Cell therapy for multiple sclerosis

Abstract

The present disclosure is directed to isolated cell populations comprising stable regulatory T cells and methods of producing the same, further wherein the isolated cell populations comprising stable CD4 + T regulatory cells (T regs) are derived from a subject suffering from multiple sclerosis wherein at least 80% of the cells are stable CD4 + T regs, comprising hypomethylated T cell specific demethylation regions (TSDRs) at the FOXP3 locus, where MHC comprises HLADRB1 * 15: 01 or HLADRA * 01: 01.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application 63 / 397,715, filed on August 12, 2022; U.S. Provisional Application 63 / 412,142, filed on September 30, 2022; U.S. Provisional Application 63 / 415,362, filed on October 12, 2022; U.S. Provisional Application 63 / 417,465, filed on October 19, 2022; and U.S. Provisional Application 63 / 432,304, filed on December 13, 2022. The entire content of each of these U.S. Provisional Applications is incorporated herein by reference.

[0003] Reference to Electronic Sequence Listing

[0004] The entire content of the electronic sequence listing (filename: ABTH_003_04WO_SeqList_ST26.xml; size: 129,941 bytes; creation date: August 10, 2023) is incorporated herein by reference. Background of the Invention

[0005] Myelin, a key component of a properly functioning central nervous system (CNS), wraps around and insulates nerve fibers to protect them and enable proper transmission of electrical signals. In the CNS of patients with multiple sclerosis, myelin is damaged by the body's own immune cells, exposing the nerve fibers. This myelin damage disrupts the neural circuits in the brain, hinders communication between the brain and the rest of the body, and makes the nerves vulnerable to cell death and neurodegeneration, causing long - term and often irreversible effects. Progressive multiple sclerosis is characterized by a progressive increase in clinical disability that is not related to acute relapsing pathology. There is currently no approved therapy for non - relapsing progressive MS. The human leukocyte antigen (HLA) DRB1*1501 haplotype is strongly associated with MS, and it is estimated that at least 45,000 patients in the United States currently have the DRB1*1501 haplotype and have non - relapsing progressive MS. Regulatory T cells have the potential to treat this and other autoimmune diseases because they can selectively target diseased cell types and tissues, thereby generating a local immune response through an antigen - specific mechanism. Summary of the Invention

[0006] In some aspects, the present disclosure provides a separated cell population comprising stable CD4+ T regulatory 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) at the FOXP3 locus. In some embodiments, these cells comprise an exogenous human T cell receptor (TCR) that specifically binds a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC). In some embodiments, the MBP peptide is the MBP 83-99 peptide comprising the amino acid sequence of SEQ ID NO:61. In some embodiments, the MHC comprises HLA-DRB1*15:01. In some embodiments, the MHC further comprises HLA-DRA*01:01. In some embodiments, the exogenous TCR comprises: (a) a TCR α-chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3; and (b) a TCR β-chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:8, and a CDR3 having the amino acid sequence of SEQ ID NO:9. In some embodiments, the TCR α-chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:4. In some embodiments, the TCR α-chain variable region comprises SEQ ID NO:4 or consists of the same. In some embodiments, the TCR α-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:5. In some embodiments, the TCR α-chain comprises SEQ ID NO:5 or consists of the same. In some embodiments, the TCR α-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:73. In some embodiments, the TCR α-chain comprises SEQ ID NO:73 or consists of the same. In some embodiments, the TCR α-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:91. In some embodiments, the TCR α-chain comprises SEQ ID NO:91 or consists of the same. In some embodiments, the TCR β-chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:10. In some embodiments, the TCR β-chain variable region comprises SEQ ID NO:10 or consists of the same.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: 11. In some embodiments, the TCR β chain comprises or consists of SEQ ID NO: 11. 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: 75. In some embodiments, the TCR β 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 that comprises 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 that comprises 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 that comprises 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 α chain variable region that comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 18, a CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a CDR3 containing the amino acid sequence of SEQ ID NO: 20; and (b) a TCR β chain variable region that comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 24, a CDR2 containing the amino acid sequence of SEQ ID NO: 25, and a CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the TCR α chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 21. In some embodiments, the TCR α chain variable region comprises or consists of SEQ ID NO: 21.In some embodiments, the TCR α-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:22. In some embodiments, the TCR α-chain comprises or consists of SEQ ID NO:22. In some embodiments, the TCR α-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:80. In some embodiments, the variable region of the TCR α-chain comprises or consists of SEQ ID NO:80. In some embodiments, the variable region of the TCR β-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:27. In some embodiments, the variable region of the TCR β-chain comprises or consists of SEQ ID NO:27. In some embodiments, the TCR β-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:28. In some embodiments, the TCR β-chain comprises or consists of SEQ ID NO:28. In some embodiments, the TCR β-chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity 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 that comprises 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 that comprises 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+ T regs do not express the 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+ T regs 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 comprises at least 4×10 7 stable CD4+ Tregs. In some embodiments, the isolated population comprises 4×10 7 to 1×10 10 stable CD4+ Tregs. In some embodiments, fewer than 25%, fewer than 20%, fewer than 15%, fewer than 10%, fewer than 5%, fewer than 2%, fewer than 1%, fewer than 0.5%, fewer than 0.1% or fewer than 0.01% of the cells in the isolated population are conventional CD4 + T cells. In some embodiments, fewer than 60%, fewer than 50%, fewer than 40%, fewer than 30%, fewer than 20%, fewer than 10%, fewer than 5% or fewer 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, fewer than 2%, fewer than 1%, fewer than 0.5%, fewer than 0.1% or fewer than 0.01% of the cells in the isolated population are CD8 + T cells. In some embodiments, the isolated population does not contain a detectable percentage of CD8+ T cells 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, after cryopreservation freeze-thaw cycles, the percentage of stable CD4 + Tregs containing a hypomethylated TSDR at the FOXP3 locus is reduced by 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. In some embodiments, stable CD4 +Tregs exhibit one or more functions selected from the following: (a) regulatory cytokine secretion activity; (b) expression of activation markers associated with regulatory T cells; and / or (c) inhibitory activity. In some embodiments, 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. In some embodiments, the single polypeptide comprises a TCR α chain and a TCR β chain, and wherein the polypeptide comprises a self-cleaving peptide sequence positioned between the TCR α chain and the TCR β chain. In some embodiments, 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. In some embodiments, the exogenous TCR comprises a TCR α chain containing a constant region that comprises 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 comprises a TCR β chain containing a constant region that comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO:60. In some embodiments, the exogenous human TCR comprises one or more amino acid substitutions to cysteine residues in the constant regions of the TCR α chain and the TCR β chain, and wherein these cysteine residues are capable of forming one or more disulfide bonds. In some embodiments, relative to the constant region of the TCR α chain comprising the amino acid sequence of SEQ ID NO:58, the constant region of the TCR α chain comprises a T48C amino acid substitution, and wherein relative to the constant region of the TCR β chain comprising the amino acid sequence of SEQ ID NO:59 or 60, the constant region of the TCR β chain comprises an S57C amino acid substitution. In some embodiments, the stable CD4+ Tregs have a human leukocyte antigen (HLA)-DR15 haplotype. In some embodiments, the stable CD4+ Tregs comprise the HLA-DRB1*15:01 allele. In some embodiments, the stable CD4+ Tregs further comprise the HLA-DRB5*01:01 allele.

[0007] In some aspects, provided herein is a method of generating a cell population comprising stable CD4 + regulatory T cells (Tregs), the method comprising: (a) removing CD8 + cells, CD19 + cells, and optionally CD14 + cells from a biological sample obtained from a subject having multiple sclerosis to produce a depleted biological sample; (b) enriching CD25 + cells in the depleted biological sample to produce an enriched population; (c) isolating CD4 + CD25 +CD127 - / lo cells; (d) amplifying and enriching the population to generate an amplified and enriched cell population; and (e) quantifying the methylation status of the T cell-specific demethylated region (TSDR) at the FOXP3 locus in the cell population, wherein at least 80% of the cells are stable CD4 + T regs, thereby generating a cell population comprising stable CD4 + T regs. In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4 + T regs that contain hypomethylated TSDRs at the FOXP3 locus. In some embodiments, if the percentage of cells containing hypomethylated TSDRs at the FOXP3 locus is 80% or greater, the isolated population is selected for therapeutic use. In some embodiments, step (c) is performed at least twice. In some embodiments, the method further comprises activating the cell population of step (c). In some embodiments, activation comprises culturing the cell population with anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the second activation step is performed between 4 and 8 days after the first activation step. In some embodiments, the cells are expanded in a medium comprising IL-2 and TNFα. In some embodiments, activation and expansion comprise culturing the cell population for at least 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, activation and expansion comprise culturing the cell population for no more than 15, 14, 13, or 12 days.

