in T cell activation, proliferation, differentiation, apoptosis or cytokine release.

[0099] An exogenous TCR can be any TCR introduced into a regulatory T cell, wherein the TCR is not endogenous to the regulatory T cell (i.e., naturally present 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 present in the genome of the regulatory T cell). In some embodiments, the nucleic acid is an engineered nucleic acid, such as a recombinant or synthetic nucleic acid.

[0100] TCR specifically binds to a target peptide compounded with MHC. If TCR has a higher binding affinity to a target peptide compounded with MHC relative to a non-target peptide compounded with MHC, it is considered that TCR "specifically" binds to a target peptide compounded with MHC. TCR can bind to a target peptide compounded with MHC with a binding affinity of at least 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M or 10-10M (e.g., 10-4M to 10-10M). In some embodiments, if the T cell expressing TCR becomes activated (e.g., as assessed by increased CD69 expression) when contacting a target peptide compounded with MHC, or becomes more highly activated relative to a non-target peptide compounded with MHC, it is considered that TCR "specifically" binds to a target peptide compounded with MHC. In some embodiments, the peptide is presented by a cell expressing MHC.

[0101] The exogenous TCR can be a human TCR. In some embodiments, the TCR is a TCR from a monkey, a mouse, a rat, or any other animal.

[0102] The target peptide can 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, hemorrhagic nephritis 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), such as MBP (83-99). A target peptide associated with type 1 diabetes may be a peptide belonging to glutamate decarboxylase (e.g., GAD65), such as GAD65 (555-567) or GAD65 (339-352), or proinsulin. In some embodiments, the target peptide is a peptide overexpressed in a cell population associated with an autoimmune disease relative to a control (e.g., relative to a cell population not associated with an autoimmune disease).

[0103] In some embodiments, the target peptide undergoes an increased autoimmune response in a subject with an autoimmune disease relative to a control. In some embodiments, the target peptide is a peptide present at the site of the autoimmune disease within the subject relative to an unaffected site within the same subject. In some embodiments, the target peptide is specifically presented by an MHC allele associated with the presence of an autoimmune disease in the subject.

[0104] In some embodiments, the target peptide is a peptide overexpressed in the cells of a subject with an autoimmune disease relative to a control (e.g., relative to a healthy subject). If the expression of the target peptide in a cell (e.g., a cell associated 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 cell relative to a control cell (e.g., a cell population not associated with an autoimmune disease), then the target peptide is considered to be overexpressed in the cell. In some embodiments, if the expression of the target peptide in the cells of a 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 a subject with an autoimmune disease relative to a healthy subject (e.g., a subject not suffering from an autoimmune disease), then the target peptide is overexpressed in the cells of a subject with an autoimmune disease.

[0105] In some embodiments, the target peptide is a peptide that is highly expressed in cells at a disease site in a subject with an autoimmune disease relative to a control (e.g., expression of the target peptide in unaffected / non-disease sites of the subject). A target peptide is considered to be highly expressed in cells at a disease site if its expression in cells at a disease site (e.g., cells associated 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 cell relative to a control cell (e.g., a cell not at a disease site).

[0106] In some embodiments, the target peptide is a peptide complexed with an MHC having an HLA haplotype associated with an autoimmune disease.

[0107] 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.

[0108] TCR may include a joint domain positioned between the α chain and the β chain. In some embodiments, the joint domain includes a self-cleaving peptide sequence (for example, a self-cleaving peptide sequence positioned between the α chain and the β chain). The self-cleaving peptide sequence is a peptide sequence that uses a non-classical mechanism to induce a polypeptide to separate into two peptides. In some embodiments, the self-cleaving peptide sequence can induce ribosome jumping during polypeptide translation. In some embodiments, the length of the self-cleaving peptide sequence is 10-30, 10-25, 15-30, 15-25 or 18-22 amino acids. In some embodiments, the self-cleaving peptide sequence can be a 2A peptide sequence. The 2A peptide sequence can include, for example, a 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), an E2A (derived from equine rhinitis A virus), an F2A (derived from foot-and-mouth disease virus) or a T2A (derived from Thosea asigna virus 2A) peptide sequence. The T2A peptide sequence may comprise, for example, an amino acid sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 5). The P2A peptide sequence may comprise, for example, an amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 6). The E2A peptide sequence may comprise, for example, an amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 7). The F2A peptide sequence may comprise, for example, an amino acid sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 8).

[0109] 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 α chain and a second polypeptide comprising a β chain.

[0110] In some embodiments, the exogenous TCR comprises one or more cysteine ​​residues present in the α chain of the TCR, which are capable of forming 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 constant region of the α chain of the TCR, which are capable of forming one or more disulfide bonds with one or more cysteine ​​residues in the constant region of the β chain of the TCR.

[0111] In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 48 to introduce cysteine ​​(e.g., T48C). In some embodiments, relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 57 to introduce cysteine ​​(e.g., S57C) amino acid substitution. In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 48 to introduce cysteine ​​(e.g., T48C), and relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 57 to introduce cysteine ​​(e.g., S57C) amino acid substitution, wherein the cysteine ​​residue at position 48 of the α chain is capable of forming a disulfide bond with the cysteine ​​residue at position 57 of the β chain.

[0112] In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 45 to introduce cysteine ​​(e.g., T45C). In some embodiments, relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 77 to introduce cysteine ​​(e.g., S77C) amino acid substitution. In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 45 to introduce cysteine ​​(e.g., T45C), and relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 77 to introduce cysteine ​​(e.g., S77C) amino acid substitution, wherein the cysteine ​​residue at position 45 of the α chain is capable of forming a disulfide bond with the cysteine ​​residue at position 77 of the β chain.

[0113] In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 10 to introduce cysteine ​​(e.g., Y10C). In some embodiments, relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 17 to introduce cysteine ​​(e.g., S17C) amino acid substitution. In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 10 to introduce cysteine ​​(e.g., Y10C), and relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 17 to introduce cysteine ​​(e.g., S17C) amino acid substitution, wherein the cysteine ​​residue at position 10 of the α chain is capable of forming a disulfide bond with the cysteine ​​residue at position 17 of the β chain.

[0114] In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 45 to introduce cysteine ​​(e.g., T45C). In some embodiments, relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 59 to introduce cysteine ​​(e.g., D59C) amino acid substitution. In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 45 to introduce cysteine ​​(e.g., T45C), and relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 59 to introduce cysteine ​​(e.g., D59C) amino acid substitution, wherein the cysteine ​​residue at position 45 of the α chain is capable of forming a disulfide bond with the cysteine ​​residue at position 59 of the β chain.

[0115] In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 15 to introduce cysteine ​​(e.g., S15C). In some embodiments, relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 15 to introduce cysteine ​​(e.g., E15C) amino acid substitution. In some embodiments, relative to the TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 1, the TCR α chain constant region comprises an amino acid substitution at position 15 to introduce cysteine ​​(e.g., S15C), and relative to the TCR β chain constant region comprising the amino acid sequence of SEQ ID NO: 3, the TCR β chain constant region comprises an amino acid substitution at position 15 to introduce cysteine ​​(e.g., E15C) amino acid substitution, wherein the cysteine ​​residue at position 15 of the α chain is capable of forming a disulfide bond with the cysteine ​​residue at position 15 of the β chain.

[0116]

[0117]

[0118] Exemplary exogenous human TCRs that can be introduced into the isolated cell populations described herein are known in the art. For example, TCRs known to target MS-associated antigens (such as MBP or MOG) include, but are 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-associated 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).

[0119] Nucleic acid encoding TCR

[0120] In some embodiments, the present disclosure provides nucleic acids encoding TCRs (e.g., exogenous TCRs). Nucleic acids may be or may 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.

[0121] Nucleic acid used herein is generally engineered nucleic acid. Engineered nucleic acid is a polynucleotide not present in nature (e.g., at least two nucleotides covalently linked together, and in some cases, containing a phosphodiester bond, referred to as a phosphodiester backbone). Engineered nucleic acid includes recombinant nucleic acid and synthetic nucleic acid. Recombinant nucleic acid is a molecule constructed by connecting nucleic acids (e.g., isolated nucleic acid, synthetic nucleic acid, or a combination thereof) from two different organisms (e.g., humans and mice). Synthetic nucleic acid is a molecule amplified or chemically synthesized or otherwise synthesized. Synthetic nucleic acid includes nucleic acids that are chemically modified or otherwise modified but can be base paired (bound) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules produced by replication of any of the above.

[0122] The engineered nucleic acids of the present disclosure can be generated using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, GIBSON is used. The nucleic acids were generated by cloning (see, eg, Gibson, DG et al. Nature Methods, 343-345, 2009; and Gibson, DG et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). Three enzyme activities are typically used in a single-tube reaction: 5′ exonuclease, 3′ extension activity of DNA polymerase, and DNA ligase activity. The 5′ exonuclease activity engulfs the 5′ end sequence and exposes the complementary sequence for annealing. The polymerase activity then fills the gap on the annealing domain. DNA ligase then closes the nick and covalently links the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in GoldenGate Assembly, and therefore result in a higher percentage of correct assembly. MegaGate molecular cloning can also be used. MegaGate is a toxin-free Gateway technology that eliminates the ccdb toxin used in Gateway recombinase cloning, and instead utilizes large-range nuclease-mediated digestion to eliminate background vectors during cloning (see, e.g., Kramme C. et al. STAR Protoc. October 22, 2021; 2(4): 100907, which is incorporated herein by reference. According to the present disclosure, other methods for producing engineered polynucleotides can be used.

[0123] In some embodiments, the present disclosure provides an expression cassette comprising an open reading frame, which comprises a nucleic acid encoding an exogenous TCR operably connected to a promoter. A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds to initiate transcription. A promoter is generally located directly upstream of the transcription start site (at the 5' end). 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 connected. In some embodiments, the promoter is an inducible promoter. An inducible promoter can be regulated in vivo by, for example, chemicals, temperature, or light.

[0124] An open reading frame is a stretch 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, such as a protein. An open reading frame is operably linked to a promoter if the promoter regulates the transcription of the open reading frame.

[0125] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding an exogenous TCR or an expression vector comprising the nucleic acid. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. For example, the vector can be a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a retroviral vector or a baculovirus vector. The viral vector provides efficient delivery of exogenous TCR to the regulatory T cells of the present disclosure. Exemplary viral vectors can be derived from lentiviruses; retroviruses (e.g., Retroviridae viral vectors); adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48); parvoviruses (e.g., adeno-associated viruses); coronaviruses; negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza virus); rhabdoviruses (e.g., rabies and vesicular stomatitis virus); paramyxoviruses (e.g., measles and Sendai virus); positive-strand RNA viruses, such as picornaviruses and alphaviruses; and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus, replication-defective herpes virus) 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 virus, D virus, oncorretrovirus, HTLV-BLV group, alpha retrovirus, gamma retrovirus, foamy virus, 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 ape leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus.

[0126] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector of the present disclosure comprises lentiviral gag, pol and rev genes and two long terminal repeats (LTRs) located on both sides of the expression cassette comprising a nucleic acid encoding an exogenous TCR. For safety reasons, the vector will not include any other active lentiviral genes, such as vpr, vif, vpu, nef, tat. In some embodiments, these genes have been removed or otherwise inactivated.

[0127] 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 greatly reduced or completely eliminated. This feature greatly minimizes the risk of replication-competent recombinants (RCRs). In addition, it reduces the risk that cell coding sequences located near the vector integration site will be abnormally expressed. In addition, the SIN design reduces the possibility of interference between the LTR and the promoter driving transgene expression.

[0128] 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 the vector RNA). Thus, during reverse transcription, this deletion is transferred to the 5'LTR of the proviral DNA. However, the elements in the LTR involved in the polyadenylation of the viral RNA are not modified. Together, this reduces or eliminates the production of full-length vector RNA in the transduced cells.

[0129] 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.

[0130] In some embodiments, the nucleic acid is codon optimized. Codon optimization methods are known in the art. In some embodiments, codon optimization can be used to match the codon frequencies in the target 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 construction or expression; customize transcription and translation control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove or tidy up protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust translation rate to allow each domain of the protein to fold properly; or reduce or eliminate problematic secondary structures in polynucleotides.

[0131] In some embodiments, the nucleic acid encoding TCR comprises a promoter operably connected to the coding sequence encoding exogenous human TCR. The promoter can be a viral promoter or a natural TCR 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 1α (EF-1α) promoter and a MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution) (see, e.g., Gill, DR. et al. Gene Ther. 2001; 8: 1539–46 and Astrakhan, A. et al. Blood 2012; 119: 4395–4407).

[0132] 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 post-transcriptional regulatory element (oPRE), a woodchuck hepatitis virus post-transcriptional 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 Vol. 16, pp. 605–619 (2009).

