Cellular Therapy for Multiple Sclerosis

Abstract

The present disclosure relates to an isolated cell population comprising stable regulatory T cells and methods for producing the same. In some embodiments, disclosed herein is an isolated population of cells comprising stable CD4+ regulatory T cells (Tregs) derived from a subject with multiple sclerosis, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus. In some embodiments, the cells comprise an exogenous human T cell receptor (TCR) that specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 397,715, filed August 12, 2022, U.S. Provisional Application No. 63 / 412142, filed September 30, 2022, U.S. Provisional Application No. 63 / 415362, filed October 12, 2022, U.S. Provisional Application No. 63 / 417465, filed October 19, 2022, and U.S. Provisional Application No. 63 / 432304, filed December 13, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (file name: ABTH_003_04WO_SeqList_ST26.xml, file size: 129,941 bytes, created date: August 10, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Myelin is a critical component of a functioning central nervous system (CNS), enveloping and insulating nerve fibers, protecting them and allowing for the proper transmission of electrical signals. In the CNS of patients with multiple sclerosis, myelin is damaged by the body's own immune cells, thereby exposing nerve fibers. This myelin damage disrupts neural circuits within the brain, impeding communication between the brain and the rest of the body, and rendering nerves vulnerable to cell death and neurodegeneration, with long-term, often irreversible, effects. Progressive multiple sclerosis is characterized by increasing clinical disability independent of acute relapsing disease. There is no approved treatment for non-relapsing progressive MS. Human leukocyte antigen (HLA) DRB1 * The 1501 haplotype is strongly associated with MS and is currently the most common DRB1 haplotype in the United States. *It is estimated that at least 45,000 patients with the 1501 haplotype suffer from non-relapsing progressive MS. Regulatory T cells have the potential to treat this and other autoimmune diseases because they can selectively target affected cell types and tissues and generate local immune responses through antigen-specific mechanisms. Summary of the Invention [Means for solving the problem]

[0004] In some embodiments, disclosed herein is an isolated population of cells comprising stable CD4+ regulatory T cells (Tregs) from a subject with multiple sclerosis, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylation region (TSDR) in the FOXP3 locus. In some embodiments, the cells comprise an exogenous human T cell receptor (TCR) that specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC). In some embodiments, the MBP peptide is MBP83-99 peptide comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the MHC is HLA-DRB1 * In some embodiments, the MHC comprises HLA-DRA *01:01. In some embodiments, the exogenous TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a TCR beta chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the TCR alpha chain variable region comprises or consists of SEQ ID NO: 4. In some embodiments, the TCR alpha chain comprises an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 5. In some embodiments, the TCR alpha chain comprises or consists of SEQ ID NO: 5. In some embodiments, the TCR alpha chain comprises an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 73. In some embodiments, the TCR alpha chain comprises or consists of SEQ ID NO: 73. In some embodiments, the TCR alpha chain comprises an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 91. In some embodiments, the TCR alpha chain comprises or consists of SEQ ID NO: 91. In some embodiments, the TCR beta chain variable region comprises an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the TCR beta chain variable region comprises or consists of SEQ ID NO: 10. In some embodiments, the TCR beta chain comprises an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the TCR beta chain comprises or consists of SEQ ID NO: 11. In some embodiments, the TCR beta chain comprises an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 75. In some embodiments, the TCR beta chain comprises or consists of SEQ ID NO: 75.In some embodiments, the TCR β chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:93. In some embodiments, the TCR β chain comprises or consists of SEQ ID NO:93. In some embodiments, the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:17. In some embodiments, the single polypeptide comprises or consists of SEQ ID NO:17. In some embodiments, the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:79. In some embodiments, the single polypeptide comprises or consists of SEQ ID NO:79. In some embodiments, the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:97. In some embodiments, the single polypeptide comprises or consists of SEQ ID NO:97. In some embodiments, the single polypeptide comprises the amino acid sequence of SEQ ID NO:79 and is encoded by a nucleotide sequence having at least 90%, 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:87. In some embodiments, the exogenous TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR3 comprising the amino acid sequence of SEQ ID NO:20, and (B) a TCR beta chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:24, a CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a CDR3 comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:21. In some embodiments, the TCR alpha chain variable region comprises or consists of SEQ ID NO:21.In some embodiments, the TCR alpha chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the TCR alpha chain comprises or consists of SEQ ID NO: 22. In some embodiments, the TCR alpha chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 80. In some embodiments, the TCR alpha chain variable region comprises or consists of SEQ ID NO: 80. In some embodiments, the TCR beta chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 27. In some embodiments, the TCR beta chain variable region comprises or consists of SEQ ID NO: 27. In some embodiments, the TCR beta chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 28. In some embodiments, the TCR beta chain comprises or consists of SEQ ID NO: 28. In some embodiments, the TCR β chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 82. In some embodiments, the TCR β chain comprises or consists of SEQ ID NO: 82. In some embodiments, the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the single polypeptide comprises or consists of SEQ ID NO: 31. In some embodiments, the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the single polypeptide comprises or consists of SEQ ID NO: 86. In some embodiments, the single polypeptide comprises the amino acid sequence of SEQ ID NO: 86 and is encoded by a nucleotide sequence having at least 90%, 95%, or 100% identity to the nucleotide sequence of SEQ ID NO: 88.In some embodiments, the stable CD4+ Tregs do not express FOXP3 protein from the engineered FOXP3 locus. In some embodiments, the TSDR is the CNS2 region of FOXP3. In some embodiments, the MHC is MHC class I or MHC class II. In some embodiments, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells are stable CD4+ Tregs that contain a hypomethylated TSDR at the endogenous FOXP3 locus. 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 - / lo FOXP3 + In some embodiments, the isolated population is at least 4 x 10 7 In some embodiments, the isolated population comprises 4 x 10 stable CD4+ Tregs. 7 ~1×10 10 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+ Tregs. +T cells. In some embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the conventional T cells comprise an exogenous human TCR. In some embodiments, the ratio of stable CD4+ Tregs to conventional T cells in the isolated population is at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, at least 500:1, at least 1000:1, or at least 10000:1. In some embodiments, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated population are CD8 + In some embodiments, the isolated population does not contain a percentage of CD8+ T cells detectable by fluorescence-activated cell sorting (FACS). In some embodiments, at least 10% of the cells express an 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 an exogenous human TCR. In some embodiments, a stable CD4 T cell comprising a hypomethylated TSDR at the FOXP3 locus is expressed. + The percentage of Tregs decreases by no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% after a cryopreservation freeze-thaw cycle. +

[0013] The isolated cell population of any one of the preceding embodiments, wherein the Tregs exhibit one or more functions selected from (a) regulatory cytokine secretion activity, (b) expression of activation markers associated with regulatory T cells, and / or (c) suppressive activity. In some embodiments, the exogenous TCR is encoded as a single polypeptide, optionally comprising an N-terminal β domain and a C-terminal α domain. In some embodiments, the single polypeptide comprises a TCR α chain and a TCR β chain, wherein the polypeptide comprises a self-cleaving peptide sequence located between the TCR α chain and the TCR β chain. In some embodiments, the self-cleaving peptide sequence is a 2A peptide sequence, optionally the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence. In some embodiments, the exogenous TCR comprises a TCR α chain comprising a constant region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the exogenous TCR is at least 90%, 95%, or 100% homologous to the amino acid sequence of SEQ ID NO:60. In some embodiments, the exogenous human TCR comprises a TCR β chain comprising a constant region comprising an amino acid sequence having identity to a TCR α chain constant region and a TCR β chain constant region, wherein the cysteine residues are capable of forming one or more disulfide bonds. In some embodiments, the TCR α chain constant region comprises a T48C amino acid substitution relative to a TCR α chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and 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: 59 or 60. In some embodiments, the stable CD4+ Tregs have a human leukocyte antigen (HLA)-DR15 haplotype. In some embodiments, the stable CD4+ Tregs have an HLA-DRB1 haplotype. * In some embodiments, the stable CD4+ Tregs comprise the HLA-DRB5 allele. * Further includes the 01:01 allele.

[0005] In some embodiments, disclosed herein are stable CD4 + A method for producing a population of cells comprising regulatory T cells (Tregs), comprising: (a) isolating CD8 + cells, CD19 + cells, and optionally CD14 + (b) removing the cells to produce a depleted biosample; and (b) isolating the depleted biosample with CD25 + (c) enriching the enriched population for CD4 + CD25 + CD127 - / lo (d) isolating the enriched population to produce an enriched population of expanded cells; and (e) quantifying the methylation status of a T cell-specific demethylated region (TSDR) at the FOXP3 locus in the cell population, wherein at least 80% of the cells are stable CD4 T cells containing a hypomethylated TSDR at the FOXP3 locus. + Tregs, thereby stabilizing CD4 + and generating a cell population comprising Tregs. In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4 T cells comprising a hypomethylated TSDR at the FOXP3 locus. +The isolated population is selected for therapeutic use if the percentage of cells containing hypomethylated TSDR at the FOXP3 locus is 80% or greater. In some embodiments, step (c) is performed at least twice. In some embodiments, the method further comprises activating the population of cells of step (c). In some embodiments, activating comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the second activation step is performed 4 to 8 days after the first activation step. In some embodiments, the cells are expanded in a culture medium containing IL-2 and TNFα. In some embodiments, activating and expanding comprises culturing the population of cells for at least 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, activating and expanding comprises culturing the population of cells for no more than 15, 14, 13, or 12 days.

[0006] In some embodiments, disclosed herein are engineered stable CD4 + 1. A method for producing a population of cells comprising Tregs, comprising: (a) isolating CD8 + cells, CD19 + cells, and optionally CD14 + (b) removing the cells to produce a depleted biosample; and (b) isolating the depleted biosample with CD25 + (c) enriching the enriched population for CD4 + CD25 + CD127 - / lo(d) isolating the cells; (d) delivering a vector comprising a nucleic acid encoding an exogenous human T cell receptor (TCR) to the isolated population of (c) to produce a population of engineered cells; (e) expanding the population of engineered cells to produce an expanded population of engineered cells; and (f) quantifying the methylation status of a T cell-specific demethylated region (TSDR) at the FOXP3 locus in the expanded population of engineered cells, wherein at least 80% of the cells are stable CD4 T cells comprising a hypomethylated TSDR at the FOXP3 locus. + In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4 Tregs comprising a hypomethylated TSDR at the FOXP3 locus. + Tregs. In some embodiments, step (c) is performed at least twice. In some embodiments, the isolated population is selected for therapeutic use when the percentage of cells containing hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95%, or more. In some embodiments, the method further comprises activating the population of engineered cells of step (c). In some embodiments, activating comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the activation step is performed at least twice. In some embodiments, the second activation step is performed 4-8 days after the first activation step. In some embodiments, the cells are expanded in a culture medium containing IL-2 and TNFα. In some embodiments, the expanded population comprises at least 1 x 10 7 In some embodiments, the methylation status of the TSDR in step (f) is assessed at least 24 hours after a cryopreservation freeze-thaw cycle. In some embodiments, the CD4 + CD25 + / high CD127 - / lo The percentage of regulatory T cells is assessed at least 24 hours after the cryopreservation freeze-thaw cycle. In some embodiments, less than 5%, less than 4%, or less than 3% of the cells in the depleted biological sample are CD8+ cells, CD19 + cells, and / or CD14 + In some embodiments, the population of cells comprises CD8+ cells. In some embodiments, 2% or less of the population of cells are CD8+ cells. In some embodiments, 20% or less of the population of cells are conventional T cells. In some embodiments, the population of cells in step (c) comprises CD25 high CD45RA - Cells and CD25 + CD45RA + cells, including CD25 + CD45RA - In some embodiments, the isolating step (c) comprises isolating (i) CD4 + (ii) identifying a first subpopulation of cells; and (iii) isolating CD25 from the first subpopulation. + / high CD127 - / lo (iii) identifying a second subpopulation of cells; and (iv) isolating CD25 from the second subpopulation. high CD45RA - and CD25 + / high CD45RA + Select cells and CD25 + CD45RA - thereby excluding CD4 + In some embodiments, the TSDR is the CNS2 region of FOXP3. In some embodiments, the subject has a human leukocyte antigen (HLA)-DR15 haplotype. In some embodiments, the regulatory T cells are HLA-DRB1 *The human subject comprises a 15:01 allele. In some embodiments, the subject has progressive multiple sclerosis or relapsing-remitting multiple sclerosis. In some embodiments, the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis. In some embodiments, the human subject is a male subject. In some embodiments, the human subject is a female subject. In some embodiments, delivering a vector comprising a nucleic acid encoding an exogenous human TCR comprises transducing the vector into a cell population. In some embodiments, the exogenous TCR binds to an MBP83-99 peptide complexed with MHC. In some embodiments, the exogenous TCR is an exogenous TCR defined in any one of the above aspects or embodiments. In some embodiments, the exogenous human TCR is encoded as a single polypeptide comprising a TCR α chain and a TCR β chain. In some embodiments, the polypeptide comprises an N-terminal TCR β chain and a C-terminal TCR α chain. In some embodiments, the polypeptide comprises a self-cleaving peptide sequence located between the TCR α chain and the TCR β 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. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is a VSVg-pseudotyped, self-inactivating third-generation lentiviral vector. In some embodiments, the nucleic acid comprises a promoter operably linked to a coding sequence encoding an exogenous human TCR, optionally the promoter is an EF-1 alpha promoter or an MND promoter. In some embodiments, the nucleic acid further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally a WPRE-mut6. In some embodiments, the coding sequence is codon-optimized.

[0007] In some aspects, disclosed herein is an engineered polynucleotide encoding a polypeptide comprising a TCR alpha chain and a TCR beta chain, wherein (a) the TCR alpha chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) the TCR beta chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 4, and the TCR beta chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the TCR alpha chain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 73, and the TCR beta chain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 75. In some embodiments, the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 79. In some embodiments, the engineered polypeptide comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:87.

[0008] In some aspects, disclosed herein is an engineered polynucleotide encoding a polypeptide comprising a TCR alpha chain and a TCR beta chain, wherein (a) the TCR alpha chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and (b) the TCR beta chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 21, and the TCR beta chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 27. In some embodiments, the TCR alpha chain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 80, and the TCR beta chain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 82. In some embodiments, the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 86. In some embodiments, the engineered polypeptide comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:88.

[0009] In some aspects, disclosed herein is an expression cassette comprising the polynucleotide of any one of the preceding aspects or embodiments operably linked to a promoter, wherein optionally the promoter is an EF-1α promoter or an MND promoter. In some embodiments, the expression cassette further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally a WPRE-mut6.

[0010] In some aspects disclosed herein, a vector comprises an expression cassette of any of the above aspects or embodiments. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is a VSVg-pseudotyped self-inactivating third-generation lentiviral vector.

[0011] In some aspects, disclosed herein is an engineered polypeptide comprising a human TCR alpha chain and a human TCR beta chain, wherein (a) the TCR alpha chain is at least 95% identical to SEQ ID NO: 73 and the TCR beta chain is at least 95% identical to SEQ ID NO: 75, or (b) the TCR alpha chain is at least 95% identical to SEQ ID NO: 80 and the TCR beta chain is at least 95% identical to SEQ ID NO: 82. In some embodiments of the engineered polypeptide, (a) the TCR alpha chain comprises or consists of SEQ ID NO: 73 and the TCR beta chain comprises or consists of SEQ ID NO: 75, or (b) the TCR alpha chain comprises or consists of SEQ ID NO: 80 and the TCR beta chain comprises or consists of SEQ ID NO: 82. In some embodiments, the engineered polypeptide comprises, from N-terminus to C-terminus, a TCR beta chain, a self-cleaving peptide sequence, and a TCR alpha chain. In some embodiments, the self-cleaving peptide is a P2A peptide sequence. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 79 or 86. In some embodiments, the engineered polypeptide comprises an amino acid sequence that comprises or consists of SEQ ID NO: 79 or 86.

[0012] In some aspects, disclosed herein is a pharmaceutical composition comprising the isolated population of any one of the preceding aspects or embodiments and a pharmaceutically acceptable excipient.

[0013] In some aspects, disclosed herein is a pharmaceutical composition comprising the isolated population of any one of the preceding aspects or embodiments and a cryopreservative.

[0014] In some aspects, disclosed herein are methods of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of any of the above aspects or embodiments or an isolated population of any one of the above aspects or embodiments. In some embodiments, the pharmaceutical composition is administered in an amount effective to alleviate one or more symptoms of multiple sclerosis. In some embodiments, the administering comprises intravenous administration. In some embodiments, the administering comprises one or more injections. In some embodiments, the cells of the isolated population are autologous to the subject. In some embodiments, the subject has progressive multiple sclerosis or relapsing-remitting multiple sclerosis. In some embodiments, the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis. [Brief explanation of the drawings]

[0015] [Figure 1A] An exemplary method for producing a cell population containing stable regulatory T cells is provided. Figure 1A shows a schematic diagram of the overall process for isolating regulatory T cells with a CD4+CD45RA+CD25+ / highCD127lo / - phenotype. Figure 1B shows data illustrating a fluorescence-activated cell sorting (FACS) strategy for enriching a cell population for cells with a CD45RA+CD4+CD25+CD127lo / - phenotype from a CD25-enriched sample to produce an isolated cell population containing stable regulatory T cells. As shown in Figure 1A, this sorting strategy is used in two consecutive steps to achieve a high level of purity. [Figure 1B]An exemplary method for producing a cell population containing stable regulatory T cells is provided. Figure 1A shows a schematic diagram of the overall process for isolating regulatory T cells with a CD4+CD45RA+CD25+ / highCD127lo / - phenotype. Figure 1B shows data illustrating a fluorescence-activated cell sorting (FACS) strategy for enriching a cell population for cells with a CD45RA+CD4+CD25+CD127lo / - phenotype from a CD25-enriched sample to produce an isolated cell population containing stable regulatory T cells. As shown in Figure 1A, this sorting strategy is used in two consecutive steps to achieve a high level of purity. [Figure 2] 1 shows a graph depicting the recovery of regulatory T cells after depletion and CD25 enrichment steps in an exemplary method for producing an isolated cell population comprising stable regulatory T cells. [Figure 3A] 1 shows data showing the relative abundance of various cell phenotypes after depletion and CD25 enrichment steps in an exemplary method for producing an isolated cell population comprising stable regulatory T cells. [Figure 3B] 1 shows data showing the relative abundance of various cell phenotypes after depletion and CD25 enrichment steps in an exemplary method for producing an isolated cell population comprising stable regulatory T cells. [Figure 4] Graph showing proliferation of cells after transduction with exogenous TCR. Non-transduced cells ("non-transduced") are included as a control. [Figure 5-1] 1 shows a graph depicting regulatory T cell phenotype and TCR expression in cell populations produced by exemplary methods of the present disclosure. [Figure 5-2] 1 shows a graph depicting regulatory T cell phenotype and TCR expression in cell populations produced by exemplary methods of the present disclosure. [Figure 6]

[0023] Figure 1 shows data demonstrating that regulatory T cells in cell populations produced by exemplary methods of the present disclosure maintain hypomethylated T cell-specific demethylated regions (TSDRs). [Figure 7] 1 shows a graph illustrating activation of lentiviral-transduced regulatory T cells of the present disclosure after cryopreservation freeze-thaw cycles. [Figure 8]Graph of the gating strategy for the purification of the CD4+ / CD45RA+CD127- / lowCD25+ cell population is shown. [Figure 9] Figure 10 shows the percent TSDR hypomethylation of different runs refined by the gating strategy shown in Figure 8 . [Figure 10A] Expression of CTLA-4 (Figure 10A), CD69 (Figure 10B), TGFβ-1 (Figure 10C), and IL-10 (Figure 10D) after incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 10B] Expression of CTLA-4 (Figure 10A), CD69 (Figure 10B), TGFβ-1 (Figure 10C), and IL-10 (Figure 10D) after incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 10C] Expression of CTLA-4 (Figure 10A), CD69 (Figure 10B), TGFβ-1 (Figure 10C), and IL-10 (Figure 10D) after incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 10D] Expression of CTLA-4 (Figure 10A), CD69 (Figure 10B), TGFβ-1 (Figure 10C), and IL-10 (Figure 10D) after incubation of cell populations in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles is shown. [Figure 11] Shows the change in percent TSDR hypomethylation over time for growth of three different MS donors. [Figure 12A] Population doubling levels (FIGS. 12A and 12B) and percent TSDR hypomethylation for CD45RA+ and CD45RA- cells over 8 days of expansion are shown. The spike-in demonstrates that CD45RA+ can expand within the CD45RA- cell population. [Figure 12B]Population doubling levels (FIGS. 12A and 12B) and percent TSDR hypomethylation for CD45RA+ and CD45RA- cells over 8 days of expansion are shown. The spike-in demonstrates that CD45RA+ can expand within the CD45RA- cell population. [Figure 12C] Population doubling levels (FIGS. 12A and 12B) and percent TSDR hypomethylation for CD45RA+ and CD45RA- cells over 8 days of expansion are shown. The spike-in demonstrates that CD45RA+ can expand within the CD45RA- cell population. [Figure 13A] The gating strategy for CD25+CD127- / loCD45- and CD25+ / highCD127-loCD45- selection is shown. [Figure 13B] The gating strategy for CD25+CD127- / loCD45- and CD25+ / highCD127-loCD45- selection is shown. [Figure 13C] The gating strategy for CD25+CD127- / loCD45- and CD25+ / highCD127-loCD45- selection is shown. [Figure 14A] The histogram approach shown in Figures 13A-13C is shown. [Figure 14B] The histogram approach shown in Figures 13A-13C is shown. [Figure 14C] The histogram approach shown in Figures 13A-13C is shown. [Figure 14D] The histogram approach shown in Figures 13A-13C is shown. [Figure 14E] The histogram approach shown in Figures 13A-13C is shown. [Figure 15] Percent TSDR hypomethylation over the course of expansion for the same donor based on different selection strategies is shown. [Figure 16] Cell proliferation after restimulation on days 6, 8, or 10 is shown. [Figure 17] The effect of TNF-α, a second stimulation, or both on fold proliferation is shown. [Figure 18A] The viability and purity of cells expanded by both TNF-α and a second stimulus are shown. [Figure 18B] The viability and purity of cells expanded by both TNF-α and a second stimulus are shown. [Figure 19A] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19B] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19C] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19D] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 19E] FIG. 13 shows an alternative histogram approach for the gating strategy shown in FIGS. 13A-13C. [Figure 20] Percent TSDR hypomethylation over the course of expansion for the same donor based on different selection strategies is shown. [Figure 21] Percent TSDR hypomethylation for three different donors based on the gating strategies shown in Figures 13A-13C and 19A-19E is shown. [Figure 22A] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 22B] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 22C] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 22D] Shown are the percentage TSDR hypomethylation and percentage FOXP3+ cells by proportion of Tregs in the initial population based on two studies. [Figure 23-1] The percentage of FOXP3+ and FOXP3- cells that expressed IL-2 and IFN-γ after activation with PMA and ionomycin is shown. [Figure 23-2] The percentage of FOXP3+ and FOXP3- cells that expressed IL-2 and IFN-γ after activation with PMA and ionomycin is shown. [Figure 24] Expression of CD3, cMYC, and GFP in transduced cells is shown. [Figure 25A] The percentage of cells that were positive for CD3 (Figure 25A), GFP (Figure 25B), and cMYC (Figure 25C) after transduction with different TCR constructs is shown. [Figure 25B] The percentage of cells that were positive for CD3 (Figure 25A), GFP (Figure 25B), and cMYC (Figure 25C) after transduction with different TCR constructs is shown. [Figure 25C] The percentage of cells that were positive for CD3 (Figure 25A), GFP (Figure 25B), and cMYC (Figure 25C) after transduction with different TCR constructs is shown. [Figure 26] 1 shows a graph demonstrating the ability of lentiviral-transduced null Jurkat cells to express MPB-specific TCR on the cell surface. [Figure 27A] 1 shows a graph depicting activation of lentivirally transduced Jurkat cells. [Figure 27B] 1 shows a graph depicting activation of lentivirally transduced Jurkat cells. [Figure 27C] 1 shows a graph depicting activation of lentivirally transduced Jurkat cells. [Figure 28A] 1 shows a graph depicting activation of lentivirally transduced conventional T cells. [Figure 28B] 1 shows a graph depicting activation of lentivirally transduced conventional T cells. [Figure 28C] 1 shows a graph depicting activation of lentivirally transduced conventional T cells. [Figure 29A]1 shows a graph depicting activation of lentivirally transduced regulatory T cells of the present disclosure. [Figure 29B] 1 shows a graph depicting activation of lentivirally transduced regulatory T cells of the present disclosure. [Figure 29C] 1 shows a graph depicting activation of lentivirally transduced regulatory T cells of the present disclosure. [Figure 30A] 1 shows a graph demonstrating the ability of lentiviral-transduced regulatory T cells of the present disclosure to suppress conventional T cells. [Figure 30B] 1 shows a graph demonstrating the ability of lentiviral-transduced regulatory T cells of the present disclosure to suppress conventional T cells. [Figure 30C] 1 shows a graph demonstrating the ability of lentiviral-transduced regulatory T cells of the present disclosure to suppress conventional T cells. [Figure 30D] 1 shows a graph demonstrating the ability of lentiviral-transduced regulatory T cells of the present disclosure to suppress conventional T cells. [Figure 31A] 1 shows the suppression of TCR-A expressing polyclonal conventional T cells by TCR-A expressing regulatory T cells. [Figure 31B] 1 shows the suppression of TCR-A expressing polyclonal conventional T cells by TCR-A expressing regulatory T cells. [Figure 31C] 1 shows the suppression of TCR-A expressing polyclonal conventional T cells by TCR-A expressing regulatory T cells. [Figure 32A] Graph showing activation of null genotypes transduced with engineered TCRs. [Figure 32B] Graph showing activation of null genotypes transduced with engineered TCRs. [Figure 33A] 1 shows a graph depicting activation of conventional T cells transduced with engineered TCRs. [Figure 33B] 1 shows a graph depicting activation of conventional T cells transduced with engineered TCRs. [Figure 33C] 1 shows a graph depicting activation of conventional T cells transduced with engineered TCRs. [Figure 34A]Figure 18 shows expression data for cells expressing engineered TCRs. Figure 18A shows flow cytometry data for Vβ2 expression and CD69 expression. Figures 18B-18C show quantification of Vβ2 expression in cells expressing engineered TCRs. [Figure 34B] Figure 18 shows expression data for cells expressing engineered TCRs. Figure 18A shows flow cytometry data for Vβ2 expression and CD69 expression. Figures 18B-18C show quantification of Vβ2 expression in cells expressing engineered TCRs. [Figure 34C] Figure 18 shows expression data for cells expressing engineered TCRs. Figure 18A shows flow cytometry data for Vβ2 expression and CD69 expression. Figures 18B-18C show quantification of Vβ2 expression in cells expressing engineered TCRs. [Figure 35A] Graphs showing activation (Figures 35A-B) and Vβ2 expression (Figure 35C) for Tregs expressing engineered MBP-specific TCRs are shown. Figures 35A and 35B show graphs showing activation of Tregs expressing engineered MBP-specific TCRs when contacted with BLS2b cells (B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and MBP83-99 peptide. Tregs in Figure 35A express TCR-E engineered v1, TCR-E engineered v2, or TCR-A. Tregs in Figure 35B express TCR-F engineered v1, TCR-F engineered v2, or TCR-B. Expression of the activation marker (CD69) increased with increasing doses of MBP83-99 peptide. Figure 35C shows the percentage of Tregs expressing MBP-specific TCRs that express Vβ2 under unstimulated conditions. [Figure 35B]Graphs showing activation (Figures 35A-B) and Vβ2 expression (Figure 35C) for Tregs expressing engineered MBP-specific TCRs are shown. Figures 35A and 35B show graphs showing activation of Tregs expressing engineered MBP-specific TCRs when contacted with BLS2b cells (B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and MBP83-99 peptide. Tregs in Figure 35A express TCR-E engineered v1, TCR-E engineered v2, or TCR-A. Tregs in Figure 35B express TCR-F engineered v1, TCR-F engineered v2, or TCR-B. Expression of the activation marker (CD69) increased with increasing doses of MBP83-99 peptide. Figure 35C shows the percentage of Tregs expressing MBP-specific TCRs that express Vβ2 under unstimulated conditions. [Figure 35C] Graphs showing activation (Figures 35A-B) and Vβ2 expression (Figure 35C) for Tregs expressing engineered MBP-specific TCRs are shown. Figures 35A and 35B show graphs showing activation of Tregs expressing engineered MBP-specific TCRs when contacted with BLS2b cells (B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and MBP83-99 peptide. Tregs in Figure 35A express TCR-E engineered v1, TCR-E engineered v2, or TCR-A. Tregs in Figure 35B express TCR-F engineered v1, TCR-F engineered v2, or TCR-B. Expression of the activation marker (CD69) increased with increasing doses of MBP83-99 peptide. Figure 35C shows the percentage of Tregs expressing MBP-specific TCRs that express Vβ2 under unstimulated conditions. [Figure 36A] Graphs are shown showing activation of suppressive capacity (measured by 4-1BB expression) in engineered TCR-transduced regulatory T cells when contacted with BLS2b cells (B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and MBP83-99 peptide. [Figure 36B]Graphs are shown showing activation of suppressive capacity (measured by 4-1BB expression) in engineered TCR-transduced regulatory T cells when contacted with BLS2b cells (B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and MBP83-99 peptide. [Figure 37A] Figure 1 shows the ability of TCR-Ev2-transduced T cells to suppress conventional T cells. [Figure 37B] Figure 1 shows the ability of TCR-Ev2-transduced T cells to suppress conventional T cells. [Figure 37C] Figure 1 shows the ability of TCR-Ev2-transduced T cells to suppress conventional T cells. [Figure 38] 1 shows a graph illustrating the stability of regulatory T cells of the present disclosure in the presence of pro-inflammatory cytokines. Cells were grown in the presence of 1,000 units / mL of IL-2 and a mixture of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, IL-17A, IFN-β, IFN-γ, TGF-β1, and TNF-α) at concentrations of 1 to 10,000 pg / mL each. The percentage of regulatory T cells with CD4+ / FOXP3+ expression remained constant for at least 23 days in the presence of pro-inflammatory cytokines (up to 1,000 pg / mL of each cytokine in the mixture). [Figure 39A] Shown are the percentage of FOXP3+ cells and the percentage of TSDR hypomethylation after incubation with the cytokine mixture used in Figure 38 (Mix-8) or IL-1β, IL-6, IL-12, IL-17A, IFNγ, and TNF-α (Mix-6). [Figure 39B] Shown are the percentage of FOXP3+ cells and the percentage of TSDR hypomethylation after incubation with the cytokine mixture used in Figure 38 (Mix-8) or IL-1β, IL-6, IL-12, IL-17A, IFNγ, and TNF-α (Mix-6). [Figure 40] 1 provides a schematic diagram of the human / mouse hybrid MBP-targeting T cell receptor (TCR) of the present disclosure. [Figure 41]Provide an EAE testing protocol. [Figure 42] We provide flow cytometry data demonstrating that human / mouse hybrid TCRs are highly expressed in mouse regulatory T cells (Tregs) after transduction. [Figure 43A] We provide data demonstrating that mouse FOXP3-GFP+ donor Tregs transduced with a human / mouse hybrid TCR are enriched in the central nervous system (CNS) of recipient mice experiencing active EAE. [Figure 43B] We provide data suggesting that murine FOXP3-GFP+ donor Tregs transduced with a human / mouse hybrid TCR can influence EAE disease severity. DETAILED DESCRIPTION OF THE INVENTION