[0008] In some aspects, provided herein is a method of generating a cell population comprising engineered stable CD4 + T regs, the method comprising: (a) removing CD8 + cells, CD19 + cells, and optionally CD14 + cells from a biological sample obtained from a subject with multiple sclerosis to generate a depleted biological sample; (b) enriching CD25 + cells in the depleted biological sample to generate an enriched population; (c) isolating CD4 + CD25 + CD127 - / locells; (d) delivering a vector comprising a nucleic acid encoding an exogenous human T cell receptor (TCR) to the isolated population of (c) to generate an engineered cell population; (e) expanding the engineered cell population to generate an expanded engineered cell population; and (f) quantifying the methylation status of the T cell-specific demethylated region (TSDR) at the FOXP3 locus in the expanded engineered cell population, wherein at least 80% of the cells are stable CD4 + Tregs. In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4 + Tregs. In some embodiments, step (c) is performed at least twice. In some embodiments, the isolated population is selected for therapeutic use if the percentage of cells containing a hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95%, or higher. In some embodiments of the method, further comprising activating the engineered cell population of step (c). In some embodiments, activation comprises culturing the cell population with anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the activation step is performed at least twice. In some embodiments, the second activation step is performed between 4 and 8 days after the first activation step. In some embodiments, the cells are expanded in a medium comprising IL-2 and TNFα. In some embodiments, the expanded population comprises at least 1×10 7 engineered stable CD4+ Tregs. In some embodiments, the methylation status of the TSDR in step (f) is evaluated at least 24 hours after a cryopreservation freeze-thaw cycle. In some embodiments, the CD4 + CD25 + / 高 CD127 - / lo percentage of regulatory T cells is evaluated at least 24 hours after a cryopreservation freeze-thaw cycle. In some embodiments, less than 5%, less than 4%, or less than 3% of the cells of the depleted biological sample contain CD8 + cells, CD19 + cells, and / or CD14 + cells. In some embodiments, 2% or less of the cell population is CD8+ cells. In some embodiments, 20% or less of the cell population is conventional T cells. In some embodiments, the cell population in step (c) comprises CD25 高 CD45RA - cells and CD25 + CD45RA + cells and does not contain CD25 + CD45RA -cells. In some embodiments, the isolation of (c) comprises: (i) identifying a first subset of CD4 + cells from the enriched population of step (b); (ii) identifying a second subset of CD25 + / 高 CD127 - / lo cells from the first subset; and (iii) selecting CD25 高 CD45RA - and CD25 + / 高 CD45RA + cells for isolation and excluding CD25 + CD45RA - thereby isolating CD4 +T reg population. In some embodiments, the TSDR is the CNS2 region of FOXP3. In some embodiments, the subject has the human leukocyte antigen (HLA)-DR15 haplotype. In some embodiments, the regulatory T cells comprise the HLA-DRB1*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 a cell population with the vector. In some embodiments, the exogenous TCR binds to the MBP83-99 peptide complexed with MHC. In some embodiments, the exogenous TCR is the exogenous TCR defined in any of the above aspects or embodiments. In some embodiments, the exogenous human TCR is encoded as a single polypeptide comprising a TCRα chain and a TCRβ chain. In some embodiments, the polypeptide comprises an N-terminal TCRβ chain and a C-terminal TCRα chain. In some embodiments, the polypeptide comprises a self-cleaving peptide sequence positioned between the TCRα chain and the TCRβ 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 the coding sequence encoding the exogenous human TCR, optionally wherein the promoter is an EF-1α promoter or an MND promoter. In some embodiments, the nucleic acid further comprises an enhancer element, an optionally optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), also optionally WPRE-mut6. In some embodiments, the coding sequence is codon-optimized.

[0009] In some aspects, an engineered polynucleotide is disclosed herein that encodes a polypeptide comprising a TCRα chain and a TCRβ chain, wherein (a) the TCRα chain comprises a variable region that comprises a CDR1 having the amino acid sequence of SEQ ID NO:1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3; and (b) the TCRβ chain comprises a variable region that comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:8, and a CDR3 having the amino acid sequence of SEQ ID NO:9. In some embodiments, the TCRα chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:4, and the TCRβ chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:10. In some embodiments, the TCRα chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:73, and the TCRβ chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:75. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:79. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:87.

[0010] In some aspects, an engineered polynucleotide is disclosed herein that encodes a polypeptide comprising a TCRα chain and a TCRβ chain, wherein (a) the TCRα chain comprises a variable region that comprises a CDR1 having the amino acid sequence of SEQ ID NO:18, a CDR2 having the amino acid sequence of SEQ ID NO:19, and a CDR3 having the amino acid sequence of SEQ ID NO:20; and (b) the TCRβ chain comprises a variable region that comprises a CDR1 having the amino acid sequence of SEQ ID NO:24, a CDR2 having the amino acid sequence of SEQ ID NO:25, and a CDR3 having the amino acid sequence of SEQ ID NO:26. In some embodiments, the TCRα chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:21, and the TCRβ chain variable region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:27. In some embodiments, the TCRα chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:80, and the TCRβ chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:82. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:86. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:88.

[0011] In some aspects, the present disclosure provides an expression cassette comprising a polynucleotide of any of the foregoing aspects or embodiments operably linked to a promoter, optionally wherein the promoter is an EF-1α promoter or an MND promoter. In some embodiments, the expression cassette comprises an enhancer element, an optionally optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and also optionally WPRE-mut6.

[0012] In some aspects, the present disclosure provides a vector comprising an expression cassette of any of the foregoing 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.

[0013] In some aspects, the present disclosure provides an engineered polypeptide comprising a human TCRα chain and a human TCRβ chain, wherein (a) the TCRα chain is at least 95% identical to SEQ ID NO:73 and the TCRβ chain is at least 95% identical to SEQ ID NO:75; or (b) the TCRα chain is at least 95% identical to SEQ ID NO:80 and the TCRβ chain is at least 95% identical to SEQ ID NO:82. In some embodiments of the engineered polypeptide, wherein (a) the TCRα chain comprises or consists of SEQ ID NO:73 and the TCRβ chain comprises or consists of SEQ ID NO:75; or (b) the TCRα chain comprises or consists of SEQ ID NO:80 and the TCRβ chain comprises or consists of SEQ ID NO:82. In some embodiments, the engineered polypeptide comprises, from the N-terminus to the C-terminus: a TCRβ chain, a self-cleaving peptide sequence, and a TCRα chain. In some embodiments, the self-cleaving peptide is a P2A sequence. In some embodiments, the engineering comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:79 or 86. In some embodiments, the engineering comprises or consists of the amino acid sequence of SEQ ID NO:79 or 86.

[0014] In some aspects, the present disclosure provides a pharmaceutical composition comprising a separated population of any of the foregoing aspects or embodiments and a pharmaceutically acceptable excipient.

[0015] In some aspects, the present disclosure provides a pharmaceutical composition comprising a separated population of any of the foregoing aspects or embodiments and a cryopreservative.

[0016] In some aspects, the present disclosure provides a method of treating a subject in need of treatment for multiple sclerosis, the method comprising administering to the subject a pharmaceutical composition of any one 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 effective amount to alleviate one or more symptoms of multiple sclerosis. In some embodiments, the administration comprises intravenous administration. In some embodiments, the administration comprises one or more infusions. 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 DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A to 1B Exemplary methods of generating a cell population comprising stable regulatory T cells are provided. Figure 1A Illustrates the overall process of isolating regulatory T cells with a CD4 + CD45RA + CD25 + / 高 CD127 lo / - phenotype. Figure 1B Illustrates data depicting a fluorescence-activated cell sorting (FACS) strategy for enriching a cell population with a CD45RA + CD4 + CD25 + CD127 lo / - phenotype from a CD25-enriched sample to generate an isolated cell population comprising stable regulatory T cells. As Figure 1A shown, this sorting strategy is employed in two consecutive steps to obtain a high level of purity.

[0018] Figure 2 Illustrates a graph depicting the recovery of regulatory T cells after depletion and CD25 enrichment steps in an exemplary method of generating an isolated cell population comprising stable regulatory T cells.

[0019] Figures 3A to 3B Illustrates data depicting the relative amounts of various cell phenotypes after depletion and CD25 enrichment steps in an exemplary method of generating an isolated cell population comprising stable regulatory T cells.

[0020] Figure 4 Illustrates a graph depicting the growth of cells after transduction with an exogenous TCR. Untransduced cells (“untransduced”) are used as a control.

[0021] Figure 5A graph depicting the phenotype and TCR expression of regulatory T cells in a cell population generated by an exemplary method of the present disclosure.

[0022] Figure 6 Data demonstrating that regulatory T cells in a cell population generated by an exemplary method of the present disclosure maintain hypomethylated T cell-specific demethylated regions (TSDR).

[0023] Figure 7 A graph depicting the activation of lentivirus-transduced regulatory T cells of the present disclosure after cryopreservation freeze-thaw cycles.

[0024] Figure 8 Shows for CD4 + CD45RA + CD127 - / 低 CD25 + A graph of the gating strategy for purification of a purified cell population.

[0025] Figure 9 Shows by Figure 8 The percentage of TSDR hypomethylation for different runs purified by the gating strategy depicted in.

[0026] Figures 10A to 10D Shows the expression of CTLA-4 ( Figure 10A ), CD69 ( Figure 10B ), TGFβ-1 ( Figure 10C ), and IL-10 ( Figure 10D ) after incubation of the cell population in the presence and absence of anti-CD3 and anti-CD28 beads after cryopreservation freeze-thaw cycles.