[0133] Isolated cell populations

[0134] In some embodiments, the present disclosure provides isolated cell populations and compositions thereof, which contain stable CD4+ T regulatory cells derived from subjects with autoimmune diseases. In some embodiments, the present disclosure provides isolated cell populations and compositions thereof, which contain stable CD4+ T regulatory cells derived from subjects with autoimmune diseases, which are engineered to express exogenous human TCRs. In some embodiments, at least 80% of the cells are stable CD4+ T regs containing a low-methylated T cell-specific demethylation region (TSDR) at the FOXP3 locus.

[0135] The methylation state of TSDR at the FOXP3 locus can be evaluated by methods known in the art. For example, in some embodiments, the methylation state of TSDR at the FOXP3 locus is evaluated by bisulfite treatment and digital droplet PCR (ddPCR) using methylation-specific primers and probes. In some embodiments, the methylation state of TSDR at the FOXP3 locus is evaluated by a single cell sequencing method.

[0136] The cell colony of separation is a cell colony taken out from the human body, or a cell colony taken out from a sample obtained from the human body. Therefore, it is considered to be "separated" from the human body. Cell colonies can be separated from a subject (e.g., obtained from a subject), or, for example, using any known cell collection method such as apheresis, a cell colony is separated from a biological sample obtained from a subject. The cell colony of the separation of the present disclosure can be subjected to the methods described herein to produce a cell colony of separation, relative to a cell colony directly obtained from a subject or, for example, obtained from a biological sample obtained from a subject by apheresis, the cell colony of the separation comprises a higher number of regulatory T cells (e.g., stable regulatory T cells).

[0137] In some embodiments, the isolated cell population comprises stable CD4+ T regulatory cells, wherein at least 80% of the cells are stable CD4+ T regs comprising a low methylated 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+ T regs comprising a low methylated TSDR at the FOXP3 locus. In some embodiments, the isolated cell population comprises stable CD4+ T regulatory 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+ T regs comprising hypomethylated TSDR at the FOXP3 locus. In some embodiments, between 80% and 90%, between 85% and 95%, between 80% and 85%, between 85% and 90%, between 90% and 95% of the cells are stable CD4+ T regs comprising 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 comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

[0138] In some embodiments, the isolated population comprises CD25 + / 高 CD4 + CD127 - / 低 In some embodiments, the isolated cell population comprises CD25 + / 高 CD4+ CD127 - / 低 FOXP3 + In some embodiments, the isolated cell population comprises CD25 + / 高 CD4 + CD127 - / lo FOXP3 + CD45RA + Regulatory T cells.

[0139] 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. + / 高 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 + / 高 CD4 + CD127 - / lo FOXP3 + Regulatory T cells.

[0140] 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 comprises 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. + / 高 CD4 + CD127 - / lo CD45RA + Regulatory T cells.

[0141] As described herein, the present disclosure provides a cell colony comprising a separation of stable T regulatory cells, which comprises a hypomethylated TSDR at the FOXP3 locus. Although in some embodiments, the isolated colony described herein is engineered to express exogenous TCR, the FOXP3 locus remains unmodified. Therefore, the cells of the isolated colony described herein do not include an engineered FOXP3 locus. "Engineering FOXP3 locus" refers to any engineered modification (e.g., by artificial modification) intended to change the expression of FOXP3. Such engineered modifications include, but are not limited to, introducing a FOXP3 transgenic, introducing a modified promoter and / or using an exogenous agent (e.g., a gene editing system, a small molecule or a peptide) intended to activate the expression of FOXP3.

[0142] In some embodiments, the isolated cell population comprises at least 1×10 2 At least 1×10 3 At least 1×10 4 At least 1×10 5 At least 1×10 6 At least 1×10 7 At least 1×10 8 At least 1×10 9 or at least 1×10 10 In some embodiments, the isolated cell population comprises 1×10 2 Up to 1×10 10 pcs, 1×10 3 Up to 1×10 10 pcs, 1×10 4 Up to 1×10 10 pcs, 1×10 5 Up to 1×10 10 pcs, 1×10 6 Up to 1×10 10 pcs, 1×10 7 Up to 1×10 10 pcs, 1×10 8 Up to 1×10 10 pcs, 1×10 5 Up to 1×10 9 pcs, 1×10 6 Up to 1×10 8 pcs, 1×10 7 Up to 1×10 10 or 1×10 4 Up to 1×10 6 In some embodiments, the isolated cell population comprises 1×10 6 Up to 1×10 10In some embodiments, the isolated cell population comprises 1×10 7 pcs, 2x10 7 pcs, 3×10 7 pcs, 4×10 7 pcs, 5x10 7 pcs, 6×10 7 pcs, 7×10 7 pcs, 8x10 7 or 9×10 7 In some embodiments, the isolated cell population comprises 1×10 7 Up to 1×10 10 pcs, 2×10 7 Up to 1×10 10 pcs, 3×10 7 Up to 1×10 10 pcs, 4×10 7 Up to 1×10 10 pcs, 5×10 7 Up to 1×10 10 pcs, 6×10 7 Up to 1×10 10 pcs, 7×10 7 Up to 1×10 10 8×10 7 Up to 1×10 10 or 9×10 7 Up to 1×10 10 Stable regulatory T cells.

[0143] In some embodiments, the isolated cell population comprises stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus, wherein the stable T regulatory cells retain markers of stability (i.e., maintaining hypomethylated TSDR at the FOXP3 locus and / or maintaining CD25 + / 高 CD4 + CD127 - / lo FOXP3 +Protein expression profile). Proinflammatory conditions refer to cells and factors known to drive inflammatory immune responses, and may include proinflammatory cytokines (e.g., IL-17, IL-22, IL-21, IFNγ, IL-12, TNFα, IL-1β, IL-6, IL-1, GM-CSF, and other cells known in the art), immune effector cells (e.g., conventional CD4+ T cells, CD8+ effector T cells, granulocytes, etc.) and other proinflammatory mediators (e.g., prostaglandins, thrombin, histamine, and matrix proteases). Proinflammatory conditions may be in vitro or in vivo. In some embodiments, the stable regulatory T cells in the isolated cell population maintain low methylation TSDR at the endogenous FOXP3 locus in the presence of proinflammatory conditions for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days.

[0144] In some embodiments, the isolated cell population comprises stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus, wherein the stable T regulatory cells retain markers of stability over time (i.e., maintaining hypomethylated TSDR at the FOXP3 locus and / or maintaining CD25 + / 高 CD4 + CD127 - / lo FOXP3 +For example, in some embodiments, the stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after isolation from a biological sample for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after activation and / or expansion for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after transduction with an exogenous TCR for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after cryopreservation freeze-thaw cycles for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days.

[0145] In some embodiments, stable T regulatory cells maintain CD25 ex vivo after isolation from a biological sample. + / 高 CD4 + CD127 - / lo FOXP3 + for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, stable T regulatory cells maintain CD25 ex vivo after activation and / or expansion. +高 CD4 + CD127 - / lo FOXP3 + for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, stable T regulatory cells maintain CD25 ex vivo after transduction with exogenous TCRs. +高 CD4 + CD127 - / lo FOXP3 +for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the stable T regulatory cells maintain CD25 ex vivo after cryopreservation freeze-thaw cycles. +高 CD4 + CD127 - / lo FOXP3 + of a protein expression profile for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days.

[0146] In some embodiments, at least 50% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 5 days. In some embodiments, at least 50% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 10 days. In some embodiments, at least 50% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 15 days. In some embodiments, at least 60% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 5 days. In some embodiments, at least 60% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 10 days. In some embodiments, at least 60% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 15 days. In some embodiments, at least 70% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 5 days. In some embodiments, at least 70% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 10 days. In some embodiments, at least 70% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 15 days. In some embodiments, at least 80% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 5 days. In some embodiments, at least 80% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 10 days. In some embodiments, at least 80% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus after separation for at least 15 days. In some embodiments, at least 90% of the cells of the isolated cell population comprise 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 hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 90% of the cells of the isolated cell population comprise hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation.

[0147] In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed at multiple (e.g., 2 or more) time points during the separation and production process. In some embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% between these 2 or more time points.

[0148] For example, in some embodiments, hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after isolation from the biological sample, and is assessed again within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus decreases by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, 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% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus decreases by no 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 T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample.

[0149] In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after separation from the biological sample, and is reassessed within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or amplification. In some embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or amplification. In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after separation from the biological sample, and is reassessed within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or amplification. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, 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% within 5, 6, 7, 8, 9, 10 or 11 days after activation and / or expansion. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no 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 T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion.

[0150] In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after separation from the biological sample, and is reassessed within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after separation from the biological sample, and is reassessed within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, 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% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no 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 T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR.

[0151] In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after separation from the biological sample, and is reassessed within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In some embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after separation from the biological sample, and is reassessed within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, 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% within 5, 6, 7, 8, 9, 10 or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 10% within 5, 6, 7, 8, 9, 10 or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0152] In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after transduction with an exogenous human TCR, and reassessed within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the hypomethylation of TSDR at the FOXP3 locus is assessed within 1 to 3 days after separation from a biological sample, and reassessed within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, 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% within 5, 6, 7, 8, 9, 10 or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells comprising hypomethylated TSDR at the FOXP3 locus is reduced by no more than 10% within 5, 6, 7, 8, 9, 10 or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0153] In some embodiments, after the stable regulatory T cells are transduced with an exogenous human TCR, the percentage of cells in the isolated cell population containing stable regulatory T cells is reduced by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percentage point during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion. In some embodiments, after the stable regulatory T cells are transduced with an exogenous human TCR, the percentage of cells in the isolated cell population containing stable regulatory T cells is increased by 1, 2, 3, 4, or 5 percentage points during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion. In some embodiments, after the stable regulatory T cells are transduced with an exogenous human TCR, the percentage of cells in the isolated cell population containing stable regulatory T cells is reduced by no more than 10 percentage points during at least 5 days of expansion. In some embodiments, after transducing stable regulatory T cells with exogenous human TCRs, during at least 10 days of expansion, the cell percentage of the isolated cell population containing stable regulatory T cells is reduced by no more than 10 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCRs, during at least 15 days of expansion, the cell percentage of the isolated cell population containing stable regulatory T cells is reduced by no more than 10 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCRs, during at least 5 days of expansion, the cell percentage of the isolated cell population containing stable regulatory T cells is reduced by no more than 5 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCRs, during at least 10 days of expansion, the cell percentage of the isolated cell population containing stable regulatory T cells is reduced by no more than 5 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCRs, during at least 15 days of expansion, the cell percentage of the isolated cell population containing stable regulatory T cells is reduced by no more than 5 percentage points. In some embodiments, after the stable regulatory T cells are transduced with an exogenous human TCR, the percentage of cells in the isolated cell population containing stable regulatory T cells decreases by no more than 1 percentage point during at least 5 days of expansion. In some embodiments, after the stable regulatory T cells are transduced with an exogenous human TCR, the percentage of cells in the isolated cell population containing stable regulatory T cells decreases by no more than 1 percentage point during at least 10 days of expansion. In some embodiments, after the stable regulatory T cells are transduced with an exogenous human TCR, the percentage of cells in the isolated cell population containing stable regulatory T cells decreases by no more than 1 percentage point during at least 15 days of expansion.

[0154] The percentage of stable regulatory T cells relative to total cells in the cell colony comprising the separation of regulatory T cells can be assessed in about 1 hour, about 6 hours, about 12, about 24 hours, about 36, about 48, about 72 hours, about 96 hours or about 120 hours after transducing cells with exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in the cell colony comprising the separation of regulatory T cells can be assessed in 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, 13 or 14 days after transducing cells with exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in the cell colony comprising the separation of regulatory T cells can be assessed in about 1-21 days, 1-7 days, 4-14 days, 4-7 days, 7-10 days, 7-14 days, 10-21 days or 14-21 days after transducing cells with exogenous human TCR.

[0155] 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 colony express exogenous TCR (for example, after the isolated cell colony is transduced with exogenous TCR). In some embodiments, 10%-60% or 20%-50% of the regulatory T cells of the isolated cell colony express 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 colony express exogenous TCR (for example, after the isolated cell colony is transduced with exogenous TCR). In some embodiments, 10%-60% or 20%-50% of the stable regulatory T cells of the isolated cell population express the exogenous TCR.

[0156] In some embodiments, the TSDR at the endogenous FOXP3 locus of the 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 the stable regulatory T cells of the isolated cell population of regulatory T cells remains hypomethylated after cryopreservation freeze-thaw cycles.