[0016] overview The present disclosure provides methods and compositions for treating multiple sclerosis through the generation and use of stable regulatory T cells. The present disclosure also provides methods and compositions for treating multiple sclerosis through the generation and use of engineered stable regulatory T cells comprising myelin basic protein (MBP)-specific T cell receptors (TCRs). Such engineered regulatory T cells can specifically target distinct cell types and tissues associated with degraded myelin, for example, to prevent immune-mediated destruction, restore homeostasis, and promote repair in affected tissues. In addition, the engineered regulatory T cells of the present disclosure suppress local inflammation in the target and do not cause systemic immunosuppression. Furthermore, the engineered regulatory T cells described herein are stable (e.g., committed to a Treg phenotype) thymic-derived regulatory T cells that can persist in vivo for extended periods and may provide therapeutic benefit for months or years after a single administration. These stable regulatory T cells are also, in some embodiments, resistant to pro-inflammatory triggers (e.g., pro-inflammatory cytokines). In some embodiments, the stable regulatory T cells of the present disclosure are autologous, meaning that they are obtained directly from the patient's own cells, engineered to express an exogenous antigen-specific TCR, and then administered back to the patient. The use of autologous cells minimizes the risk of rejection by the patient (e.g., graft-versus-host disease).

[0017] Isolating a highly stable and pure population of engineered regulatory T cells produced in the thymus and obtaining a sufficiently large and pure population of the engineered regulatory T cells to treat patients has proven difficult because the population of stable thymic regulatory T cells is very small relative to the total population of lymphocytes and because there are no cell surface markers associated exclusively with regulatory T cells. Although isolated polyclonal regulatory T cells have proven effective in clinical treatment, when engineering regulatory T cells with exogenous TCRs, a higher level of purity and stability is required because sufficient numbers of exogenous TCR-engineered conventional T cells and unstable regulatory T cells (i.e., peripheral Tregs) can have deleterious effects by triggering rather than suppressing immune responses at the site of autoimmune disease.

[0018] These stably engineered regulatory T cells and methods of production offer advantages over some current methods of producing regulatory T cells for cell therapy. Alternative approaches have been used to generate populations of engineered regulatory T cells or regulatory-like T cells, but these cells have drawbacks compared to the populations of cells described herein.

[0019] One possible approach is to increase the proportion of regulatory T cells by driving FOXP3 expression through manipulation of conventional T cells or induced stem cells, either through manipulation of the FOXP3 promoter or through the introduction of an ectopic FOXP3 gene into the cells. FOXP3 expression confers a regulatory-like phenotype. However, FOXP3 is only one of several genes with upregulated expression in stable regulatory T cells, and these regulatory-like T cells have been shown to differ from natural Tregs. Indeed, when CD4+ T cells were edited to exhibit a Treg-like phenotype by introducing an exogenous promoter upstream of FOXP3, the engineered cells were shown to have reduced suppressive capacity compared to natural Tregs (Buckner, Science Translational Medicine, 2022), suggesting that these cells would not exhibit the same level of efficacy as true stable regulatory T cells when administered in a clinical setting.

[0020] Conversion of conventional CD4+ T cells has also been attempted, for example, by stimulation with TGF-β and IL-2, retinoic acid, short-chain fatty acids, and TGF-β, or rapamycin. However, these induced regulatory T cells lack the epigenetic changes associated with stable regulatory T cells, particularly regulatory T cell-specific demethylation of the conserved noncoding sequence 2 (CNS2) of the FOXP3 gene, and are therefore functionally unstable and do not retain a stable phenotype when administered in a clinical setting. Furthermore, proteomic analysis has shown that these induced Treg-like cells show little overlap in protein expression profiles with stable thymic Tregs and instead share signaling and metabolic proteins with conventional T cells (Mensink et al., Sci Rep. 2022).

[0021] Contemplated herein are populations of cells and strategies for isolating highly purified and stable thymic regulatory T cells engineered with an exogenous TCR, of sufficient purity and in sufficient numbers to be administered to a subject in therapeutic amounts. In some embodiments, the optimal population of regulatory T cells is one that expresses CD25 T cells after isolation and prior to introduction of an exogenous TCR. + / high CD4 + CD127 - / lo CD45RA + , as this phenotype is associated with thymic naive regulatory T cells. In some embodiments, the optimal population of regulatory T cells is CD25 T cells after isolation and prior to introduction of an exogenous TCR. high CD4 + CD127 - / lo These cells further comprise CD45RA, as this phenotype is associated with thymic antigen-experienced regulatory T cells. These cells also exhibit a stable TSDR phenotype after introduction of an exogenous TCR, indicating no expansion of non-regulatory T cell subpopulations or loss of regulatory T cell stability (e.g., no change in cell fate—cells remain terminally differentiated).

[0022] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document.

[0023] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0024] Also, unless expressly indicated to the contrary, it is to be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0025] In the claims and the above specification, all transitional phrases such as "comprise," "including," "carry," "have," "include," "involving," "hold," "comprise," and the like, are to be understood to be open-ended, i.e., to mean including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" must be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedure.

[0026] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0027] Where a range of values is provided, each value between and including the upper and lower endpoints of the range is specifically contemplated and described herein.

[0028] regulatory T cells In some embodiments, the present disclosure provides stable CD4 + Provided are isolated populations of cells, including regulatory T cells, as well as compositions thereof. These isolated populations may be used, for example, to treat multiple sclerosis in a subject in need thereof.

[0029] The terms "regulatory T cells," "T regulatory cells," and "Tregs" are used interchangeably herein and are intended to refer to other cell populations of the immune system (e.g., conventional CD4 + T cells, effector CD8 + Regulatory T cells are T cells that suppress the effector functions of T cells (e.g., T cells, antigen-presenting cells, and / or granulocytes). Regulatory T cells are defined by the expression of the cell surface markers CD4 and CD25, as well as the expression of the transcription factor FOXP3. Furthermore, regulatory T cells do not express, or express at low levels, the cell surface marker CD127. Thus, regulatory T cells have the following protein expression profile: CD4+ CD25 high / + CD127 low / - FOXP3 + It is characterized by:

[0030] Throughout this disclosure, expression of the indicated proteins by a cell or population of cells is indicated by a "+" (e.g., CD4 + ), "-" (e.g., CD127 - ), "high" (e.g., CD25 High ), "int" or "intermediate" (e.g., CD25 int ), "low" (e.g., CD127 low ), "High / +" (e.g., CD25 High / + ), or "low / -" (e.g., CD127 low / - ) As used herein, various expression indices refer to the presence or absence (e.g., "+" or "-", respectively) of the indicated protein, or the relative level of protein expression as measured by conventional protein expression assays (e.g., flow cytometry, fluorescence activated cell sorting (FACS), or Western blot). Unless otherwise indicated, protein expression described throughout this application is determined by FACS. (a) Positive ( + ) indicator refers to the detectable level of expression of the indicated protein by FACS. Positive ( + ) may be further divided into populations of "low" and / or "high" subpopulations. (b) A subpopulation of "low" cells expresses the indicated protein, but at a lower level (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower) than other cells in the population, or at a lower level (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower), e.g., CD4 + express the indicated proteins at levels lower than expression in control cell populations (compared to conventional T cells or CD8+ effector T cells). (c) A subpopulation of "high" cells has a higher level (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher) or (e.g., CD4 + express the indicated proteins at levels higher than expression in control cell populations (compared to conventional T cells or CD8+ effector T cells). (d) Negative ( - ) indication refers to the absence of expression of the indicated protein, or an expression level of the indicated protein that is below the limit of detection for a particular detection assay (e.g., below the limit of detection for a particular fluorescent antibody and / or flow cytometer). (e) " - / Low The designation " refers to a cell population that includes cells that are (-) for the indicated protein and cells that express "low" levels of the indicated protein. (f) + / High The designation " refers to a cell population containing cells that are (+) for the indicated protein, including cells that express high levels of the indicated protein.

[0031] In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + / high In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD4 + In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD45RA + In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are FOXP3 + is.

[0032] In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + / high CD4 + CD127 - / lowRegulatory T cells are cells that express CD25 or express it at high levels, express CD4, and do not express CD127 or express it at low levels. In some embodiments, regulatory T cells do not express CD127. In some embodiments, regulatory T cells (e.g., stable regulatory T cells) express CD25 + / high CD4 + CD127 - / low CD45RA + Regulatory T cells are cells. Thus, regulatory T cells express CD25 or express it at high levels, express CD4 and CD45RA, and do not express CD127 or express it at low levels. In some embodiments, regulatory T cells express FOXP3. In some embodiments, regulatory T cells express CD25 + / high CD4 + CD127 - / low FOXP3 + Thus, regulatory T cells express CD25 or express it at high levels, express CD4 and FOXP3, and do not express CD127 or express it at low levels. In some embodiments, regulatory T cells express CD25 + / high CD4 + CD45RA+CD127 - / low FOXP3 + Thus, regulatory T cells express CD25 or express high levels of CD25, CD4, CD45RA, and FOXP3, and do not express CD127 or express low levels of CD127.

[0033] During thymic regulatory T cell development, the genome organizer SATB1 (special AT-rich sequence-binding protein) mediates CD4 expression to open chromatin and activation super-enhancers associated with many regulatory T cell signature genes, such as FOXP3, IL2RA, (CD25), CTLA4, IKZF2 (HELIOS), and IFZF4 (EOS). + CD8 +Binding to specific genomic sites from the thymus stage. SATB1 and MLL4 (myeloid / lymphoid or mixed lineage leukemia 4), enzymes involved in enhancer priming, generally occupy a newly identified conserved enhancer region in the FOXP3 locus, termed conserved noncoding sequence 0 (CNS0), followed by activation of enhancers and then promoters in CNS3 and CNS2. This results in stable hypomethylation and expression of FOXP3 and other regulatory T cell-related genes, thereby resulting in a stable regulatory T cell phenotype (Piotrowska, et al.; Int J Mol Sci. 2021).

[0034] After hematopoietic progression, populations of T cells can transition between one phenotype and another under various conditions (see, e.g., Kitagawa et al., Nat Immunol. 2017 Feb;18(2):173-183). As used herein, "stable regulatory T cells" are regulatory T cells that contain a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus. In some embodiments, stable regulatory T cells are further defined by expression of CD4, CD25, and / or FOXP3. Tregs derived from the thymus (e.g., thymic Tregs) are stable, and regulatory T cells that can no longer transition between T cell phenotypes due to changes in the methylation status of one or more loci in the FOXP3 locus are also considered stable. In contrast, peripheral Tregs, which are not stable and can be induced in response to pro-inflammatory conditions, do not exhibit stable hypomethylation of the TSDR region of the FOXP3 locus. Hypomethylation of the TSDR, an evolutionarily conserved CpG-rich regulatory element of the FOXP3 gene, is associated with FOXP3 expression. The TSDR of the endogenous FOXP3 locus is hypomethylated when one or more methylated cytosines in the TSDR are substituted with cytosine by removing the methyl group. Therefore, the stability of regulatory T cells is determined by the presence of a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus.

[0035] In some embodiments, measuring the methylation status of the TSDR of the FOXP3 locus is as described in Kressler et al., "Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Regulatory T Phenotype," Frontiers in Immunology, January 7, 2021; or Schreiber, et al., "The Regulatory T-Specific Demethylated Region Stabilizes Foxp3 Expression Independent of NF-κB Signaling," PLOS One, February 5, 2014. In some embodiments, the TSDR of the FOXP3 locus is selected from conserved non-coding sequence 0 (CNS0), CNS3, and CNS2. In some embodiments, the TSDR of the FOXP3 locus is CNS2.

[0036] In some embodiments, stable regulatory T cells maintain a hypomethylated T-regulatory cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus in the presence of pro-inflammatory conditions (e.g., in the presence of one or more pro-inflammatory cytokines). In some embodiments, stable regulatory T cells comprise a hypomethylated T-regulatory cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus in the presence of pro-inflammatory conditions for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, or 25 days.

[0037] In some embodiments, the stable regulatory T cells further exhibit one or more of the following functions: (i) regulatory cytokine secretion activity (e.g., secretion of IL-10, TGFβ, and IL-35); (ii) expression or activation markers associated with regulatory T cells (e.g., expression of CD69, 4-1BB, CD25, CD71, and / or CTLA-4); and / or (iii) suppressive activity (e.g., the ability of the stable regulatory T cells to suppress the activation and / or proliferation of other effector cells of the immune system).

[0038] Thus, in some embodiments, the isolated cell populations provided herein comprise stable regulatory T cells that comprise expression of a hypomethylated TSDR of the FOXP3 locus, CD4, CD25, and FOXP3, and / or exhibit cytokine secretion, activation, and / or suppressive activity.

[0039] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after isolation from a biological sample. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after isolation from a biological sample. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for 1 to 20 days, 1 to 10 days, 1 to 5 days, 5 to 30 days, 5 to 20 days, 10 to 40 days, or 25 to 50 days after isolation from the biological sample.

[0040] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after transduction with a nucleic acid encoding an exogenous TCR. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with a nucleic acid encoding an exogenous TCR. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after transduction with a nucleic acid encoding an exogenous TCR. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the locus for 1 to 20 days, 1 to 10 days, 1 to 5 days, 5 to 30 days, 5 to 20 days, 10 to 40 days, or 25 to 50 days after transduction with a nucleic acid encoding an exogenous TCR.

[0041] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain hypomethylated TSDR at the FOXP3 locus over time after cryopreservation. For example, in some embodiments, the isolated cell populations described herein are cryopreserved and then thawed for use and / or analysis, which is referred to herein as cryopreservation freeze-thaw cycling. In such embodiments, the stable regulatory T cells of the isolated cell population maintain hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after cryopreservation freeze-thaw cycling. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after cryopreservation freeze-thaw cycling. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the locus for 1-20 days, 1-10 days, 1-5 days, 5-30 days, 5-20 days, 10-40 days, or 25-50 days after a cryopreservation freeze-thaw cycle.

[0042] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after administration to a subject. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after administration to a subject. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after administration to a subject. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for 1 to 20 days, 1 to 10 days, 1 to 5 days, 5 to 30 days, 5 to 20 days, 10 to 40 days, or 25 to 50 days after administration to a subject.

[0043] In some embodiments, the isolated population described herein is an autologous cell population.In this context, the term autologous refers to the cells obtained from the same subject that they are subsequently administered to.For example, a cell population can be obtained from a subject, subjected to the method described herein, and then administered to the same subject (the subject from whom the cell population was originally obtained) to treat multiple sclerosis.In such embodiments, the cell population administered to a subject comprises autologous regulatory T cells.

[0044] In some embodiments, the isolated population described herein is an allogeneic cell population.In this context, the term allogeneic refers to the cells obtained from one subject and then administered to another subject.For example, a cell population can be obtained from a subject, subjected to the method described herein, and then administered to another subject to treat multiple sclerosis.

[0045] In some embodiments, the isolated population of cells described herein (e.g., an isolated population comprising stable CD4+ regulatory T cells) is isolated from a biological sample obtained from a subject diagnosed with or suspected of having multiple sclerosis.

[0046] Exogenous TCR and manipulated regulatory T cells In some embodiments, the present disclosure provides an isolated population of cells comprising regulatory T cells (e.g., stable regulatory T cells) that comprise an exogenous human T cell receptor (TCR) that binds to a target peptide. This binding occurs when the target peptide is complexed with a major histocompatibility complex (MHC) (e.g., MHC class I or MHC class II). Such populations are referred to herein as "engineered regulatory T cells" or "stable engineered regulatory T cells."

[0047] T cell receptors (TCRs) are transmembrane heterodimers containing α and β chains linked by disulfide bonds. Within these chains are complementarity-determining regions (CDRs), which determine the target peptide to which the TCR binds. TCRs activate T cells that contain them, resulting in an exaggerated immune response. Antigen-presenting cells digest specific proteins (antigens) and present their fragments (peptides) on major histocompatibility complexes (MHC). This peptide-MHC (pMHC) complex binds to the TCR, while other costimulatory molecules are activated, leading to T cell activation, proliferation, differentiation, apoptosis, or cytokine release.

[0048] An exogenous TCR can be any TCR introduced into a regulatory T cell, where the TCR is not endogenous to the regulatory T cell (i.e., naturally occurring in the regulatory T cell). For example, in some embodiments, the exogenous TCR is encoded by a nucleic acid that is not endogenous to the regulatory T cell (i.e., not naturally occurring in the genome of the regulatory T cell). In some embodiments, the nucleic acid is an engineered nucleic acid, e.g., a recombinant or synthetic nucleic acid.

[0049] A TCR specifically binds to a target peptide complexed with MHC. A TCR is considered to "specifically" bind to a target peptide complexed with MHC if the TCR has a higher binding affinity for a non-target peptide complexed with MHC compared to the target peptide complexed with MHC. A TCR binds to a target peptide complexed with MHC with at least 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M (e.g., 10 -4 M~10 -10M) with a binding affinity of 100-150%. In some embodiments, a TCR is considered to "specifically" bind a target peptide complexed with MHC when a T cell expressing the TCR is activated or more highly activated upon contact with the target peptide complexed with MHC (e.g., as assessed by increased CD69 expression) compared to a non-target peptide complexed with MHC. In some embodiments, the peptide is presented by a cell expressing MHC.

[0050] The exogenous TCR may be a human TCR. In some embodiments, the TCR is from a monkey, a mouse, a rat, or any other animal.

[0051] The target peptide may be a peptide associated with multiple sclerosis. In some embodiments, the target peptide associated with multiple sclerosis may be a peptide belonging to myelin basic protein (MBP) (e.g., MBP(83-99)). In some embodiments, the target peptide is a peptide that is overexpressed in a population of cells associated with multiple sclerosis compared to a control (e.g., compared to a population of cells not associated with multiple sclerosis).

[0052] In some embodiments, the target peptide is a peptide that is likely to increase the autoimmune response in subjects with multiple sclerosis compared to controls. In some embodiments, the target peptide is a peptide that is present at a site of autoimmune disease in a subject compared to an unaffected site in the same subject. In some embodiments, the target peptide is specifically presented by an MHC allele associated with the presence of autoimmune disease in a subject.

[0053] In some embodiments, the target peptide is a peptide that is overexpressed in cells of a subject with multiple sclerosis compared to a control (e.g., compared to a healthy subject). A target peptide is considered to be overexpressed in a cell (e.g., associated with multiple sclerosis) if the expression of the target peptide in the cell is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher in the cell compared to control cells (e.g., a population of cells not associated with an autoimmune disease). In some embodiments, a target peptide is overexpressed in cells of a subject with multiple sclerosis if expression of the target peptide in cells of the subject with multiple sclerosis is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher in the subject with multiple sclerosis compared to a healthy subject (e.g., a subject without an autoimmune disease).

[0054] In some embodiments, the target peptide is a peptide that is highly expressed in cells at disease sites in subjects with multiple sclerosis compared to controls (e.g., target peptide expression in unaffected / non-diseased sites in a subject). A target peptide is considered to be highly expressed in cells at disease sites (e.g., associated with multiple sclerosis) if expression of the target peptide in cells at disease sites is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher in cells at disease sites compared to control cells (e.g., cells at non-diseased sites).