[0027] Figure 11 Shows the change over time in the percentage of TSDR hypomethylation for the expansion of three different MS donors.

[0028] Figures 12A to 12C Shows the population doubling levels ( + and - ) and the percentage of TSDR hypomethylation of CD45RA Figure 12A and Figure 12B cells during an eight-day expansion. The labeling indicates that CD45RA + cells can expand within the CD45RA - population.

[0029] Figures 13A to 13C Shows for CD25 + CD127 - / lo CD45 - and CD25 + / 高 CD127 -loCD45 - Selected gating strategy.

[0030] Figures 14A to 14E Shows Figures 13A to 13C The histogram method shown in

[0031] Figure 15 Shows the percentage of TSDR hypomethylation during expansion of the same donor based on different sorting strategies.

[0032] Figure 16 Shows the expansion of cells after restimulation on day 6, day 8, or day 10.

[0033] Figure 17 Shows the effect of the fold expansion of TNF-α, second stimulation, or both.

[0034] Figures 18A to 18B Shows the viability and purity of cells expanded by both TNF-α and second stimulation.

[0035] Figures 19A to 19E Shows for Figures 13A to 13C An alternative histogram method for the gating strategy shown in

[0036] Figure 20 Shows the percentage of TSDR hypomethylation during expansion of the same donor based on different sorting strategies.

[0037] Figure 21 Shows based on Figures 13A to 13C and Figures 19A to 19E The percentage of TSDR hypomethylation of three different donors for the gating strategy depicted in

[0038] Figures 22A to 22D Shows the percentage of TSDR hypomethylation and percentage of FOXP3+ cells according to the proportion of Tregs in the initial population based on two trials.

[0039] Figure 23 Shows FOXP3 expressing IL-2 and IFN-γ after activation with PMA and ionomycin + and FOXP3 - Ratio.

[0040] Figure 24 Shows the expression of CD3, cMYC, and GFP in transduced cells.

[0041] Figures 25A to 25C Shows the percentage of cells positive for CD3 ( Figure 25A ), GFP ( Figure 25B ), and cMYC ( Figure 25C ) after transduction with different TCR constructs.

[0042] Figure 26 A figure depicting the ability of lentivirally transduced Jurkat cells to express an MPB-specific TCR at the cell surface is shown.

[0043] Figures 27A to 27C A figure depicting the activation of lentivirally transduced Jurkat cells is shown.

[0044] Figures 28A to 28C A figure depicting the activation of lentivirally transduced conventional T cells is shown.

[0045] Figures 29A to 29C A figure depicting the activation of the lentivirally transduced regulatory T cells of the present disclosure is shown.

[0046] Figures 30A to 30D A figure depicting the ability of the lentivirally transduced regulatory T cells of the present disclosure to inhibit conventional T cells is shown.

[0047] Figures 31A to 31C Inhibition of polyclonal conventional T cells expressing TCR-A by regulatory T cells expressing TCR-A is shown.

[0048] Figures 32A to 32B A figure depicting the activation of Jurkat transduced with an engineered TCR is shown.

[0049] Figures 33A to 33C A figure depicting the activation of conventional T cells transduced with an engineered TCR is shown.

[0050] Figures 34A to 34C Expression data of cells expressing an engineered TCR are shown. Figure 18A Flow cytometry data of Vβ2 expression and CD69 expression are shown. Figure 18B To Figure 18C shows the quantification of Vβ2 expression in cells expressing an engineered TCR.

[0051] Figures 35A to 35C A figure depicting the activation ( Figures 35A to 35B ) and Vβ2 expression ( Figure 35C ) of Tregs expressing an engineered MBP-specific TCR is shown. Figure 35A and Figure 35B A figure depicting the activation of Tregs expressing an engineered MBP-specific TCR when contacted with BLS2b cells (B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and the MBP 83-99 peptide is shown. Figure 35A The Tregs in Figure 35BThe Tregs therein express TCR-F engineered v1, TCR-F engineered v2, or TCR-B. The expression of the activation marker (CD69) increases with the increase in the dose of MBP 83-99 peptide. Figure 35C Shows the proportion of Tregs expressing MBP-specific TCR under non-stimulated conditions, which Tregs express Vβ2.

[0052] Figures 36A to 36B Shows a graph depicting the activation of the inhibitory ability in regulatory T cells transduced with engineered TCRs (as measured by 4-1BB expression) when in contact with BLS2b cells (B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and MBP 83-99 peptide.

[0053] Figures 37A to 37C Shows the ability of T cells transduced with TCR-E v2 to inhibit conventional T cells.

[0054] Figure 38 Shows a graph depicting the stability of the regulatory T cells of the present disclosure in the presence of pro-inflammatory cytokines. Cells were expanded in the presence of a mixture of 1000 units / mL of IL-2 and pro-inflammatory cytokines (IL-1β, IL-6, IL-12, IL-17A, IFN-β, IFN-γ, TGF-β1, and TNF-α) each at a concentration of 1 pg / ml to 10,000 pg / ml. The percentage of regulatory T cells with CD4 + / FOXP3 + expression remained constant in the presence of pro-inflammatory cytokines (each cytokine in the mixture at a concentration up to 1,000 pg / mL) for at least 23 days.

[0055] Figures 39A to 39B Shows after incubation with Figure 38 the cytokine mixture (mixture-8) used in + or with IL-1β, IL-6, IL-12, IL-17A, IFN-γ, and TNF-α (mixture-6) the percentage of FOXP3

[0056] Figure 40 Provides a schematic diagram of the human / mouse chimeric MBP-targeted T cell receptor (TCR) of the present disclosure.

[0057] Figure 41 Provides an EAE research protocol.

[0058] Figure 42 Provides flow cytometry data demonstrating high expression of the human / mouse chimeric TCR in murine regulatory T cells (Tregs) after transduction.

[0059] Figure 43A Provided data demonstrating the enrichment of murine FOXP3-GFP-expressing human / mouse hybrid TCR-transduced donor Tregs in the central nervous system (CNS) of recipient mice experiencing active EAE. +

[0060] Figure 43B Provided data indicating that murine FOXP3-GFP-expressing human / mouse hybrid TCR-transduced donor Tregs are capable of influencing EAE disease severity. + DETAILED DESCRIPTION

[0061] Overview

[0062] The present disclosure provides methods and compositions for treating multiple sclerosis by generating and utilizing stable regulatory T cells. The present disclosure also provides methods and compositions for treating multiple sclerosis by generating and utilizing engineered stable regulatory T cells comprising a myelin basic protein (MBP)-specific T cell receptor (TCR). For example, such engineered regulatory T cells are capable of specifically targeting discrete cell types and tissues associated with degraded myelin in order to prevent immune-mediated damage, restore homeostasis, and promote repair in affected tissues. Additionally, the engineered regulatory T cells of the present disclosure inhibit local inflammation at the target and do not cause systemic immunosuppression. Furthermore, the engineered regulatory T cells described herein are stable (e.g., committed to the Treg phenotype), thymus-derived regulatory T cells that are capable of persisting in vivo for extended periods and can provide therapeutic benefits for months or years following a single dose. In some embodiments, these stable regulatory T cells are also resistant to proinflammatory triggers (e.g., proinflammatory cytokines). In some embodiments, the stable regulatory T cells of the present disclosure are autologous cells, meaning that they are directly obtained from the patient's own cells, engineered to express an exogenous antigen-specific TCR, and subsequently administered back to the patient. The use of autologous cells minimizes the risk of patient rejection (e.g., graft-versus-host disease).

[0063] ​​Isolating a highly stable and pure population of engineered regulatory T cells generated in the thymus and obtaining a sufficiently large and pure population of the engineered regulatory T cells for treating a patient has proven challenging, both because the stable thymic regulatory T cell population is very small relative to the total population of lymphocytes and because there are no cell surface markers associated only with regulatory T cells. Although isolated polyclonal regulatory T cells have been shown to be effective in clinical therapies, higher levels of purity and stability are required when engineering regulatory T cells with exogenous TCRs because conventional T cells engineered with exogenous TCRs and unstable regulatory T cells (i.e., peripheral Tregs), if present in sufficient numbers, can have a deleterious effect by eliciting rather than suppressing an immune response at the site of an autoimmune disease.

[0064] These stable engineered regulatory T cells and methods of production provide advantages over several existing methods for generating regulatory T cells for cell therapy. Alternative methods have been employed to generate populations of engineered regulatory T cells or regulatory-like T cells, but these cells have disadvantages relative to the cell populations described herein.

[0065] One possible method is to engineer conventional T cells or induced stem cells (by engineering the FOXP3 promoter or introducing an ectopic FOXP3 gene into the cells) to drive the expression of FOXP3, thereby increasing the proportion of regulatory-like T cells. FOXP3 expression confers a regulatory-like phenotype. However, FOXP3 is only one of several genes that are upregulated in stable regulatory T cells, and studies have shown that these regulatory-like T cells are different from natural Tregs. In fact, when CD4+ T cells are edited by introducing an exogenous promoter upstream of FOXP3 to display a Treg-like phenotype, the engineered cells have been shown to have reduced inhibitory capacity relative to natural Tregs (Buckner, Science Translational Medicine, 2022), indicating that these cells will not exhibit the same level of efficacy as true stable regulatory T cells when administered in a clinical setting.