[0157] In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) of the separated cell colonies exhibit one or more cell functions, which are associated with regulatory T cells when activated by binding to pMHC. Non-limiting examples of such cell functions include cytokine secretion activity, expression and inhibitory activity of certain activation markers. Cytokine secretion activity refers only to the secretion of certain anti-inflammatory cytokines (such as IL-10, TGFβ and IL-35). Activation markers include but are not limited to CD69, 4-1BB, CD25, CD71 or CTLA-4. For example, when regulatory T cells contact a target peptide compounded with MHC, they express one or more of these markers.

[0158] Suppressive activity refers to the inhibition of activation, proliferation and cytokine production of non-regulatory T cells (e.g., CD8+ T cells and CD4+ conventional T cells), in part to inhibit the immune system from becoming overactive. When contacted with a target peptide complexed with MHC that is bound to an exogenous TCR expressed by a regulatory T cell, the regulatory T cell exhibits suppressive activity. In some embodiments, the target peptide is presented by a cell expressing MHC. For example, in some embodiments, regulatory T cells inhibit activation, proliferation and cytokine production of conventional T cells that are specific to a shared target peptide complexed with MHC (e.g., a shared target peptide presented by an antigen presenting cell (APC)). In some embodiments, relative to a control (e.g., a non-regulatory T cell in the absence of a regulatory T cell), regulatory T cells inhibit 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%.

[0159] In some embodiments, relative to a control (e.g., conventional T cells in the absence of regulatory T cells), regulatory T cells inhibit IFN-γ production of conventional T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. In some embodiments, relative to a control, regulatory T cells inhibit IFN-γ production of conventional T cells by 50%-99%, 75%-99%, or 80%-100% (e.g., when present in a population of regulatory T cells comprising a 1:1 to 1:8 ratio compared to conventional T cells). In some embodiments, relative to a control (e.g., conventional T cells in the absence of regulatory T cells), regulatory T cells inhibit CD71 production of conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%. In some embodiments, the regulatory T cells inhibit CD71 production by conventional T cells by 20%-90% or 30%-80% relative to the control (e.g., when present in a population of regulatory T cells comprising a 1:1 to 1:8 ratio compared to conventional T cells). In some embodiments, the regulatory T cells inhibit CD25 production by conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% relative to the control (e.g., conventional T cells without regulatory T cells). In some embodiments, the regulatory T cells inhibit CD25 production by conventional T cells by 40%-70% relative to the control (e.g., when present in a population of regulatory T cells comprising a 1:1 to 1:8 ratio compared to conventional T cells).

[0160] In some embodiments, when contacted with a target peptide (e.g., a target peptide presented by an APC) in combination with an exogenous TCR expressed by a regulatory T cell and compounded with MHC, the regulatory T cell exhibits cytokine secretion activity (e.g., secretion of IL-10). In some embodiments, when contacted with a target peptide compounded with MHC in combination with an exogenous TCR expressed by a regulatory T cell, the regulatory T cell exhibits the expression of activation markers. For example, in some embodiments, when contacted with a target peptide compounded with MHC in combination with an exogenous TCR expressed by a regulatory T cell, the regulatory T cell exhibits the expression of CD69, 4-1BB, CD25, CD71, and / or CTLA-4. In some embodiments, when contacted with a target peptide compounded with MHC in combination with an exogenous TCR expressed by a regulatory T cell, the regulatory T cell exhibits inhibitory activity. For example, in some embodiments, regulatory T cells inhibit the activation of conventional T cells that are specific to a shared target peptide compounded with MHC.

[0161] The cellular function of stable regulatory T cells can be assessed at about 1 hour, about 6 hours, about 12, about 24 hours, about 36, about 48, about 72 hours, about 96 hours, or about 120 hours after cells are transduced with exogenous human TCRs. The cellular function of stable regulatory T cells can be assessed at 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, 13, or 14 days after cells are transduced with exogenous human TCRs. The cellular function of stable regulatory T cells can be assessed at about 1-21 days, 1-7 days, 4-14 days, 4-7 days, 7-10 days, 7-14 days, 10-21 days, or 14-21 days after cells are transduced with exogenous human TCRs.

[0162] In some embodiments, transduced regulatory T cells (e.g., transduced regulatory T cells of a separated colony) retain their cell functionality (e.g., ability to be activated) after cryopreservation freeze-thaw cycles. In some embodiments, transduced regulatory T cells can be activated and / or amplified after cryopreservation freeze-thaw cycles. In some embodiments, regulatory T cells can exhibit cytokine secretion activity, expression of certain activation markers, and / or inhibitory activity after cryopreservation freeze-thaw cycles.

[0163] 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 cell population is CD25 + / 高 CD4 + CD127 - / lo .

[0164] The cell colony comprising the separation of stable regulatory T cells may include a small amount 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 cell colony comprising the separation of 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 cell colony comprising the separation of 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 of the cell colonies of the separation of regulatory T cells are non-regulatory T cells comprising 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 of the cell colonies of the separation of 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 of the cell colonies of the separation of 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 of the cell population separated containing regulatory T cells are conventional T cells containing exogenous human TCR. In some embodiments, the cell population separated containing regulatory T cells comprises undetectable amounts of conventional T cells. In some embodiments, the cell population separated containing regulatory T cells comprises undetectable amounts of conventional CD4+ T cells. In some embodiments, the cell population separated containing regulatory T cells comprises undetectable amounts of B cells. In some embodiments, the cell population separated containing regulatory T cells comprises undetectable amounts of NK T cells. In some embodiments, the cell population separated containing regulatory T cells comprises undetectable amounts of CD14+ cells. In some embodiments, the cell population separated containing regulatory T cells comprises undetectable amounts 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 number of CD8+ T cells.

[0165] Conventional T cells or other non-regulatory T cells can be assessed for the percentage of total cells (or relative to stable regulatory T cells) at about 1 hour, about 6 hours, about 12, about 24 hours, about 36, about 48, about 72 hours, about 96 hours or about 120 hours after transducing cells with exogenous human TCR. Conventional T cells or other non-regulatory T cells can be assessed for the percentage of total cells (or relative to stable regulatory T cells) at 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, 13 or 14 days after transducing cells with exogenous human TCR. Conventional T cells or other non-regulatory T cells can be assessed for the percentage of total cells (or relative to stable regulatory T cells) at about 1-21 days, 1-7 days, 4-14 days, 4-7 days, 7-10 days, 7-14 days, 10-21 days or 14-21 days after transducing cells with 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) can be assessed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after the cryopreservation freeze-thaw cycle.

[0166] In some embodiments, in an isolated cell population comprising regulatory T cells, the ratio of regulatory T cells to conventional 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.

[0167] Methods for generating stable regulatory T cells

[0168] In some embodiments, the present disclosure provides a method for producing a cell colony of separation, and these separated cell colonies include stable CD4+ T regulatory cells derived from a subject suffering from an autoimmune disease. The conventional method for producing antigen-specific regulatory T cells cannot produce clinical scale and clinical-grade manufacturing of a cell colony of stable antigen-specific regulatory T cells comprising a high concentration. Although polyclonal T cells have been successfully produced and applied in a clinical setting, the use of stable thymic regulatory T cells engineered with exogenous TCRs can cause increased safety risks, which requires increased cell purity and regulatory T cell stability. It is not desirable to be bound by theory, stable thymic regulatory T cells account for a small part of leukocytes, and if the regulatory T cell colony of separation is not highly purified, other types of leukocytes will be engineered with exogenous TCRs. If the number is large enough, these engineered non-regulatory T cells can have a harmful effect by causing rather than suppressing immune responses at the site of autoimmune disease. By integrating and modifying several biomarker selection methods, including an unexpectedly effective dual selection method using CD25 and in some cases CD45RA, the methods designed herein provide the art with tools to generate antigen-specific regulatory T cell populations with high yields and high relative concentrations of stable TCR-transduced regulatory T cells.

[0169] In some embodiments, the methods of the present disclosure involve removing conventional T cells and selecting for CD25 + / 高 CD4 + CD127 - / lo In some embodiments, the methods of the present disclosure involve removing conventional T cells, selecting cells with CD25 + / 高 CD4 + CD127 - / lo In some embodiments, the method of generating 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.

[0170] Figure 1A-1B An example method of the present disclosure is provided in Figure 1AIn the embodiments provided in , 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 (targeting non-CD4 conventional T cells), anti-CD19 (targeting B cells), and anti-CD14 (targeting monocytes) antibodies; 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., CD25-PE-biotin antibody) and a secondary molecule (e.g., anti-biotin microbeads) to produce a cell population enriched in CD25. In some embodiments, a CD25-PE-biotin antibody is used. The CD25-PE-biotin antibody comprises an anti-CD25 antibody attached to a tandem conjugate of phycoerythrin (PE) and biotin. In some embodiments, this enrichment step may be repeated more than once (e.g., twice or more). Then, as Figure 1A and Figure 1B As described, these CD25-enriched cultures are further processed by sorting the cells using a TCR gating strategy using fluorescence activated cell sorting (FACS) or Tyto sorting (Miltenyi). Cells are first labeled with anti-CD45RA, anti-CD4, and anti-CD127 antibodies. Cells are then selected in multiple sorting steps. In each step, in some embodiments, the labeled CD25 is first identified by the gating strategy. + / 高 CD4 + CD127 - / lo The gating strategy involves first identifying singlets, then live cells, then CD4+ cells, and then CD25 + / 高 CD127 - / 低 cells, and then identify CD25 高 CD45RA and CD25 + / 高 CD45RA + Collect identified cell populations (e.g., containing CD4 + CD25 高 CD127 - / 低 CD45RA - Protein expression profiles of cells and CD4 + CD25 + CD127 - / 低 CD45RA + In some embodiments, the sorting procedure is performed twice to optimize the purity of the cells.

[0171] In some embodiments, the present disclosure provides a method for producing an isolated population comprising stable CD4+ T regulatory cells, wherein the method comprises (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 from the enriched population. - / lo In some embodiments, the present disclosure provides a method for producing an isolated population comprising stable CD4+ T regulatory cells, wherein the method comprises (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 CD25+ cells in the depleted biological sample to produce an enriched population; (c) isolating CD4+CD25+CD127 from the enriched population. - / lo cells; and; and (d) engineering the cell population 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.

[0172] In some embodiments, the method further comprises the methylation state of the T cell-specific demethylation region (TSDR) at the FOXP3 locus in the quantitative cell population, wherein at least 80% of the cells are stable CD4+ T regs comprising low methylated TSDR at the FOXP3 locus. In some embodiments, the methylation state of the TSDR is quantified after the separation step of (c). In some embodiments, the methylation state of the TSDR is quantified after the separation step of (d).

[0173] In some embodiments, CD4+CD25+CD127 is isolated from the enriched population. - / lo The steps of cells include: (i) identifying CD4 + (ii) identifying CD25 from the first subpopulation + / 高 CD127 - / lo a second subset of cells; and (iii) selecting CD25 高 CD45RA - and CD25 + / 高 CD45RA + Cells were isolated and CD25 + CD45RA - , thereby isolating a stable CD4+ T reg population.

[0174] In some embodiments, the method provided herein also includes separating biological samples comprising regulatory T cells from human subjects suffering from autoimmune diseases. In some embodiments, sample separation is performed using a single sampling technique (e.g., a leukocyte single sampling technique for separating leukocytes). In some embodiments, separating biological samples comprising regulatory T cells from human subjects suffering from autoimmune diseases is performed by taking out blood from the subject and separating the blood into plasma and cells. In some embodiments, single sampling techniques involve taking out whole blood from the subject and separating whole blood to remove desired cell types (e.g., T cells). In some embodiments, the separation step of single sampling is performed using continuous flow centrifugation or intermittent flow centrifugation.

[0175] Cells expressing specific biomarkers (e.g., CD8+ cells, CD19+ cells, CD14+ cells, and / or CD56+ cells) can 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 ordinary skill in the art to remove cells expressing specific biomarkers. In some embodiments, cells expressing specific biomarkers can be removed from a sample by labeling those 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 specific biomarkers).

[0176] A depleted biological sample is a biological sample (e.g., isolated from a human subject with an autoimmune disease) that has been treated 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 generates a depleted biological sample in which less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, or less than 3% of the total cells contain 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 generates a depleted biological sample in which less than 0.5% of the total cells contain CD8+ cells.

[0177] Cell selection techniques can be used to select cells with a specific phenotype (e.g., cells expressing specific biomarkers such as CD4, CD25, or CD45RA; or cells that do not express or low express specific biomarkers such as CD127). Cell selection techniques can be FACS techniques, Tyto devices, antibody pull-down assay techniques, magnetic cell separation techniques, or any other method known to those of ordinary skill in the art for selecting cells expressing specific biomarkers. In some embodiments, cell selection techniques include labeling cells expressing a specific biomarker with a biotinylated antibody targeting a specific biomarker (e.g., CD25), followed by a pull-down assay (e.g., with anti-biotin microbeads or streptavidin complexes). In some embodiments, cell selection techniques include labeling cells expressing a specific biomarker with an antibody targeting a specific biomarker (e.g., CD25, CD4, and / or CD45RA). FACS can then be used to select cells labeled with antibodies targeting a specific biomarker from a sample (e.g., a population rich in CD25).