[0055] In some embodiments, the target peptide is a peptide complexed with an MHC having an HLA haplotype associated with multiple sclerosis. In some embodiments, the HLA haplotype is HLA-DRB1 * In some embodiments, the HLA haplotype is HLA-DRA * It's 01.01.

[0056] 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.

[0057] The TCR may include a linker domain located between the α chain and the β chain. In some embodiments, the linker domain includes a self-cleaving peptide sequence (e.g., a self-cleaving peptide sequence located between the α chain and the β chain). The self-cleaving peptide sequence is a peptide sequence that induces separation of a polypeptide into two peptides using a non-classical mechanism. In some embodiments, the self-cleaving peptide sequence may induce ribosomal skipping during translation of the polypeptide. In some embodiments, the self-cleaving peptide sequence is 10-30, 10-25, 15-30, 15-25, or 18-22 amino acids in length. In some embodiments, the self-cleaving peptide sequence may be a 2A peptide sequence. The 2A peptide sequence may include, for example, the DXEXNPGP (SEQ ID NO: 102) amino acid motif (wherein X can be any amino acid). In some embodiments, the 2A peptide sequence is a P2A (derived from porcine teschovirus-1 2A), E2A (derived from equine rhinitis A virus), F2A (derived from foot-and-mouth disease virus), or T2A (derived from Thosea asigna virus 2A) peptide sequence. The T2A peptide sequence may include, for example, the amino acid sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 103). The P2A peptide sequence may include, for example, the amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 104). The E2A peptide sequence may include, for example, the amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 105). The F2A peptide sequence may include, for example, the amino acid sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 106).

[0058] In some embodiments, the exogenous TCR comprises two or more polypeptides, for example, in some embodiments, the exogenous TCR comprises a first polypeptide comprising an alpha chain and a second polypeptide comprising a beta chain.

[0059] In some embodiments, the exogenous TCR comprises one or more cysteine residues present in the α chain of the TCR that can form one or more disulfide bonds with one or more cysteine residues in the β chain of the TCR. In some embodiments, the exogenous TCR comprises one or more cysteine residues present in the α chain constant region of the TCR that can form one or more disulfide bonds with one or more cysteine residues in the β chain constant region of the TCR.

[0060] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 48 of the alpha chain can form a disulfide bond with the cysteine residue at position 57 of the beta chain.

[0061] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and the TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 45 of the alpha chain can form a disulfide bond with the cysteine residue at position 77 of the beta chain.

[0062] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and the TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 10 of the alpha chain can form a disulfide bond with the cysteine residue at position 17 of the beta chain.

[0063] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and the TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 45 of the alpha chain can form a disulfide bond with the cysteine residue at position 59 of the beta chain.

[0064] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and the TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 15 of the alpha chain can form a disulfide bond with the cysteine residue at position 15 of the beta chain.

[0065] [Table 28]

[0066] In some embodiments, the TCR comprises one or more amino acid sequences set forth in Table A (e.g., one or more amino acid sequences belonging to any one of TCR-A, TCR-B, TCR-C, TCR-D, TCR-E, or TCR-F). The TCR may comprise any alpha chain CDR1, CDR2, or CDR3 amino acid sequence provided in Table 1. In some embodiments, the alpha chain CDR1 of the TCR is any one of SEQ ID NOs: 1, 18, 32, or 46. In some embodiments, the alpha chain CDR2 of the TCR is any one of SEQ ID NOs: 2, 19, 33, or 47. In some embodiments, the alpha chain CDR3 of the TCR is any one of SEQ ID NOs: 3, 20, 34, or 48. The TCR may comprise any β chain CDR1, CDR2, or CDR3 amino acid sequence provided in Table A. In some embodiments, the β chain CDR1 of the TCR is any one of SEQ ID NOs: 7, 24, 38, or 52. In some embodiments, the β chain CDR2 of the TCR is any one of SEQ ID NOs: 8, 25, 39, or 53. In some embodiments, the β chain CDR3 of the TCR is any one of SEQ ID NOs: 9, 26, 40, or 54.

[0067] In some embodiments, the alpha chain variable region of the TCR comprises an amino acid sequence that is at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 4, 21, 35, or 49. In some embodiments, the beta chain variable region of the TCR comprises an amino acid sequence that is at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, 14, 27, 41, or 55. In some embodiments, the alpha chain of the TCR comprises an amino acid sequence that is at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, 22, 36, 50, or 73. In some embodiments, the beta chain of the TCR comprises an amino acid sequence that is at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, 15, 28, 42, 56, or 75.

[0068] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a TCR beta chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 consisting of SEQ ID NO: 1, a CDR2 consisting of SEQ ID NO: 2, and a CDR3 consisting of SEQ ID NO: 3, and (b) a TCR beta chain variable region comprising a CDR1 consisting of SEQ ID NO: 7, a CDR2 consisting of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0069] In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the TCR alpha chain variable region comprises SEQ ID NO: 4. In some embodiments, the TCR alpha chain variable region consists of SEQ ID NO: 4. In some embodiments, the TCR beta chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the TCR beta chain variable region comprises SEQ ID NO: 10. In some embodiments, the TCR beta chain variable region consists of SEQ ID NO: 10.

[0070] In some embodiments, the exogenous human TCR comprises (a) a TCR α chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 4, and (b) a TCR β chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the exogenous human TCR comprises (a) a TCR α chain variable region comprising SEQ ID NO: 4, and (b) a TCR β chain variable region comprising SEQ ID NO: 10. In some embodiments, the exogenous human TCR comprises (a) a TCR α chain variable region consisting of SEQ ID NO: 4, and (b) a TCR β chain variable region consisting of SEQ ID NO: 10.

[0071] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 5, and (b) a TCR beta chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising SEQ ID NO: 5, and (b) a TCR beta chain comprising SEQ ID NO: 11. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain consisting of SEQ ID NO: 5, and (b) a TCR beta chain consisting of SEQ ID NO: 11.

[0072] In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 17. In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising SEQ ID NO: 17. In some embodiments, the exogenous human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 17.

[0073] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 73, and (b) a TCR beta chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 75. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising SEQ ID NO: 73, and (b) a TCR beta chain comprising SEQ ID NO: 75. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain consisting of SEQ ID NO: 73, and (b) a TCR beta chain consisting of SEQ ID NO: 75.

[0074] In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 79. In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising SEQ ID NO: 79. In some embodiments, the exogenous human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 79.

[0075] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and (b) a TCR beta chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 consisting of SEQ ID NO: 18, a CDR2 consisting of SEQ ID NO: 19, and a CDR3 consisting of SEQ ID NO: 20, and (b) a TCR beta chain variable region comprising a CDR1 consisting of SEQ ID NO: 24, a CDR2 consisting of SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0076] In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the TCR alpha chain variable region comprises SEQ ID NO: 21. In some embodiments, the TCR alpha chain variable region consists of SEQ ID NO: 21. In some embodiments, the TCR beta chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 27. In some embodiments, the TCR beta chain variable region comprises SEQ ID NO: 27. In some embodiments, the TCR beta chain variable region consists of SEQ ID NO: 27.

[0077] In some embodiments, the exogenous human TCR comprises (a) a TCR α chain variable region comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 21, and (b) a TCR β chain variable region comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 27. In some embodiments, the exogenous human TCR comprises (a) a TCR α chain variable region comprising SEQ ID NO: 21, and (b) a TCR β chain variable region comprising SEQ ID NO: 27. In some embodiments, the exogenous human TCR comprises (a) a TCR α chain variable region consisting of SEQ ID NO: 21, and (b) a TCR β chain variable region consisting of SEQ ID NO: 27.

[0078] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 22, and (b) a TCR beta chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 28. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising SEQ ID NO: 22, and (b) a TCR beta chain comprising SEQ ID NO: 28. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain consisting of SEQ ID NO: 22, and (b) a TCR beta chain consisting of SEQ ID NO: 28.

[0079] In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising SEQ ID NO: 31. In some embodiments, the exogenous human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 31.

[0080] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 80, and (b) a TCR beta chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 82. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising SEQ ID NO: 80, and (b) a TCR beta chain comprising SEQ ID NO: 82. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain consisting of SEQ ID NO: 80, and (b) a TCR beta chain consisting of SEQ ID NO: 82.

[0081] In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 86. In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising SEQ ID NO: 86. In some embodiments, the exogenous human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 86.

[0082] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34, and (b) a TCR beta chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 consisting of SEQ ID NO: 32, a CDR2 consisting of SEQ ID NO: 33, and a CDR3 consisting of SEQ ID NO: 34, and (b) a TCR beta chain variable region comprising a CDR1 consisting of SEQ ID NO: 38, a CDR2 consisting of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 40.

[0083] In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 35. In some embodiments, the TCR alpha chain variable region comprises SEQ ID NO: 35. In some embodiments, the TCR alpha chain variable region consists of SEQ ID NO: 35. In some embodiments, the TCR beta chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 41. In some embodiments, the TCR beta chain variable region comprises SEQ ID NO: 41. In some embodiments, the TCR beta chain variable region consists of SEQ ID NO: 41.

[0084] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 35, and (b) a TCR beta chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 41. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising SEQ ID NO: 35, and (b) a TCR beta chain variable region comprising SEQ ID NO: 41. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region consisting of SEQ ID NO: 35, and (b) a TCR beta chain variable region consisting of SEQ ID NO: 41.

[0085] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 36, and (b) a TCR beta chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 42. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising SEQ ID NO: 36, and (b) a TCR beta chain comprising SEQ ID NO: 42. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain consisting of SEQ ID NO: 36, and (b) a TCR beta chain consisting of SEQ ID NO: 42.

[0086] In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 45. In some embodiments, the exogenous human TCR is expressed as a single polypeptide comprising SEQ ID NO: 45. In some embodiments, the exogenous human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 45.

[0087] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 48, and (b) a TCR beta chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising a CDR1 consisting of SEQ ID NO: 46, a CDR2 consisting of SEQ ID NO: 47, and a CDR3 consisting of SEQ ID NO: 48, and (b) a TCR beta chain variable region comprising a CDR1 consisting of SEQ ID NO: 52, a CDR2 consisting of SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 54.

[0088] In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 49. In some embodiments, the TCR alpha chain variable region comprises SEQ ID NO: 49. In some embodiments, the TCR alpha chain variable region consists of SEQ ID NO: 49. In some embodiments, the TCR beta chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 55. In some embodiments, the TCR beta chain variable region comprises SEQ ID NO: 55. In some embodiments, the TCR beta chain variable region consists of SEQ ID NO: 55.

[0089] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 49, and (b) a TCR beta chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 55. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region comprising SEQ ID NO: 49, and (b) a TCR beta chain variable region comprising SEQ ID NO: 55. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain variable region consisting of SEQ ID NO: 49, and (b) a TCR beta chain variable region consisting of SEQ ID NO: 55.

[0090] In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 50, and (b) a TCR beta chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 56. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain comprising SEQ ID NO: 50, and (b) a TCR beta chain comprising SEQ ID NO: 56. In some embodiments, the exogenous human TCR comprises (a) a TCR alpha chain consisting of SEQ ID NO: 50, and (b) a TCR beta chain consisting of SEQ ID NO: 56.

[0091] [Table A-1]

[0092] [Table A-2]

[0093] [Table A-3]

[0094] [Table A-4]

[0095] [Table A-5]

[0096] [Table A-6]

[0097] [Table A-7]

[0098] [Table A-8]

[0099] [Table A-9]

[0100] [Table A-10]

[0101] [Table A-11]

[0102] In some embodiments, the exogenous TCR comprises two or more polypeptides, for example, in some embodiments, the exogenous TCR comprises a first polypeptide comprising an alpha chain and a second polypeptide comprising a beta chain.

[0103] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0104] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0105] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0106] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0107] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0108] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0109] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0110] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0111] In some embodiments, the T cell receptor comprises (a) an alpha chain comprising an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 113, and / or (b) a beta chain comprising an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 119, and the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0112] In some embodiments, the amino acid sequence of the alpha chain is at least about 85% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the alpha chain is at least about 90% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the alpha chain is at least about 95% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the alpha chain is at least about 96% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the alpha chain is at least about 97% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the alpha chain is at least about 98% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the alpha chain is at least about 99% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the alpha chain is at least about 100% identical to the amino acid sequence of SEQ ID NO: 113.

[0113] In some embodiments, the amino acid sequence of the alpha chain is at least about 85% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the alpha chain is at least about 90% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the alpha chain is at least about 95% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the alpha chain is at least about 96% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the alpha chain is at least about 97% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the alpha chain is at least about 98% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the alpha chain is at least about 99% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the alpha chain is at least about 100% identical to the amino acid sequence of SEQ ID NO: 114.

[0114] In some embodiments, the amino acid sequence of the β chain has at least about 85% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β chain has at least about 90% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β chain has at least about 95% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β chain has at least about 96% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β chain has at least about 97% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β chain has at least about 98% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β chain has at least about 99% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β chain has at least about 100% identity to the amino acid sequence of SEQ ID NO: 119.

[0115] In some embodiments, the amino acid sequence of the β chain has at least about 85% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β chain has at least about 90% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β chain has at least about 95% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β chain has at least about 96% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β chain has at least about 97% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β chain has at least about 98% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β chain has at least about 99% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β chain has at least about 100% identity to the amino acid sequence of SEQ ID NO: 120.

[0116] In some embodiments, the amino acid sequence of the β chain has at least about 85% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β chain has at least about 90% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β chain has at least about 95% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β chain has at least about 96% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β chain has at least about 97% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β chain has at least about 98% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β chain has at least about 99% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β chain has at least about 100% identity to the amino acid sequence of SEQ ID NO: 121.

[0117] In some embodiments, the amino acid sequence of the β chain has at least about 85% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β chain has at least about 90% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β chain has at least about 95% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β chain has at least about 96% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β chain has at least about 97% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β chain has at least about 98% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β chain has at least about 99% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β chain has at least about 100% identity to the amino acid sequence of SEQ ID NO: 122.

[0118] In some embodiments, the T cell receptor comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the T cell receptor comprises an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO:108.

[0119] In some embodiments, the TCR comprises one or more amino acid sequences set forth in Table B. The TCR may comprise any alpha chain CDR1, CDR2, or CDR3 amino acid sequence provided in Table B. In some embodiments, the alpha CDR1 of the TCR comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the alpha CDR2 of the TCR comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the alpha CDR3 of the TCR comprises the amino acid sequence of SEQ ID NO: 111. The TCR may comprise any β chain CDR1, CDR2, or CDR3 amino acid sequence provided in Table B. In some embodiments, the β CDR1 of the TCR comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, the β CDR2 of the TCR comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, the β CDR3 of the TCR comprises the amino acid sequence of SEQ ID NO: 117.

[0120] In some embodiments, the alpha chain constant region of the TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the beta chain constant region of the TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 118.

[0121] In some embodiments, the α chain constant region of the TCR is a murine constant region. In some embodiments, the β chain constant region of the TCR is a murine constant region.

[0122] In some embodiments, the alpha chain constant region of the TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the beta chain constant region of the TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 124.

[0123] In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the engineered polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 108.

[0124] In some embodiments, the present disclosure provides engineered polypeptides comprising the TCR sequences described herein.

[0125] In some embodiments, the disclosure provides an engineered polypeptide comprising a human TCR alpha chain and a human TCR beta chain, wherein the TCR alpha chain is at least 95% identical to SEQ ID NO: 73 and the TCR beta chain is at least 95% identical to SEQ ID NO: 75, or wherein the TCR alpha chain is at least 95% identical to SEQ ID NO: 80 and the TCR beta chain is at least 95% identical to SEQ ID NO: 82. In some embodiments, the TCR alpha chain comprises or consists of SEQ ID NO: 73 and the TCR beta chain comprises or consists of SEQ ID NO: 75; or the TCR alpha chain comprises or consists of SEQ ID NO: 80 and the TCR beta chain comprises or consists of SEQ ID NO: 82.

[0126] In some embodiments, the engineered polypeptides provided herein comprise, from N- to C-terminus, a TCR beta chain, a self-cleaving peptide sequence, and a TCR alpha chain. In some embodiments, the self-cleaving peptide is the P2A sequence (SEQ ID NO: 104).

[0127] In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 79. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 100% identical to SEQ ID NO: 79. In some embodiments, the engineered polypeptide comprises SEQ ID NO: 79. In some embodiments, the engineered polypeptide consists of SEQ ID NO: 79.

[0128] In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 100% identical to SEQ ID NO: 86. In some embodiments, the engineered polypeptide comprises SEQ ID NO: 86. In some embodiments, the engineered polypeptide consists of SEQ ID NO: 86.

[0129] In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the engineered polypeptide comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 113.

[0130] In some embodiments, the amino acid sequence of the polypeptide further comprises an amino-terminal leader sequence, optionally METLLGVSLV ILWLQLARVN (SEQ ID NO: 100).

[0131] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:114.

[0132] In some embodiments, the engineered polypeptide encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO:119.

[0133] In some embodiments, the amino acid sequence of the polypeptide further comprises an amino-terminal leader sequence, optionally METLLGVSLV ILWLQLARVN (SEQ ID NO: 100). In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 121.

[0134] In some embodiments, the amino acid sequence of the polypeptide further comprises a carboxy-terminal P2A overhang sequence, optionally ATNFSLLKQAGDVEENPG (SEQ ID NO: 104).

[0135] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:120.

[0136] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:122.

[0137] [Table B-1]

[0138] [Table B-2]

[0139] [Table B-3]

[0140] Nucleic acid encoding a TCR In some embodiments, the present disclosure provides a nucleic acid encoding a TCR (e.g., an exogenous TCR). The nucleic acid may be or include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., messenger RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeras.

[0141] As used herein, a nucleic acid generally refers to an engineered nucleic acid. An engineered nucleic acid is a non-naturally occurring polynucleotide (e.g., at least two nucleotides covalently linked to each other, optionally containing phosphodiester bonds, referred to as a phosphodiester backbone). Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that are amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that are chemically or otherwise modified but are still capable of base pairing (binding) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules resulting from replication of any of the above.

[0142] The engineered nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009, and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: a 5' exonuclease, a 3' extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity chews back the 5' terminal sequence, exposing complementary sequences for annealing. Polymerase activity then fills in the gaps in the annealed domains. DNA ligase then seals the nicks, covalently linking the DNA fragments to each other. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. The MegaGate molecular cloning method may also be used. MegaGate is a less toxic Gateway technology that eliminates the ccdb toxin used in Gateway recombinase cloning and instead utilizes meganuclease-mediated digestion to eliminate background vectors during cloning (see, for example, Kramme C. et al. STAR Protoc. 2021 Oct. 22;2(4):100907, incorporated herein by reference). Other methods of producing engineered polynucleotides may also be used in accordance with the present disclosure.

[0143] In some embodiments, the present disclosure provides an expression cassette comprising an open reading frame comprising a nucleic acid encoding an exogenous TCR operably linked to a promoter. A promoter is a nucleotide sequence (e.g., ATG) at which RNA polymerase binds to initially transcribe. A promoter is typically located immediately upstream (5' end) of the transcription start site. In some embodiments, the promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably linked. In some embodiments, the promoter is an inducible promoter. An inducible promoter may be regulated in vivo, for example, by chemical agents, temperature, or light.

[0144] An open reading frame is a series of consecutive codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide, e.g., a protein. An open reading frame is operably linked to a promoter if the promoter controls transcription of the open reading frame.

[0145] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding an exogenous TCR or an expression vector comprising the same. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. For example, the vector may be a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpesvirus vector, a retroviral vector, or a baculoviral vector. Viral vectors provide efficient delivery of exogenous TCRs to regulatory T cells of the present disclosure. Exemplary viral vectors can be derived from negative-stranded RNA viruses such as lentiviruses, retroviruses (e.g., Retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpesviruses), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus.Examples of retroviruses include avian leukosis-sarcoma, avian C virus, mammalian C, B, and D viruses, oncoretroviruses, HTLV-BLV complex, alpharetroviruses, gammaretroviruses, Spumaretrovirus genus, murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0146] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector of the present disclosure comprises two long terminal repeats (LTRs) flanking an expression cassette containing the lentiviral gag, pol, and rev genes, and a nucleic acid encoding an exogenous TCR. For safety, the vector does not contain any other active lentiviral genes, such as vpr, vif, vpu, nef, and tat. In some embodiments, these genes are deleted or otherwise inactivated.

[0147] In some embodiments, the lentiviral vector is a self-inactivating vector. A self-inactivating vector is a vector in which the production of full-length vector RNA in transduced cells is significantly reduced or completely eliminated. This feature greatly minimizes the risk of the emergence of replication-competent recombinants (RCRs). Furthermore, this feature reduces the risk of abnormal expression of cellular coding sequences located adjacent to the vector integration site. Furthermore, the SIN design reduces the possibility of interference between the LTR and the promoter driving the expression of the transgene.

[0148] Self-inactivation is preferably achieved by introducing a deletion in the U3 region of the 3'LTR of the vector DNA, i.e., the DNA used to produce vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5'LTR of the proviral DNA. However, the LTR elements involved in the polyadenylation of viral RNA are not modified. Together, this reduces or eliminates the production of full-length vector RNA in transduced cells.

[0149] 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.

[0150] In some embodiments, nucleic acids are codon-optimized. Codon optimization methods are known in the art. In some embodiments, codon optimization can be used to match the codon frequency in target organisms and host organisms to ensure proper folding; bias GC content to increase RNA (e.g., mRNA) stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in encoded proteins; add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust translation speed to ensure that various domains of proteins fold properly; or reduce or eliminate problematic secondary structures in polynucleotides.

[0151] In some embodiments, the nucleic acid encoding a TCR comprises a promoter operably linked to a coding sequence encoding an exogenous human TCR. The promoter may be a viral promoter or a native promoter. In some embodiments, the promoter is a constitutively active promoter or an inducible promoter. In some embodiments, the promoter is a eukaryotic translation elongation factor 1 alpha (EF-1 alpha) promoter and an MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution) (see, e.g., Gill, D.R. et al. Gene Ther. 2001;8:1539-46 and Astrakhan, A. et al. Blood 2012;119:4395-4407).

[0152] The vector may also include a stop codon and / or an expression enhancer element. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used. In some embodiments, the enhancer element is an optimized posttranscriptional regulatory element (oPRE), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The WPRE may be a wild-type WPRE or a WPRE mutant sequence (e.g., WPRE-mut6). In some embodiments, the WPRE is as described in Zanta-Boussif, MA et al., Gene Therapy volume 16, pp. 605-619 (2009).

[0153] In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 96% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 97% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having at least 99% identity to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the engineered polynucleotide comprises an open reading frame comprising a nucleotide sequence having 100% identity to the nucleotide sequence of SEQ ID NO: 87.

[0154] In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:79. In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO:79.

[0155] In some embodiments, the amino acid sequence of the polypeptide encoded by the polynucleotide further comprises a carboxy-terminal self-cleaving peptide sequence. The self-cleaving peptide sequence may be selected from P2A, E2A, F2A, and T2A. In some embodiments, the self-cleaving peptide sequence is ATNFSLLKQAGDVEENPG (SEQ ID NO: 104).

[0156] Isolated cell populations In some embodiments, the present disclosure provides stable CD4 +

[0003] In some embodiments, the present disclosure provides an isolated population of cells comprising regulatory T cells, as well as compositions thereof. In some embodiments, the present disclosure provides a stable CD4 T cell derived from a subject with multiple sclerosis that has been engineered to express an exogenous human TCR. + Provided are isolated populations of cells comprising regulatory T cells, as well as compositions thereof. In some embodiments, at least 80% of the cells are stable CD4 T cells comprising a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus. + It is Treg.

[0157] The methylation status of TSDR in FOXP3 locus can be evaluated by means known in the art.For example, in some embodiments, the methylation status of TSDR in FOXP3 locus is evaluated by bisulfite treatment and digital droplet PCR (ddPCR) using methylation-specific primers and probes.In some embodiments, the methylation status of TSDR in FOXP3 locus is evaluated by single-cell sequencing method.

[0158] An isolated cell population is a cell population removed from a human body or from a sample obtained from a human body. Therefore, it is considered "isolated" from a human body. A population of cells may be isolated (e.g., obtained) from a subject or from a biological sample obtained from a subject using any known cell collection method, such as apheresis. The isolated cell population of the present disclosure may be subjected to the methods described herein to produce an isolated cell population with a higher number of regulatory T cells (e.g., stable regulatory T cells) than a population of cells obtained directly from a subject or from a biological sample obtained from a subject, for example, by apheresis.

[0159] In some embodiments, the isolated cell population is a stable CD4 +Contains regulatory T cells, with at least 80% of cells containing a stable CD4 T cell-specific demethylated region (TSDR) in 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 cells comprising a hypomethylated TSDR at the FOXP3 locus. + In some embodiments, the isolated cell population comprises stable CD4+ regulatory T cells, wherein 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+ regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus. + In some embodiments, 80% to 90%, 85% to 95%, 80% to 85%, 85% to 90%, or 90% to 95% of the cells are stable CD4 T cells comprising a hypomethylated TSDR at the FOXP3 locus. + In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the isolated cell population are stable regulatory T cells that comprise a hypomethylated TSDR at the endogenous FOXP3 locus.

[0160] In some embodiments, the isolated population is CD25 + / high CD4 + CD127 - / low In some embodiments, the isolated cell population comprises CD25 regulatory T cells. + / high CD4 + CD127 - / low FOXP3 + In some embodiments, the isolated cell population comprises CD25 regulatory T cells. + / high CD4 + CD127 - / loFOXP3 + CD45RA + Includes regulatory T cells.