[0066] Attempts have also been made to convert conventional CD4+ T cells, for example by stimulation with TGF-β and IL-2, stimulation with retinoic acid, short-chain fatty acids and TGF-β, or stimulation with rapamycin. However, these induced regulatory T cells lack the epigenetic changes associated with stable regulatory T cells, in particular the regulatory T cell-specific demethylation at the conserved non-coding sequence 2 (CNS2) of the FOXP3 gene, and are therefore functionally unstable and will not retain a stable phenotype when administered in a clinical setting. In addition, proteomic analysis has shown that these induced Treg-like cells have a very small overlap with stable thymic Tregs in protein expression profiles and, conversely, share signaling and metabolic proteins with conventional T cells (Mensink et al., Sci Rep. 2022).

[0067] The present disclosure contemplates a cell population and a strategy for isolating the cell population, the cell population comprising highly pure and stable thymic regulatory T cells engineered with an exogenous TCR, having sufficient purity and sufficient quantity to be administered to a subject in a therapeutically effective amount. In some embodiments, the optimal population of the regulatory T cells, after isolation and prior to introduction of the exogenous TCR, is CD25 + / 高 CD4 + CD127 - / lo CD45RA + , as this phenotype is associated with thymic naive regulatory T cells. In some embodiments, the optimal population of the regulatory T cells, after isolation and prior to introduction of the exogenous TCR, further comprises CD25 高 CD4 + CD127 - / lo CD45RA - , as this phenotype is associated with thymic antigen-experienced regulatory T cells. After introduction of the exogenous TCR, these cells also exhibit a stable TSDR phenotype, indicating no overgrowth of non-regulatory T cell subsets or loss of regulatory T cell stability (e.g., cell fate is not altered - cells remain terminally differentiated).

[0068] All references, patents, and patent applications cited herein are incorporated by reference in their entirety for the subject matter to which each is cited, in some cases covering the entire document.

[0069] As used in the specification and claims, the indefinite articles "a" and "an" shall be understood to mean "at least one" unless expressly stated to the contrary.

[0070] It should also be understood that, unless expressly stated to the contrary, in any method that includes more than one step or act as claimed herein, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0071] In the claims and the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like are to be understood to be open-ended, i.e., meaning including but not limited to. As set forth in Section 2111.03 of the United States Patent and Trademark Office's Manual of Patent Examining Procedure, only the transitional phrases "consisting of" and "consisting essentially of" should be closed or semi-closed transitional phrases, respectively.

[0072] The terms "about" and "substantially" preceding a numerical value mean ±10% of the recited numerical value.

[0073] Where a numerical range is provided, each numerical value between and including the upper and lower limits of that range is specifically contemplated and described herein.

[0074] Regulatory T cells

[0075] In some embodiments, the present disclosure provides isolated cell populations and compositions thereof, the isolated cell populations comprising stable CD4 + T regulatory cells derived from a subject having multiple sclerosis. These isolated populations can be used, for example, to treat multiple sclerosis in a subject in need thereof.

[0076] The terms "regulatory T cell," "T regulatory cell," and "Treg" are used interchangeably herein and refer to T cells that inhibit the effector functions of other cell populations of the immune system (e.g., conventional CD4 + T cells, effector CD8 + T cells, antigen-presenting cells, and / or granulocytes). Regulatory T cells are defined by the expression of the cell surface markers CD4 and CD25 and the expression of the transcription factor FOXP3. Further, regulatory T cells do not express or express at low levels the cell surface marker CD127. Thus, regulatory T cells are characterized by the following protein expression profile: CD4 + CD25 高 / + CD127 低 / - FOXP3 + .

[0077] Throughout the present disclosure, the expression of a protein by a cell or cell population may include reference to various expression indicators, such as "+" (e.g., CD4 + ), "-" (e.g., CD127 -) "high" (e.g., CD25 高 ) "int" or "medium" (e.g., CD25 int ) "low" (e.g., CD127 低 ) "high / +" (e.g., CD25 高 / + ) or "low / -" (e.g., CD127 低 / - ). In this context, the various expression indicators refer to the presence or absence of the indicated protein (e.g., "+" or "-", respectively) or the relative level of protein expression measured by conventional protein expression assays (e.g., flow cytometry, fluorescence-activated cell sorting (FACS), or Western blotting). Unless otherwise specified, protein expression as described throughout this application is determined by FACS.

[0078] (a) A positive ( + ) indicator refers to the level of expression of the indicated protein detectable by FACS. A cell population positive ( + ) for a specific protein can be further divided into "low"

[0079] and / or "high" subpopulations.

[0080] (b) A "low" cell subpopulation expresses the indicated protein, but its expression level is lower than that of other cells in the population (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than that of other cells in the population) or lower than the expression level in a control cell population (e.g., relative to CD4 + naive T cells or CD8+ effector T cells).

[0081] (c) A "high" cell subpopulation expresses the indicated protein at a level higher than that of other cells in the population (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than that of other cells in the population) or higher than the expression level in a control cell population (e.g., relative to CD4 + naive T cells or CD8+ effector T cells).

[0082] (d) A negative ( - ) indicator refers to the absence of expression of the indicated protein, or an expression level of the indicated protein below the detection limit of a specific detection assay (e.g., below the detection limit of a specific fluorescent antibody and / or flow cytometer).

[0083] (e) The indicator " - / 低 " refers to a cell population containing cells that are (-) for the indicated protein and cells that express the indicated protein at a "low" level.

[0084] (f) The indicator "+ / 高 "refers to a cell population that includes cells that are (+) for the indicated protein, including cells that express a high level of the indicated protein.

[0085] In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + / 高 In some embodiments, 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 + .

[0086] In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + / 高 CD4 + CD127 - / 低 cells. That is, regulatory T cells express or express a high level of CD25, express CD4, and do not express or express a low level of CD127. In some embodiments, regulatory T cells do not express CD127. In some embodiments, regulatory T cells (e.g., stable regulatory T cells) are CD25 + / 高 CD4 + CD127 - / 低 CD45RA + cells. Thus, regulatory T cells express or express a high level of CD25, express CD4 and CD45RA, and do not express or express a low level of CD127. In some embodiments, regulatory T cells express FOXP3. In some embodiments, regulatory T cells are CD25 + / 高 CD4 + CD127 - / 低 FOXP3 + cells. Thus, regulatory T cells express or express a high level of CD25, express CD4 and FOXP3, and do not express or express a low level of CD127. In some embodiments, regulatory T cells are CD25 + / 高 CD4 + CD45RA+CD127 - / 低 FOXP3 + cells. Thus, regulatory T cells express or express a high level of CD25, express CD4, CD45RA and FOXP3, and do not express or express a low level of CD127.

[0087] In thymic regulatory T cell development, the genomic organizer SATB1 (special AT-rich sequence-binding protein) binds to specific genomic loci from the CD4 + CD8 + thymocyte stage to open chromatin and activate super-enhancers associated with many regulatory T cell signature genes, such as FOXP3, IL2RA, (CD25), CTLA4, IKZF2 (HELIOS), and IFZF4 (EOS). The enhancer-initiation enzymes SATB1 and MLL4 (myeloid / lymphoid or mixed-lineage leukemia 4) typically occupy a newly identified conserved enhancer region at the FOXP3 locus, named conserved non-coding sequence 0 (CNS0), subsequently activating the enhancers at CNS3 and CNS2, and then the promoter. This results in stable hypomethylation and expression of FOXP3 and other regulatory T cell-related genes, leading to a stable regulatory T cell phenotype (Piotrowska et al.; Int J MolSci. 2021).

[0088] After hematopoietic development, T cell populations are capable of transitioning between one phenotype and another under various conditions (see, for example, Kitagawa et al., Nat Immunol. February 2017; 18(2):173-183). As used herein, a "stable regulatory T cell" is a regulatory T cell that contains a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, stable regulatory T cells are also defined by the expression of CD4, CD25, and / or FOXP3. Tregs derived from the thymus (e.g., thymic T regs) are stable, and regulatory T cells that are no longer able to transition between T cell phenotypes due to a change in the methylation status of one or more loci in the FOXP3 locus are also considered stable. In contrast, unstable peripheral Tregs that can be induced in response to a pro-inflammatory state 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 the expression of FOXP3. Hypomethylation of the TSDR of the endogenous FOXP3 locus occurs when the methyl group from one or more methylated cytosines in the TSDR has been removed to replace the methylated cytosine with cytosine. Thus, the stability of T regulatory cells is determined by the presence of a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus.

[0089] In some embodiments, the methylation status of the TSDR of the FOXP3 locus is measured as described in the following references: 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 Independently 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.

[0090] In some embodiments, stable regulatory T cells maintain a hypomethylated regulatory T 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 contain a hypomethylated regulatory T 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.