[0178] The CD25 enrichment step is followed by a multi-step sorting process to select for CD25 + / 高 CD4 + CD127 - / lo Regulatory T cells. In some embodiments, the sorting step is performed on a Tyto device (Miltenyi Biopharmaceuticals). Those skilled in the art will appreciate that software for visualizing the sorted cell populations enables data to be displayed in a variety of ways, including dot plots and histograms. When a population is identified based on the presence or absence of a single protein, a dot plot or histogram can be used for identification. When a population needs to be identified based on the presence or absence of two proteins (e.g., CD25 and CD45RA), a dot plot should be used for identification.

[0179] In some embodiments, before the first sorting step, the CD25-enriched cell population is separated into multiple cell populations, and the first sorting step is performed simultaneously and / or sequentially in multiple subpopulations. This provides for accelerated processing of the large number of cells that need to be processed in order to isolate a sufficient number of small proportions of CD25 + / 高 CD4 + CD127 - / lo In some embodiments, the cell population enriched in CD25 is separated into 2, 3, 4, 5, or 6 or more subpopulations, each of which is sorted in the first sorting step. In some embodiments, after the first sorting step, the sorted subpopulations are combined before the second sorting step.

[0180] In some embodiments, the present disclosure provides a method for producing an isolated population comprising stable CD4+ T regulatory cells, wherein the method comprises (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+ T regulatory cells from the enriched population. + CD25 + CD127 - / lo cells, wherein the separation comprises (i) identifying CD4 + (ii) identifying CD25 from the first subpopulation + / 高 CD127 - / lo a second subset of cells; and (iii) selecting CD25 高 CD45RA - and CD25 + / 高 CD45RA + Cells were isolated and CD25 + CD45RA - , thereby isolating a stable CD4+ T reg population.

[0181] In some embodiments, CD25 is selected from a population enriched for CD25. + / 高 CD4 + CD127 - / lo The method includes identifying a first subset of CD4+ cells from a population enriched in CD25. Then, CD25 + / 高 CD127 - / lo Finally, CD25 高 CD45RA - and CD25 + / 高 CD45RA + Isolate cell populations.

[0182] 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.

[0183] 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% of CD25. + / 高 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% of CD25 + / 高 CD4 + CD127 - / lo FoxP3 + Regulatory T cells.

[0184] In some embodiments, the population selected for isolation comprises CD25 高 CD4 + CD127 - / 低 CD45RA - (CD25 高 CD45RA - ) and CD25 + / 高 CD4 + CD127 - / 低 CD45RA + (CD25 + / 高 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 高 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 + / 高 CD45RA + It should be understood that the final population of CD25 高 CD45RA - and CD25 + / 高 CD45RA + The percentage of cells totals to equal about 80%, 90%, or 100% of the total cell population.

[0185] In some embodiments, the population selected for isolation comprises less than 10% CD25 + CD4 + CD127 - / 低 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 - / 低 CD45RA cells.

[0186] In some embodiments, the methods described herein produce an isolated cell population, wherein at least 80% of the cells are stable CD4+ T regs comprising a low methylated 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+ T regs comprising a low methylated TSDR at the FOXP3 locus. In some embodiments, the isolated cell population comprises stable CD4+ T regulatory 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+ T regs comprising hypomethylated TSDR at the FOXP3 locus. In some embodiments, between 80% and 90%, between 85% and 95%, between 80% and 85%, between 85% and 90%, between 90% and 95% of the cells are stable CD4+ T regs comprising hypomethylated TSDR at the FOXP3 locus.

[0187] In some embodiments, if at least 80% of the cells are stable CD4+ T regs comprising a low methylated T cell-specific demethylated region (TSDR) at the FOXP3 locus, the isolated cell population produced by the methods described herein is selected for therapeutic use. In some embodiments, 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+ T regs comprising a low methylated TSDR at the FOXP3 locus, the isolated cell population is selected for therapeutic use. In some embodiments, 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+ T regs comprising hypomethylated TSDR at the FOXP3 locus, the isolated cell population is selected for therapeutic use. In some embodiments, if between 80% and 90%, between 85% and 95%, between 80% and 85%, between 85% and 90%, between 90% and 95% of the cells are stable CD4+ T regs comprising hypomethylated TSDR at the FOXP3 locus, the isolated cell population is selected for therapeutic use.

[0188] In some embodiments, the method provided herein also includes introducing an exogenous TCR specifically binding to a target peptide compounded with MHC into a cell colony comprising stable CD4+ T regulatory cells. In some embodiments, introducing exogenous TCR includes transfecting a cell colony with a nucleic acid encoding an exogenous TCR. In some embodiments, introducing exogenous TCR includes transducing a cell colony with a nucleic acid encoding an exogenous TCR. The nucleic acid encoding an exogenous TCR can be an RNA (e.g., mRNA) molecule or a DNA molecule. In some embodiments, nucleic acid is a vector or plasmid. In some embodiments, a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a retroviral vector or a baculovirus vector is used to deliver nucleic acid to a cell colony.

[0189] In some embodiments, the method or process of producing a cell colony of regulatory T cells also includes activation and / or amplification of the regulatory T cells of the colony (e.g., after deletion, enrichment, and engineering steps described herein). In some embodiments, regulatory T cells are activated before transfection or transduction of regulatory T cells with exogenous TCR. In some embodiments, regulatory T cells are activated with anti-CD28 and / or anti-CD8 antibodies. In some embodiments, activation of regulatory T cells includes contacting regulatory T cells with antigens that specifically bind to exogenous TCRs belonging to separated cell colonies. In some embodiments, antibodies (e.g., anti-CD3, anti-CD28, and / or TCR-specific antibodies) are conjugated or compounded with beads (e.g., magnetic beads or polymer beads). In some embodiments, antibodies are covalently attached to polymer matrices.

[0190] In some embodiments, the methods provided herein further comprise a second activation and / or amplification step. In some embodiments, the second activation and / or amplification step is performed 5 days, 6 days or 7 days after the initial activation and / or amplification step.

[0191] In some embodiments, activation and / or amplification of regulatory T cells include culturing a cell colony separated in a cell culture medium. In some embodiments, the culture medium includes IL-2. In some embodiments, the culture medium includes about 100IU / ml, 200IU / ml, 300IU / ml, 400IU / ml, 500IU / ml, 600IU / ml, 700IU / ml, 800IU / ml, 900IU / ml, 1,000IU / ml, 1200IU / ml, 1400IU / ml, 1600IU / ml, 1800IU / ml, 2000IU / ml, 3000IU / ml or 4000IU / ml IL-2. In some embodiments, the culture medium includes 500-1500IU / ml or 800-1200IU / ml IL-2. In some embodiments, the culture medium includes about 1,000IU / ml IL-2. In some embodiments, the culture medium comprises 1,000 IU / ml IL-2. In some embodiments, the culture medium further comprises TNFα. In some embodiments, the culture medium comprises 500 IU / ml, 750 IU / ml, 1000 IU / ml, 1500 IU / ml, 1750 IU / ml, 2000 IU / ml, 2250 IU / ml, 2500 IU / ml or about 3000 IU / ml TNFα. In some embodiments, the culture medium comprises 2000-3000 IU / ml or 2250-2750 IU / ml TNFα. In some embodiments, the culture medium comprises about 2500 IU / ml TNFα. In some embodiments, the culture medium comprises 2500 IU / ml TNFα.

[0192] In some embodiments, activating and expanding regulatory T cells comprises culturing the cells of the population for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, activating and expanding regulatory T cells comprises culturing the cells of the population for 2-14 days, 2-10 days, 2-5 days, 5-10 days, or 5-14 days. In some embodiments, activating and expanding regulatory T cells comprises culturing the cells of the population for no more than 15 days, 14 days, 13 days, or 12 days. In some embodiments, after activation and / or expansion, the isolated cell population comprises at least 1×10 2 At least 1×10 3 At least 1×10 4 At least 1×10 5 At least 1×10 6 At least 1×10 7 At least 1×10 8 At least 1×10 9 or at least 1×10 10 Stable CD25 + / 高 CD4 + CD127 - / lo Regulatory T cells, these cells contain hypomethylated TSDR at the endogenous FOXP3 locus.

[0193] In some embodiments, the method or process of producing a cell population of regulatory T cells may further include cryopreserving the cell population. In some embodiments, the method or process of producing a cell population of regulatory T cells may further include thawing the cryopreserved cell population. A cryopreservation freeze-thaw cycle refers to a process of cryopreserving a cell population and thawing the cryopreserved cell population at a later time.

[0194] Pharmaceutical composition

[0195] In some embodiments, the disclosure provides a pharmaceutical composition comprising a cell colony of an isolated regulatory T cell (e.g., a stable regulatory T cell) as described herein. In some embodiments, the pharmaceutical composition comprises a cell colony of a regulatory T cell (e.g., a stable regulatory T cell) as described herein and a pharmaceutically acceptable excipient.

[0196] 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 suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens.

[0197] Pharmaceutical composition should be sterile and stable under manufacturing and storage conditions usually.Nontoxic parenteral acceptable diluent or solvent can be used to prepare sterile injectable formulations.The pharmaceutical composition used according to the present invention can include pharmaceutically acceptable dispersants, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts or stabilizers, which are nontoxic to the subject at the dosage and concentration used.In some embodiments, the pharmaceutical composition can include organic solvents, such as but not limited to methyl acetate, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), dimethoxyethane (DME) and dimethylacetamide, including their mixture or combination.

[0198] The pharmaceutical composition may include an effective amount of stable regulatory T cells that are sufficient to induce a desired biological response. For example, in some embodiments, an effective amount of stable regulatory T cells as described herein may refer to a cell quantity sufficient to improve the symptoms associated with an autoimmune disease (e.g., progressive multiple sclerosis). As will be appreciated by those skilled in the art, the effective amount of a solution or preparation provided herein may vary according to various factors, e.g., a desired biological response, e.g., a particular disease treated, a particular symptom to be alleviated, a cell or tissue targeted, and age, sex, and general health of the subject.

[0199] In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising hypomethylated TSDR at the endogenous FOXP3 locus) comprises at least 1×10 2 At least 1×10 3 At least 1×10 4 At least 1×10 5 At least 1×10 6 At least 1×10 7 At least 1×10 8 At least 1×10 9 or at least 1×10 10 In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising hypomethylated TSDR at the endogenous FOXP3 locus) is 1×10 2 Up to 1×10 10 pcs, 1×10 3 Up to 1×10 10 pcs, 1×10 4 Up to 1×10 10 pcs, 1×10 5 Up to 1×10 10 pcs, 1×10 6 Up to 1×10 10 pcs, 1×107 Up to 1×10 10 pcs, 1×10 8 Up to 1×10 10 pcs, 1×10 9 Up to 1×10 10 In some embodiments, the effective amount of stable regulatory T cells comprises 1×10 7 pcs, 2x10 7 pcs, 3×10 7 pcs, 4×10 7 pcs, 5x10 7 pcs, 6×10 7 pcs, 7×10 7 pcs, 8x10 7 or 9×10 7 In some embodiments, the effective amount of stable regulatory T cells comprises 1×10 7 Up to 1×10 10 pcs, 2×10 7 Up to 1×10 10 pcs, 3×10 7 Up to 1×10 10 pcs, 4×10 7 Up to 1×10 10 pcs, 5×10 7 Up to 1×10 10 pcs, 6×10 7 Up to 1×10 10 pcs, 7×10 7 Up to 1×10 10 8×10 7 Up to 1×10 10 or 9×10 7 Up to 1×10 10 Stable regulatory T cells.

[0200] In some embodiments, the isolated cell population (e.g., intended for use in a pharmaceutical composition) is cryopreserved (e.g., subjected to one or more cryopreservation freeze-thaw cycles). That is, the isolated cell population produced herein can be combined with a cryoprotectant that lowers the melting temperature by forming a chemical bond with water and increasing the total concentration of solutes in the system. Non-limiting examples of cryoprotectants include glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, and propylene glycol. Although traditional methods that typically include the use of serum and DMSO can be used, the present disclosure also contemplates the use of cryopreservation medium manufactured under cGMP conditions and formulated as serum-free and non-animal-derived (e.g., using <10% DMSO in the freezing mixture). Other cryopreservation techniques are also provided herein, including more advanced cooling techniques, for example, vitrification of cells without the use of cryoprotectants. See, for example, Shinshu University. "A new way to'freeze'cells promises to transform the common cell-freezing practice." ScienceDaily.com April 2019. This ultra-fast cooling process utilizes inkjet cell printing to cool at a rate of 10,000 degrees Celsius per second, bringing the cells close to vitrification.