[0161] 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+ / highCD4+CD127- / lo regulatory T cells. 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+ / highCD4+CD127- / lo regulatory T cells. + / high CD4 + CD127 - / low FOXP3 + Includes regulatory T cells.

[0162] In some embodiments, at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the regulatory T cells in the isolated population are CD45RA + In some embodiments, the isolated cell population is at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25 + / high CD4 + CD127 - / low CD45RA + Includes regulatory T cells.

[0163] As described herein, the present disclosure provides an isolated cell population comprising stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated population described herein is engineered to express an exogenous TCR, but the FOXP3 locus remains unmodified. Thus, the cells of the isolated population described herein do not comprise an engineered FOXP3 locus. An "engineered FOXP3 locus" refers to any engineered modification (e.g., human modification) intended to alter the expression of FOXP3. Such engineered modifications include, but are not limited to, the introduction of a FOXP3 transgene, the introduction of a modified promoter, and / or the use of an exogenous agent (e.g., a gene editing system, a small molecule, or a peptide) intended to activate the expression of FOXP3.

[0164] In some embodiments, the isolated cell population comprises at least 1 x 10 2 , at least 1 x 10 3 , at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , or at least 1 × 10 10 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 2 ~1×10 10 , 1×10 3 ~1×10 10 , 1×10 4 ~1×10 10 , 1×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 10 , 1×10 8 ~1×10 10 , 1×10 5 ~1×10 9 , 1×10 6 ~1×10 8 , 1×107 ~1×10 10 or 1×10 4 ~1×10 6 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 6 ~1×10 10 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9×10 7 In some embodiments, the isolated cell population comprises 1 x 10 stable regulatory T cells. 7 ~1×10 10 , 2 × 10 7 ~1×10 10 , 3×10 7 ~1×10 10 , 4×10 7 ~1×10 10 , 5×10 7 ~1×10 10 , 6×10 7 ~1×10 10 , 7×10 7 ~1×10 10 , 8×10 7 ~1×10 10 , or 9×10 7 ~1×10 10 Contains stable regulatory T cells.

[0165] In some embodiments, the isolated cell population comprises stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus, wherein the stable regulatory T cells retain markers of stability in the presence of a pro-inflammatory condition (i.e., maintain a hypomethylated TSDR at the FOXP3 locus and / or express CD25 + / high CD4 + CD127 - / low FOXP3 +(Maintaining a protein expression profile of). A pro-inflammatory state refers to cells and factors known to drive an inflammatory immune response and can include pro-inflammatory cytokines (e.g., IL-17, IL-22, IL-21, IFNγ, IL-12, TNFα, IL-1β, IL-6, IL-1, GM-CSF, and others known in the art), immune effector cells (e.g., conventional CD4+ T cells, CD8+ effector T cells, granulocytes, etc.), and other pro-inflammatory mediators (e.g., prostaglandins, thrombin, histamine, and matrix proteases). A pro-inflammatory state can be in vitro or in vivo. In some embodiments, stable regulatory T cells within the isolated cell population maintain a hypomethylated TSDR at the endogenous FOXP3 locus in the presence of pro-inflammatory conditions (for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, or 25 days).

[0166] In some embodiments, the isolated cell population comprises stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus, where the stable regulatory T cells retain markers of stability over time (i.e., maintain a hypomethylated TSDR at the FOXP3 locus and / or express CD25 + / high CD4 + CD127 - / low FOXP3 +(Maintain a protein expression profile of . For example, in some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. In some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after activation and / or expansion. In some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with an exogenous TCR. In some embodiments, stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after a cryopreservation freeze-thaw cycle.

[0167] In some embodiments, stable regulatory T cells are expressed ex vivo on CD25 T cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. + / high CD4 + CD127 - / low FOXP3 + In some embodiments, stable regulatory T cells maintain a protein expression profile of CD25 ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after activation and / or expansion. + / high CD4 + CD127 - / low FOXP3 +In some embodiments, stable regulatory T cells maintain a protein expression profile of CD25 ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with an exogenous TCR. + / high CD4 + CD127 - / low FOXP3 + In some embodiments, stable regulatory T cells maintain a protein expression profile of CD25 ex vivo for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after a cryopreservation freeze-thaw cycle. + / high CD4 + CD127 - / low FOXP3 + Maintain the protein expression profile of

[0168] In some embodiments, at least 50% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 50% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 50% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 60% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 60% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 60% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 70% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 70% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 70% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 80% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 80% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 80% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 90% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation.In some embodiments, at least 90% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation, hi some embodiments, at least 90% of the cells of the isolated cell population comprise a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation.

[0169] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed at multiple (e.g., two or more) time points during the isolation and production process. In some embodiments, the percentage of stable regulatory T cells containing hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% between these two or more time points.

[0170] For example, in some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from the biological sample, and again within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11%, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample.

[0171] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In some embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11%, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion.

[0172] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11%, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR.

[0173] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of stable regulatory T cells containing a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0174] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after transduction with an exogenous human TCR, and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of stable regulatory T cells containing a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample, and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable regulatory T cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0175] In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percentage point(s) for at least 5, 6, 7, 8, 9, 10, or 11 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells increases by 1, 2, 3, 4, or 5 percentage point(s) for at least 5, 6, 7, 8, 9, 10, or 11 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 15 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points during at least 15 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percent during at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR. In some embodiments, the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 15 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0176] The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed about 1, about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of the cells with an exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction of the cells with an exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed about 1-21, 1-7, 4-14, 4-7, 7-10, 7-14, 10-21, or 14-21 days after transduction of the cells with an exogenous human TCR.

[0177] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the regulatory T cells of the isolated cell population express the exogenous TCR (e.g., after transduction of the isolated cell population with an exogenous TCR). In some embodiments, 10% to 60% or 20% to 50% of the regulatory T cells of the isolated cell population express the exogenous TCR. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the stable regulatory T cells of the isolated cell population express the exogenous TCR (e.g., after transduction of the isolated cell population with an exogenous TCR). In some embodiments, 10% to 60% or 20% to 50% of the stable regulatory T cells of the isolated cell population express an exogenous TCR.

[0178] In some embodiments, the TSDR at the endogenous FOXP3 locus of stable regulatory T cells of the isolated cell population of regulatory T cells remains hypomethylated until the isolated cell population is administered to a subject. In some embodiments, the TSDR at the endogenous FOXP3 locus of stable regulatory T cells of the isolated cell population of regulatory T cells remains hypomethylated after a cryopreservation freeze-thaw cycle.

[0179] In some embodiments, the regulatory T cells (e.g., stable regulatory T cells) of the isolated cell population exhibit one or more cellular functions associated with regulatory T cells when activated by binding to pMHC. Non-limiting examples of such cellular functions include cytokine secretion activity, expression of specific activation markers, and suppressive activity. Cytokine secretion activity simply refers to the secretion of specific anti-inflammatory cytokines (e.g., IL-10, TGFβ, and IL-35). Activation markers include, but are not limited to, CD69, 4-1BB, CD25, CD71, or CTLA-4. Regulatory T cells express one or more of these markers, for example, upon contact with a target peptide complexed with MHC.

[0180] Suppressive activity refers to the suppression of activation, proliferation, and cytokine production of non-regulatory T cells (e.g., CD8+ T cells and CD4+ conventional T cells) to partially suppress overactivity of the immune system. Regulatory T cells exhibit suppressive activity when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. In some embodiments, the target peptide is presented by a cell expressing MHC. For example, in some embodiments, regulatory T cells suppress the activation, proliferation, and cytokine production of conventional T cells that have specificity for a shared target peptide complexed with MHC, e.g., a shared target peptide presented by an antigen-presenting cell (APC). In some embodiments, regulatory T cells suppress the proliferation and growth of non-regulatory T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% compared to a control (e.g., non-regulatory T cells in the absence of regulatory T cells).

[0181] In some embodiments, regulatory T cells suppress IFN-γ production from conventional T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% compared to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress IFN-γ production from conventional T cells by 50% to 99%, 75% to 99%, or 80% to 100% compared to a control (e.g., when present in a population comprising regulatory T cells at a ratio of 1:1 to 1:8 compared to conventional T cells). In some embodiments, regulatory T cells suppress CD71 production from conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% compared to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress CD71 production by conventional T cells by 20% to 90% or 30% to 80% compared to a control (e.g., when present in a population containing regulatory T cells at a ratio of 1:1 to 1:8 compared to conventional T cells). In some embodiments, regulatory T cells suppress CD25 production from conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% compared to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress CD25 production by conventional T cells by 40% to 70% compared to a control (e.g., when present in a population containing regulatory T cells at a ratio of 1:1 to 1:8 compared to conventional T cells).

[0182] In some embodiments, regulatory T cells exhibit cytokine secretion activity (e.g., secretion of IL-10) when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells, e.g., a target peptide presented by an APC. In some embodiments, regulatory T cells exhibit expression of activation markers when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. For example, in some embodiments, regulatory T cells exhibit expression of CD69, 4-1BB, CD25, CD71, and / or CTLA-4 when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. In some embodiments, regulatory T cells exhibit suppressive activity when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. For example, in some embodiments, regulatory T cells suppress the activation of conventional T cells with specificity for a shared target peptide complexed with MHC.

[0183] The cellular function of stable regulatory T cells may be assessed about 1, about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of the cells with an exogenous human TCR. The cellular function of stable regulatory T cells may be assessed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction of the cells with an exogenous human TCR. The cellular function of stable regulatory T cells may be assessed about 1-21, 1-7, 4-14, 4-7, 7-10, 7-14, 10-21, or 14-21 days after transduction of the cells with an exogenous human TCR.

[0184] In some embodiments, transduced regulatory T cells (e.g., isolated populations of transduced regulatory T cells) retain their cellular function (e.g., the ability to be activated) after cryopreservation freeze-thaw cycling. In some embodiments, transduced regulatory T cells can be activated and / or expanded after cryopreservation freeze-thaw cycling. In some embodiments, regulatory T cells can exhibit cytokine secretion activity, expression of specific activation markers, and / or suppressive activity after cryopreservation freeze-thaw cycling.

[0185] In some embodiments, prior to activation and / or transduction with an exogenous human T cell receptor, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of cells are CD25+ / highCD4+CD127- / lo.

[0186] An isolated cell population comprising stable regulatory T cells may also contain a small number of non-regulatory T cells (e.g., conventional T cells). Non-regulatory T cells may be NK T cells, B cells, CD8+ T cells, neutrophils, eosinophils, CD14+ cells, or conventional (CD4+) T cells derived from peripheral blood and lymph nodes. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising stable regulatory T cells are non-regulatory T cells. In some embodiments, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 10%, about 2% to about 5%, or about 5% to about 10% of the cells in an isolated cell population comprising regulatory T cells are non-regulatory T cells. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising regulatory T cells are non-regulatory T cells comprising an exogenous human TCR. Conventional T cells generally produce IL-2 and other interleukin factors. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising regulatory T cells are conventional T cells. In some embodiments, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 10%, about 2% to about 5%, or about 5% to about 10% of the cells in an isolated cell population comprising regulatory T cells are conventional T cells. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in an isolated cell population comprising regulatory T cells are conventional T cells comprising an exogenous human TCR. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of conventional T cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of conventional CD4+ T cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of B cells.In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable amount of NK T cells. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable amount of CD14+ cells. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable number of eosinophils. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable number of neutrophils. In some embodiments, the isolated cell population comprising regulatory T cells comprises an undetectable amount of CD8+ T cells.

[0187] The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or stable regulatory T cells) may be assessed about 1, about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of the cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or stable regulatory T cells) may be assessed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction of the cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or stable regulatory T cells) may be assessed about 1-21, 1-7, 4-14, 4-7, 7-10, 7-14, 10-21, or 14-21 days after transduction of the cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) may be assessed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after a cryopreservation freeze-thaw cycle.

[0188] In some embodiments, the ratio of regulatory T cells to conventional T cells in the isolated population of cells comprising regulatory T cells is at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 100:1.

[0189] Methods for producing stable regulatory T cells In some embodiments, the present disclosure provides a method for producing an isolated population of cells containing stable CD4+ regulatory T cells derived from a subject with multiple sclerosis. Previous methods for producing antigen-specific regulatory T cells have not resulted in clinical-scale and clinical-grade production of cell populations containing high concentrations of stable antigen-specific regulatory T cells. While polyclonal T cells have been successfully produced and administered in clinical settings, the use of stable thymic regulatory T cells engineered with exogenous TCRs poses increased safety risks and requires improved cell purity and regulatory T cell stability. Without wishing to be bound by theory, stable thymic regulatory T cells comprise a small percentage of leukocytes, and if the isolated regulatory T cell population is not highly pure, other types of leukocytes are engineered with exogenous TCRs. In sufficient numbers, these engineered non-regulatory T cells may have a deleterious effect by triggering rather than suppressing immune responses at the site of autoimmune disease. Through the integration and modification of several biomarker selection processes, including an unexpectedly effective dual selection process using CD25, and in some cases CD45RA, the methods designed herein provide the field with the tools to generate populations of antigen-specific regulatory T cells with high yields and high relative concentrations of stable TCR-transduced regulatory T cells.

[0190] In some embodiments, the methods of the disclosure include depleting conventional T cells and selecting for cells with a CD25+ / highCD4+CD127- / lo phenotype to produce a cell population comprising stable regulatory T cells. In some embodiments, the methods of the disclosure include depleting conventional T cells, selecting for cells with a CD25+ / highCD4+CD127- / lo phenotype to produce a cell population comprising stable regulatory T cells. + / high CD4 + CD127 - / lowand engineering the stable regulatory T cells to comprise an exogenous human TCR that specifically binds to a target peptide complexed with MHC. In some embodiments, a method for producing a cell population of stable regulatory T cells comprises isolating a biological sample comprising regulatory T cells from a human subject with multiple sclerosis.

[0191] An exemplary method of the present disclosure is provided in FIGS. 1A-1B. In the embodiment provided in FIG. 1A, a biological sample (e.g., a blood sample) is first isolated from a human subject with multiple sclerosis using apheresis. Cells of the biological sample are labeled with an anti-CD8 antibody (which targets non-CD4 conventional T cells), an anti-CD19 antibody (which targets B cells), and an anti-CD14 antibody (which targets monocytes), and the labeled cells are removed from the biological sample to produce a depleted biological sample. In some embodiments, the volume of the depleted biological sample is then reduced (e.g., by removing water / liquid) to aid in downstream processing of the sample. The sample is then labeled with an anti-CD25 antibody (e.g., a CD25-PE-biotin antibody) and a secondary molecule (e.g., an anti-biotin microbead) to produce a CD25-enriched cell population. In some embodiments, a CD25-PE-biotin antibody is used. The CD25-PE-biotin antibody comprises an anti-CD25 antibody bound to a tandem conjugate of phycoerythrin (PE) and biotin. In some embodiments, this enrichment step may be repeated two or more times (e.g., two or more times). These CD25 cultures are then further processed by sorting the cells using a TCR gating strategy utilizing fluorescence-activated cell sorting (FACS) or Tyto sorting (Miltenyi) as described in Figures 1A and 1B. First, the cells are labeled with anti-CD45RA, anti-CD4, and anti-CD127 antibodies. The cells are then selected through multiple sorting steps. In each step, in some embodiments, the labeled CD25+ / highCD4+CD127- / lo cells are first identified as singlets, then as live cells, and then as CD4 + The cells were then isolated and then CD25 + / high CD127 - / lowIdentify cells and then CD25 high CD45RA - and CD25 + / high CD45RA + The cells are first identified by a gating strategy that involves identifying a specific population of cells (e.g., CD4 + CD25 high CD127 - / low CD45RA - Cells containing the protein expression profile of CD4 + CD25 + CD127 - / low CD45RA + In some embodiments, the sorting procedure is performed twice to optimize cell purity.

[0192] In some embodiments, the present disclosure provides a stable CD4 + A method for producing an isolated population comprising regulatory T cells, comprising: (a) isolating CD8 T cells from a biological sample obtained from a subject with multiple sclerosis; + Cells and CD19 + (b) removing the cells to produce a depleted biosample; and (b) isolating the depleted biosample with CD25 + (c) enriching for CD4 cells to produce an enriched population; and + CD25 + CD127 - / low In some embodiments, the present disclosure provides a method for isolating a stable CD4 + A method for producing an isolated population comprising regulatory T cells, comprising: (a) isolating CD8 T cells from a biological sample obtained from a subject with multiple sclerosis; + Cells and CD19 + (b) removing the cells to produce a depleted biosample; and (b) isolating the depleted biosample with CD25 + (c) enriching the enriched population for CD4 + CD25 + CD127 - / lowand (d) engineering the population of cells of (c) to express an exogenous human T cell receptor (TCR) that specifically binds to a target peptide complexed with MHC. In some embodiments, step (a) comprises isolating CD14 from the biological sample. + In some embodiments, step (a) further comprises removing CD56 cells from the biological sample. + Further comprising removing the cells.

[0193] In some embodiments, the method further comprises quantifying the methylation status of a T cell-specific demethylated region (TSDR) at the FOXP3 locus in the population of cells, wherein at least 80% of the cells are stable CD4 T cells comprising a hypomethylated TSDR at the FOXP3 locus. + In some embodiments, the methylation status of the TSDR is quantified after the isolation step (c). In some embodiments, the methylation status of the TSDR is quantified after the isolation step (d).

[0194] In some embodiments, the enriched population is enriched for CD4 + CD25 + CD127 - / low The step of isolating cells comprises (i) isolating CD4 + (ii) identifying a first subpopulation of cells; and (iii) isolating CD25 from the first subpopulation. + / high CD127 - / low (iii) identifying a second subpopulation of cells; and (iv) isolating CD25highCD45RA- and CD25 from the second subpopulation. + / high CD45RA + Select cells and CD25 + CD45RA - thereby excluding stable CD4 + The present invention provides a method comprising isolating a population of Tregs.

[0195] In some embodiments, the methods provided herein further include isolating a biological sample comprising regulatory T cells from a human subject with multiple sclerosis. In some embodiments, sample isolation is performed using an apheresis technique (e.g., a leukapheresis technique for isolating leukocytes). In some embodiments, isolating a biological sample comprising regulatory T cells from a human subject with multiple sclerosis is performed by removing blood from the subject and separating the blood into plasma and cells. In some embodiments, the apheresis technique involves removing whole blood from the subject and separating the whole blood to remove desired cell types (e.g., T cells). In some embodiments, the separation step of apheresis is performed using continuous flow centrifugation or intermittent flow centrifugation.

[0196] Cells expressing specific biomarkers (e.g., CD8 + cells, CD19 + cells, CD14 + cells, and / or CD56 + Cells) may be removed from a sample (e.g., a biological sample) using FACS, antibody pull-down assay techniques, or any other method known to one of skill in the art that removes cells expressing specific biomarkers. In some embodiments, cells expressing specific biomarkers may be removed from a sample by labeling the cells with microbeads (e.g., anti-CD8, anti-CD14, anti-CD19, and / or anti-CD56 microbeads) and then subjecting the sample to FACS (to remove cells expressing the specific biomarkers).

[0197] Depleted biospecimens are CD8 + cells, CD19 + cells, CD14 + cells, and / or CD56 + In some embodiments, the biological sample is a biological sample that has been treated to remove CD8 cells (e.g., isolated from a human subject with multiple sclerosis). + cells, CD19 + cells, CD14+ cells, and / or CD56 + By removing cells, CD8 + cells, CD19 + cells, CD14 + cells, and / or CD56 + A depleted biological sample is generated having less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, or less than 3% of its total cells containing CD8 cells. + cells, CD19 + cells, CD14 + cells, and / or CD56 + By removing cells, CD8 + A depleted biological sample is generated having less than 0.5% of its total cells containing cells.

[0198] Cell selection techniques may be used to select cells with a particular phenotype (e.g., cells expressing a particular biomarker, such as CD4, CD25, or CD45RA, or cells with no or low expression of a specific biomarker, such as CD127). The cell selection technique may be FACS technology, a Tyto device, an antibody pull-down assay technique, a magnetic cell separation technique, or any other method of selecting cells expressing a particular biomarker known to those skilled in the art. In some embodiments, the cell selection technique involves labeling cells expressing a particular biomarker (e.g., CD25) with a biotinylated antibody targeting that biomarker, and then performing a pull-down assay (e.g., using anti-biotin microbeads or streptavidin conjugates). In some embodiments, the cell selection technique involves labeling cells expressing a particular biomarker (e.g., CD25, CD4, and / or CD45RA) with an antibody targeting that biomarker. Cells labeled with the antibody targeting the particular biomarker can then be selected from a sample (e.g., a CD25-enriched population) using FACS.

[0199] Then, after the CD25 enrichment step, + / high CD4 +CD127 - / low A multi-step selection process is followed to select regulatory T cells. In some embodiments, the selection process is carried out on a Tyto device (Miltenyi). Those skilled in the art will understand that software for visualizing selected cell populations can display data in various ways, including dot plots and histograms. When a population is identified based on the presence or absence of a single protein, the identification can be carried out using dot plots or histograms. When a population needs to be identified based on the presence or absence of two proteins (for example, CD25 and CD45RA), the identification should be carried out using dot plots.

[0200] In some embodiments, prior to the first sorting step, the CD25 enriched cell population is separated into multiple subpopulations of cells, and the first sorting step is performed simultaneously and / or sequentially on the multiple subpopulations. + / high CD4 + CD127 - / low This provides rapid processing of large numbers of cells that need to be processed to isolate a sufficient number of cells that are a small percentage of the total. In some embodiments, the CD25-enriched cell population is separated into two, three, four, five, six, or more subpopulations, each of which is sorted in a first sorting step. In some embodiments, after the first sorting step, the sorted subpopulations are combined before a second sorting step.

[0201] In some embodiments, the present disclosure provides a stable CD4 + A method for producing an isolated population comprising regulatory T cells, comprising: (a) isolating CD8 T cells from a biological sample obtained from a subject with multiple sclerosis; + Cells and CD19 + (b) removing the cells to produce a depleted biosample; and (b) isolating the depleted biosample with CD25 + (c) enriching for CD4 cells to produce an enriched population; and + CD25 + CD127 - / lowand isolating cells, the isolating comprising (i) isolating CD4 + (ii) identifying a first subpopulation of cells; and (iii) isolating CD25 from the first subpopulation. + / high CD127 - / low (iii) identifying a second subpopulation of cells; and (iv) isolating CD25 from the second subpopulation. high CD45RA - and CD25 + / high CD45RA + Select cells and CD25 + CD45RA - and thereby isolating a population of stable CD4+ Tregs.

[0202] In some embodiments, CD25 is isolated from the CD25 enriched population. + / high CD4 + CD127 - / low Selecting cells from the CD25 enriched population to identify a first subpopulation, CD4 + Then, CD25 + / high CD127 - / low A second subpopulation of cells is identified from the first subpopulation. high CD45RA and CD25 + / high CD45RA + A population of cells is selected from the second subpopulation for isolation.

[0203] 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.

[0204] In some embodiments, the second subpopulation is at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD25 + / high CD4 + CD127 - / lowIn some embodiments, the second subpopulation comprises at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD25 + / high CD4 + CD127 - / low FoxP3 + Includes regulatory T cells.

[0205] In some embodiments, the population selected for isolation is CD25 high CD4 + CD127 - / low CD45RA(CD25 high CD45RA - ) and CD25 + / high CD4 + CD127 - / low CD45RA + (CD25 + / high CD45RA + In some embodiments, the population selected for isolation comprises at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% CD25 regulatory T cells. high CD45RA -In some embodiments, the population selected for isolation comprises at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% CD25 cells. + / high CD45RA + CD25 in the final population high CD45RA and CD25 + / high CD45RA + It is understood that the percentages of cells total approximately equal 80%, 90%, or 100% of the total cell population.

[0206] In some embodiments, the population selected for isolation has less than 10% CD25 + CD4 + CD127 - / low In some embodiments, the population selected for isolation comprises less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% CD25 + CD4 + CD127 - / low CD45RA - Contains cells.

[0207] In some embodiments, the methods described herein involve the generation of stable CD4 T cells in which at least 80% of the cells contain a hypomethylated T cell-specific demethylated region (TSDR) in 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 cells comprising a hypomethylated TSDR at the FOXP3 locus. + In some embodiments, the isolated cell population is a stable CD4 Treg. +Contains regulatory T cells, and 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 cells containing hypomethylated TSDR at the FOXP3 locus + In some embodiments, 80% to 90%, 85% to 95%, 80% to 85%, 85% to 90%, or 90% to 95% of the cells are stable CD4 T cells comprising a hypomethylated TSDR at the FOXP3 locus. + It is Treg.

[0208] In some embodiments, at least 80% of the cells are stable CD4 cells containing a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus. + If Tregs, the isolated population of cells produced by the methods described herein are selected for therapeutic use. In some embodiments, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4 Tregs comprising a hypomethylated TSDR at the FOXP3 locus. + If Tregs, the isolated population of cells is selected for therapeutic use. In some embodiments, 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 cells containing a hypomethylated TSDR at the FOXP3 locus. + If Tregs, the isolated population of cells is selected for therapeutic use. In some embodiments, 80% to 90%, 85% to 95%, 80% to 85%, 85% to 90%, or 90% to 95% of the cells are stable CD4 T cells containing a hypomethylated TSDR at the FOXP3 locus. + If Tregs, the isolated population of cells is selected for therapeutic use.