[0091] In some embodiments, stable regulatory T cells also exhibit one or more of the following functions: (i) regulatory cytokine secretion activity (e.g., secretion of IL-10, TGFβ, and IL-35); (ii) expression or activation markers associated with regulatory T cells (e.g., expression of CD69, 4-1BB, CD25, CD71, and / or CTLA-4); and / or (iii) inhibitory activity (e.g., the ability of stable regulatory T cells to inhibit the activation and / or proliferation of other effector cells of the immune system).

[0092] Thus, in some embodiments, the isolated cell populations provided herein comprise stable T regulatory cells that comprise hypomethylated TSDR at the FOXP3 locus, expression of CD4, CD25, and FOXP3, and / or exhibit cytokine secretion, activation, and / or inhibitory activity.

[0093] 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 isolation from a biological sample. For example, in some embodiments, the stable regulatory T cells of the isolated cell population maintain hypomethylated TSDR at the FOXP3 locus for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days after isolation from a biological sample. 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 isolation from a biological sample. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 locus for 1 - 20 days, 1 - 10 days, 1 - 5 days, 5 - 30 days, 5 - 20 days, 10 - 40 days, or 25 - 50 days after isolation from a biological sample.

[0094] 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 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 hypomethylated TSDR at the FOXP3 locus for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days after transduction with a nucleic acid encoding an exogenous TCR. 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 transduction with a nucleic acid encoding an exogenous TCR. 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 transduction with a nucleic acid encoding an exogenous TCR.

[0095] 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 a cryopreservation freeze-thaw cycle. In such embodiments, the stable regulatory T cells of the isolated cell populations maintain hypomethylated TSDR at the FOXP3 locus after a cryopreservation freeze-thaw cycle 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. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 locus after a cryopreservation freeze-thaw cycle for more than 5 days, more than 10 days, more than 15 days, or more than 20 days. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the locus after a cryopreservation freeze-thaw cycle for 1-20 days, 1-10 days, 1-5 days, 5-30 days, 5-20 days, 10-40 days, or 25-50 days.

[0096] 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 administration to a subject. For example, in some embodiments, the stable regulatory T cells of the isolated cell populations maintain hypomethylated TSDR at the FOXP3 locus after administration to a subject 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. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 locus after administration to a subject for more than 5 days, more than 10 days, more than 15 days, or more than 20 days. In some embodiments, the stable regulatory T cells maintain hypomethylated TSDR at the FOXP3 locus after administration to a subject for 1-20 days, 1-10 days, 1-5 days, 5-30 days, 5-20 days, 10-40 days, or 25-50 days.

[0097] In some embodiments, the isolated population described herein is an autologous cell population. In this context, the term autologous refers to cells obtained from the same subject to whom they are subsequently administered. For example, a cell population can be obtained from a subject, subjected to the methods described herein, and then administered to the same subject (from whom the cell population was initially obtained) to treat multiple sclerosis. In such embodiments, the cell population administered to the subject contains autologous regulatory T cells.

[0098] In some embodiments, the isolated populations described herein are allogeneic cell populations. In this context, the term allogeneic refers to cells obtained from one subject and then administered to another subject. For example, a cell population can be obtained from a subject, subjected to the methods described herein, and then administered to another subject for the treatment of multiple sclerosis.

[0099] In some embodiments, the isolated cell populations described herein (e.g., an isolated population comprising stable CD4+ T regulatory cells) are isolated from a biological sample obtained from a subject diagnosed with or suspected of having multiple sclerosis.

[0100] Exogenous TCR and engineered regulatory T cells

[0101] In some embodiments, the present disclosure provides an isolated cell population 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".

[0102] The T cell receptor (TCR) is a transmembrane heterodimer that includes an α chain and a β chain linked by a disulfide bond. Within these chains are complementarity-determining regions (CDRs) that determine the target peptide to which the TCR will bind. The TCR activates the T cell in which it resides, resulting in an excessive immune response. Antigen-presenting cells digest certain proteins (antigens) and display their fragments (peptides) on the major histocompatibility complex (MHC). This peptide-MHC (pMHC) complex binds to the TCR, and other co-stimulatory molecules are activated, resulting in T cell activation, proliferation, differentiation, apoptosis, or cytokine release.

[0103] The exogenous TCR can be any TCR that is introduced into a regulatory T cell, where the TCR is not endogenous to the regulatory T cell (i.e., not naturally present in the regulatory T cell). For example, in some embodiments, the exogenous TCR is encoded by a nucleic acid that is not endogenous to the regulatory T cell (i.e., not naturally present in the genome of the regulatory T cell). In some embodiments, the nucleic acid is an engineered nucleic acid, such as a recombinant or synthetic nucleic acid.

[0104] The TCR specifically binds to a target peptide complexed with an MHC. A TCR is considered to "specifically" bind to a target peptide complexed with an MHC if it has a higher binding affinity for the target peptide complexed with the MHC than for a non-target peptide complexed with the MHC. The TCR can have a binding affinity of 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 to 10 -10 binds to a target peptide complexed with MHC with a binding affinity of M). In some embodiments, a TCR is considered to "specifically" bind to a target peptide complexed with MHC if a T cell expressing the TCR becomes activated (e.g., as assessed by increased CD69 expression) upon contact with the target peptide complexed with MHC, or becomes more highly activated relative to a non-target peptide complexed with MHC. In some embodiments, the peptide is presented by a cell expressing MHC.

[0105] The exogenous TCR can be a human TCR. In some embodiments, the TCR is from a monkey, mouse, rat, or any other animal.

[0106] The target peptide can be a peptide associated with multiple sclerosis. In some embodiments, the target peptide associated with multiple sclerosis can be a peptide belonging to myelin basic protein (MBP), such as MBP(83 - 99). In some embodiments, the target peptide is overexpressed in a cell population associated with multiple sclerosis relative to a control (e.g., relative to a cell population not associated with multiple sclerosis).

[0107] In some embodiments, the target peptide undergoes an increased autoimmune response in a subject with multiple sclerosis relative to a control. In some embodiments, the target peptide is a peptide present at an autoimmune disease site within the same subject relative to an unaffected site within the subject. In some embodiments, the target peptide is specifically presented by an MHC allele associated with the presence of an autoimmune disease in the subject.

[0108] In some embodiments, the target peptide is a peptide that is overexpressed in the cells of a subject with multiple sclerosis relative to a control (e.g., relative to a healthy subject). A target peptide is considered to be overexpressed in a cell if its expression in the cell (e.g., a cell associated 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 cell relative to a control cell (e.g., a population of cells not associated with an autoimmune disease). In some embodiments, a target peptide is overexpressed in the cells of a subject with multiple sclerosis if its expression in the 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 relative to a healthy subject (e.g., a subject not having an autoimmune disease).

[0109] In some embodiments, the target peptide is a peptide that is highly expressed in the cells at the disease site in a subject with multiple sclerosis relative to a control (e.g., the expression of the target peptide in an unaffected / non-disease site of the subject). A target peptide is considered to be highly expressed in the cells at the disease site if its expression in the cells at the disease site (e.g., a cell associated 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 cells relative to a control cell (e.g., a cell not at the disease site).

[0110] 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*15.01. In some embodiments, the HLA haplotype is HLA-DRA*01.01.

[0111] 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.

[0112] The TCR may include a linker domain positioned 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 positioned between the α-chain and the β-chain). A self-cleaving peptide sequence is a peptide sequence that induces the separation of a polypeptide into two peptides using a non-classical mechanism. In some embodiments, the self-cleaving peptide sequence may induce ribosome skipping during polypeptide translation. In some embodiments, the length of the self-cleaving peptide sequence is 10-30, 10-25, 15-30, 15-25, or 18-22 amino acids. In some embodiments, the self-cleaving peptide sequence may be a 2A peptide sequence. The 2A peptide sequence may include, for example, the DXEXNPGP (SEQ ID NO:102) amino acid motif, where X can be any amino acid. In some embodiments, the 2A peptide sequence is the 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 EGRGSLLTCGDVEENPGP (SEQ ID NO:103). The P2A peptide sequence may include, for example, the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO:104). The E2A peptide sequence may include, for example, the amino acid sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO:105). The F2A peptide sequence may include, for example, the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:106).

[0113] In some embodiments, the exogenous TCR includes two or more polypeptides. For example, in some embodiments, the exogenous TCR includes a first polypeptide containing the α-chain and a second polypeptide containing the β-chain.

[0114] In some embodiments, the exogenous TCR includes one or more cysteine residues present in the α-chain of the TCR that are capable of forming one or more disulfide bonds with one or more cysteine residues in the β-chain of the TCR. In some embodiments, the exogenous TCR includes one or more cysteine residues present in the constant region of the α-chain of the TCR that are capable of forming one or more disulfide bonds with one or more cysteine residues in the constant region of the β-chain of the TCR.

[0115] In some embodiments, relative to the TCRα chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCRα chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C). In some embodiments, relative to the TCRβ chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCRβ chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution. In some embodiments, relative to the TCRα chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCRα chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C), and relative to the TCRβ chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCRβ chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution, wherein the cysteine residue at position 48 of the α chain is capable of forming a disulfide bond with the cysteine residue at position 57 of the β chain.