[0201] Treatment

[0202] In some embodiments, the present disclosure provides methods for treating autoimmune diseases in subjects in need, including administering cell colonies or compositions thereof comprising stable CD4+ T regulatory cells. In some embodiments, autoimmune diseases are selected from multiple sclerosis (e.g., progressive multiple sclerosis), type 1 diabetes, and inclusion body myositis. In some embodiments, the present disclosure provides methods for administering cell colonies (and related pharmaceutical compositions) comprising regulatory T cells as described herein to subjects (e.g., subjects with autoimmune diseases). In some embodiments, the present disclosure provides methods for administering cell colonies or pharmaceutical compositions comprising regulatory T cells as described herein to subjects in an effective amount to alleviate one or more symptoms of autoimmune diseases. In some embodiments, the present disclosure provides methods for treating autoimmune diseases in subjects, including administering cell colonies or pharmaceutical compositions comprising regulatory T cells as described herein to the subject. In some embodiments, at least a portion of the cell colony is an autologous cell (i.e., obtained from the same subject subsequently administered). In some embodiments, a cell population is isolated from a subject, subjected to a method for producing a cell population as described herein (e.g., to increase 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.

[0203] In some embodiments, the autoimmune disease is selected from Sjögren's syndrome, hemorrhagic nephritis 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 associated with an autoimmune disease. In some embodiments, the autoimmune disease is selected from multiple sclerosis, type 1 diabetes, inclusion body myositis.

[0204] In some embodiments, treatment (or treatment) disease refers to a clinical intervention intended to reverse, alleviate, delay the onset of an autoimmune disease or one or more of its symptoms or suppress its progress. In some embodiments, treatment can be administered after one or more symptoms occur and / or after the disease is diagnosed. In other embodiments, treatment can be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to suppress the onset or progress of a disease. For example, treatment can be administered to susceptible individuals before the onset of symptoms (for example, due to understanding genetic factors). Treatment can also be continued after symptoms have subsided, for example, to prevent or delay the recurrence of symptoms.

[0205] 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 subject or a 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 can be male or female.

[0206] Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intravenous, subcutaneous, intravenous, intrathecal administration directly delivered to selected organs (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intramuscular, intradermal, intratumoral and other parenteral administration routes. If desired, administration routes can be combined. In some embodiments, the isolated population described herein or its composition is administered intravenously.

[0207] The cell colony or pharmaceutical composition comprising regulatory T cells can be administered as a bolus administration. In some embodiments, the administration of the cell colony or pharmaceutical composition comprising regulatory T cells comprises one or more infusions of the isolated cell colony or pharmaceutical composition to the subject (e.g., infusion of cells through a central line, similar to a blood transfusion).

[0208] Alternative numbering schemes

[0209] Additional numbered embodiments of the present disclosure are as follows:

[0210] Embodiment 1. A separated cell population comprising regulatory T cells, wherein: the regulatory T cells of the separated cell population comprise stable regulatory T cells, which (a) comprise an exogenous human T cell receptor (TCR) that specifically binds a target peptide complexed with a major histocompatibility complex (MHC), and (b) comprise a low-methylated regulatory T cell-specific demethylation region (TSDR) at the endogenous FOXP3 locus, at least 80% of the cells of the separated cell population are CD25+ / high CD4+CD127- / lo regulatory T cells, and less than 10% of the cells of the separated cell population express FOXP3 protein from an engineered FOXP3 locus.

[0211] Embodiment 2. The isolated cell population according to embodiment 1, wherein the MHC is MHC class I or MHC class II.

[0212] Embodiment 3. An isolated cell population according to 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.

[0213] Embodiment 4. An isolated cell population according to 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.

[0214] Embodiment 5. An isolated cell population according to any 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+ / high CD4+CD127- / lo regulatory T cells.

[0215] Embodiment 6. An isolated cell population according to any one of the preceding embodiments, wherein at least 70% of the cells of the isolated cell population are CD25+ / high CD4+CD127- / loFOXP3+ regulatory T cells.

[0216] Embodiment 7. An isolated cell population according to 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+ / high CD4+CD127- / loFOXP3+ regulatory T cells.

[0217] Embodiment 8. An isolated cell population according to any of the preceding embodiments, wherein after transduction of the stable regulatory T cells with the exogenous human TCR, the percentage of cells in the isolated cell population comprising stable regulatory T cells decreases by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 percentage points during an expansion period of at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

[0218] Embodiment 9. An isolated cell population according to embodiment 8, wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population containing stable regulatory T cells decreases by no more than 10 percentage points during at least 5 days of expansion, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population containing stable regulatory T cells decreases by no more than 10 percentage points during at least 10 days of expansion.

[0219] Embodiment 10. An isolated cell population according to embodiment 8, wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 5 percentage points during at least 5 days of expansion, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 5 percentage points during at least 10 days of expansion.

[0220] Embodiment 11. An isolated cell population according to embodiment 8, wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population containing stable regulatory T cells decreases by no more than 1 percentage point during at least 5 days of expansion, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population containing stable regulatory T cells decreases by no more than 1 percentage point during at least 10 days of expansion.

[0221] Embodiment 12. An isolated cell population according to any of the preceding embodiments, wherein after transduction of the stable regulatory T cells with the exogenous human TCR, the percentage of cells in the isolated cell population comprising stable regulatory T cells decreases by no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% during an expansion period of at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

[0222] Embodiment 13. An isolated cell population according to embodiment 12, wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 10% during at least 5 days of expansion, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 10% during at least 10 days of expansion.

[0223] Embodiment 14. An isolated cell population according to embodiment 12, wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 5% during an expansion period of at least 5 days, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 5% during an expansion period of at least 10 days.

[0224] Embodiment 15. An isolated cell population according to embodiment 12, wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 1% during at least 5 days of expansion, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, the percentage of cells of the isolated cell population comprising stable regulatory T cells decreases by no more than 1% during at least 10 days of expansion.

[0225] Embodiment 16. An isolated cell population according to any one of the preceding embodiments, comprising at least 1×10 7 stable regulatory T cells.

[0226] Embodiment 17. The isolated cell population according to Embodiment 16, comprising 1×107 to 1×109 stable regulatory T cells.

[0227] Embodiment 18. The isolated cell population according to any 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 an engineered FOXP3 locus.

[0228] Embodiment 19. An isolated cell population according to any 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.

[0229] Embodiment 20. An isolated cell population according to 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.

[0230] Embodiment 21. An isolated cell population according to any 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 contain an engineered FOXP3 gene or an engineered FOXP3 protein.

[0231] Embodiment 22. An isolated cell population according to any 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 an exogenous FOXP3 protein.

[0232] Embodiment 23. An isolated cell population according to any 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.

[0233] Embodiment 24. An isolated cell population according to any 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.

[0234] Embodiment 25. An isolated cell population according to any 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 in the isolated cell population overexpress functional FOXP3 protein.

[0235] Embodiment 26. An isolated cell population according to any 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 in the isolated cell population are conventional T cells.

[0236] Embodiment 27. An isolated cell population according to any 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 of the isolated cell population are conventional T cells.

[0237] Embodiment 28. An isolated cell population according to Embodiment 26 or 27, wherein the conventional T cells of the isolated population comprise the exogenous human TCR.

[0238] Embodiment 29. An isolated cell population according to Embodiment 28, wherein about 10%, about 15%, about 20%, about 30%, about 40%, about 50% or about 60% of conventional T cells contain the exogenous TCR.

[0239] Embodiment 30. An isolated cell population according to any 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.

[0240] Embodiment 31. An isolated cell population according to any 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.

[0241] Embodiment 32. The isolated cell population according to embodiment 31, wherein the CD8+ T cells are undetectable, optionally by fluorescence activated cell sorting (FACS).

[0242] Embodiment 33. An isolated cell population according to any of the preceding embodiments, wherein at least 10% of the regulatory T cells of the isolated cell population express the exogenous human TCR.

[0243] Embodiment 34. An isolated cell population according to 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 the exogenous human TCR.

[0244] Embodiment 35. An isolated cell population according to any of the preceding embodiments, wherein the percentage of CD25+ / highCD4+CD127- / lo regulatory T cells is assessed between 1 hour and 120 days after transduction.

[0245] Embodiment 36. An isolated cell population according to any one of embodiments 1 to 34, wherein the percentage of CD25+ / high CD4+CD127- / lo regulatory T cells is assessed at least 1 hour, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours or 120 hours after transduction.

[0246] Embodiment 37. An isolated cell population according to 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.

[0247] Embodiment 38. An isolated cell population according to any one of embodiments 1 to 34, wherein the percentage of CD25+ / highCD4+CD127- / lo regulatory T cells is assessed at least 10 days, 15 days, 30 days, 60 days or 120 days after transduction.

[0248] Embodiment 39. An isolated cell population according to any 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.

[0249] Embodiment 40. An isolated cell population according to any of the preceding embodiments, wherein the stable regulatory T cells of the isolated cell population exhibit one or more functions selected from the following: (a) cytokine secretion activity; (b) expression of activation markers associated with regulatory T cells; and / or (c) inhibitory activity.

[0250] Embodiment 41. The isolated cell population of Embodiment 40, wherein the one or more functions are assessed between 1 hour and 14 days after transduction.

[0251] Embodiment 42. An isolated cell population according to embodiment 40, wherein the one or more functions are assessed at least 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours or 120 hours after transduction.

[0252] Embodiment 43. An isolated cell population according to Embodiment 40, wherein the one or more functions are assessed at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after transduction.

[0253] Embodiment 44. An isolated cell population according to 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.

[0254] Embodiment 45. The isolated cell population according to any of the preceding embodiments, wherein at least 10% of the stable regulatory T cells are CD45RA+.

[0255] Embodiment 46. An isolated cell population according to 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+.

[0256] Embodiment 47. An isolated cell population according to any of the preceding embodiments, wherein at least 10% of the cells of the isolated cell population are CD25+ / high CD4+CD127- / loCD45RA+ prior to activation and transduction with the exogenous human TCR.

[0257] Embodiment 48. An isolated cell population according to any of the preceding embodiments, wherein prior to activation and transduction with the exogenous human TCR, 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 in the isolated cell population are CD25+ / high CD4+CD127- / loCD45RA+.

[0258] Embodiment 49. An isolated cell population according to any of the preceding embodiments, wherein the exogenous human TCR is encoded as a single polypeptide.

[0259] Embodiment 50. An isolated cell population according to Embodiment 49, wherein the exogenous human TCR comprises an N-terminal β chain and a C-terminal α chain.

[0260] Embodiment 51. An isolated cell population according to embodiment 49 or 50, wherein the polypeptide comprises an α chain and a β chain, and wherein the polypeptide comprises a self-cleaving peptide sequence positioned between the α chain and the β chain.

[0261] Embodiment 52. An isolated cell population according to Embodiment 51, wherein the self-cleaving peptide sequence is a 2A peptide sequence.

[0262] Embodiment 53. An isolated cell population according to embodiment 52, wherein the 2A peptide sequence is a P2A, E2A, F2A or T2A peptide sequence.

[0263] Embodiment 54. An isolated cell population according to any of the preceding embodiments, wherein 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, and wherein these cysteine ​​residues are capable of forming one or more disulfide bonds.

[0264] Embodiment 55. An isolated cell population according to Embodiment 54, wherein relative to a TCR α chain constant region comprising the amino acid sequence of SEQ ID NO:1, the TCR α chain constant region comprises a T48C amino acid substitution, and wherein relative to a TCR β chain constant region comprising the amino acid sequence of SEQ ID NO:3, the TCR β chain constant region comprises a S57C amino acid substitution.

[0265] Embodiment 56. A method for producing an isolated cell population according to any one of the preceding embodiments, the method comprising: (a) isolating a biological sample containing regulatory T cells from a human subject suffering from 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 cell population enriched in CD25; (d) selecting CD25+ / high CD4+CD127- / loCD45RA+ cells from the CD25-enriched cell population to produce one or more positive fractions; (e) selecting CD25+high CD4+CD127- / loCD45RA+ cells from the one or more positive fractions to produce a cell population containing stable regulatory T cells; and (f) engineering the cell population containing stable regulatory T cells so that these stable regulatory T cells contain the exogenous human TCR, thereby producing the isolated cell population.

[0266] Embodiment 57. A method according to 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.

[0267] Embodiment 58. The method of embodiment 56 or 57, wherein less than 0.5% of the cells of the depleted sample comprise CD8+ cells.

[0268] Embodiment 59. A method according to any one of Embodiments 56 to 58, wherein the selection in (d) comprises identifying CD4+CD45RA+ cells, and 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 the one or more positive fractions.