[0209] In some embodiments, the methods provided herein involve activating an exogenous TCR that specifically binds a target peptide complexed with MHC by activating a stable CD4 + The method further comprises introducing the exogenous TCR into a population of cells comprising T regulatory cells. In some embodiments, introducing the exogenous TCR comprises transfecting the cell population with a nucleic acid encoding the exogenous TCR. In some embodiments, introducing the exogenous TCR comprises transducing the cell population with a nucleic acid encoding the exogenous TCR. The nucleic acid encoding the exogenous TCR may be an RNA (e.g., mRNA) molecule or a DNA molecule. In some embodiments, the nucleic acid is a vector or a plasmid. In some embodiments, the nucleic acid is delivered to the population of cells using a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, or a baculoviral vector.

[0210] In some embodiments, the method or process for producing a cell population of regulatory T cells further comprises activating and / or expanding the population of regulatory T cells (e.g., after the depletion, enrichment, and manipulation steps described herein). In some embodiments, the regulatory T cells are activated prior to transfection or transduction of the regulatory T cells with an exogenous TCR. In some embodiments, the regulatory T cells are activated with an anti-CD28 antibody and / or an anti-CD8 antibody. In some embodiments, activating the regulatory T cells comprises contacting the regulatory T cells with an antigen that specifically binds to an exogenous TCR belonging to the isolated cell population. In some embodiments, the antibody (e.g., anti-CD3, anti-CD28, and / or a TCR-specific antibody) is conjugated or complexed to beads, e.g., magnetic beads or polymer beads. In some embodiments, the antibody is covalently bound to a polymer matrix.

[0211] In some embodiments, the methods provided herein further comprise a second activation and / or expansion step, hi some embodiments, the second activation and / or expansion step is performed 5, 6, or 7 days after the first activation and / or expansion step.

[0212] In some embodiments, activating and / or expanding regulatory T cells comprises culturing the isolated cell population in a cell culture medium. In some embodiments, the medium comprises IL-2. In some embodiments, the medium comprises approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, 1,400, 1,600, 1,800, 2,000, 3,000, or 4,000 IU / mL of IL-2. In some embodiments, the medium comprises 500-1,500 or 800-1,200 IU / mL of IL-2. In some embodiments, the medium comprises approximately 1,000 IU / mL of IL-2. In some embodiments, the medium comprises 1,000 IU / mL of IL-2. In some embodiments, the medium further comprises TNFα. In some embodiments, the medium contains about 500, 750, 1000, 1500, 1750, 2000, 2250, 2500, or about 3000 IU / mL of TNFα. In some embodiments, the medium contains 2000-3000 or 2250-2750 IU / mL of TNFα. In some embodiments, the medium contains about 2500 IU / mL of TNFα. In some embodiments, the medium contains 2500 IU / mL of TNFα.

[0213] In some embodiments, activating and expanding the regulatory T cells comprises culturing the cells of the population for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, activating and expanding the regulatory T cells comprises culturing the cells of the population for 2-14, 2-10, 2-5, 5-10, or 5-14 days. In some embodiments, activating and expanding the regulatory T cells comprises culturing the cells of the population for 15, 14, 13, or 12 days or less. In some embodiments, after activation and / or expansion, the isolated cell population comprises at least 1 x 10 regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus. 2 , at least 1 x 10 3 , at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , or at least 1 × 10 10 Stable CD25 + / high CD4 + CD127 - / low Includes regulatory T cells.

[0214] In some embodiments, the method or process for producing a cell population of regulatory T cells may further comprise the step of cryopreserving the cell population. In some embodiments, the method or process for producing a cell population of regulatory T cells may further comprise the step of thawing the cryopreserved cell population. A cryopreservation freeze-thaw cycle refers to the process of cryopreserving a cell population and subsequently thawing the cryopreserved cell population.

[0215] Pharmaceutical Composition In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated cell population of regulatory T cells (e.g., stable regulatory T cells) described herein. In some embodiments, the pharmaceutical composition comprises a cell population of regulatory T cells (e.g., stable regulatory T cells) described herein and a pharmaceutically acceptable excipient.

[0216] As used herein, a pharmaceutically acceptable excipient may also be referred to as a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, or a pharmaceutically acceptable adjuvant. The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of appropriate dosage and treatment regimens for using the particular compositions described herein in various treatment regimens.

[0217] Pharmaceutical compositions should typically be sterile and stable under the conditions of manufacture and storage. Sterile injectable formulations may be prepared using non-toxic parenterally acceptable diluents or solvents. Pharmaceutical compositions for use according to the present invention may contain pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers, which are non-toxic to subjects at the dosages and concentrations used. In some embodiments, the pharmaceutical composition may contain an organic solvent, such as, but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide (including mixtures or combinations thereof).

[0218] The pharmaceutical composition may comprise an effective amount of stable regulatory T cells sufficient to induce a desired biological response. For example, in some embodiments, the effective amount of stable regulatory T cells described herein may refer to a number of cells sufficient to improve symptoms associated with multiple sclerosis (e.g., progressive multiple sclerosis). As will be understood by those skilled in the art, the effective amount of the solution or preparation provided herein may vary depending on various factors, such as the desired biological response, for example, the specific disease to be treated, the specific symptoms to be alleviated, the cells or tissues to be targeted, and the age, sex, and general health of the subject.

[0219] In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus) is at least 1 x 10 2 , at least 1 x 10 3 , at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , or at least 1 × 10 10 In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus) is between 1x10 and 1x10, 1x10 to 1x10, 1x10 to 1x10, 1x10 4 ~1x10 10 , 1x10 5 ~1x10 10 , 1x10 6 ~1x10 10 , 1x10 7 ~1x10 10 , 1x10 8 ~1x10 10 , 1x10 9 ~1x10 10 In some embodiments, an effective amount of stable regulatory T cells is 1 x 10 7 , 2x10 7 , 3x107 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , or 9x10 7 In some embodiments, the effective amount of stable regulatory T cells comprises 1 x 10 stable regulatory T cells. 7 ~1x10 10 , 2x10 7 ~1x10 10 , 3x10 7 ~1x10 10 , 4x10 7 ~1x10 10 , 5x10 7 ~1x10 10 , 6x10 7 ~1x10 10 , 7x10 7 ~1x10 10 , 8x10 7 ~1x10 10 , or 9x10 7 ~1x10 10 Contains stable regulatory T cells.

[0220] In some embodiments, isolated cell populations (e.g., intended for use in pharmaceutical compositions) are cryopreserved (e.g., subjected to one or more cryopreservation freeze-thaw cycles). That is, the isolated cell populations produced herein may be combined with a cryoprotectant that forms chemical bonds with water and lowers the melting temperature by increasing the total concentration of solutes in the system. Non-limiting examples of cryoprotectants include glycerol, dimethyl sulfoxide (DMSO), ethanediol, and propanediol. While conventional methods, often involving the use of serum and DMSO, may be used, the present disclosure also contemplates the use of freezing media manufactured under cGMP conditions and formulated to be serum-free and non-animal in origin (e.g., using <10% DMSO in the freezing cocktail). Other cryopreservation techniques are also provided herein, including more advanced cooling techniques that vitrify cells without the use of cryoprotectants, for example. See, for example, Shinshu University, "A new way to 'freeze' cells promises to transform the common cell-freezing practice." ScienceDaily.com, April 2019. This process of ultra-rapid cooling utilizes inkjet cell printing to cool cells at a rate of 10,000°C / second, causing near-vitrification of the cells.

[0221] Treatment method In some embodiments, the present disclosure provides a stable CD4 +

[0010] Methods for treating multiple sclerosis in a subject in need thereof are provided, comprising administering an isolated population of cells comprising regulatory T cells, or a composition thereof. In some embodiments, the present disclosure provides methods for administering to a subject (e.g., a subject with multiple sclerosis) an isolated cell population comprising regulatory T cells (and related pharmaceutical compositions) described herein.

[0222] Multiple sclerosis typically begins with relapses and remissions of symptoms in relapsing-remitting multiple sclerosis (RRMS). Approximately 80–85% of MS patients are initially diagnosed with this form of the disease. There are over 15 disease-modifying therapies for RRMS. In progressive MS, there is a slow, steady progression of the disease. Progressive MS can be relapsing (sometimes called secondary progressive MS) or non-relapsing (sometimes called primary progressive MS). Progressive multiple sclerosis is characterized by increasing clinical disability independent of acute relapsing episodes. Currently, there is no available treatment for progressive multiple sclerosis. More than 250,000 patients suffer from progressive multiple sclerosis. Furthermore, while currently approved disease-modifying therapies are effective in reducing the occurrence of relapses in RRMS, even RRMS subjects experience relapse-independent progression (PIRA), which is unsuccessful treatment (Ransohoff, 2023).

[0223] In progressive MS, compartmentalized inflammation, primarily localized to meningeal lymphoid aggregates, drives ongoing inflammatory demyelination behind an intact blood-brain barrier during progressive multiple sclerosis (Lassman, Frontiers in Immunology, 2019). Inflammatory T cells in stable, persistent aggregates release inflammatory mediators that activate macrophages. This high density of meningeal lymphocytes and inflammation has been shown to be associated with elevated levels of subpial demyelination in the cortex and increased white matter lesions (Ahmed et al., JCI Insight. 2022;7(5)). Macrophages at the lesion edge continuously degrade myelin. PIRA in RRMS subjects has the same mechanistic basis as progression in progressive MS (Ransohoff, 2023).

[0224] Without wishing to be bound by theory, it is contemplated herein that targeting stably engineered regulatory T cells to the meninges by engineering them to bind to MBP presented by cells in the meninges of subjects with multiple sclerosis will reduce, halt, or reverse the progression of multiple sclerosis and progressive multiple sclerosis, particularly by reducing the inflammatory response in the meninges.

[0225] In some embodiments, the present disclosure provides a method of administering to a subject a cell population or pharmaceutical composition comprising a regulatory T cell described herein in an amount effective to alleviate one or more symptoms of multiple sclerosis, including, but not limited to, fatigue, blurred vision, muscle tissue paralysis, muscle spasms, muscle rigidity, muscle weakness, mobility disorders, pain (e.g., muscle pain), depression, anxiety, sexual disorders, bladder disorders, bowel disorders, speech disorders, and difficulty swallowing.

[0226] In some embodiments, the present disclosure provides a method of treating multiple sclerosis in a subject, the method comprising administering to the subject a cell population or pharmaceutical composition comprising regulatory T cells described herein. In some embodiments, at least a portion of the population of cells are autologous cells (i.e., obtained from the same subject to whom they are subsequently administered). In some embodiments, the population of cells is isolated from the subject, subjected to a method of producing a cell population 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 the disease.

[0227] In some embodiments, treating a disease (or treatment thereof) refers to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of multiple sclerosis or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have occurred and / or after the disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or inhibit the onset or progression of the disease. For example, treatment may be administered to a susceptible individual before the onset of symptoms (e.g., to identify genetic factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0228] A subject refers to an individual organism, e.g., an individual human. In some embodiments, the subject is a human subject, such as a male or female subject. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be male or female.

[0229] Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intravenous, subcutaneous, intravenous, intrathecal administration, direct delivery to a selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intramuscular, intradermal, intratumoral, and other parenteral routes of administration. If desired, administration routes may be combined. In some embodiments, the isolated populations or compositions thereof described herein are administered intravenously.

[0230] The cell population or pharmaceutical composition comprising regulatory T cells may be administered as a bolus dose. In some embodiments, administration of the cell population or pharmaceutical composition comprising regulatory T cells comprises one or more infusions of the isolated cell population or pharmaceutical composition into the subject (e.g., the cells are infused through a central line, similar to a blood transfusion).

[0231] Further Numbered Embodiments Further numbered embodiments of the present disclosure are provided as follows:

[0232] Embodiment 1. An isolated cell population comprising regulatory T cells, wherein the regulatory T cells of the isolated cell population (a) comprise an exogenous human T cell receptor (TCR) that specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC), and (b) comprise stable regulatory T cells that comprise a hypomethylated regulatory T cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus, wherein at least 80% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / lo regulatory T cells, and wherein less than 10% of the cells of the isolated cell population express FOXP3 protein from an engineered FOXP3 locus.

[0233] Embodiment 2. The isolated population of embodiment 1, wherein the MBP peptide is an MBP83-99 peptide comprising the amino acid sequence of SEQ ID NO:61.

[0234] Embodiment 3. The MHC is HLA-DRB1 * 3. The isolated population of embodiment 1 or 2, comprising 15:01.

[0235] Embodiment 4. The MHC is HLA-DRA * 4. The isolated population of embodiment 3, further comprising:

[0236] Embodiment 5. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR comprises a TCR alpha chain comprising: (a) a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 2; and / or (c) a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 3.

[0237] Embodiment 6. The isolated population of embodiment 5, wherein the exogenous TCR comprises a TCR alpha chain variable region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:4.

[0238] Embodiment 7. The isolated population of embodiment 5, wherein the exogenous TCR comprises a TCR alpha chain comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 5 or 73.

[0239] Embodiment 8. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR comprises a TCR β chain comprising a CDR1-β sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR2-β sequence comprising the amino acid sequence of SEQ ID NO:8, and / or a CDR3-β sequence comprising the amino acid sequence of SEQ ID NO:9.

[0240] Embodiment 9. The isolated population of embodiment 8, wherein the exogenous TCR comprises a TCR beta chain variable region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 10.

[0241] Embodiment 10. The isolated population of embodiment 8, wherein the exogenous TCR comprises a TCR beta chain comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11 or 75.

[0242] Embodiment 11. The isolated population of any one of embodiments, wherein the exogenous TCR comprises a TCR alpha chain comprising: (a) a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 18; (b) a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 19; and / or (c) a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 20.

[0243] Embodiment 12. The isolated population of embodiment 11, wherein the exogenous TCR comprises a TCR alpha chain variable region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 21.

[0244] Embodiment 13. The isolated population of embodiment 11, wherein the exogenous TCR comprises a TCR alpha chain comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 22 or 80.

[0245] Embodiment 14. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR comprises a TCR β chain comprising a CDR1-β sequence comprising the amino acid sequence of SEQ ID NO: 24, a CDR2-β sequence comprising the amino acid sequence of SEQ ID NO: 25, and / or a CDR3-β sequence comprising the amino acid sequence of SEQ ID NO: 26.

[0246] Embodiment 15. The isolated population of embodiment 14, wherein the exogenous TCR comprises a TCR beta chain variable region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 27.

[0247] Embodiment 16. The isolated population of embodiment 14, wherein the exogenous TCR comprises a TCR beta chain comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 28 or 82.

[0248] Embodiment 17. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR comprises a TCR alpha chain comprising: (a) a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 32; (b) a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 33; and / or (c) a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 34.

[0249] Embodiment 18. The isolated population of embodiment 17, wherein the exogenous TCR comprises a TCR alpha chain variable region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 35.

[0250] Embodiment 19. The isolated population of embodiment 17, wherein the exogenous TCR comprises a TCR alpha chain comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 36.

[0251] Embodiment 20. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR comprises a TCR β chain comprising a CDR1-β sequence comprising the amino acid sequence of SEQ ID NO: 38, a CDR2-β sequence comprising the amino acid sequence of SEQ ID NO: 39, and / or a CDR3-β sequence comprising the amino acid sequence of SEQ ID NO: 40.

[0252] Embodiment 21. The isolated population of embodiment 20, wherein the exogenous TCR comprises a TCR beta chain variable region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 41.

[0253] Embodiment 22. The isolated population of embodiment 20, wherein the exogenous TCR comprises a TCR beta chain comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 42.

[0254] Embodiment 23. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR is encoded as a single polypeptide, and optionally, the polypeptide comprises an N-terminal β domain and a C-terminal α domain.

[0255] Embodiment 24. The isolated population of embodiment 23, wherein the single polypeptide comprises a TCR alpha chain and a TCR beta chain, and the polypeptide comprises a self-cleaving peptide sequence located between the TCR alpha chain and the TCR beta chain.

[0256] Embodiment 25. The isolated population of embodiment 24, wherein the self-cleaving peptide sequence is a 2A peptide sequence, and optionally the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence.

[0257] Embodiment 26. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR comprises a TCR alpha chain comprising a constant region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 58.

[0258] Embodiment 27. The isolated population of any one of the preceding embodiments, wherein the exogenous TCR comprises a TCR beta chain comprising a constant region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 59 or 60.

[0259] Embodiment 28. The isolated population of any one of the preceding embodiments, wherein the exogenous human TCR comprises one or more amino acid substitutions to cysteine residues in the TCR alpha chain constant region and the TCR beta chain constant region, wherein the cysteine residues are capable of forming one or more disulfide bonds.

[0260] Embodiment 29. The isolated population of any one of the preceding embodiments, wherein the TCR alpha chain constant region comprises a T48C amino acid substitution relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and the TCR beta chain constant region comprises a S57C amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60.

[0261] Embodiment 30. The isolated cell population of any one of the preceding embodiments, wherein at least 70% of the cells of the isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

[0262] Embodiment 31. The isolated population of embodiment 30, 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.

[0263] Embodiment 32. The isolated cell population of any one of the preceding embodiments, wherein at least 85%, at least 90%, or at least 95% of the cells of the isolated cell population are CD25+ / highCD4+CD127- / lo regulatory T cells.

[0264] Embodiment 33. At least 70% of the cells of the isolated cell population are CD25 + / high CD4 + CD127 - / low FOXP3 + 10. The isolated cell population of any one of the preceding embodiments, wherein the cell population is regulatory T cells.

[0265] Embodiment 34. At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells of the isolated cell population are CD25 + / high CD4 + CD127 - / low FOXP3 + 34. The isolated cell population of embodiment 33, which is regulatory T cells.

[0266] Embodiment 35. The isolated cell population of any one of the preceding embodiments, wherein the percentage of cells of the isolated cell population comprising stable regulatory T cells does not decrease by more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percentage point(s) during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0267] Embodiment 36. The isolated cell population of embodiment 35, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0268] Embodiment 37. The isolated cell population of embodiment 35, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 5 percentage points for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0269] Embodiment 38. The isolated cell population of embodiment 35, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1 percentage point for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0270] Embodiment 39. The isolated cell population of any one of the preceding embodiments, wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0271] Embodiment 40. The isolated cell population of embodiment 39, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10% for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 10% for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0272] Embodiment 41. The isolated cell population of embodiment 39, wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 5% for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population comprising stable regulatory T cells does not decrease by more than 5% for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0273] Embodiment 42. The isolated cell population of embodiment 39, wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1% for at least 5 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR, or wherein the percentage of cells in the isolated cell population that comprise stable regulatory T cells does not decrease by more than 1% for at least 10 days of expansion after transduction of the stable regulatory T cells with an exogenous human TCR.

[0274] Embodiment 43. At least 1 × 10 7 10. The isolated cell population of any one of the preceding embodiments, comprising stable regulatory T cells.

[0275] Embodiment 44. The isolated cell population of embodiment 43, comprising 1 x 10 to 1 x 10 stable regulatory T cells.

[0276] Embodiment 45. The isolated cell population of any one of the preceding embodiments, wherein less than 5%, less than 2%, or less than 1% of the cells of the isolated cell population express FOXP3 protein from the engineered FOXP3 locus.

[0277] Embodiment 46. The isolated cell population of any one of the preceding embodiments, wherein less than 10% of the cells of the isolated cell population express FOXP3 protein from (a) an endogenous open reading frame operably linked to an engineered FOXP3 promoter, or (b) an exogenous FOXP3 transgene.

[0278] Embodiment 47 The isolated cell population of any one of the preceding embodiments, wherein at least 90%, at least 95%, or 100% of the cells of the isolated cell population comprise an unmodified endogenous FOXP3 locus.

[0279] Embodiment 48. The isolated cell population of any one of the preceding embodiments, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% of the cells of the isolated cell population comprise an engineered FOXP3 gene or engineered FOXP3 protein.

[0280] Embodiment 49 The isolated cell population of any one of the preceding embodiments, wherein at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the cells of the isolated cell population do not contain an exogenous FOXP3 gene or exogenous FOXP3 protein.

[0281] Embodiment 50 The isolated cell population of any one of the preceding embodiments, wherein at least 90%, at least 95%, or 100% of the cells of the isolated cell population do not exhibit ectopic FOXP3 expression.

[0282] Embodiment 51 The isolated cell population of any one of the preceding embodiments, wherein at least 90%, at least 95%, or 100% of the cells of the isolated cell population express physiological levels of functional FOXP3 protein.

[0283] Embodiment 52. The isolated cell population of any one of the preceding embodiments, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% of the cells of the isolated cell population overexpress functional FOXP3 protein.

[0284] Embodiment 53. The isolated cell population of any one of the preceding embodiments, wherein less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells of the isolated cell population are conventional T cells.

[0285] Embodiment 54. The isolated cell population of any one of the preceding embodiments, wherein about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 5%, or about 5% to about 10% of the cells of the isolated cell population are conventional T cells.

[0286] Embodiment 55. The isolated cell population of embodiment 53 or 54, wherein the conventional T cells of the isolated population comprise an exogenous human TCR.

[0287] Embodiment 56. The isolated cell population of embodiment 55, wherein about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, or about 60% of the conventional T cells comprise an exogenous TCR.

[0288] Embodiment 57. The isolated cell population of any one of the preceding embodiments, wherein the ratio of regulatory T cells to conventional T cells in the isolated cell population is at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 100:1.

[0289] Embodiment 58 The isolated cell population of any one of the preceding embodiments, wherein less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells of the isolated cell population are CD8+ T cells.

[0290] Embodiment 59. The isolated cell population of embodiment 58, wherein CD8+ T cells are undetectable, optionally undetectable by fluorescence-activated cell sorting (FACS).

[0291] Embodiment 60 The isolated cell population of any one of the preceding embodiments, wherein at least 10% of the regulatory T cells of the isolated cell population express an exogenous human TCR.

[0292] Embodiment 61. The isolated cell population of embodiment 60, wherein at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the regulatory T cells of the isolated cell population express an exogenous human TCR.

[0293] Embodiment 62.CD25 + / high CD4 + CD127 - / low

[0023] 2. The isolated cell population of any one of the preceding embodiments, wherein the percentage of regulatory T cells is assessed between 1 hour and 120 days after transduction.

[0294] Embodiment 63.CD25 + / high CD4 + CD127 - / low 63. The isolated cell population of any one of embodiments 1-62, wherein the percentage of regulatory T cells is assessed at least 1, 12, 24, 48, 72, 96, or 120 hours after transduction.

[0295] Embodiment 64. CD25 + / high CD4 + CD127 - / low 63. The isolated cell population of any one of embodiments 1-62, wherein the percentage of regulatory T cells is assessed 7-14 days after transduction.

[0296] Embodiment 65.CD25 + / high CD4 + CD127 - / low 63. The isolated cell population of any one of embodiments 1-62, wherein the percentage of regulatory T cells is assessed at least 10, 15, 30, 60, or 120 days after transduction.

[0297] Embodiment 66. The isolated cell population of any one of the preceding embodiments, wherein the TSDR at the endogenous FOXP3 locus remains hypomethylated after cryopreservation freeze-thaw cycles, at least until the cells of the isolated cell population are administered to a subject.

[0298] Embodiment 67. The isolated cell population of any one of the preceding embodiments, wherein the stable regulatory T cells of the isolated cell population exhibit one or more functions selected from: (a) cytokine secretion activity, (b) expression of activation markers associated with regulatory T cells, and (c) suppressive activity.

[0299] Embodiment 68. The isolated cell population of embodiment 67, wherein one or more functions are assessed 1 hour to 14 days after transduction.

[0300] Embodiment 69. The isolated cell population of embodiment 67, wherein one or more functions are assessed at least 12, 24, 48, 72, 96, or 120 hours after transduction.

[0301] Embodiment 70. The isolated cell population of embodiment 67, wherein the one or more functions are assessed at least 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction.

[0302] Embodiment 71. An isolated cell population according to any one of embodiments 67 to 70, wherein one or more functions are assessed after cryopreservation of the isolated cell population, optionally at least 24 hours after cryopreservation.

[0303] Embodiment 72 The isolated cell population of any one of the preceding embodiments, wherein at least 10% of the stable regulatory T cells are CD45RA+.

[0304] Embodiment 73. 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 + 73. The isolated cell population of embodiment 72, wherein

[0305] Embodiment 74. At least 10% of the cells of the isolated cell population express CD25 prior to activation and transduction with an exogenous human TCR. + / high CD4 + CD127 - / low CD45RA + 10. The isolated cell population of any one of the preceding embodiments, wherein

[0306] Embodiment 75. At least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells of the isolated cell population are CD25 TCR-positive prior to activation and transduction with an exogenous human TCR. + / high CD4 + CD127 - / low CD45RA + 10. The isolated cell population of any one of the preceding embodiments, wherein

[0307] Embodiment 76.CD25 + / high CD4 + CD127 - / low 10. The isolated population of any one of the preceding embodiments, wherein the regulatory T cells have a human leukocyte antigen (HLA)-DR15 haplotype.

[0308] Embodiment 77.CD25 + / high CD4 + CD127 - / lowRegulatory T cells express HLA-DRB1 * 77. The isolated population of embodiment 76, comprising the 15:01 allele.

[0309] Embodiment 78.CD25 + / high CD4 + CD127 - / low Regulatory T cells express HLA-DRB5 * 78. The isolated population of embodiment 77, further comprising the 01:01 allele.