[0116] In some embodiments, relative to the TCRα chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCRα chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C). In some embodiments, relative to the TCRβ chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCRβ chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution. In some embodiments, relative to the TCRα chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCRα chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C), and relative to the TCRβ chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCRβ chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution, wherein the cysteine residue at position 45 of the α chain is capable of forming a disulfide bond with the cysteine residue at position 77 of the β chain.

[0117] In some embodiments, relative to the TCR α-chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCR α-chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C). In some embodiments, relative to the TCR β-chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCR β-chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution. In some embodiments, relative to the TCR α-chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCR α-chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C), and relative to the TCR β-chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCR β-chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution, wherein the cysteine residue at position 10 of the α-chain is capable of forming a disulfide bond with the cysteine residue at position 17 of the β-chain.

[0118] In some embodiments, relative to the TCR α-chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCR α-chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C). In some embodiments, relative to the TCR β-chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCR β-chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution. In some embodiments, relative to the TCR α-chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCR α-chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C), and relative to the TCR β-chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCR β-chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution, wherein the cysteine residue at position 45 of the α-chain is capable of forming a disulfide bond with the cysteine residue at position 59 of the β-chain.

[0119] In some embodiments, relative to the TCR α-chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCR α-chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C). In some embodiments, relative to the TCR β-chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCR β-chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution. In some embodiments, relative to the TCR α-chain constant region comprising the amino acid sequence of SEQ ID NO:58, the TCR α-chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C), and relative to the TCR β-chain constant region comprising the amino acid sequence of SEQ ID NO:60, the TCR β-chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution, wherein the cysteine residue at position 15 of the α-chain is capable of forming a disulfide bond with the cysteine residue at position 15 of the β-chain.

[0120]

[0121]

[0122] In some embodiments, the TCR comprises one or more amino acid sequences as described in Table A (e.g., one or more amino acid sequences belonging to any of TCR-A, TCR-B, TCR-C, TCR-D, TCR-E, or TCR-F). The TCR may comprise the amino acid sequence of any α-chain CDR1, CDR2, or CDR3 provided in Table 1. In some embodiments, the α-chain CDR1 of the TCR is any one of SEQ ID NO:1, 18, 32, or 46. In some embodiments, the α-chain CDR2 of the TCR is any one of SEQ ID NO:2, 19, 33, or 47. In some embodiments, the α-chain CDR3 of the TCR is any one of SEQ ID NO:3, 20, 34, or 48. The TCR may comprise the amino acid sequence of any β-chain CDR1, CDR2, or CDR3 provided in Table A. In some embodiments, the β-chain CDR1 of the TCR is any one of SEQ ID NO:7, 24, 38, or 52. In some embodiments, the β-chain CDR2 of the TCR is any one of SEQ ID NO:8, 25, 39, or 53. In some embodiments, the β-chain CDR3 of the TCR is any one of SEQ ID NO:9, 26, 40, or 54.

[0123] In some embodiments, the variable region of the α-chain of the TCR comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 4, 21, 35 or 49. In some embodiments, the variable region of the β-chain of the TCR comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 10, 14, 27, 41 or 55. In some embodiments, the α-chain of the TCR comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 5, 22, 36, 50 or 73. In some embodiments, the β-chain of the TCR comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 11, 15, 28, 42, 56 or 75.

[0124] In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain that comprises a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3; and (b) a variable region of the TCR β-chain that comprises a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8, and a CDR3 having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain that comprises 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 variable region of the TCR β-chain that comprises a CDR1 consisting of SEQ ID NO: 7, a CDR2 consisting of SEQ ID NO: 8, and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

[0125] In some embodiments, the variable region of the TCR α-chain comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 4. In some embodiments, the variable region of the TCR α-chain comprises SEQ ID NO: 4. In some embodiments, the variable region of the TCR α-chain consists of SEQ ID NO: 4. In some embodiments, the variable region of the TCR β-chain comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 10. In some embodiments, the variable region of the TCR β-chain comprises SEQ ID NO: 10. In some embodiments, the variable region of the TCR β-chain consists of SEQ ID NO: 10.

[0126] In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain comprising an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:4; and (b) a variable region of the TCR β-chain comprising an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:10. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain comprising SEQ ID NO:4; and (b) a variable region of the TCR β-chain comprising SEQ ID NO:10. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain consisting of SEQ ID NO:4; and (b) a variable region of the TCR β-chain consisting of SEQ ID NO:10.

[0127] In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:5; and (b) a TCR β-chain comprising an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:11. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising SEQ ID NO:5; and (b) a TCR β-chain comprising SEQ ID NO:11. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain consisting of SEQ ID NO:5; and (b) a TCR β-chain consisting of SEQ ID NO:11.

[0128] In some embodiments, the exogenous human TCR is expressed as a single polypeptide having 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.

[0129] In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO:73; and (b) a TCR β-chain comprising an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO:75. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising SEQ ID NO:73; and (b) a TCR β-chain comprising SEQ ID NO:75. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain consisting of SEQ ID NO:73; and (b) a TCR β-chain consisting of SEQ ID NO:75.

[0130] In some embodiments, the exogenous human TCR is expressed as a single polypeptide having 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.

[0131] In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:18, a CDR2 having the amino acid sequence of SEQ ID NO:19, and a CDR3 having the amino acid sequence of SEQ ID NO:20; and (b) a TCR β-chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:24, a CDR2 having the amino acid sequence of SEQ ID NO:25, and a CDR3 having the amino acid sequence of SEQ ID NO:26. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-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 β-chain variable region comprising a CDR1 consisting of SEQ ID NO:24, a CDR2 consisting of SEQ ID NO:25, and a CDR3 having the amino acid sequence of SEQ ID NO:26.

[0132] In some embodiments, the variable region of the TCR α chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 21. In some embodiments, the variable region of the TCR α chain comprises SEQ ID NO: 21. In some embodiments, the variable region of the TCR α chain consists of SEQ ID NO: 21. In some embodiments, the variable region of the TCR β chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 27. In some embodiments, the variable region of the TCR β chain comprises SEQ ID NO: 27. In some embodiments, the variable region of the TCR β chain consists of SEQ ID NO: 27.

[0133] In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α chain that comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 21; and (b) a variable region of the TCR β chain that comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 27. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α chain that comprises SEQ ID NO: 21; and (b) a variable region of the TCR β chain that comprises SEQ ID NO: 27. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α chain that consists of SEQ ID NO: 21; and (b) a variable region of the TCR β chain that consists of SEQ ID NO: 27.

[0134] In some embodiments, the exogenous human TCR comprises: (a) a TCR α chain that comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 22; and (b) a TCR β chain that comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 28. In some embodiments, the exogenous human TCR comprises: (a) a TCR α chain that comprises SEQ ID NO: 22; and (b) a TCR β chain that comprises SEQ ID NO: 28. In some embodiments, the exogenous human TCR comprises: (a) a TCR α chain that consists of SEQ ID NO: 22; and (b) a TCR β chain that consists of SEQ ID NO: 28.

[0135] In some embodiments, the exogenous human TCR is expressed as a single polypeptide having 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.

[0136] In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:80; and (b) a TCR β-chain comprising an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:82. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising SEQ ID NO:80; and (b) a TCR β-chain comprising SEQ ID NO:82. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain consisting of SEQ ID NO:80; and (b) a TCR β-chain consisting of SEQ ID NO:82.

[0137] In some embodiments, the exogenous human TCR is expressed as a single polypeptide having 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.

[0138] In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain comprising CDR1 having the amino acid sequence of SEQ ID NO:32, CDR2 having the amino acid sequence of SEQ ID NO:33, and CDR3 having the amino acid sequence of SEQ ID NO:34; and (b) a variable region of the TCR β-chain comprising CDR1 having the amino acid sequence of SEQ ID NO:38, CDR2 having the amino acid sequence of SEQ ID NO:39, and CDR3 having the amino acid sequence of SEQ ID NO:40. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain comprising CDR1 consisting of SEQ ID NO:32, CDR2 consisting of SEQ ID NO:33, and CDR3 consisting of SEQ ID NO:34; and (b) a variable region of the TCR β-chain comprising CDR1 consisting of SEQ ID NO:38, CDR2 consisting of SEQ ID NO:39, and CDR3 having the amino acid sequence of SEQ ID NO:40.

[0139] In some embodiments, the variable region of the TCR α-chain comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 35. In some embodiments, the variable region of the TCR α-chain comprises SEQ ID NO: 35. In some embodiments, the variable region of the TCR α-chain consists of SEQ ID NO: 35. In some embodiments, the variable region of the TCR β-chain comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 41. In some embodiments, the variable region of the TCR β-chain comprises SEQ ID NO: 41. In some embodiments, the variable region of the TCR β-chain consists of SEQ ID NO: 41.

[0140] In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 35; and (b) a variable region of the TCR β-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 41. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain comprising SEQ ID NO: 35; and (b) a variable region of the TCR β-chain comprising SEQ ID NO: 41. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain consisting of SEQ ID NO: 35; and (b) a variable region of the TCR β-chain consisting of SEQ ID NO: 41.