[0269] Embodiment 60. A method according to embodiment 59, wherein the selection in (e) comprises selecting and identifying CD4+CD45RA+ cells, and 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.

[0270] Embodiment 61. The method according to any one of embodiments 56 to 60, wherein the autoimmune disease is multiple sclerosis, optionally progressive multiple sclerosis, type 1 diabetes or inclusion body myositis.

[0271] Embodiment 62. A method according to embodiment 61, wherein the human subject has a genetic HLA haplotype of DR2a / b, DR3 or DR4.

[0272] Embodiment 63. A method according to any one of Embodiments 56 to 62, wherein the human subject is a male subject.

[0273] Embodiment 64. The method of any one of Embodiments 56 to 62, wherein the human subject is a female subject.

[0274] Embodiment 65. A method according to any one of Embodiments 56 to 64, wherein engineering the isolated cell population comprises transducing the cell population with a nucleic acid encoding the exogenous human TCR.

[0275] Embodiment 66. A method according to any one of Embodiments 56 to 65, which further comprises activating and expanding cells of the isolated cell population to produce a cell population comprising at least 1×107 stable CD25+ / high CD4+CD127- / lo regulatory T cells, which regulatory T cells comprise hypomethylated TSDR at the endogenous FOXP3 locus.

[0276] Embodiment 67. A method according to Embodiment 66, wherein the activation and expansion comprises culturing the cells of the isolated cell population for at least 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

[0277] Embodiment 68. A method according to Embodiment 67, wherein the activation and expansion comprises culturing the cells of the isolated cell population for no more than 15 days, 14 days, 13 days or 12 days.

[0278] Embodiment 69. The method according to any one of Embodiments 65 to 68, wherein the nucleic acid is a vector.

[0279] Embodiment 70. The method according to embodiment 69, wherein the vector is a viral vector.

[0280] Embodiment 71. The method according to Embodiment 70, wherein the viral vector is a lentiviral vector.

[0281] Embodiment 72. A method according to any one of Embodiments 65 to 71, 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.

[0282] 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 WPRE-mut6.

[0283] Embodiment 74. A method according to embodiment 72 or 73, wherein the coding sequence is codon optimized.

[0284] Embodiment 75. The vector according to any one of Embodiments 69 to 74.

[0285] Embodiment 76. A pharmaceutical composition comprising the isolated cell population according to any of the preceding embodiments and a pharmaceutically acceptable excipient.

[0286] Embodiment 77. A composition comprising an isolated cell population according to any of the preceding embodiments and a cryopreservative.

[0287] Embodiment 78. A method comprising administering the pharmaceutical composition of Embodiment 76 to a subject, wherein the subject has an autoimmune disease.

[0288] Embodiment 79. The method according to embodiment 78, wherein the autoimmune disease is multiple sclerosis, optionally progressive multiple sclerosis, type 1 diabetes or inclusion body myositis.

[0289] 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.

[0290] Embodiment 81. A method comprising administering to a subject an isolated cell population according to any one of the preceding embodiments.

[0291] Embodiment 82. The method of embodiment 81, wherein the subject suffers from an autoimmune disease.

[0292] Embodiment 83. The method according to embodiment 82, wherein the autoimmune disease is multiple sclerosis, optionally progressive multiple sclerosis, type 1 diabetes or inclusion body myositis.

[0293] Embodiment 84. The method of any one of Embodiments 81 to 83, wherein the administration comprises intravenous administration.

[0294] Embodiment 85. The method of any one of Embodiments 81 to 84, wherein the administration comprises one or more infusions.

[0295] Embodiment 86. A method according to any one of Embodiments 81 to 85, wherein the cells of the isolated cell population are autologous to the subject.

[0296] Example

[0297] Example 1. Manufacturing process for generating autologous stable regulatory T cells expressing exogenous TCRs

[0298] This example shows the generation of a cell population containing stable thymic-derived regulatory T cells expressing exogenous TCRs. The overall enrichment and sorting strategy is shown in Figure 1A to Figure 1B middle.

[0299] Leukocyte apheresis, depletion and enrichment

[0300] First, leukapheresis from human subjects was collected, transported to a central processing facility, and kept 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 mixture of magnetic microbeads (CD8, CD14, and CD19 microbeads; Miltenyi Biotec) using an automated cell processing system.

[0301] Using magnetic cell separation technology, cytotoxic T cells (CD8+), B cells (CD19+), and monocytes (CD14+) were depleted before enriching CD25+ cells. The volume of the depleted biological sample was reduced by centrifugation. The depleted biological sample was then labeled with CD25-PE-biotin antibody (Miltenyi Biotec) 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 Biotec). The CD25+ cells were then isolated using magnetic cell separation technology to produce a CD25-enriched sample.

[0302] Figure 2 Shown are the viable cells (VC) of all cells after apheresis, removal of CD8+, CD19+, and CD14+ cells (depleted sample), and selection of CD25+ (CD25-enriched sample) for Run 1. After the CD25 selection step to generate the CD25-enriched sample, approximately 98% of the total cells of the population were CD3+ cells, and approximately 95% of the cells were CD4+ T cells.

[0303] Table 1 shows the apheresis cell populations as well as the CD4+ cell and naive Treg cell composition after depletion and enrichment steps for 5 additional manufacturing runs.

[0304] Table 1: Composition of cell populations during depletion and enrichment processes

[0305] cell Run 2 Run 3 Run 4 Run 5 Run 6 Donor gender 33 59 29 37 26 Donor age M M M M M Donor health status (MS / non-MS) MS Healthy donor Healthy donor Healthy donor Healthy donor VC apheresis 1.1E+10 1.1E+10 8.7E+09 1.5E+10 1.1E+10 Percentage of CD4 T cells in apheresis 38％ 30％ 22％ 36％ 49％ Percentage of naive Treg in apheresis 1％ 1％ 1％ 1％ 1％ VC after depletion 3.3E+09 2.6E+09 1.9E+09 4.6E+09 4.0E+09 Percentage of CD4 T cells after depletion 83％ 59％ 65％ 89％ 88％ Percentage of naive Treg after depletion 1％ 3％ 4％ 3％ 2％ VC after enrichment 3.5E+09 2.4E+08 3.0E+08 4.6E+08 2.0E+08 Percentage of CD4 T cells after enrichment 95％ 96％ 93％ 96％ 97％ Percentage of naive Treg after enrichment 10％ 19％ 21％ 18％ 19％

[0306] Figure 3A Shown are viable counts (VC) of naive regulatory T cells (specifically, CD4+CD25+ / highCD127- / loCD45RA+ cells) after apheresis, removal of CD8+, CD19+ and CD14+ cells (depleted samples) and selection of CD25+ (CD25-enriched samples) for Run 1. Figure 3B The recovery of naive Tregs (CD4+CD25+ / highCD127- / loCD45RA+ cells) after each step is shown, with approximately 80% of CD4+CD25+ / highCD127- / loCD45RA cells recovered after the depletion step and 75% of CD4+CD25+ / highCD127- / loCD45RA+ cells recovered after the CD25 enrichment step.

[0307] Table 2 shows the VC of naive Tregs after apheresis, depletion and enrichment, as well as the recovery of naive Tregs from the previous step after depletion and enrichment.

[0308] Table 2: Total viable count (TVC) of naive Tregs and percent recovery after each step of the manufacturing process

[0309]

[0310] Gating strategy

[0311] The CD25-enriched samples were then stained with fluorescently labeled anti-CD45RA, anti-CD4, and anti-CD127 antibodies and sorted by fluorescence-activated cell sorting (FACS) for CD4 + CD25 + / 高 CD127 - / lo CD45RA +Phenotypic sorting is performed to generate a cell population containing stable regulatory T cells. FACS is performed in two rounds, the first round is a subtraction fraction sorting, and the second round is a graded purity sorting. For the subtraction sorting, the CD25-enriched sample is separated into multiple fractions, and these multiple fractions are sorted through multiple cell sorters. After the subtraction sorting, the fractions are combined for the second round of purity sorting. The purpose of graded sorting of cells in subtraction and / or purity sorting is to speed up the sorting process by sorting on multiple machines, thereby ensuring that the cells remain viable. The gating strategy used for subtraction and purity sorting is the same. For the gating strategy, a dot plot-based approach is used, in which CD45RA is first selected + CD4 + Cells were then isolated from CD45RA + CD4 + Select CD25 + / 高 CD127 - / lo cells to optimize the purity of stable regulatory T cells in the population as determined by the TSDR phenotype. Figure 1B As shown, this FACS gating strategy first plots CD45RA on a dot plot. + Cells vs CD4 + cells and select these cells based on population distribution using a box gate. From the selected cell population, CD25 + / 高 Cells vs CD127 - / lo Cells were plotted on dot plots and gated using a polygonal gate to select CD4 + CD25 + / 高 CD127 - / lo CD45RA + This FACS strategy was developed to optimize the selection of stable naive Tregs based on their TSDR hypomethylation phenotype. CD4 + CD25 + / 高 CD127 - / lo CD45RA + The composition of the cell population after cell population is provided in Table 3.

[0312] Table 3. Composition of cell population after FACS

[0313]

[0314] The purity and recovery of naive Tregs during the sorting process for five additional manufacturing runs are shown in Table 4.

[0315] Table 4: Purity and recovery of naive Tregs after FACS sorting

[0316]

[0317] Regulatory T cells were activated for approximately 48 hours using T cell TransAct activation reagent (Miltenyi Biopharmaceuticals) 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), referred to as 'TCR-A'. The vector construct comprises an N-terminal TCRβ chain and a C-terminal TCRα chain with a linker domain comprising a GSG amino acid sequence followed by a P2A self-cleaving peptide. The TCRβ-GSG-P2A-TCRα fusion construct was expressed under the EF1α promoter. Figure 5 The proportion of transduced cells measured by FACS analysis using anti-Vβ antibody is shown in FIG.

[0318] The transduced Tregs were then expanded in T cell expansion medium supplemented with 1,000 IU / ml IL-2 until harvest (day 9, day 10, or day 11). Cell counts, size, population doublings, and viability of untransduced and transduced Tregs were assessed and are shown for Run 1 ( Figure 4 ). Notably, the transduced Tregs shared similar growth curves with non-transduced Tregs, suggesting that exogenous TCRs do not impair cell growth and proliferation.

[0319] The population doubling level and the percentage of vigor of 5 additional manufacturing runs are shown in Table 5. The target dose percentage achieved by the whole process of all 6 manufacturing runs is also shown. The target dose percentage is calculated in the following manner: the total final number of the original Treg is determined based on the initial number and population doubling level of the original Treg before amplification after the sorting process, and the transduction efficiency is assumed to be 25%. The target dose percentage is calculated based on the target dose of 100 million transduced cells. As shown in the figure, the manufacturing process in this embodiment obtains a Treg product with high purity and enough cells to be administered to the patient from a single leukocyte apheresis solution (leukopak). As used herein, PDL is the population doubling level used throughout. TDN represents the cells after transduction.

[0320] Table 5: Properties of the cells produced

[0321] sky PDL(TDN) Vitality percentage Percentage of target dose Run 2 9 6.8 94％ 370％ Run 3 9 7.8 97％ 420％ Run 4 9 6.4 94％ 450％ Run 5 9 6.6 96％ 1400％ Run 6 9 6.5 not applicable 482％

[0322] Transduced cells were collected from day 0 to day 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 treated with bisulfite. Digital droplet PCR (ddPCR) assay using methylation-specific primers and probes was then used to quantify unmethylated and methylated sequences. Figure 6 The TSDR hypomethylation status of the collected cells for all 6 post-transduction manufacturing runs is shown in . During the expansion period, the TSDR hypomethylation level 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 of all 6 donor samples processed using the method described in this example.

[0323] These data demonstrate that the obtained regulatory T cells are stable, thymus-derived regulatory T cells.

[0324] Table 6: TSDR status of cells generated using the method of this example

[0325] TSDR hypomethylation % Donor gender Day 0 Day 3 Day 6 Day 9 Run 1 M not applicable not applicable 93％ 93％ Run 2 M 96％ not applicable 94％ 90％ Run 3 M 94％ not applicable 90％ 86％ Run 4 M 90％ not applicable 89％ 88％ Run 5 M 95％ 95％ 94％ not applicable Run 6 M not applicable 98％ 99％ 99％

[0326] Activity of Tregs after cryopreservation freeze-thaw cycles

[0327] Regulatory T cells produced according to the methods of this example, either untransduced or transduced with a lentiviral vector encoding a TCR described herein, were cryopreserved for storage. The cells were frozen and stored in a vapor phase nitrogen storage freezer.