[0310] Embodiment 79. A method of producing an isolated cell population of any one of the preceding embodiments, comprising: (a) isolating a biological sample comprising regulatory T cells from a human subject with multiple sclerosis, optionally progressive multiple sclerosis; (b) isolating from the biological sample CD8+ cells, CD19+ cells, and optionally CD14+ cells. + (c) removing the cells to produce a depleted biological sample; and (d) detecting CD25 + / high (d) selecting the cells to produce a CD25 enriched cell population; and (e) isolating the CD25 enriched cell population from the CD25 enriched cell population. + / high CD4 + CD127 - / low CD45RA + (e) selecting the cells to produce one or more positive fractions; and (f) detecting CD25 from the one or more positive fractions. + / high CD4 + CD127 - / low CD45RA + (f) selecting the cells to produce a cell population comprising stable regulatory T cells; and (f) engineering the cell population comprising stable regulatory T cells such that the stable regulatory T cells comprise an exogenous human TCR, thereby producing an isolated population of cells.

[0311] Embodiment 80. The method of embodiment 79, 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.

[0312] Embodiment 81. Less than 0.5% of the cells in the depleted sample are CD8 +81. The method of any one of embodiments 79-80, comprising cells.

[0313] In embodiment 82, (d) selecting is performed by CD4 + CD45RA + The cells were then identified and the identified CD4 + CD45RA + CD25 from cells + / high CD127 - / low Identify cells and identify CD25 + / high CD4 + CD127 - / low CD45RA + Identifying a first population of cells and then isolating CD25 from the first subpopulation. + / high CD4 + CD127 - / low CD45RA + 82. The method of any one of embodiments 79-81, comprising selecting the first population of cells to produce one or more positive fractions.

[0314] In embodiment 83, (e) selecting is performed by CD4 + CD45RA + The cells were then identified and the identified CD4 + CD45RA + CD25 from cells + / high CD127 - / low Identify cells and identify CD25 + / high CD4 + CD127 - / low CD45RA + Identifying a second population of cells and then isolating CD25 from the second subpopulation. + / high CD4 + CD127 - / low CD45RA + 83. The method of any one of embodiments 79-82, comprising selecting the second population of cells to produce a cell population comprising stable regulatory T cells.

[0315] Embodiment 84. The method of any one of embodiments 79 to 83, wherein the subject has a human leukocyte antigen (HLA)-DR15 haplotype.

[0316] Embodiment 85. Regulatory T cells are HLA-DRB1 * 85. The method of embodiment 84, comprising the 15:01 allele.

[0317] Embodiment 86. The method of any one of embodiments 79 to 85, wherein the subject is a male subject.

[0318] Embodiment 87. The method of any one of embodiments 79 to 85, wherein the subject is a female subject.

[0319] Embodiment 88. The method of any one of embodiments 79 to 87, wherein manipulating the isolated cell population comprises transducing the cell population with a nucleic acid encoding an exogenous human TCR.

[0320] Embodiment 89. Cells of the isolated cell population are activated and expanded to produce at least 1 x 10 stable CD25 cells containing a hypomethylated TSDR at the endogenous FOXP3 locus. + / high CD4 + CD127 - / low 89. The method of any one of embodiments 79-88, further comprising producing a cell population comprising regulatory T cells.

[0321] Embodiment 90. The method of embodiment 89, wherein the activating and expanding comprises culturing the cells of the isolated cell population for at least 5, 6, 7, 8, 9, or 10 days.

[0322] Embodiment 91. The method of embodiment 89, wherein activating and expanding comprises culturing the cells of the isolated cell population for no more than 15, 14, 13, or 12 days.

[0323] Embodiment 92. The method of any one of embodiments 88 to 91, wherein the nucleic acid is a vector.

[0324] Embodiment 93. The method of embodiment 92, wherein the vector is a viral vector.

[0325] Embodiment 94. The method of embodiment 93, wherein the viral vector is a lentiviral vector.

[0326] Embodiment 95. The method of any one of embodiments 88 to 94, wherein the nucleic acid comprises a promoter operably linked to a coding sequence encoding an exogenous human TCR, and optionally the promoter is an EF-1α promoter or an MND promoter.

[0327] Embodiment 96. The method of any one of embodiments 88 to 95, wherein the nucleic acid further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally a WPRE-mut6.

[0328] Embodiment 97. The method of any one of embodiments 88 to 96, wherein the coding sequence is codon-optimized.

[0329] Embodiment 98. A vector according to any one of embodiments 88 to 97.

[0330] Embodiment 99. A pharmaceutical composition comprising the isolated cell population of any one of embodiments 1 to 78 and a pharmaceutically acceptable excipient.

[0331] Embodiment 100. A composition comprising the isolated cell population of any one of embodiments 1 to 78 and a cryopreservative.

[0332] Embodiment 101. A method comprising administering to a subject the pharmaceutical composition of embodiment 99, wherein the subject has multiple sclerosis.

[0333] Embodiment 102. The method of embodiment 101, wherein the subject has progressive multiple sclerosis.

[0334] Embodiment 103. The method of embodiment 102, wherein the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

[0335] Embodiment 104. The method of any one of embodiments 101-103, wherein the pharmaceutical composition is administered in an amount effective to alleviate one or more symptoms of multiple sclerosis.

[0336] Embodiment 105. The method of any one of embodiments 101 to 104, wherein the isolated population is obtained from a subject.

[0337] Embodiment 106. The method of any one of embodiments 101 to 105, wherein the subject has an HLA-DR15 haplotype.

[0338] Embodiment 107. The subject has HLA-DRB1 * 107. The method of embodiment 106, wherein the patient has the 15:01 allele.

[0339] Embodiment 108. The subject has HLA-DRB5 * 108. The method of embodiment 106 or 107, having the 01:01 allele.

[0340] Embodiment 109. A method comprising administering to a subject the isolated population of any one of embodiments 1 to 78.

[0341] Embodiment 110. The method of embodiment 109, wherein the subject has multiple sclerosis.

[0342] Embodiment 111. The method of embodiment 110, wherein the subject has progressive multiple sclerosis.

[0343] Embodiment 112. The method of embodiment 111, wherein the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

[0344] Embodiment 113. The method of any one of embodiments 101 to 112, wherein the isolated population is obtained from a subject.

[0345] Embodiment 114. The method of any one of embodiments 101 to 113, wherein the subject has an HLA-DR15 haplotype.

[0346] Embodiment 115. The subject has HLA-DRB1 * 115. The method of embodiment 114, wherein the patient has the 15:01 allele.

[0347] Embodiment 116. The subject has HLA-DRB5 * 116. The method of embodiment 114 or 115, wherein the patient has the 01:01 allele.

[0348] Embodiment 117. The method of any one of embodiments 101 to 116, wherein administering comprises intravenous administration.

[0349] Embodiment 118. The method of any one of embodiments 101 to 117, wherein administering comprises one or more injections.

[0350] Embodiment 133. An engineered polynucleotide comprising an open reading frame comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 107.

[0351] Embodiment 134. The engineered polynucleotide of embodiment 133, wherein the nucleotide sequence of the open reading frame has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 107.

[0352] Embodiment 135. The engineered polynucleotide of embodiment 134, wherein the nucleotide sequence of the open reading frame has 100% identity to the nucleotide sequence of SEQ ID NO: 107.

[0353] Embodiment 136. An engineered polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 108.

[0354] Embodiment 137. The engineered polynucleotide of embodiment 136, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 108.

[0355] Embodiment 138. The engineered polynucleotide of embodiment 136 or 137, wherein the amino acid sequence of the polypeptide comprises a first cysteine at a position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 108, and / or a second cysteine at a position corresponding to position 513 of the amino acid sequence of SEQ ID NO: 108.

[0356] Embodiment 139. The engineered polynucleotide of embodiment 138, wherein the amino acid sequence of the polypeptide has 100% identity to the amino acid sequence of SEQ ID NO: 108.

[0357] Embodiment 140. The engineered polynucleotide of any one of embodiments 133 to 139, wherein the open reading frame encodes a human / mouse hybrid T cell receptor (TCR), e.g., wherein the variable region is a human variable region and the constant domain is a mouse constant domain.

[0358] Embodiment 141. The engineered polynucleotide of embodiment 140, wherein the human / mouse hybrid TCR comprises an alpha chain comprising the amino acid sequences of SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111.

[0359] Embodiment 142. The engineered polynucleotide of embodiment 141, wherein the human / mouse hybrid TCR comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 112.

[0360] Embodiment 143. The engineered polynucleotide of embodiment 141, wherein the human / mouse hybrid TCR comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 114.

[0361] Embodiment 144. The engineered polynucleotide of embodiment 141, wherein the human / mouse hybrid TCR comprises an alpha constant domain comprising the amino acid sequence of SEQ ID NO: 123.

[0362] Embodiment 145. The engineered polynucleotide of any one of embodiments 140 to 144, wherein the human / mouse hybrid TCR comprises a β chain comprising the amino acid sequences of SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0363] Embodiment 146. The engineered polynucleotide of embodiment 145, wherein the human / mouse hybrid TCR comprises a beta chain comprising the amino acid sequence of SEQ ID NO: 118.

[0364] Embodiment 147. The engineered polynucleotide of embodiment 145, wherein the human / mouse hybrid TCR comprises a beta chain comprising the amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.

[0365] Embodiment 148. The engineered polynucleotide of embodiment 145, wherein the human / mouse hybrid TCR comprises a β constant domain comprising the amino acid sequence of SEQ ID NO: 124.

[0366] Embodiment 149. The engineered polynucleotide of any one of embodiments 140 to 148, wherein the TCR specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0367] Embodiment 150. An engineered polynucleotide comprising an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:113.

[0368] Embodiment 151. The engineered polynucleotide of embodiment 150, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 113.

[0369] Embodiment 152. The engineered polynucleotide of embodiment 151, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.

[0370] Embodiment 153. The engineered polynucleotide of any one of embodiments 150 to 152, wherein the amino acid sequence of the polypeptide further comprises an amino-terminal leader sequence, optionally METLLGVSLV ILWLQLARVN (SEQ ID NO: 100).

[0371] Embodiment 154. The engineered polynucleotide of embodiment 153, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 114.

[0372] Embodiment 155. An engineered polynucleotide comprising an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119.

[0373] Embodiment 156. The engineered polynucleotide of embodiment 155, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 119.

[0374] Embodiment 157. The engineered polynucleotide of embodiment 156, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 119.

[0375] Embodiment 158. The engineered polynucleotide of any one of embodiments 155 to 157, wherein the amino acid sequence of the polypeptide further comprises an amino-terminal leader sequence, optionally METLLGVSLV ILWLQLARVN (SEQ ID NO: 100).

[0376] Embodiment 159. The engineered polynucleotide of embodiment 158, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 121.

[0377] Embodiment 160. The engineered polynucleotide of any one of embodiments 155 to 159, wherein the amino acid sequence of the polypeptide is optionally selected from P2A, E2A, F2A, and T2A, and optionally further comprises a carboxy-terminal self-cleaving peptide sequence comprising the sequence GSG ATNFSLLKQA GDVEENPG (SEQ ID NO: 104).

[0378] Embodiment 161. The engineered polynucleotide of any one of embodiments 155 to 157, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 120.

[0379] Embodiment 162. The engineered polynucleotide of any one of embodiments 155 to 157, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 122.

[0380] Embodiment 163. The engineered polynucleotide of any one of the preceding embodiments, wherein the engineered polynucleotide is a messenger ribonucleic acid (mRNA).

[0381] Embodiment 164. The engineered polynucleotide of any one of embodiments 133 to 162, wherein the engineered polynucleotide is deoxyribonucleic acid (DNA).

[0382] Embodiment 165. The engineered polynucleotide of embodiment 164, further comprising a promoter operably linked to the open reading frame.

[0383] Embodiment 166. The engineered polynucleotide of embodiment 165, wherein the promoter is an EF-1α promoter or an MND promoter.

[0384] Embodiment 179. An engineered polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 108.

[0385] Embodiment 180. The engineered polypeptide of embodiment 179, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 108.

[0386] Embodiment 181. The engineered polypeptide of embodiment 180, wherein the amino acid sequence of the polypeptide has 100% identity to the amino acid sequence of SEQ ID NO: 108.

[0387] Embodiment 182. An engineered polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 113.

[0388] Embodiment 183. The engineered polypeptide of embodiment 182, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 113.

[0389] Embodiment 184. The engineered polypeptide of embodiment 183, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.

[0390] Embodiment 185. The engineered polypeptide of any one of embodiments 182 to 184, wherein the amino acid sequence of the polypeptide further comprises an amino-terminal leader sequence, optionally METLLGVSLV ILWLQLARVN (SEQ ID NO: 100).

[0391] Embodiment 186. The engineered polypeptide of embodiment 185, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 114.

[0392] Embodiment 187. An engineered polypeptide encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119, wherein the amino acid sequence of the polypeptide comprises a cysteine at a position corresponding to position 175 of the amino acid sequence of SEQ ID NO: 119.

[0393] Embodiment 188. The engineered polypeptide of embodiment 187, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 119.

[0394] Embodiment 189. The engineered polypeptide of embodiment 188, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 119.

[0395] Embodiment 190. The engineered polypeptide of any one of embodiments 187 to 189, wherein the amino acid sequence of the polypeptide further comprises an amino-terminal leader sequence, optionally METLLGVSLV ILWLQLARVN (SEQ ID NO: 100).

[0396] Embodiment 191. The engineered polypeptide of embodiment 190, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 121.

[0397] Embodiment 192. The engineered polypeptide of any one of embodiments 187 to 191, wherein the amino acid sequence of the polypeptide further comprises a carboxy-terminal P2A overhang sequence, optionally GSG ATNFSLLKQA GDVEENPG (SEQ ID NO: 104).

[0398] Embodiment 193. The engineered polypeptide of any one of embodiments 187 to 189, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 120.

[0399] Embodiment 194. The engineered polypeptide of any one of embodiments 187 to 189, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 122.

[0400] Embodiment 195. A T cell receptor in which (a) the amino acid sequence of the alpha chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 114, and / or (b) the amino acid sequence of the beta chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.

[0401] Embodiment 196. The T cell receptor of embodiment 195, wherein (a) the amino acid sequence of the alpha chain has 100% identity to the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 114, and / or (b) the amino acid sequence of the beta chain has 100% identity to the amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.

[0402] Embodiment 197. A T cell receptor comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 108.

[0403] Embodiment 198. The T cell receptor of embodiment 197, wherein the amino acid sequence of the T cell receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 108.

[0404] Embodiment 199. The T cell receptor of embodiment 198, wherein the amino acid sequence of the T cell receptor has 100% identity to the amino acid sequence of SEQ ID NO: 108.

[0405] Embodiment 200. A T cell receptor encoded by an engineered polynucleotide according to any one of embodiments 123 to 166.

[0406] Embodiment 201. A T cell receptor comprising an engineered polypeptide according to any one of embodiments 173 to 194.

[0407] Embodiment 202. An isolated population according to any one of embodiments 1 to 74, comprising an engineered polypeptide according to any one of embodiments 173 to 194. [Example]

[0408] Example 1. Manufacturing method for the production of autologous stable regulatory T cells expressing exogenous TCRs The generation of a cell population containing stable thymus-derived regulatory T cells expressing an exogenous TCR is demonstrated in this example. The overall enrichment and sorting strategy is shown in Figures 1A-1B.

[0409] Leukapheresis, depletion, and enrichment First, leukapheresis transfusions from human subjects were collected, transported to a central processing facility, and held at 4°C until processing approximately 24 hours after collection. The incoming leukapheresis product was washed in phosphate-buffered saline containing 0.5% human serum albumin (HSA) to reduce platelet counts and then labeled with a cocktail of magnetic microbeads (CD8, CD14, and CD19 microbeads; Miltenyi) using an automated cell processing system.

[0410] Using magnetic cell separation technology, cytotoxic T cells (CD8 + ), B cells (CD19 + ), and monocytes (CD14 + ) and then CD25 +The cells were concentrated. The volume of the depleted biosample was reduced by centrifugation. The depleted biosample was then labeled with CD25-PE-biotin antibody (Miltenyi) according to the manufacturer's recommendations. The cells were then washed with phosphate-buffered saline containing 0.5% HSA and labeled with anti-biotin microbeads (Miltenyi). Magnetic cell separation technology was then used to separate CD25 + The cells were isolated to produce a CD25 enriched sample.

[0411] Figure 2 shows apheresis for Run 1, CD8 + , CD19 + , and CD14 + Depletion of cells (depleted samples), and CD25 + Figure 1 shows the viable cells (VC) of all cells after selection for CD3 (CD25 enriched sample). Approximately 98% of all cells in the population were CD3 after the CD25 selection step to generate the CD25 enriched sample. + Approximately 95% of the cells are CD4+ T cells.

[0412] Table 1 shows the CD4+ saturation of the apheresis cell populations after the depletion and enrichment steps for five additional manufacturing runs. + The composition of T cells and naive Treg cells is shown.

[0413] [Table 1]

[0414] Figure 3A shows the apheresis and CD8 + naive regulatory T cells (specifically, CD4 T cells) after selection for CD25+ (CD25-enriched samples), removal of CD19+, and CD14+ cells (depleted samples), and CD25+ (CD25-enriched samples). + CD25 +Figure 3B shows the viable count (VC) of naive regulatory T cells (CD4+CD25+ / highCD127- / loCD45RA+ cells) after each step. Approximately 80% of CD4+CD25+ / highCD127- / loCD45RA+ cells were recovered after the depletion step, and 75% of CD4+CD25+ / highCD127- / loCD45RA+ cells were recovered after the CD25 enrichment step.

[0415] Table 2 shows the VC of naive regulatory T cells after apheresis, depletion, and enrichment, as well as the recovery rate from the previous step of naive regulatory T cells after depletion and enrichment.

[0416] [Table 2]

[0417] Gating Strategy The CD25-enriched samples were then stained with fluorescently labeled anti-CD45RA, anti-CD4, and anti-CD127 antibodies to detect CD4 + CD25 + / high CD127 - / lo CD45RA + Phenotypes were sorted by fluorescence-activated cell sorting (FACS) to generate cell populations containing stable regulatory T cells. FACS was performed in two rounds: a first debulk fraction sort and a second fractional purity sort. For debulk sorting, the CD25-enriched sample was divided into multiple fractions that were sorted across multiple cell sorters. After debulk sorting, the fractions were combined for the second round of purity sorting. The purpose of differentially sorting cells in debulk and / or purity sorting is to expedite the sorting process by distributing the sorting across multiple machines and ensure that cells remain viable. The gating strategies used for debulk and purity sorting were identical. For the gating strategy, CD45RA + CD4 + Cells were first selected and then CD25 + / high CD127 - / lo CD45RA cells+ CD4 + We used a dot plot-based approach to select from a cell population to optimize the purity of stable regulatory T cells in the population as determined by their TSDR phenotype. As shown in Figure 1B, this FACS gating strategy begins with first selecting CD45RA on a dot plot. + vs. CD4 + The cells were plotted and selected based on their population distribution using a box gate. From the selected population of cells, CD25 + / high Cells vs. CD127 - / lo Plot the cells and gate using a polygon gate to identify CD4 + CD25 + / high CD127 - / lo CD45RA + A population of cells was selected. This FACS strategy was developed to optimize the selection of stable naive regulatory T cells based on the TSDR hypomethylation phenotype. CD4 for run 1 + CD25 + / high CD127 - / lo CD45RA + The composition of the cell population before sorting, after debulking, and after production of the cell population is provided in Table 3.

[0418] [Table 3]

[0419] The purity and recovery of naive Tregs during the selection process for five additional manufacturing runs are shown in Table 4.

[0420] [Table 4]

[0421] Regulatory T cells were activated using T cell TransAct Activation Reagent (Miltenyi) for approximately 48 hours after FACS. The activated regulatory T cells were then transduced with a third-generation VSV-pseudotyped, self-inactivating lentiviral vector encoding an exogenous TCR that specifically binds to a target peptide associated with multiple sclerosis (e.g., progressive multiple sclerosis), designated "TCR-A." The vector construct contains an N-terminal TCR β chain and a C-terminal TCR α chain, with a linker domain containing a GSG amino acid sequence followed by a P2A self-cleaving peptide. The TCR β-GSG-P2A-TCR α fusion construct was expressed under the EF1α promoter. The percentage of transduced cells, measured by FACS analysis using an anti-Vβ antibody, is shown in Figure 5.

[0422] The transduced regulatory T cells were then expanded in T cell expansion medium supplemented with 1,000 IU / mL of IL-2 until harvest (day 9, 10, or 11). The cell number, size, population doubling, and viability of non-transduced and transduced regulatory T cells were assessed and are shown for run 1 (Figure 4). Notably, the transduced regulatory T cells shared a similar growth profile to the non-transduced regulatory T cells, suggesting that the exogenous TCR did not impair cell growth and proliferation.

[0423] The population doubling levels and % viability for five additional manufacturing runs are shown in Table 5. The % target dose achieved for the entire process for all six manufacturing runs is also shown. The % target dose was calculated by determining the final total number of naive Tregs based on the initial number and population doubling level of naive Tregs before expansion after the selection process, assuming a transduction efficiency of 25%. The % target dose was calculated based on a target dose of 100 million transduced cells. As shown, the manufacturing process in this example achieves a Treg product with high purity and sufficient cells to dose a patient from a single leukopack. As used herein, PDL is the population doubling level used throughout. TDN refers to transduced cells.

[0424] [Table 5]

[0425] Transduced cells were collected on days 0–11 of the activation and expansion process, and TSDR hypomethylation at the FOXP3 CNS2 locus was measured by ddPCR assay. Genomic DNA (gDNA) was obtained from enriched regulatory T cells and bisulfite-treated. A digital droplet PCR (ddPCR) assay using methylation-specific primers and probes was then employed to quantify unmethylated and methylated sequences. The TSDR hypomethylation status of cells collected for all six transduced manufacturing runs is shown in Figure 6. During expansion, TSDR hypomethylation levels did not decrease by more than 10% for any manufacturing run and remained above 80% for all manufacturing runs. Table 6 below shows the TSDR hypomethylation status for all six donor samples processed using the method described in this example.

[0426] These data indicate that the resulting regulatory T cells are stable, thymus-derived regulatory T cells.

[0427] [Table 6]

[0428] Treg activity after cryopreservation freeze-thaw cycles Regulatory T cells produced by the methods of this example and either untransduced or transduced with lentiviral vectors encoding the TCRs described herein were cryopreserved for storage. Cells were frozen and stored in a vapor-phase nitrogen storage freezer.

[0429] After a period of refrigeration, the frozen cell population was thawed. The cell population was divided into two experimental groups. The first experimental group was activated with anti-CD3 and anti-CD28 antibodies (transduced (TDN)), whereas the second experimental group was not activated (untransduced (UNT)). Both groups were grown in medium containing IL-2. After incubation, IL-10 levels were measured after 1, 2, and 3 days.

[0430] The increase in IL-10 levels for each of the three-day runs for cells activated with anti-CD3 and anti-CD28 antibodies is shown in Figure 7. The cells showed increased secretion of IL-10 cytokine over the three days, demonstrating that the cell populations were able to be activated and retain their cellular function after cryopreservation freeze-thaw cycles (Figure 7). Cells that were not activated did not show an increase in IL-10 levels. This data further demonstrates that regulatory T cells retained function.

[0431] Example 2. Gating strategy optimized for GMP conditions A second gating strategy was developed using a 1D histogram-based approach rather than a 2D dot-based approach to reduce user error in a GMP manufacturing setting. Cells were prepared as in Example 1 up to the FACS sorting step. In the FACS sorting step, the gating scheme used, shown in Figure 8, was a one-dimensional histogram plot-based approach in which CD25-enriched cells were sequentially gated for CD4+, CD45RA+, CD127- / lo, and CD25+ / high using the histogram (CD4+ > CD45RA+ (histo) > CD127- / lo (histo) 40% > CD25Hi (histo) 70%). For runs with the same run number as in Example 1, both gating strategies were performed in parallel on the same donor sample. Cells were transduced with TCR-A or a disulfide-modified, codon-optimized version called "TCR-E v2."

[0432] The characteristics of the cells before sorting are shown in Table 7, and the viability of the cells after each step of the manufacturing process is shown in Table 8.

[0433] [Table 7]

[0434] [Table 8]

[0435] The purity and recovery of naive Tregs during the sorting process for each run is shown in Table 9.

[0436] [Table 9]

[0437] The characteristics of the produced cells are shown in Table 10 below.

[0438] [Table 10]

[0439] The TSDR status of the cells is shown in Table 11 below and Figure 9. One run with a TSDR level above 100% is due to the donor being female. Because the TSDR assay measures the FoxP3 locus on the X chromosome, and females have one silenced (methylated) copy of the X chromosome, the TSDR demethylation level is doubled for female subjects to obtain the percentage of cells with a hypomethylated FoxP3 locus.

[0440] [Table 11]

[0441] Treg activity after cryopreservation freeze-thaw cycles Regulatory T cells produced by the method in this example for runs 5-7, either untransduced or transduced with the indicated lentiviral vectors, were cryopreserved for storage. Cells were frozen and stored in a vapor-phase nitrogen storage freezer.