[0141] In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 36; and (b) a TCR β-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 42. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising SEQ ID NO: 36; and (b) a TCR β-chain comprising SEQ ID NO: 42. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain consisting of SEQ ID NO: 36; and (b) a TCR β-chain consisting of SEQ ID NO: 42.

[0142] In some embodiments, the exogenous human TCR is expressed as a single polypeptide having 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.

[0143] In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 46, a CDR2 having the amino acid sequence of SEQ ID NO: 47, and a CDR3 having the amino acid sequence of SEQ ID NO: 48; and (b) a TCR β-chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 52, a CDR2 having the amino acid sequence of SEQ ID NO: 53, and a CDR3 having the amino acid sequence of SEQ ID NO: 54. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-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 β-chain variable region comprising a CDR1 consisting of SEQ ID NO: 52, a CDR2 consisting of SEQ ID NO: 53, and a CDR3 having the amino acid sequence of SEQ ID NO: 54.

[0144] In some embodiments, the TCR α-chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 49. In some embodiments, the TCR α-chain variable region comprises SEQ ID NO: 49. In some embodiments, the TCR α-chain variable region consists of SEQ ID NO: 49. In some embodiments, the TCR β-chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 55. In some embodiments, the TCR β-chain variable region comprises SEQ ID NO: 55. In some embodiments, the TCR β-chain variable region consists of SEQ ID NO: 55.

[0145] In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 49; and (b) a variable region of the TCR β-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 55. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain comprising SEQ ID NO: 49; and (b) a variable region of the TCR β-chain comprising SEQ ID NO: 55. In some embodiments, the exogenous human TCR comprises: (a) a variable region of the TCR α-chain consisting of SEQ ID NO: 49; and (b) a variable region of the TCR β-chain consisting of SEQ ID NO: 55.

[0146] In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 50; and (b) a TCR β-chain, which comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 56. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain comprising SEQ ID NO: 50; and (b) a TCR β-chain comprising SEQ ID NO: 56. In some embodiments, the exogenous human TCR comprises: (a) a TCR α-chain consisting of SEQ ID NO: 50; and (b) a TCR β-chain consisting of SEQ ID NO: 56.

[0147] Table A: TCR sequences

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] In some embodiments, the exogenous TCR comprises two or more polypeptides. For example, in some embodiments, the exogenous TCR comprises a first polypeptide containing an α chain and a second polypeptide containing a β chain.

[0159] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0160] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0161] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0162] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0163] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0164] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0165] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0166] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0167] In some embodiments, the T cell receptor comprises: (a) an α 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 β chain comprising an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 119, wherein the T cell receptor specifically binds to a myelin basic protein (MBP) peptide complexed with a major histocompatibility complex (MHC).

[0168] In some embodiments, the amino acid sequence of the α-chain has at least 85% identity with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the α-chain has at least 90% identity with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the α-chain has at least 95% identity with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the α-chain has at least 96% identity with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the α-chain has at least 97% identity with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the α-chain has at least 98% identity with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the α-chain has at least 99% identity with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the amino acid sequence of the α-chain has 100% identity with the amino acid sequence of SEQ ID NO: 113.

[0169] In some embodiments, the amino acid sequence of the α-chain has at least 85% identity with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the α-chain has at least 90% identity with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the α-chain has at least 95% identity with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the α-chain has at least 96% identity with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the α-chain has at least 97% identity with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the α-chain has at least 98% identity with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the α-chain has at least 99% identity with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the α-chain has 100% identity with the amino acid sequence of SEQ ID NO: 114.

[0170] In some embodiments, the amino acid sequence of the β-chain has at least 85% identity with the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β-chain has at least 90% identity with the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β-chain has at least 95% identity with the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β-chain has at least 96% identity with the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β-chain has at least 97% identity with the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β-chain has at least 98% identity with the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β-chain has at least 99% identity with the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the β-chain has 100% identity with the amino acid sequence of SEQ ID NO: 119.

[0171] In some embodiments, the amino acid sequence of the β-chain has at least 85% identity with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β-chain has at least 90% identity with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β-chain has at least 95% identity with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β-chain has at least 96% identity with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β-chain has at least 97% identity with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β-chain has at least 98% identity with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β-chain has at least 99% identity with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the β-chain has 100% identity with the amino acid sequence of SEQ ID NO: 120.

[0172] In some embodiments, the amino acid sequence of the β-chain has at least 85% identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β-chain has at least 90% identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β-chain has at least 95% identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β-chain has at least 96% identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β-chain has at least 97% identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β-chain has at least 98% identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β-chain has at least 99% identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the β-chain has 100% identity with the amino acid sequence of SEQ ID NO: 121.

[0173] In some embodiments, the amino acid sequence of the β-chain has at least 85% identity with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β-chain has at least 90% identity with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β-chain has at least 95% identity with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β-chain has at least 96% identity with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β-chain has at least 97% identity with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β-chain has at least 98% identity with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β-chain has at least 99% identity with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the β-chain has 100% identity with the amino acid sequence of SEQ ID NO: 122.

[0174] 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.

[0175] In some embodiments, the TCR comprises one or more amino acid sequences as set forth in Table B. The TCR can comprise the amino acid sequence of any α-chain CDR1, CDR2, or CDR3 provided in Table B. In some embodiments, the αCDR1 of the TCR comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the αCDR2 of the TCR comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the αCDR3 of the TCR comprises the amino acid sequence of SEQ ID NO: 111. The TCR can comprise the amino acid sequence of any β-chain CDR1, CDR2, or CDR3 provided in Table B. In some embodiments, the βCDR1 of the TCR comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, the βCDR2 of the TCR comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, the βCDR3 of the TCR comprises the amino acid sequence of SEQ ID NO: 117.

[0176] In some embodiments, the variable region of the α-chain 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 variable region of the β-chain 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.

[0177] In some embodiments, the variable region of the α-chain of the TCR is a murine constant region. In some embodiments, the variable region of the β-chain of the TCR is a murine constant region.

[0178] In some embodiments, the constant region of the α-chain 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 constant region of the β-chain 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.

[0179] 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.

[0180] In some embodiments, the present disclosure provides an engineered polypeptide comprising a TCR sequence described herein.

[0181] In some embodiments, the present disclosure provides an engineered polypeptide comprising a human TCR α-chain and a human TCR β-chain, wherein the TCR α-chain is at least 95% identical to SEQ ID NO:73 and the TCR β-chain is at least 95% identical to SEQ ID NO:75; or the TCR α-chain is at least 95% identical to SEQ ID NO:80 and the TCR β-chain is at least 95% identical to SEQ ID NO:82. In some embodiments, the TCR α-chain comprises SEQ ID NO:73 or consists of SEQ ID NO:73 and the TCR β-chain comprises SEQ ID NO:75 or consists of SEQ ID NO:75; or the TCR α-chain comprises SEQ ID NO:80 or consists of SEQ ID NO:80 and the TCR β-chain comprises SEQ ID NO:82 or consists of SEQ ID NO:82.

[0182] In some embodiments, the engineered polypeptide provided herein comprises, from the N-terminus to the C-terminus: a TCR β-chain, a self-cleaving peptide sequence, and a TCR α-chain. In some embodiments, the self-cleaving peptide is the P2A sequence (SEQ ID NO:104).

[0183] 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.

[0184] 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.

[0185] 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:113. 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:113. 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:113. 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:113. 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:113. 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:113. 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:113. 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:113. 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:113.

[0186] In some embodiments, the amino acid sequence of the polypeptide further comprises an amino-terminal leader sequence, optionally METLLGVSLV ILWLQLARVN (SEQ ID NO:100).

[0187] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 114.

[0188] In some embodiments, the engineered polypeptide encodes a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119. 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: 119. 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: 119. 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: 119. 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: 119. 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: 119. 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: 119. 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: 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.

[0189] 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.

[0190] In some embodiments, the amino acid sequence of the polypeptide further comprises a carboxy-terminal P2A linker sequence, optionally ATNFSLLKQAGDVEENPG (SEQ ID NO: 104).

[0191] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 120.

[0192] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 122.

[0193] Table B: Additional exemplary TCR sequences

[0194]

[0195]

[0196]

[0197] Nucleic acids encoding TCR

[0198] In some embodiments, the present disclosure provides nucleic acids encoding a TCR (e.g., an exogenous TCR). The nucleic acid can be or can 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.

[0199] The nucleic acids used herein are generally engineered nucleic acids. Engineered nucleic acids are polynucleotides that do not exist in nature (e.g., at least two nucleotides covalently linked together and, in some cases, containing a phosphodiester bond, referred to as a phosphodiester backbone). Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by ligating 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 chemically synthesized or synthesized by other means. Synthetic nucleic acids include nucleic acids that are chemically modified or otherwise modified but can base pair (hybridize) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules produced by replication of any of the foregoing.