[0328] After a period of refrigeration, the frozen cell colony was thawed. The cell colony was divided into two experimental groups. The first experimental group was activated with anti-CD3 and anti-CD28 antibodies (transduced (TDN)), while the second experimental group was not activated (untransduced (UNT)). Both groups were expanded in a culture medium containing IL-2. After incubation, IL-10 levels were measured after 1 day, 2 days and 3 days.

[0329] Figure 7 The increase in IL-10 levels per run over a three-day period for cells activated with anti-CD3 and anti-CD28 antibodies is shown in FIG. The cells exhibited increased secretion of the IL-10 cytokine over a three-day period, demonstrating that the cell population can be activated and retains its cellular functionality after cryopreservation freeze-thaw cycles ( Figure 7 ). Non-activated cells showed no increase in IL-10 levels. This data further demonstrates that regulatory T cells retain function.

[0330] Example 2. Gating strategy optimized for GMP conditions

[0331] A second gating strategy was developed using a 1-D histogram-based approach rather than a 2-D point-based approach to reduce user error in a GMP manufacturing environment. Cells were prepared as in Example 1 until the FACS sorting step. In the FACS sorting step, the gating scheme used was as follows: Figure 8 As shown, a one-dimensional histogram-based method is used in which CD25-rich cells are sorted by CD4 + 、CD45RA + 、CD127 - / lo and CD25 + / 高 Gating (CD4 + >CD45RA + (Histogram)>CD127 - / lo (Histogram) 40%>CD25 Hi (Histogram) 70%). Both gating strategies were run in parallel on the same donor sample for the same number of runs as in Example 1. Cells were transduced with TCR-A or a disulfide modified, codon optimized version (termed 'TCR-E v2').

[0332] The properties 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.

[0333] Table 7: Composition of cell populations during depletion and enrichment processes

[0334]

[0335] Table 8: VC of naive Tregs and percent recovery after each step of the manufacturing process

[0336]

[0337] Table 9 shows the purity and recovery of naive Tregs during the sorting process for each run.

[0338] Table 9: Purity and recovery of naive Tregs after FACS sorting

[0339]

[0340] The properties of the produced cells are shown in Table 10 below.

[0341] Table 10: Properties of the cells produced

[0342] TCR sky PDL(TDN) Vitality percentage Percentage of target dose Run 5 A 9 6.6 96％ 1400％ Run 6 A 9 6.5 Unavailable 482％ Run 7 Ev2 9 7.8 Unavailable 2100％ Run 8 Ev2 10 7.8 Unavailable 1200％ Run 9 Ev2 10 7.6 99％ 380％

[0343] The TSDR status of the cells is shown in Tables 11 and Fig. 9In one run, the TSDR level was over 100% because the donor was female. Since the TSDR assay measures the FoxP3 locus on the X chromosome, and females have one silent (methylated) X chromosome copy, the TSDR demethylation level was doubled for female subjects to obtain the proportion of cells with a hypomethylated FoxP3 locus.

[0344] Table 11: TSDR status of cells generated using the method of this example

[0345]

[0346] Activity of Tregs after cryopreservation freeze-thaw cycles

[0347] Regulatory T cells generated according to the methods of 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.

[0348] After refrigeration for a period of time, the frozen cell colony was thawed. The cell colony 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 activated. Both groups were expanded in a culture medium containing IL-2. After incubation, regulatory T cell activation markers and cytokine levels (IL-10, CTLA-4, TGF-β-1 and CD69) were measured after 1 day, 2 days and 3 days.

[0349] FIG. 10A to FIG. 10B Figure 3 shows the increase in the levels of each of IL-10, CTLA-4, TGF-β-1, and CD69 for all runs over a three-day period for cells activated with anti-CD3 and anti-CD28 beads. Activated cells showed an increase in IL-10 ( Fig. 10D )、CTLA-4( Fig. 10A )、TGF-β-1( Fig. 10C ) and CD69( Fig. 10B ) levels increased, demonstrating that the cell population can be activated and retain its cellular functionality after cryopreservation freeze-thaw cycles ( FIG. 10A to FIG. 10D ). Non-activated cells showed no increase in levels. This data further demonstrates that regulatory T cells retain function.

[0350] Example 3: Manufacturing strategy for increasing stringency to select stable Tregs

[0351] Challenges of generating stable Tregs from patient populations

[0352] After establishing the manufacturing process with healthy donor samples, the manufacturing process was evaluated with samples from MS donors. For some MS donors, lower initial TSDR hypomethylation levels and / or less stable TSDR hypomethylation levels during expansion (from day 0 to harvest) were observed compared to the results using healthy donor cells. Fig.11 The changes of TSDR hypomethylation with amplification of three MS donors are shown. For two of the three MS donors, the initial TSDR is less than 90%, and the TSDR decreases by more than 10% during the amplification process. This may be because there are differences in the Treg population between healthy donors and autoimmune donors (including MS donors), making the boundary between Treg and T conventional cells less clear when sorting cells. These results show that, for some donors, increasing the stringency of the gating strategy will likely improve the purity of the separated Treg population, and therefore improve the ratio of the stable Treg obtained in the process. However, increasing stringency may lead to a reduction in yield. Described in this embodiment is a more stringent gating method, which is combined with an improved amplification scheme. The improved amplification scheme enhances the proliferation of Treg, which is required to overcome the yield reduced by increasing the stringency of selecting stable Treg.

[0353] Evaluation of Treg subsets included in the process

[0354] The sorting strategies described in the above examples focus on the selection of naive Tregs. Considering that a more stringent sorting strategy is required for some donors and that such a strategy may exclude an increased proportion of naive Tregs, we evaluated the CD45RA for the selection of naive Tregs in the context of a more stringent sorting strategy. + Is the selection step necessary, or can a more stringent selection of antigen-experienced Tregs be used? + CD25 + CD127 - / lo CD45RA + Antigen-experienced Tregs are CD4 + CD25 高 CD127 - / lo CD45 - Both populations are stable Tregs that will have a stable TSDR hypomethylation phenotype. To assess whether CD45RA-based selection is required, antigen-experienced (CD45RA - ) and naive (CD45RA + )Treg and compared their expansion capacity and stability.

[0355] Cells were prepared as described in Example 1 and then subjected to the FACS sorting step. Cells were then subjected to two rounds of FACS sorting for reduction and purity. - Population and CD45RA + The population was sorted by CD4, CD25, and CD127 during the subtraction sorting. + , and then a polygonal gate was used to identify CD25 + / 高 CD127 - / lo Treg, which was set at 70% of the standard polygonal gate, was used for Treg identification and selection. Sorting was repeated, and for CD45RA - Cells, in the identification of CD25 + / 高 CD127 - / lo After Treg population, CD45RA was identified and selected - For CD45RA + Cells, in the identification of CD25 + / 高 CD127 - / lo After Treg population, CD45RA was identified and selected + Cells. In each population, naive (CD45RA + )The percentages of Tregs are shown in Table 12 below.

[0356] At the end of the sorting, 3 groups were established:

[0357] (a)CD45RA + Treg–enriched CD45RA + Treg fraction

[0358] (b)CD45RA - Treg-enriched CD45RA - Treg fraction

[0359] (c) “Spiked” Tregs – 80% enriched CD45RA - Treg fraction + 20% enriched CD45RA + Treg fraction

[0360] Table 12: Percentage of naive Tregs in each condition before expansion

[0361] condition Day 0 nTreg% CD45RA+ 94％ Spiking 20％ CD45RA- 2％

[0362] The cells were then expanded in culture as described in Example 1 for 8 days. FIG. 12A to FIG. 12C The population doubling level (PDL) and TSDR hypomethylation after cell expansion are shown. Fig. 12A and Fig. 12C As shown in -Group, CD45RA + The population showed a significant increase in expansion, as well as increased TSDR hypomethylation. This suggests that CD45RA + Naive Tregs are essential for the expansion process because they bind to CD45RA - Compared with Treg, CD45RA + Naive Tregs have a relative capacity to expand. Importantly, the “spiked” samples showed that in the presence of a large excess of CD45RA - In the case of antigen-experienced Tregs, CD45RA + Naive Tregs can still expand effectively.

[0363] Development of a more stringent gating strategy for isolating stable Tregs

[0364] Based on the above findings, an improved gating strategy was developed to increase the stringency of sorting while still capturing CD45RA + In particular, this strategy identifies antigen-experienced (CD4 + CD25 高 CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + )Treg, while specifically excluding CD4 + CD25 + CD127 - / lo CD45RA - Non-Treg. By separately identifying antigen-experienced Tregs and naive Tregs, sufficient numbers of Tregs are obtained to produce a cell therapy product from a single leukapheresis, and antigen-experienced Tregs have high suppressive capacity, which suppress any non-Tregs ex vivo and in vivo. In this method, CD4 + Cells were then identified with CD127 in combination with a critical “NOT” gate or an L-shaped gate - / lo and CD25 + (sequentially or simultaneously), as the final step of the gating scheme to select for antigen-experienced (CD4 + CD25 高 CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + ) Treg, while specifically excluding unwanted CD4+ CD25 + CD127 - / lo CD45RA - This gating method is FIG. 13A to FIG. 13C and FIG. 14A to FIG. 14E It is shown in FIG. 13A to FIG. 13C The dot plot method for the CD25 / CD127 selection step is shown, and FIG. 14A to FIG. 14E shows the method for selecting CD127 - / lo Then select CD25 + Histogram method.

[0365] To further describe the gating strategy in detail, cells enriched for CD25 were stained with fluorescently labeled antibodies that recognize human CD4, CD127, and CD45RA cell surface markers. The cells were already labeled with CD25 antibodies during the enrichment step of the process. The cells were then sorted twice using a FACS instrument using the gating scheme described below (using the same strategy in each step). For dot plot-based methods, such as FIG. 13A to FIG. 13C As shown, in the first step of the gating process, CD4 + T cells ( Fig.13A In the second step, CD25 was identified based on the two-parameter flow cytogram. + CD127 - / lo Treg cells, thereby visualizing the spatial context of the Treg cell population relative to another cell population (i.e., Tconv cells), visualizing CD25 and CD127 levels, and drawing a polygonal gate in the upper left quadrant to capture CD25 + CD127 - / lo This polygonal gate was then shifted upward to reduce the selected cell population to 70% of the original population, thereby increasing the stringency of the selection ( Fig. 13B ). In the final step, a “NOT” gate or L-shaped gate is applied, thereby visualizing CD45RA and CD25 levels to exclude CD25 from the sorting gate. + CD45RA - Cell populations were used to select naive Treg (CD4 + CD25 + CD127 - / lo CD45RA + ) and antigen-experienced Treg (CD4 + CD25 高 CD127 - / 低 CD45RA - ) cell population( Fig. 13C Although both antigen-experienced Treg populations and unstable Treg populations are CD45RA- , but they can be distinguished by CD25 levels, with antigen-experienced Tregs being CD25 高 , while the unstable Treg population is only CD25 + But it is not high. + and CD25 + (but not high), so it is used to determine CD45RA - CD25 + Still CD25 高 The threshold for 高 Defined as CD25 levels above those expressed in the naive Treg population. The L-shaped gate is critical as it allows selection of two important cell populations. 高 CD45RA - Antigen-experienced Tregs and CD25 + CD45RA + Naive Treg, excluding CD25 + CD45RA - Contaminating cells.

[0366] The histogram-based approach was implemented as an operational alternative for certain manufacturing scenarios and produced similar results to the dot plot strategy ( FIG. 14A to FIG. 14E Using sequential histogram flow plots, the cells with the lowest CD127 expression (i.e., 40 ± 2% CD127 - / lo ), followed by the highest CD25 expression (i.e., 70±2% CD25 + ) + T cells ( FIG. 14A to FIG. 14C The cell population was further refined by selecting 70 ± 2% of Treg cells in a CD25 vs. CD127 dual parameter flow plot ( Fig.14D ). Naive Tregs (CD4 + CD25 + CD127 - / lo CD45RA + ) and antigen-experienced Treg (CD4 + CD25 高 CD127 - / lo CD45RA - )( Fig.14E ).

[0367] The MS donor used for run 10 donated a second leukapheresis fluid (run 12), and the leukapheresis fluid was processed using a new sorting strategy (histogram + L-shaped gate). Except for the FACS sorting step, all other elements of the manufacturing process were the same as described in Example 1. Table 13 shows the properties of the cells after enrichment and before sorting, demonstrating that the properties of the cells are highly similar between the first two runs of the sorting step. In the context of Table 13 and all tables herein, N / A means that the data is not applicable because it was not collected.

[0368] Table 13: Properties of MS donor cells before FACS sorting

[0369]

[0370]

[0371] Table 14 shows the properties of the cells after isolation and before expansion. As expected, although the purity of Tregs after sorting remained high using stringent sorting methods, the inclusion of antigen-experienced Tregs in the sorting strategy reduced the proportion of naive Tregs relative to the original process described in Example 1. As the stringency of the gating scheme increased, the recovery of naive Tregs also decreased.