[0442] After a period of refrigeration, the frozen cell population was thawed. The cell population was divided into two experimental groups. The first experimental group was activated with anti-CD3 and anti-CD28 antibodies, while the second experimental group was not. Both groups were grown in medium containing IL-2. After incubation, regulatory T cell activation markers and cytokine levels were measured at 1, 2, and 3 days (IL-10, CTLA-4, TGF-β-1, and CD69).

[0443] Figures 10A-10B show the increased levels of IL-10, CTLA-4, TGF-β-1, and CD69 for all runs over three days for cells activated with anti-CD3 and anti-CD28 beads. Activated cells showed increased levels of IL-10 (Figure 10D), CTLA-4 (Figure 10A), TGF-β-1 (Figure 10C), and CD69 (Figure 10B) over the period, demonstrating that the cell populations were activated and could retain their cellular function after cryopreservation freeze-thaw cycles (Figures 10A-10D). Non-activated cells did not show increased levels. This data further demonstrates that regulatory T cells retained function.

[0444] Example 3: High-stringency manufacturing strategies for stable Treg selection Challenges in generating stable Tregs from patient populations Following the establishment of the manufacturing process using healthy donor samples, the manufacturing process was evaluated using samples from MS donors. For certain MS donors, lower initial TSDR hypomethylation levels and / or less stable TSDR hypomethylation levels over the course of expansion (from day 0 to harvest) were observed compared to results using healthy donor cells. Figure 11 shows the change in TSDR hypomethylation over expansion for three MS donors. For two of the three MS donors, the initial TSDR was less than 90%, and the TSDR decreased by more than 10% over the course of expansion. This is likely due to differences in Treg populations between healthy donors and autoimmune donors, including MS donors, making the boundary between Treg cells and conventional T cells less clear during cell sorting. These results suggested that for some donors, increasing the stringency of the gating strategy likely improved the purity of the isolated Treg population and, therefore, the proportion of stable Tregs obtained in the process. However, increasing stringency likely resulted in a decrease in yield. This example describes a more stringent gating approach coupled with a modified expansion protocol that enhances Treg expansion, which is required to overcome the reduced yields resulting from the increased stringency of stable Treg selection.

[0445] Assessment of Treg subpopulations for inclusion in the process The selection strategy described in the above examples focuses on the selection of naive Tregs. Considering the need for a more stringent selection strategy for a particular donor and the possibility that this strategy may eliminate an increase in the proportion of naive Tregs, we evaluated whether a CD45RA+ selection step to select naive Tregs is necessary in the context of a more stringent selection strategy, or whether more stringently selected antigen-experienced Tregs can be used. Naive Tregs are CD4 + CD25 + CD127 - / low CD45RA +Antigen-experienced Tregs are CD4+CD25highCD127- / loCD45-. Both of these populations are stable Tregs with a stable TSDR hypomethylation phenotype. To assess whether CD45RA-based selection is necessary, we performed a cross-sectional analysis of antigen-experienced (CD45RA-) and naive (CD45RA) Tregs. + ) Tregs were isolated and compared for proliferation capacity and stability.

[0446] Cells were prepared prior to the FACS sorting step as described in Example 1. Cells were then subjected to two rounds of FACS sorting for debulking and purity. - and CD45RA + Both populations were sorted on CD4, CD25, and CD127 by debulk sorting. Cells were first sorted on CD4 + and then using a polygon gate set at 70% of the standard polygon gate for Treg identification and selection, CD25 + / high CD127 - / low Tregs were identified by repeated selection and CD45RA - For cells, CD25 + / high CD127 - / low Following identification of the Treg population, CD45RA - Cells were identified and selected. + For cells, CD25 + / high CD127 - / lo Following identification of the Treg population, CD45RA + Cells were identified and selected. Naive (CD45RA + ) Treg percentages are shown in Table 12 below.

[0447] At the end of selection, three groups were established. (a) CD45RA + Treg-enriched CD45RA + Treg fraction (b) CD45RA - Treg-enriched CD45RA - Treg fraction (c) “Spike-in” Treg-80% enriched CD45RA- Treg fraction + 20% enriched CD45RA + Treg fraction

[0448] [Table 12]

[0449] The cells were then expanded in culture for 8 days as described in Example 1. Figures 12A-12C show the population doubling level (PDL) and TSDR hypomethylation after cell expansion. As shown in Figures 12A and 12C, CD45RA + The CD45RA population showed increased TSDR hypomethylation along with a dramatic increase in proliferation compared to the CD45RA population. - CD45RA compared to Tregs + Due to the relative proliferation capacity of naive Tregs, CD45RA + These findings suggest that naive Tregs are required for the expansion process. Importantly, the "spike-in" samples express CD45RA + Naive Tregs express a large excess of CD45RA - We show that they can still proliferate effectively in the presence of antigen-experienced Tregs.

[0450] Development of a more stringent gating strategy for the isolation of stable Tregs In light of the above findings, CD45RA + To increase the stringency of selection while still capturing Tregs, we developed an improved gating strategy. In particular, this strategy targets antigen-experienced (CD4 + CD25 high CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + ) Tregs, while CD4 + CD25 + CD127 - / loSpecifically, CD45RA non-Tregs are excluded. By separately identifying both antigen-experienced and naive Tregs, sufficient numbers of Tregs can be obtained from a single leukopack to produce a cell therapy product. Antigen-experienced Tregs have high suppressive capacity and can suppress any non-Tregs both ex vivo and in vivo. This approach allows for the elimination of CD45RA non-Tregs. + Identify cells and subsequently (either sequentially or simultaneously) use a CD127 gating scheme in combination with a critical "not" or L-shaped gate as the final step in the gating scheme. - / lo and CD25 + Identify antigen-experienced (CD4 + CD25 highい CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + ) Tregs are selected, while unwanted CD4 + CD25 + CD127 - / lo CD45RA - This gating approach is illustrated in Figures 13A-13C and 14A-14E, where Figures 13A-13C show a dot plot approach of the CD25 / CD127 selection step and Figures 14A-14E show a dot plot approach of the CD127 selection step. - / lo , followed by CD25 + We present a histogram approach to selecting

[0451] To further illustrate the gating strategy, CD25-enriched cells were stained with fluorescently labeled antibodies that recognize human CD4, CD127, and CD45RA cell surface markers. The cells had already been labeled with CD25 antibodies in the enrichment step of the process. The cells were then sorted twice using a FACS instrument (using the same strategy in each step) using the gating scheme described below. In the dot plot-based approach shown in Figures 13A-13C, the first step of the gating process was to select CD4+ T cells were first identified from all live cell events (Figure 13A). In a second step, CD25 T cells were identified based on two-parameter flow plots. + CD127 - / low Treg cells were identified, thereby allowing the spatial context of the Treg cell population to be visualized relative to another cell population (i.e., Tconv cells), and CD25 and CD127 levels were visualized by drawing a polygon gate in the upper left quadrant to capture CD25 + CD127 - / low The cells were captured. The polygonal gate was then shifted upward to reduce the selected cell population to 70% of the original population, thereby increasing the stringency of selection (Figure 13B). In a final step, a "not" or L-shaped gate was applied to visualize CD45RA and CD25 levels and identify naive Tregs (CD4 + CD25 + CD127 - / lo CD45RA + ) and antigen-experienced Tregs (CD4 + CD25 high CD127 - / low CD45RA - ) cell populations were analyzed by CD25 + The CD45RA cell population was selected for expansion by excluding it from the sorting gate (Figure 13C). Both antigen-experienced and non-stable Treg populations expressed CD45RA - However, they can be distinguished by CD25 levels, and antigen-experienced Tregs express CD25 high and the non-stable Treg population is CD25 + Naive Tregs express only CD45RA + and CD25 + Because CD45RA cells are CD25 + or CD25 high The threshold for determining whether or not a Treg is present is determined based on the CD25 level in the naive Treg population, and highis defined as a CD25 level above that expressed in the naive Treg population. The L-shaped gate is important because it allows the selection of two important populations of cells: CD25 high CD45RA - Antigen-experienced Tregs and CD25 + CD45RA + Select naive Tregs and CD25 + CD45RA - Exclude contaminating cells.

[0452] The histogram-based approach was implemented as an operational alternative for certain manufacturing situations and yielded similar results as the dot plot strategy (Figure 14A-E). Using continuous histogram flow plots, we identified the lowest expression of CD127 (i.e., 40 ± 2% CD127 - / lo ), followed by the highest expression of CD25 (i.e., 70±2% CD25 + ) with CD4 + T cells were selected (Figures 14A-14C). The cell population was further refined by selecting 70±2% of Treg cells in a CD25 vs. CD127 parameter flow plot (Figure 14D). Naive Tregs (CD4 + CD25 + CD127 - / lo CD45RA + ) and antigen-experienced Tregs (CD4 + CD25 high CD127 - / lo CD45RA - ) were identified using the same "not" or L-shaped gate as in the dot plot-based approach (Figure 14E).

[0453] The MS donor used in run 10 provided a second leukopack (run 12), which was processed using a new sorting strategy (histogram + L-shaped gate). With the exception of the FACS sorting step, all other elements of the manufacturing process were as described in Example 1. Table 13 shows the characteristics of the cells after enrichment and before sorting, demonstrating that the cell characteristics were very similar between the two runs before the sorting step. In the context of Table 13 and all tables herein, N / A means that the data was not collected and therefore not available.

[0454] [Table 13]

[0455] Table 14 shows the characteristics of the cells after isolation and before expansion. As expected, the purity of Tregs after sorting remains high with the stringent sorting approach, but the proportion of naive Tregs is reduced by including antigen-experienced Tregs in the sorting strategy compared to the original process described in Example 1. The recovery of naive Tregs was also reduced due to the increased stringency of the gating protocol.

[0456] [Table 14]

[0457] Population doubling levels and % survival are shown in Table 15. The % target dose achieved for the entire process is also shown and was calculated similarly to Example 1, except that for the new selection strategy, both populations are selected, so the target dose calculation was based on the total number of Tregs rather than the total number of naive Tregs.

[0458] [Table 15]

[0459] Figure 15 and Table 16 below show the % TSDR hypomethylation after sorting and over the course of expansion. The L-shaped gate approach achieved an initial TSDR hypomethylation of over 95% (compared to 87.79% with the sorting approach from Example 1). Furthermore, with the L-gate-based approach, TSDR hypomethylation levels decreased by less than 10% over the course of expansion.

[0460] [Table 16]

[0461] New Reproduction Process Because the newly developed gating strategy selects fewer naive Tregs than the original process, the expansion process was modified to increase Treg expansion.

[0462] The expansion protocol was modified to include the addition of TNF-α to the expansion medium and a second anti-CD28 / anti-CD3 stimulation. First, the effect of restimulation was evaluated at various time points after transduction. For this experiment, cells were prepared according to the method described in Example 1, except that cells were gated only on CD4, CD25, and CD127, and not on CD45RA. Cells were then activated and transduced as in Example 1, and a second activation step using anti-CD3 and anti-CD28 was performed on days 6, 8, and 10 after the first activation step. This restimulation on day 6 resulted in similar proliferation as restimulation on days 7 or 8. However, advantageously, restimulation on day 6 of the expansion process was found to result in the greatest increase in proliferation when cells were harvested on day 10, crucially not requiring an extended expansion process (Figure 16). Next, the effect of adding TNF-α to the expansion protocol was evaluated. Cells prepared by the method described in this Example (including the L-shaped gate) were expanded as described in Example 1, with or without a second stimulus on day 6, and with or without 2500 IU / mL of TNF-α, to evaluate the effect of TNF-α on Treg proliferation, alone or in combination with a second stimulus. As shown in Figure 17, the addition of TNF-α throughout the expansion process, combined with a second stimulus with anti-CD3 and anti-CD28 on day 6 of the expansion process, provided the greatest increase in proliferation. However, each individual change to the process also increased proliferation. As shown in Figure 18, cells expanded in TNF-α in combination with a second stimulus exhibited the highest viability and purity at harvest compared to either the process change alone or the original expansion protocol.

[0463] Tregs generated through a novel gating and expansion process The manufacturing process described in this example was then applied to a second replicate MS donor. The MS donor from Run 11 provided an additional leukopack. This leukopack was processed using both dot plot and histogram-based stringent sorting strategies and with improvements to the growth conditions described above. In the CD127 / CD25 selection step (FIG. 19C), minor modifications were made to the histogram-based L-shaped gate approach. The lowest expression of CD127 (i.e., 30±2% CD127 - / lo ), followed by the highest expression of CD25 (i.e., 85±2% CD25 + ) with CD4 + T cells were selected. The cell population was further refined by selecting 85±2% Treg cells in a two-parameter flow plot of CD25 vs. CD127 (FIG. 19D). The L-shaped gate itself was unchanged (FIG. 19E). Prior to the sorting step, cells were treated as in Example 1 (FIGS. 19A-19E). Table 17 shows the characteristics of the cells after enrichment and before sorting, demonstrating that the characteristics of the cells from each run were very similar prior to the sorting step.

[0464] [Table 17]

[0465] The properties of the cells after sorting are shown in Table 18 below.

[0466] [Table 18]

[0467] Table 19 below shows the characteristics of the production cells. The % target dose was calculated as above.

[0468] [Table 19]

[0469] Figure 20 and Table 20 below show TSDR hypomethylation across expansion for this donor using the process described in Example 2 and both the dot plot and histogram-based L-shaped gating strategies described in this example.

[0470] [Table 20]

[0471] The comparative data presented in this example demonstrate that a strict gating strategy combined with an updated expansion process results in a highly stable Treg population and generates sufficient transduced Tregs to achieve the target dose.

[0472] This process was repeated with an additional MS donor and two additional healthy donors, with both L-shaped gating approaches, and similar results were achieved. Table 21 shows the characteristics of the cells after enrichment and before sorting.

[0473] [Table 21]

[0474] The properties of the cells after sorting are shown in Table 22 below.

[0475] [Table 22]

[0476] The characteristics of the production cells are shown below in Table 23. The target dose was calculated as described earlier in this example.

[0477] [Table 23]

[0478] The TSDR status of the cells is shown in Table 24 below and in FIG.

[0479] [Table 24]

[0480] For all donors processed using the selection and expansion strategy described in this example, a post-sorting TSDR hypomethylation status of over 90% was achieved, which did not decrease by more than 10% over the expansion process. This demonstrates that this process generates highly pure stable Treg populations from various donors. In addition, the process was also able to achieve sufficient numbers of transduced Tregs to achieve the target dose.

[0481] Example 4: Evaluation of TSDR threshold levels We investigated the threshold level of TSDR hypomethylation, which represents the level of stable Tregs required for stable Treg cell products. We first examined the percentage of stable Tregs on day 0 to maintain a stable level of Treg cells throughout the expansion process, indicating low or no growth of contaminating conventional T cells or other cellular impurities. To do this, we spiked various percentages of conventional T cells into Tregs isolated from the same healthy donor on day 0 and tracked TSDR levels throughout the expansion process. TSDR hypomethylation and FOXP3+ levels correlated with initial Treg purity; when Treg purity was above 60%, they remained stable with a 10% or less decrease in TSDR hypomethylation throughout expansion (Figures 22A-D). Next, we assessed the Treg purity of the expanded cell products for the presence of conventional T cells and the production of pro-inflammatory cytokines upon activation with PMA and ionomycin. Cryopreserved cells from the harvest were thawed into fresh medium and then stimulated with PMA and ionomycin for 4 hours at 37°C and 5% CO2. Intracellular cytokine (ICC) and IL-2 production in response to IFN-g were measured by flow cytometry. Figure 23 shows the percentage of FoxP3+ and FoxP3- cells expressing IL-2 and IFN-g after activation with PMA and ionomycin. In the absence of conventional T cells, 100% Treg products showed little or no secretion of IFN-g or IL-2, indicating that only a small number of unstable Treg cells had expanded from the initial culture. As the initial Treg purity decreased, the percentage of T cells secreting IFN-g and IL-2 cytokines increased, with the greatest increase observed when the starting Treg purity was less than 90%. This increase is likely due to an increased Tcon-to-Treg ratio. Upon activation, some conventional T cells upregulate FoxP3, which is why IFN-γ and IL-2 are produced by some FoxP3+ cells, suggesting that high TSDR levels are important for maintaining a stable population of cells that can suppress contaminating conventional T cells.

[0482] Example 5: Characterization of MBP83-99-reactive TCR constructs Three TCRs with high affinity for the MBP83-99 peptide (amino acid sequence ENPVVHFFKNIVTPRTP) were selected for evaluation, and the sequences were assembled based on the literature and the IMGT database. MBP is localized in the meninges, where inflammatory processes associated with MS pathogenesis are known to occur. Therefore, TCRs targeting the MBP peptide were selected as a therapeutic agent for multiple sclerosis.

[0483] TCRs with the α and β chains of TCR A, described in further detail below, were cloned into lentiviral vectors in α-P2A-β and β-P2Aα orientations to determine the optimal configuration of the TCR components in the vector. Each α and β chain was tagged to allow visualization of the expression level of each TCR component. Constructs were engineered with a P2A tag between the two TCR subunits and a Myc tag at the N-terminus of the downstream protein (TCRα or TCRβ) to allow detection of the downstream component. It was assumed that if the second subunit was expressed, the first encoded subunit would also be expressed. Expression of the complete ORF (EF1αTCRβ_P2A_cMyc_TCRα_IRES_EGFP, tcrβ_P2A_cMyc_TCRα_IRES_EGFP, or EF1αTCRα_P2A_cMyc_TCRβ_IRES_EGFP) was confirmed.

[0484] The constructs were transduced into TCR-null juvenile cells at an MOI of 10, 20, or 30. Cells were then incubated overnight, and fresh medium was added before staining for flow cytometry. Cells were stained with DAPI, anti-cMYC AX647, and CD3 BV786. Cell surface staining for CD3 indicates TCR assembly and expression on the cell surface. Detection of Myc indicates expression of the second expressed TCR component. GFP+ cells indicate expression of the complete construct.

[0485] As shown in Figure 24, for the TCRβ_P2A_cMyc_TCRα_IRES_EGFP construct, flow cytometry analysis demonstrated cell surface localization of CD3 in GFP-positive cells, indicating that the TCR complex was assembled at the cell surface. cMyc-TCRβ was also detected on the cell surface, indicating expression and cell surface localization of TCRα and TCRβ. However, as seen in Figures 25A-C, less CD3 (Figure 25A) and cMyc (Figure 25C) were detected on the cell surface of the TCRα_P2A_cMyc_TCRβ_IRES_EGFP construct compared to constructs with the opposite orientation of TCRα and TCRβ, suggesting insufficient TCRα expression or cell surface localization. The percentage of GFP-positive cells was also reduced (Figure 25B), suggesting reduced TCR expression with the TCRα_P2A_cMyc_TCRβ_IRES_EGFP construct compared to the TCRβ_P2A_cMyc_TCRα_IRES_EGFP construct. Based on this data, a construct was generated that had TCRβ sequentially upstream of TCRα.

[0486] Each TCR (TCR-A, TCR-B, and TCR-C) was then cloned into a vector construct containing the N-terminal TCR β chain and the C-terminal TCR α chain, along with a linker domain containing a GSG amino acid sequence followed by a P2A self-cleaving peptide. The TCR β-GSG-P2A-TCR α fusion construct was expressed under the EF1α promoter. The constructs used in Examples 5-10 also contained a GFP tag at the C-terminus of the TCR α to allow for in vitro detection.

[0487] TCR-A contained a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 4 (comprising a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 1, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 3), and a TCR beta variable domain having the amino acid sequence of SEQ ID NO: 10 (comprising a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 7, a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3-beta sequence comprising the amino acid sequence of SEQ ID NO: 9).

[0488] TCR-B comprised a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 21 (comprising a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 18, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 20), and a TCR beta variable domain having the amino acid sequence of SEQ ID NO: 27 (comprising a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 24, a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 25, and a CDR3-beta sequence comprising the amino acid sequence of SEQ ID NO: 26).

[0489] The TCR-C comprised a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 35 (comprising a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 32, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 34), and a TCR beta variable domain having the amino acid sequence of SEQ ID NO: 41 (comprising a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 38, a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3-beta sequence comprising the amino acid sequence of SEQ ID NO: 40).

[0490] The TCR was expressed in TCR-null Jurkat cells to assess the ability of the TCR to be expressed on the cell surface and activate T cells in the absence of endogenous TCR.

[0491] TCR null cuts were seeded at 1 x 105 / well in 96-well plates and transduced with lentiviral vectors encoding one of the three TCRs. Cells were then expanded, assessed for TCR expression, and harvested for downstream assays to assess function. Surface expression of each of the TCRs was verified by flow cytometry detection of CD3 expression and lentiviral GFP reporter (Figure 26). All three of the TCRs tested demonstrated surface expression in the TCR null cuts.

[0492] To test the responsiveness of the TCR to MBP complexed with MHC, transduced Jurkat cells were transduced with BLS2b cells (HLA-DRA * 01:01 and HLA-DRB1 * B cells expressing 15:01 exclusively were co-cultured overnight with MBP83-99 peptide. Peptides were titrated 4-fold at the indicated concentrations. Flow cytometry was used to measure upregulation of CD69 surface expression (Figures 27A-C). This data demonstrates that all three TCRs are expressed on the cell surface and can activate TCR-null Jurkat cells.

[0493] TCR-A and TCR-B were expressed independently in conventional CD4 T cells to assess the ability of the TCRs to activate T cells in the presence of cell surface-expressed endogenous TCRs.

[0494] For each TCR, TCR-transduced CD4 T cells were seeded at 100,000 cells / well across a row (12 wells) of a 96-well round-bottom plate. 50,000 BLS2b cells (HLA-DRA * 01:01 and HLA-DRB1 * A single-cell plate containing transduced T cells (B cells expressing 15:01 exclusively) was added to each well, and the MBP83-99 peptide was serially titrated across the plate. Cells were cultured overnight with peptide for 20 hours in a humidified 37°C, 5% CO2 incubator. The following day, cells were prepared for analysis by flow cytometry by staining with fluorescently labeled antibodies (anti-CD4 BV605, anti-CD69 BV421, anti-CD20 PE). Data were acquired on a BioRad ZE5 cytometer and analyzed with FlowJo v10. Transduced T cells were identified as CD20-, CD4+, GFP+ singlet events. T cell activation was determined by the level of CD69 expression (Figures 28A-C). This data demonstrates that both TCR-A and TCR-B can activate conventional T cells.

[0495] Example 6: Characteristics of regulatory T cells expressing exogenous TCRs Regulatory T cells transduced with lentiviral vectors encoding the TCR from Example 5 were tested in a functional assay for their ability to be activated and have suppressive function in response to the MBP83-99 peptide when presented by HLA. Tregs were isolated from PBMCs using the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (Stemcell Technologies) and transduced and expanded as described in Example 1.

[0496] To test whether transduced regulatory T cells can be activated, transduced regulatory T cells or non-transduced controls were incubated with HLA DRB1 * Mitomycin C-treated peripheral blood mononuclear cells (PBMCs) isolated from 15:01 donors were incubated with MBP83-99 peptide at concentrations ranging from 0 to 100 μM (0, 0.000001, 0.00001, 0.0001, 0.01, 0.1, 1, 10, and 100 μM) and 200 U / mL IL-2 in T cell culture medium at 37°C. The PBMC-to-regulatory T cell ratio was 4:1. Transduced regulatory T cells were differentiated from non-transduced regulatory T cells by GFP incorporated into a lentiviral vector. After incubation, regulatory T activation markers were measured by FACS one day later, and cytokine levels were measured three days later.

[0497] A representative graph of CD69 expression levels in regulatory T cell populations transduced with each of the two TCRs (TCR-A and TCR-B) and non-transduced regulatory T cells across the range of MBP83-99 peptides is provided in Figure 29A. TCR-A provided an EC50 for CD69 of 7.0 nM, and TCR-B provided an EC50 for CD69 of 7.9 nM.

[0498] A representative graph of 41-BB expression levels in regulatory T cell populations transduced with each of the two TCRs (TCR-A and TCR-B) and non-transduced regulatory T cells across the range of MBP83-99 peptides is provided in Figure 29B. TCR-A provided an EC50 for 4-1BB of 11.0 nM, and TCR-B provided an EC50 for 4-1BB of 18.0 nM.

[0499] A representative graph of IL-10 expression levels in regulatory T cell populations transduced with each of the two TCRs (TCR-A and TCR-B) and non-transduced regulatory T cells across the range of MBP83-99 peptides is provided in Figure 29C. TCR-A provided an EC50 for IL-10 of 16.1 nM, and TCR-B provided an EC50 for IL-10 of 13.7 nM.

[0500] As shown, regulatory T cells expressing either TCR-A or TCR-B exhibited elevated levels of CD69, 4-1BB, and IL-10 in response to MBP83-99 presented by PBMCs compared with regulatory T cells that did not express the exogenous TCR. These data demonstrate that transduced regulatory T cells are activated in response to MBP83-99 and exhibit desirable characteristics.

[0501] To test the ability of transduced regulatory T cells to suppress conventional T cell function, activation of conventional T cells expressing either TCR-A or TCR-B was performed in the presence of regulatory T cells expressing either TCR-A or TCR-B, and HLA DRB1 * PBMCs isolated from the 15:01 donor were stimulated with the MBP83-99 peptide. Conventional T cell activation was assessed by the presence of activation markers and cytokine release.

[0502] Regulatory T cells are identified by HLA DRB1 *Mitomycin C-treated PBMCs isolated from a 15:01 donor were incubated with MBP83-99 peptide at concentrations of 1 μM or 0.1 μM, conventional CD4 T cells expressing TCR-A or TCR-B, and 200 U / mL IL-2. The PBMC to conventional T cell ratio was 4:1. Conventional T cell suppression was measured at various ratios of regulatory T cells to conventional T cells (1:1, 1:2, 1:4, and 1:8). Cells were incubated for 4 days. After incubation, T cell activation markers were measured by FACS, and cytokine levels were measured.

[0503] Figure 30A shows the suppression of TCR-A-expressing conventional T cells by TCR-A-expressing regulatory T cells. The percent suppression of CD71, CD25, and IFN-γ is presented at 1 μM and 0.1 μM of MBP83-99 peptide. Figure 30B shows the suppression of TCR-B-expressing conventional T cells by TCR-A-expressing regulatory T cells. The percent suppression of CD71, CD25, and IFN-γ is presented at 1 μM and 0.1 μM of MBP83-99 peptide. Figure 30C shows the suppression of TCR-A-expressing conventional T cells by TCR-B-expressing regulatory T cells. The percent suppression of CD71, CD25, and IFN-γ is presented at 1 μM and 0.1 μM of MBP83-99 peptide. Figure 30D shows the suppression of TCR-B-expressing conventional T cells by TCR-B-expressing regulatory T cells. The percent suppression of CD71, CD25, and IFN-γ at 1 μM and 0.1 μM of MBP83-99 peptide is presented.

[0504] The ability of TCR-A-expressing regulatory T cells to suppress polyclonal (untransduced) conventional T cells was measured using the same methods: for polyclonal conventional T cells, suppression of proliferation (Figure 31A), CD25 (Figure 31B), and interferon gamma (Figure 31C) was measured at the indicated Treg to Tcon ratios.

[0505] These data demonstrate that stable regulatory T cells expressing exogenous TCRs were able to suppress conventional T cells. Specifically, regulatory T cells expressing exogenous TCRs targeting MBP83-99 were able to suppress activation and cytokine release by conventional T cells. Collectively, these data suggest that cell populations, including regulatory T cells, can suppress immune responses via MBP-specific mechanisms and therefore should be useful for treating subjects with multiple sclerosis (e.g., progressive multiple sclerosis).

[0506] Example 7: Characterization of T cells expressing engineered T cell receptors Cysteine substitutions were introduced into the TCRα and TCRβ sequences of TCR-A and TCR-B to introduce disulfide bonds and increase TCR pairing at the cell surface. For each TCR, a T48C mutation was introduced into the TRAC constant region (SEQ ID NO:58) and an S57C mutation was introduced into the TRBC2 constant region (SEQ ID NO:60). Engineered TCR-E v1 (middle line) is an engineered version of TCR-A with an additional engineered disulfide bond (the amino acid sequence of v1 corresponds to SEQ ID NO:79), and engineered TCR-E v2 has the amino acid sequence of SEQ ID NO:79 and is encoded by the codon-optimized nucleic acid sequence of SEQ ID NO:87. Engineered TCR-Fv 1 (middle line) is an engineered version of TCR-B with an additional engineered disulfide bond (the amino acid sequence of v1 corresponds to SEQ ID NO:86), and modified TCR-Fv 2 (top line) has the amino acid sequence of SEQ ID NO:86 and is encoded by the codon-optimized nucleic acid sequence of SEQ ID NO:88.

[0507] To assess the ability of the engineered TCRs to be expressed on the cell surface and activate T cells in the absence of endogenous TCR, the TCRs were expressed in TCR-null Jurkat cells.

[0508] TCR-null cells were seeded at 1 x 10 cells / well in 96-well plates and transduced with lentiviral vectors encoding one of the TCRs. Cells were then expanded and harvested for downstream assays to assess function.

[0509] To test the responsiveness of cells expressing TCR to MBP complexed with MHC, transduced Jurkat cells were transduced with BLS2b cells (HLA-DRA * 01:01 and HLA-DRB1 * Jurkats expressing each TCR were co-cultured overnight with 15:01 (B cells exclusively expressing 15:01) and MBP83-99 peptide. Peptides were titrated 4-fold at the concentrations shown in Figures 32A-32B. Flow cytometry was used to measure upregulation of CD69 surface expression (Figures 32A-32B and Table 25). These data demonstrated that Jurkats expressing each TCR responded to increasing concentrations of antigen.

[0510] [Table 25]

[0511] To assess cell surface expression of TCRs and T cell activation in the presence of endogenous TCRs, TCRs were also expressed on conventional CD4+ T cells.

[0512] CD4+ T cells transduced with each TCR were seeded at 100,000 cells / well across rows (12 wells) of a 96-well round-bottom plate. 50,000 BLS2b cells (HLA-DRA * 01:01 and HLA-DRB1 * A 15:01-expressing B cell population (B cells expressing MBP83-99 exclusively) was added to each well containing transduced T cells, and the MBP83-99 peptide was serially titrated across the plate. Cells were cultured overnight with peptide for 20 hours in a humidified 37°C, 5% CO2 incubator. The following day, cells were prepared for analysis by flow cytometry by staining with fluorescently labeled antibodies (anti-CD4 BV605, anti-CD69 BV421, anti-CD20 PE). Data were acquired on a BioRad ZE5 cytometer and analyzed with FlowJo v10. Transduced T cells were analyzed by staining with CD20 - , CD4 + , GFP +T cell activation was determined by the level of CD69 expression (Figures 33A-33C and Table 26).

[0513] [Table 26]

[0514] These data suggest that the expression levels of markers of cell activation (e.g., CD69) are significantly higher in CD4+ T cells expressing engineered TCRs (e.g., TCR-E engineered v2 and TCR-F engineered v2) compared to cells expressing the parental versions (TCR-A and TCR-B). + demonstrated to be higher in T cells.

[0515] Surface expression of each TCR was confirmed by flow cytometry detection of Vβ2 and CD69 expression (Figure 34A). Vβ2 expression correlates with surface-level expression of the TCR (Figures 34B-34C). These data demonstrate that engineered TCRs (e.g., TCR-E engineered v2 and TCR-F engineered v2) do not significantly affect the surface expression of these TCRs on conventional T cells compared to their parental versions (TCR-A and TCR-B).

[0516] Example 8. Activation of regulatory T cells expressing engineered T cell receptors Regulatory T (Treg) cells were prepared as described in Example 6. Isolated Tregs were transduced with the TCRs described herein (TCR-A, TCR-E, TCR E-v2, TCR-B, TCR-F, and TCR-Fv2). Treg cells were then transduced with HLA-DRA IgG1-associated T cells as described in Example 6. * 01:01 and HLA-DRB1 * BLS2b cells, B cells expressing only 15:01, were contacted with MBP83-99 peptide and Treg activation, as evidenced by CD69 expression, was measured. Figures 35A-35B show graphs depicting Treg activation in response to increasing doses of MBP peptide.

[0517] As shown in Figure 35A, activation of Tregs expressing TCR-E engineered v2 was 1.53-fold greater than that of Tregs expressing the parental version (TCR-A), whereas activation of Tregs expressing TCR-E engineered v1 was 1.37-fold greater than that of Tregs expressing the parental version (TCR-A).

[0518] As shown in Figure 35B, activation of Tregs expressing engineered TCR-F v2 was 1.89-fold greater than that of Tregs expressing the parental version (TCR-B), and activation of Tregs expressing engineered TCR-F v1 was 1.56-fold greater than that of Tregs expressing the parental version (TCR-B).

[0519] Surface expression of each TCR was confirmed by flow cytometry detection of Vβ2 expression. Cells transduced with TCR-E engineered v2 or TCR-F engineered v2 expressed Vβ2 at a higher percentage compared to the parental control and modified v1 versions, respectively (Figure 35C). Vβ2 expression correlates with surface level expression of the TCR.

[0520] These data demonstrate that the level of activation of Tregs transduced with engineered TCRs of the present disclosure (e.g., TCRs encoded by nucleotide sequences having 100% identity to the nucleotide sequence of SEQ ID NO: 87 or 88) in response to the target antigen MBP83-99 peptide is higher...

Claims

1. 1. An isolated population of cells comprising stable CD4+ regulatory T cells (Tregs) derived from a subject with multiple sclerosis, comprising: An isolated population, wherein at least 80% of said cells are stable CD4+ Tregs comprising a hypomethylated T cell-specific demethylated region (TSDR) in the FOXP3 locus.

2. 2. The isolated population of claim 1, wherein the cells comprise an exogenous human T cell receptor (TCR) that specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

3. 3. The isolated population of claim 2, wherein the MBP peptide is an MBP83-99 peptide comprising the amino acid sequence of SEQ ID NO:

61.

4. The MHC is HLADRB1 ＊ 4. The isolated population of claim 2 or claim 3, comprising 15:

01.

5. The MHC is HLA-DRA ＊ 5. The isolated population of claim 4, further comprising 01:

01.

6. The exogenous TCR is a. a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and b. The isolated population of any one of claims 2 to 5, comprising a TCR β chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

9.

7. The isolated population of claim 6 , wherein the TCR α chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

4.

8. 8. The isolated population of claim 6 or 7, wherein the TCR alpha chain variable region comprises or consists of SEQ ID NO:

4.

9. The isolated population of claim 6 , wherein the TCR α chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

5.

10. 10. The isolated population of claim 6 or claim 9, wherein the TCRα chain comprises or consists of SEQ ID NO:

5.

11. The isolated population of claim 6 , wherein the TCR α chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

73.

12. 12. The isolated population of claim 6 or claim 11, wherein the TCRα chain comprises or consists of SEQ ID NO:

73.

13. The isolated population of claim 6 , wherein the TCR α chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

91.

14. 14. The isolated population of claim 6 or claim 13, wherein the TCR alpha chain comprises or consists of SEQ ID NO:

91.

15. The isolated population of any one of claims 6 to 14, wherein the TCR β chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

10.

16. 16. The isolated population of any one of claims 6 to 15, wherein the TCR beta chain variable region comprises or consists of SEQ ID NO:

10.

17. 17. The isolated population of any one of claims 6 to 16, wherein the TCR β chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

11.

18. 18. The isolated population of any one of claims 6 to 17, wherein the TCR beta chain comprises or consists of SEQ ID NO:

11.

19. 17. The isolated population of any one of claims 6 to 16, wherein the TCR beta chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

75.

20. 20. The isolated population of any one of claims 6 to 16 or claim 19, wherein the TCR beta chain comprises or consists of SEQ ID NO:

75.

21. 17. The isolated population of any one of claims 6 to 16, wherein the TCR β chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

93.

22. 20. The isolated population of any one of claims 6 to 16 or claim 19, wherein the TCR beta chain comprises or consists of SEQ ID NO:

93.

23. 10. The isolated population of any one of the preceding claims, wherein the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:

17.

24. 24. The isolated population of claim 23, wherein the single polypeptide comprises or consists of SEQ ID NO:

17.

25. 10. The isolated population of any one of the preceding claims, wherein the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:

79.

26. 26. The isolated population of claim 25, wherein the single polypeptide comprises or consists of SEQ ID NO:

79.

27. 10. The isolated population of any one of the preceding claims, wherein the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:

97.

28. 28. The isolated population of claim 27, wherein the single polypeptide comprises or consists of SEQ ID NO:

97.

29. 27. The isolated population of claim 25 or 26, wherein the single polypeptide comprises the amino acid sequence of SEQ ID NO:79 and is encoded by a nucleotide sequence having at least 90%, 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:

87.

30. The exogenous TCR is a. a TCR alpha chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and b. The isolated population of any one of claims 2 to 5, comprising a TCR β chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:24, a CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

26.

31. 31. The isolated population of claim 30, wherein the TCR alpha chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

21.

32. 32. The isolated population of claim 30 or 31, wherein the TCR alpha chain variable region comprises or consists of SEQ ID NO:

21.

33. 31. The isolated population of claim 30, wherein the TCRα chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

22.

34. 34. The isolated population of claim 30 or 33, wherein the TCRα chain comprises or consists of SEQ ID NO:

22.

35. 31. The isolated population of claim 30, wherein the TCRα chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

80.

36. 36. The isolated population of claim 30 or claim 35, wherein the TCR alpha chain variable region comprises or consists of SEQ ID NO:

80.

37. 37. The isolated population of any one of claims 30 to 36, wherein the TCR beta chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

27.

38. 38. The isolated population of any one of claims 30 to 37, wherein the TCR beta chain variable region comprises or consists of SEQ ID NO:

27.

39. 39. The isolated population of any one of claims 30-38, wherein the TCR beta chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

28.

40. 40. The isolated population of any one of claims 30 to 39, wherein the TCR beta chain comprises or consists of SEQ ID NO:

28.

41. 37. The isolated population of any one of claims 30-36, wherein the TCR beta chain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

82.

42. 42. The isolated population of any one of claims 30 to 36 or claim 41, wherein the TCR beta chain comprises or consists of SEQ ID NO:

82.

43. 43. The isolated population of any one of claims 30-42, wherein the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:

31.

44. 44. The isolated population of claim 43, wherein the single polypeptide comprises or consists of SEQ ID NO:

31.

45. 43. The isolated population of any one of claims 30-42, wherein the exogenous TCR is encoded as a single polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:

86.

46. 46. The isolated population of claim 45, wherein the single polypeptide comprises or consists of SEQ ID NO:

86.

47. 47. The isolated population of claim 45 or claim 46, wherein the single polypeptide comprises the amino acid sequence of SEQ ID NO: 86 and is encoded by a nucleotide sequence having at least 90%, 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:

88.

48. 48. The isolated population of any one of claims 1 to 47, wherein said stable CD4+ Tregs do not express FOXP3 protein from an engineered FOXP3 locus.

49. 49. The isolated population of any one of claims 1 to 48, wherein the TSDR is the CNS2 region of FOXP3.

50. 50. The isolated population of any one of claims 1 to 49, wherein the MHC is MHC class I or MHC class II.

51. 51. The isolated population of any one of claims 1-50, wherein at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

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

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

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

55. 4 x 10 ７ ~1 x 10 １０ 55. The isolated population of claim 54, comprising stable CD4+ Tregs.

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

57. 57. The isolated population of claim 56, 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 said conventional T cells comprise said exogenous human TCR.

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

1.

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

60. 60. The isolated population of claim 59, wherein the isolated population does not contain a percentage of CD8+ T cells detectable by fluorescence-activated cell sorting (FACS).

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

62. 62. The isolated population of claim 61, 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.

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

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

65. 10. The isolated population of any one of the preceding claims, wherein the exogenous TCR is encoded as a single polypeptide, optionally wherein the polypeptide comprises an N-terminal β domain and a C-terminal α domain.

66. 66. The isolated population of claim 65, wherein the single polypeptide comprises a TCR alpha chain and a TCR beta chain, and the polypeptide comprises a self-cleaving peptide sequence located between the TCR alpha chain and the TCR beta chain.

67. 67. The isolated population of claim 66, wherein the self-cleaving peptide sequence is a 2A peptide sequence, optionally wherein the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence.

68. 10. The isolated population of any one of the preceding claims, wherein the exogenous TCR comprises a TCR alpha chain comprising a constant region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:

58.

69. 10. The isolated population of any one of the preceding claims, wherein the exogenous TCR comprises a TCR beta chain comprising a constant region comprising an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:

60.

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

71. The isolated population of claim 65, wherein the TCR alpha chain constant region comprises a T48C amino acid substitution relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and the TCR beta chain constant region comprises a S57C amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 59 or 60.

72. 10. The isolated population of any one of the preceding claims, wherein said stable CD4+ Tregs have the human leukocyte antigen (HLA)-DR15 haplotype.

73. The stable CD4+ Tregs are HLA-DRB1 ＊ 73. The isolated population of claim 72, comprising the 15:01 allele.

74. The stable CD4+ Tregs are HLA-DRB5 ＊ 74. The isolated population of claim 73, further comprising the 01:01 allele.

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

76. 76. The method of claim 75, wherein at least 85%, at least 90%, or at least 95% of said cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus.

77. 77. The method of claim 75 or 76, wherein the isolated population is selected for therapeutic use if the percentage of cells containing hypomethylated TSDR in the FOXP3 locus is 80% or greater.

78. 78. The method of any one of claims 75 to 77, wherein step (c) is performed at least twice.

79. 79. The method of any one of claims 75 to 78, further comprising activating the population of cells of step (c).

80. 80. The method of claim 79, wherein said activating comprises culturing said population of cells with an anti-CD3 antibody and an anti-CD28 antibody.

81. 81. The method of claim 79 or 80, wherein the second activation step is carried out 4 to 8 days after the first activation step.

82. 82. The method of claim 80 or 81, wherein the cells are grown in a culture medium comprising IL-2 and TNFα.

83. 83. The method of any one of claims 75-82, wherein said activating and expanding comprises culturing said population of cells for at least 5, 6, 7, 8, 9, 10, 11, or 12 days.

84. 84. The method of any one of claims 75-83, wherein said activating and expanding comprises culturing said population of cells for no more than 15, 14, 13, or 12 days.

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

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

87. 87. The method of claim 85 or 86, wherein step (c) is performed at least twice.

88. 88. The method of any one of claims 85-87, wherein the isolated population is selected for therapeutic use if the percentage of cells containing hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95% or more.

89. 89. The method of any one of claims 85 to 88, further comprising activating the population of engineered cells of step (c).

90. 90. The method of claim 89, wherein said activating comprises culturing said population of cells with an anti-CD3 antibody and an anti-CD28 antibody.

91. 91. The method of claim 89 or 90, wherein the activation step is performed at least twice.

92. 92. The method of claim 91, wherein the second activation step is performed 4 to 8 days after the first activation step.

93. 93. The method of any one of claims 85 to 92, wherein the cells are grown in a culture medium comprising IL-2 and TNFα.

94. the expanded population is at least 1 x 10 ７ 94. The method of any one of claims 85 to 93, comprising engineered stable CD4+ Tregs.

95. 95. The method of any one of claims 85 to 94, wherein the methylation status of the TSDR in step (f) is assessed at least 24 hours after a cryopreservation freeze-thaw cycle.

96. CD4 ＋ CD25 ＋／ｈｉｇｈ CD127 －／ｌｏ 96. The method of any one of claims 85-95, wherein the percentage of regulatory T cells is assessed at least 24 hours after a cryopreservation freeze-thaw cycle.

97. Less than 5%, less than 4%, or less than 3% of the cells in the depleted biological sample are CD8 ＋ cells, CD19 ＋ cells, and / or CD14 ＋ 97. The method of any one of claims 75 to 96, comprising cells.

98. 98. The method of any one of claims 75 to 97, wherein no more than 2% of the population of cells are CD8+ cells.

99. 99. The method of any one of claims 75 to 98, wherein no more than 20% of the population of cells are conventional T cells.

100. The population of cells in step (c) is ｈｉｇｈ CD45RA － cells and CD25+CD45RA+ cells, ＋ CD45RA － 100. The method of any one of claims 75 to 99, which is cell-free.

101. (c) said isolating i. Removing CD4 from the enriched population of step (b). ＋ identifying a first subpopulation of cells; ii. CD25 ＋／ｈｉｇｈ CD127 －／ｌｏ identifying a second subpopulation of cells from said first subpopulation; and iii. Isolating CD25 from the second subpopulation ｈｉｇｈ CD45RA － and CD25 ＋／ｈｉｇｈ CD45RA ＋ Cells were selected and CD25 ＋ CD45RA － Excluding iv. The method of any one of claims 75 to 100, whereby said population of CD4+ Tregs is isolated.

102. 102. The method of any one of claims 75 to 101, wherein the TSDR is the CNS2 region of FOXP3.

103. The method of any one of claims 75 to 102, wherein the subject has a human leukocyte antigen (HLA)-DR15 haplotype.

104. The regulatory T cells are HLA-DRB1 ＊ 104. The method of claim 103, comprising the 15:01 allele.

105. 105. The method of any one of claims 75 to 104, wherein the subject has progressive multiple sclerosis or relapsing-remitting multiple sclerosis.

106. 106. The method of claim 105, wherein the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

107. 107. The method of any one of claims 75 to 106, wherein the human subject is a male subject.

108. 108. The method of any one of claims 75 to 107, wherein the human subject is a female subject.

109. 109. The method of any one of claims 85-108, wherein said delivering said vector comprising nucleic acid encoding said exogenous human TCR comprises transducing said cell population with said vector.

110. 110. The method of any one of claims 85 to 109, wherein the exogenous TCR binds to the MBP83-99 complexed with MHC.

111. The method of any one of claims 85 to 110, wherein the exogenous TCR is the exogenous TCR of any one of claims 6 to 47.

112. The method of any one of claims 85 to 111, wherein the exogenous human TCR is encoded as a single polypeptide comprising a TCR alpha chain and a TCR beta chain.

113. 111. The method of claim 110, wherein the polypeptide comprises an N-terminal TCR beta chain and a C-terminal TCR alpha chain.

114. 114. The method of claim 110 or 113, wherein the polypeptide comprises a self-cleaving peptide sequence located between the TCR alpha chain and the TCR beta chain.

115. 115. The method of claim 114, wherein the self-cleaving peptide sequence is a 2A peptide sequence.

116. 116. The method of claim 115, wherein the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence.

117. The method of claims 85 to 116, wherein the vector is a viral vector.

118. 118. The method of claim 117, wherein the viral vector is a lentiviral vector.

119. The method of claim 118, wherein the lentiviral vector is a VSVg-pseudotyped self-inactivating third-generation lentiviral vector.

120. 120. The method of any one of claims 85-119, 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-1 alpha promoter or an MND promoter.

121. 121. The method of any one of claims 85 to 120, wherein the nucleic acid further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally a WPRE-mut6.

122. 122. The method of claim 120 or 121, wherein the coding sequence is codon-optimized.

123. 1. An engineered polynucleotide encoding a polypeptide comprising a TCR alpha chain and a TCR beta chain, the TCR alpha chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; b. An engineered polynucleotide wherein the TCR β chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

9.

124. The engineered polynucleotide of claim 123, wherein the TCR alpha chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:4, and the TCR beta chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

10.

125. 125. The engineered polynucleotide of claim 123 or 124, wherein the TCR alpha chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 73, and the TCR beta chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

75.

126. 126. The engineered polynucleotide of any one of claims 123 to 125, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

79.

127. 127. The engineered polynucleotide of any one of claims 123 to 126, comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO:

87.

128. 1. An engineered polynucleotide encoding a polypeptide comprising a TCR alpha chain and a TCR beta chain, the TCR alpha chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; b. An engineered polynucleotide wherein the TCR beta chain comprises a variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:24, a CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

26.

129. The engineered polynucleotide of claim 123, wherein the TCR alpha chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:21, and the TCR beta chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

27.

130. 125. The engineered polynucleotide of claim 123 or 124, wherein the TCR alpha chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 80, and the TCR beta chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

82.

131. 126. The engineered polynucleotide of any one of claims 123 to 125, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

86.

132. 127. The engineered polynucleotide of any one of claims 123 to 126, comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO:

88.

133. 133. An expression cassette comprising a polynucleotide according to any one of claims 123 to 132 operably linked to a promoter, optionally wherein the promoter is an EF-1α promoter or an MND promoter.

134. 134. The expression cassette of claim 133, further comprising 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.

135. A vector comprising the expression cassette of claim 133 or 134.

136. The vector of claim 135, wherein the vector is a viral vector.

137. 137. The method of claim 136, wherein the viral vector is a lentiviral vector.

138. The method of claim 137, wherein the lentiviral vector is a VSVg-pseudotyped self-inactivating third-generation lentiviral vector.

139. 1. An engineered polypeptide comprising a human TCR alpha chain and a human TCR beta chain, a. the TCR alpha chain is at least 95% identical to SEQ ID NO: 73 and the TCR beta chain is at least 95% identical to SEQ ID NO: 75, or b. An engineered polypeptide wherein said TCR alpha chain is at least 95% identical to SEQ ID NO:80 and said TCR beta chain is at least 95% identical to SEQ ID NO:

82.

140. a. the TCR alpha chain comprises or consists of SEQ ID NO: 73 and the TCR beta chain comprises or consists of SEQ ID NO: 75, or b. The engineered polypeptide of claim 139, wherein said TCR alpha chain comprises or consists of SEQ ID NO:80 and said TCR beta chain comprises or consists of SEQ ID NO:

82.

141. 141. The engineered polypeptide of claim 139 or 140, comprising, from N-terminus to C-terminus, the TCR beta chain, a self-cleaving peptide sequence, and the TCR alpha chain.

142. 142. The engineered polypeptide of claim 141, wherein the self-cleaving peptide sequence is a P2A peptide sequence.

143. 143. The engineered polypeptide of any one of claims 139-142, comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:79 or 86.

144. 143. The engineered polypeptide of any one of claims 139 to 142, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 79 or 86.

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

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

147. 146. A method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to claim 145 or an isolated population according to any one of claims 1 to 71.

148. 148. The method of claim 147, wherein the pharmaceutical composition is administered in an amount effective to alleviate one or more symptoms of multiple sclerosis.

149. 149. The method of claim 147 or 148, wherein the administering comprises intravenous administration.

150. 150. The method of any one of claims 147-149, wherein said administering comprises one or more injections.

151. 151. The method of any one of claims 147-150, wherein the cells of the isolated population are autologous to the subject.

152. 152. The method of any one of claims 147-151, wherein the subject has progressive multiple sclerosis or relapsing-remitting multiple sclerosis.

153. 153. The method of claim 152, wherein the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

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