[0372] Table 14: Properties of MS donor cells after FACS sorting

[0373]

[0374] The population doubling levels and percent viability are shown in Table 15. As in Example 1, the percentage of target dose achieved for the entire process is also shown and calculated, but for the new sorting strategy, the target dose calculation is based on the total number of Tregs rather than the total number of naive Tregs because two populations are selected.

[0375] Table 15: Properties of the cells produced

[0376] Run 10 (Example 1 Process) Run 12 (histogram + L-gate) TCR A Ev2 sky 9 10 PDL 6.4 6.3 Vitality percentage 86％ 90％ <![CDATA[Final FoxP3 + Percentage]]> 76％ 84％ <![CDATA[TCR + ]]> 39％ Not applicable (assumed to be 25%) Target dose percentage 59％ 112％

[0377] Fig.15 Table 16 below shows the TSDR hypomethylation percentage after sorting and during amplification. For the L-gate method, more than 95% of the initial TSDR hypomethylation was achieved (relative to 87.79% for the sorting method from Example 1). In addition, for the L-gate-based method, the TSDR hypomethylation level decreased by less than 10% during amplification.

[0378] Table 16 :

[0379] Run 10 (Example 1 Process) Run 12 (histogram + L-gate) Day 0 88％ 97％ Day 3 86％ not applicable Day 6 85％ 95％ Day 9 75％ not applicable Day 10 not applicable 87％

[0380] New amplification process

[0381] Since the newly developed gating strategy selects a smaller number of naive Tregs compared to the original process, the expansion process was modified to increase the expansion of Tregs.

[0382] The expansion protocol was changed to add TNF-α to the expansion medium and add a second anti-CD28 anti-CD3 stimulation. First, the effect of restimulation was evaluated at different time points after transduction. For this experiment, cells were prepared according to the method described in Example 1, except that only CD4, CD25 and CD127 were gated but not CD45RA. The cells were then activated and transduced as in Example 1, with a second activation step using anti-CD3 and anti-CD28 on days 6, 8 and 10 after the first activation step. It was found that this restimulation on day 6 produced similar amplification to restimulation on days 7 or 8, but advantageously, when cells were harvested on day 10, restimulation on day 6 of the expansion process gave the greatest increase in amplification, and the key is that this does not require an extension of the amplification process ( Fig.16 ). Next, the effect of adding TNF-α to the expansion protocol was evaluated. Cells prepared according to the method described in this example (including the L-shaped gate) were expanded as described in Example 1, with or without a second stimulation on day 6, and with or without the addition of 2500 IU / ml TNF-α to evaluate the effect of TNF-α alone or in combination with a second stimulation on Treg expansion. Fig.17 As shown, adding TNF-α throughout the expansion process while performing a second stimulation with anti-CD3 and anti-CD28 on day 6 of the expansion process gave the greatest increase in expansion. However, each individual change to the process also increased expansion. As shown in Figure 18, cells expanded in TNF-α and combined with a second stimulation showed the highest viability and purity at harvest compared to either the individual process changes or the original expansion protocol.

[0383] Tregs generated using a novel gating and expansion process

[0384] The manufacturing process described in this example was then applied to a second replicate MS donor. An additional leukocyte apheresis was donated by the MS donor from run 11. This leukocyte apheresis was processed using a rigorous dot plot and histogram based sorting strategy and modifications to the above expansion conditions. Fig.19C ), with a minor modification of the histogram-based L-shaped gate method. The cells with the lowest CD127 expression (i.e., 30 ± 2% CD127 - / lo ), followed by the highest CD25 expression (i.e., 85±2% CD25 + ) +T cells. The cell population was further refined by selecting 85 ± 2% of Treg cells in a CD25 vs. CD127 dual parameter flow plot ( Fig.19D The L-shaped door itself has not changed ( Fig.19E Prior to the sorting step, cells were treated as in Example 1 ( FIG. 19A to FIG. 19E Table 17 shows the properties of the cells after enrichment and before sorting, demonstrating that the properties of the cells from each run were highly similar before the sorting step.

[0385] Table 17: Properties of MS donor cells before FACS sorting

[0386]

[0387] The properties of the sorted cells are shown in Table 18 below:

[0388] Table 18: Properties of MS donor cells after FACS sorting

[0389] Treg number purity Number of nTregs purity Run 11 (Example 2 Process) not applicable not applicable 6.5E+06 95％ Running 13L-shaped door (dot image) 5.6E6 100 1.9E+06 34 Run 13 L-shaped gate (histogram) 4.6E6 100 1.2E+06 25

[0390] The properties of the manufactured cells are shown below in Table 19. The target dose percentages were calculated as described above.

[0391] Table 19: Properties of the cells produced

[0392]

[0393] Fig. 20 Table 20 below shows the relationship between TSDR hypomethylation and amplification for this donor using the process described in Example 2 and the L-shaped gate strategy based on dot plots and histograms described in this example.

[0394] Table 20: TSDR of MS donors using original process and L-gate

[0395] Run 11 (Example 2 Process) Running 13L-shaped door (dot image) Run 13 L-shaped gate (histogram) TSDR Initial Yield: 61% (Day 3) Yield: 94% (Day 0) Yield: 95% (Day 0) TSDR Final Yield: 42% (Day 10) Yield: 91% (Day 10) Yield: 92% (Day 10) TSDR changes -19％ -3％ -3％

[0396] The comparative data presented in this example demonstrate that a stringent 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.

[0397] This process was repeated for another MS donor and two other healthy donors using two L-shaped gating methods and similar results were obtained. Table 21 shows the properties of the cells after enrichment and before sorting.

[0398] Table 21: Composition of cell populations during depletion and enrichment processes

[0399]

[0400]

[0401] The properties of the sorted cells are shown in Table 22 below:

[0402] Table 22: VC of regulatory T cells and recovery percentage after sorting

[0403]

[0404] The properties of the cells produced are shown below in Table 23. The target dose was calculated as described earlier in this example.

[0405] Table 23: Properties of the cells produced

[0406]

[0407]

[0408] The TSDR status of the cells is shown in Tables 24 and Fig.21 middle.

[0409] Table 24: TSDR status of cells generated using the method of this example

[0410]

[0411] For all donors treated using the sorting and expansion strategy described in this example, more than 90% of TSDR hypomethylation status after sorting was achieved, which did not decrease by more than 10% during the expansion process. This shows that this process generates a high purity and stable Treg population from multiple donors. In addition, this process can also obtain a sufficient number of transduced Tregs to achieve the target dose.

[0412] Example 4: Evaluation of TSDR threshold level

[0413] We investigated the threshold level of TSDR hypomethylation necessary for stable Treg cell production, which represents the level of stable Tregs. We first asked what fraction of stable Tregs on day 0 was required to maintain stable Treg cell levels throughout expansion, suggesting low or no growth of contaminating conventional T cells or other cellular impurities. To this end, we spiked conventional T cells into Tregs at various percentages on day 0, each of which was isolated from the same healthy donor, and tracked TSDR levels throughout expansion. TSDR hypomethylation levels and FOXP3 + The level was related to the initial Treg purity and remained stable. When the Treg purity was greater than 60%, TSDR hypomethylation did not decrease by more than 10% during the entire expansion process ( FIG. 22A to FIG. 22D). Next, the Treg purity of the expanded cell products was evaluated to determine the presence of conventional T cells and the production of proinflammatory cytokines when activated with PMA and ionomycin. Cryopreserved cells from the harvest were thawed into fresh medium and then stimulated with PMA and ionomycin at 37°C and 5% CO2 for 4 hours. Intracellular cytokine (ICC) production of IFN-γ and IL-2 was measured by flow cytometry. Fig.23 FoxP3 expressing IL-2 and IFN-γ after activation with PMA and ionomycin is shown + and FoxP3 - In the absence of conventional T cells, 100% Treg products secreted little or no IFN-γ or IL-2 cytokines, indicating that the number of unstable Treg cells expanded from the initial culture was not significant. As the purity of the initial Treg decreased, the proportion of T conventional cells secreting IFN-γ and IL-2 cytokines increased, with the largest increase observed when the starting Treg purity was less than 90%. This increase may be due to an increase in the Tcon:Treg ratio. Upon activation, some conventional T cells upregulate FoxP3, which is why some FoxP3 + This suggests that high TSDR levels are critical for maintaining a stable population of cells that can suppress contaminating conventional T cells.

Claims

1. An isolated cell population comprising stable CD4+ T regulatory cells (T regs) derived from a subject suffering from an autoimmune disease, wherein at least 80% of said cells are stable CD4+ T regs comprising a hypomethylated T cell-specific demethylation region (TSDR) at the FOXP3 locus, and wherein the cell comprises an exogenous human T cell receptor (TCR) that specifically binds a target peptide complexed with a major histocompatibility complex (MHC). 2 . The isolated population of claim 1 , wherein the stable CD4+ T regs do not express FOXP3 protein from an engineered FOXP3 locus.

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

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

5. An isolated population according to 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+T regs containing hypomethylated TSDR at the endogenous FOXP3 locus.

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

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

8. An isolated population according to any one of the preceding claims comprising at least 4×10 7 A stable CD4+Treg.

9. The isolated population according to claim 8, comprising 4×10 7 to 1×10 10 A stable CD4+T reg.

10. An isolated population according to any 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. An isolated population according to 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 contain the exogenous human TCR.

12. An isolated population according to any of the preceding claims, wherein the ratio of stable CD4+T regs 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.

13. The isolated population of any of the preceding claims, wherein 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 population are CD8 + T cells.

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

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

16. An isolated population according to 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. An isolated population according to any of the preceding claims, wherein after a cryopreservation freeze-thaw cycle, the percentage of stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus is reduced by 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

18. The isolated population according to any one of the preceding claims, wherein the stable CD4+ Tregs exhibit one or more functions selected from the group consisting of: a. Regulatory cytokine secretion activity; b. expression of activation markers associated with regulatory T cells; and / or c. Inhibitory activity.

19. An isolated population according to any of the preceding claims, wherein the exogenous human TCR comprises one or more amino acid substitutions of cysteine ​​residues in the TCR α chain constant region and the TCR β chain constant region, and wherein the cysteine ​​residues are capable of forming one or more disulfide bonds.

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

3.

21. A method for producing a cell population comprising stable CD4+ T regulatory cells (T regs), comprising: a. Removal of CD8 from a biological sample obtained from a subject with an autoimmune disease + Cells, CD19 + cells and optionally CD14 + cells to produce depleted biological samples; b. enriching the CD25+ cells in the depleted biological sample to produce an enriched population; c. Isolation of CD4+CD25+CD127 from the enriched population - / lo cell; d. expanding the enriched population to produce an expanded enriched cell population; and e. quantifying the methylation status of the 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 low methylated TSDR at the FOXP3 locus, This generates a cell population containing stable CD4+ T regs.

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

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

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

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

26. The method of claim 25, wherein the activation comprises culturing the cell population with anti-CD3 antibodies and anti-CD28 antibodies.

27. The method of claim 25 or 26, wherein the second activation step is performed between 4 and 8 days after the first activation step.

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

29. The method of any one of claims 21 to 28, wherein the activation and expansion comprises culturing the cell population for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days.

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

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

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

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

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 comprising hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95% or more.

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

36. The method of claim 35, wherein the activation comprises culturing the cell population with an anti-CD3 antibody and an anti-CD28 antibody.

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

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

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

40. The method of any one of claims 31 to 39, wherein the amplified population comprises at least 1×10 7 An engineered stable CD4+ T reg.

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

43. The method of any one of claims 21 to 42, wherein 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 + cell.

44. The method of any one of claims 21 to 43, wherein 1% or less of the cell population are CD8+ cells.

45. The method of any one of claims 21 to 44, wherein 20% or less of the CD4+ T reg population are conventional T cells.

46. ​​The method according to any one of claims 21 to 45, wherein the cell population in step (c) comprises CD25 高 CD45RA - cells and CD25+CD45RA+ cells, and do not contain CD25 + CD45RA - cell.

47. The method of claims 21 to 46, wherein the separating of (c) comprises: (i) identifying CD4 + (ii) identifying CD25 + / 高 CD127 - / lo a second subpopulation of cells; and (iii) selecting CD25 高 CD45RA - and CD25 + / 高 CD45RA + Cells were isolated and CD25 + CD45RA - , thereby isolating the CD4+T reg population.

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

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

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

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

52. The method 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. The method of claim 52, wherein the polypeptide comprises an N-terminal TCRβ chain and a C-terminal TCRα chain.

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

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

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

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

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

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

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 a MND promoter.

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 WPRE-mut6.

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

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

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

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

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

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

69. The method of any one of claims 66 to 68, wherein the administration comprises one or more infusions.

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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