[0200] The engineered nucleic acids of the present disclosure can 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, nucleic acids are produced using GIBSON 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 Three enzyme activities are typically used in single-tube reactions: 5'-exonuclease, 3'-extension activity of DNA polymerase, and DNA ligase activity. The 5'-exonuclease activity chews up the 5'-end sequence and exposes the complementary sequence for annealing. Then, the polymerase activity fills in the gap on the annealed domain. Then, the DNA ligase seals the nick and covalently links the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly and thus result in a higher percentage of correct assembly. The MegaGate molecular cloning method can also be used. MegaGate is a toxin-free Gateway technology that eliminates the ccdb toxin used in Gateway recombinase cloning and instead uses meganuclease-mediated digestion during cloning to eliminate background vectors (see, e.g., Kramme C. et al. STAR Protoc. October 22, 2021; 2(4):100907, which is incorporated herein by reference). Other methods for generating engineered polynucleotides can be used according to the present disclosure.

[0201] In some embodiments, the present disclosure provides an expression cassette comprising an open reading frame that comprises a nucleic acid encoding a foreign TCR operably linked to a promoter. A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds to initiate transcription. The promoter is typically located immediately upstream (at the 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 is regulatable in vivo by, for example, a chemical agent, temperature, or light.

[0202] An open reading frame is a continuous stretch of codons that begins with a start codon (e.g., ATG), ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide, such as a protein. An open reading frame is operably linked to a promoter if the promoter regulates the transcription of the open reading frame.

[0203] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding an exogenous TCR or an expression vector comprising the nucleic acid. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. For example, the vector can be a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a retroviral vector, or a baculovirus vector. The viral vector provides efficient delivery of the exogenous TCR into the regulatory T cells of the present disclosure. Exemplary viral vectors can be derived from lentiviruses; retroviruses (e.g., retroviridae viral vectors); adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48); parvoviruses (e.g., adeno-associated viruses); coronaviruses; negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses); rhabdoviruses (e.g., rabies and vesicular stomatitis viruses); paramyxoviruses (e.g., measles and Sendai viruses); positive-strand RNA viruses, such as picornaviruses and alphaviruses; and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-defective herpesviruses), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, human papillomaviruses, human foamy viruses, and hepatitis viruses. Examples of retroviruses include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncogenic retroviruses, HTLV-BLV group, alpha-retroviruses, gamma-retroviruses, foamy viruses, murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor viruses, bovine leukemia viruses, feline leukemia viruses, feline sarcoma viruses, avian leukemia viruses, human T-cell leukemia viruses, baboon endogenous viruses, gibbon ape leukemia viruses, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0204] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector of the present disclosure comprises lentiviral gag, pol, and rev genes and two long terminal repeats (LTRs) flanking an expression cassette comprising a nucleic acid encoding an exogenous TCR. For safety reasons, the vector will not include any other active lentiviral genes, such as vpr, vif, vpu, nef, tat. In some embodiments, these genes have been removed or otherwise inactivated.

[0205] In some embodiments, the lentiviral vector is a self-inactivating vector. A self-inactivating vector is a vector in which the production of full-length vector RNA in transduced cells is greatly reduced or completely eliminated. This feature greatly minimizes the risk that replication-competent recombinants (RCRs) will arise. In addition, it reduces the risk that cellular coding sequences located near the vector integration site will be aberrantly expressed. Further, the SIN design reduces the likelihood of interference between the LTR and the promoter driving transgene expression.

[0206] Self-inactivation is preferably achieved by introducing deletions 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 elements in the LTR involved in polyadenylation of viral RNA are not modified. This together reduces or eliminates the production of full-length vector RNA in transduced cells.

[0207] 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.

[0208] In some embodiments, the nucleic acid is codon-optimized. Codon optimization methods are known in the art. In some embodiments, codon optimization can be used to match codon frequencies in the target and host organisms to ensure proper folding; bias the GC content to increase RNA (e.g., mRNA) stability or reduce secondary structure; minimize tandem repeat codons or runs of bases that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove, or streamline protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust the translation rate to allow the individual domains of the protein to fold properly; or reduce or eliminate problematic secondary structure present within the polynucleotide.

[0209] In some embodiments, the nucleic acid encoding the TCR comprises a promoter operably linked to a coding sequence encoding an exogenous human TCR. The promoter can be a viral promoter or a native TCR promoter. In some embodiments, the promoter is a constitutively active promoter or an inducible promoter. In some embodiments, the promoter is the eukaryotic translation elongation factor 1α (EF-1α) promoter and the MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site replaced) (see, for example, Gill, D.R. et al. Gene Ther. 2001; 8:1539–46 and Astrakhan, A. et al. Blood 2012; 119:4395-4407).

[0210] 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 can be used. In some embodiments, the enhancer element is an optimized post-transcriptional regulatory element (oPRE), the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The WPRE can 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, M.A. et al., Gene Therapy Vol. 16, pp. 605-619 (2009).

[0211] In some embodiments, the engineered polynucleotide comprises an open reading frame that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises a nucleotide sequence having 100% identity to the nucleotide sequence of SEQ ID NO:87.

[0212] In some embodiments, the engineered polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80% identity 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 at least 85% identity 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 at least 90% identity 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 at least 95% identity 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 at least 96% identity 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 at least 97% identity 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 at least 98% identity 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 at least 99% identity 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.

[0213] 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 can be selected from P2A, E2A, F2A, and T2A. In some embodiments, the self-cleaving peptide sequence is the sequence of ATNFSLLKQAGDVEENPG (SEQ ID NO:104).

[0214] Isolated cell populations

[0215] In some embodiments, the present disclosure provides isolated cell populations and compositions thereof, the isolated cell populations comprising stable CD4 + T regulatory cells derived from a subject with multiple sclerosis. In some embodiments, the present disclosure provides isolated cell populations and compositions thereof, the isolated cell populations comprising stable CD4 + T regulatory cells derived from a subject with multiple sclerosis, which are engineered to express an exogenous human TCR. In some embodiments, at least 80% of the cells are stable CD4 + T regs comprising a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus.

[0216] The methylation status of the TSDR at the FOXP3 locus can be evaluated by methods known in the art. For example, in some embodiments, the methylation status of the TSDR at the FOXP3 locus is evaluated by bisulfite treatment and digital droplet PCR (ddPCR) using methylation-specific primers and probes. In some embodiments, the methylation status of the TSDR at the FOXP3 locus is evaluated by single-cell sequencing methods.

[0217] An isolated cell population is a population of cells removed from the human body or a population of cells removed from a sample obtained from the human body. Thus, it is considered to be "isolated" from the human body. A cell population can be isolated (e.g., obtained from a subject), or a cell population can be isolated from a biological sample obtained from a subject, for example, using any known cell collection method such as apheresis. The isolated cell populations of the present disclosure can be subjected to the methods described herein to produce isolated cell populations that contain a higher number of regulatory T cells (e.g., stable regulatory T cells) relative to cell populations obtained directly from a subject or from a biological sample obtained from a subject, for example, by apheresis.

[0218] In some embodiments, the isolated cell population contains stable CD4 + T regulatory cells, wherein at least 80% of the cells are stable CD4 + T cells that contain a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells are stable CD4 + T reg cells that contain a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated cell population contains stable CD4+ T regulatory cells, wherein 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells are stable CD4 + T reg cells that contain a hypomethylated TSDR at the FOXP3 locus. In some embodiments, between 80% and 90%, between 85% and 95%, between 80% and 85%, between 85% and 90%, between 90% and 95% of the cells are stable CD4 +Tregs. 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 contain a hypomethylated TSDR at the endogenous FOXP3 locus.

[0219] In some embodiments, the isolated population contains CD25 + / 高 CD4 + CD127 - / 低 Regulatory T cells. In some embodiments, the isolated cell population contains CD25 + / 高 CD4 + CD127 - / 低 FOXP3 + Regulatory T cells. In some embodiments, the isolated cell population contains CD25 + / 高 CD4 + CD127 - / 低 FOXP3 + CD45RA + Regulatory T cells.

[0220] In some embodiments, the isolated cell population contains 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+ / hi CD4+ CD127− / lo regulatory T cells. In some embodiments, the isolated cell population contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25 + / 高 CD4 + CD127 - / 低 FOXP3 + Regulatory T cells.

[0221] 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 contains at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25 + / 高 CD4+ CD127 - / 低 CD45RA + Regulatory T cells.

[0222] As described herein, the present disclosure provides isolated cell populations comprising stable T regulatory cells that comprise a hypomethylated TSDR at the FOXP3 locus. While in some embodiments the isolated populations described herein are engineered to express an exogenous TCR, the FOXP3 locus remains unmodified. Thus, the cells of the isolated populations described herein do not comprise an engineered FOXP3 locus. An “engineered FOXP3 locus” refers to any engineered modification (e.g., by artificial modification) designed to alter the expression of FOXP3. Such engineered modifications include, but are not limited to, introduction of a FOXP3 transgene, introduction of a modified promoter, and / or use of an exogenous agent (e.g., a gene editing system, a small molecule, or a peptide) designed to activate the expression of FOXP3.

[0223] In some embodiments, the isolated cell population comprises at least 1×10 2 cells, at least 1×10 3 cells, at least 1×10 4 cells, at least 1×10 5 cells, at least 1×10 6 cells, at least 1×10 7 cells, at least 1×10 8 cells, at least 1×10 9 cells, or at least 1×10 10 cells of stable regulatory T cells. In some embodiments, the isolated cell population comprises 1×10 2 to 1×10 10 cells, 1×10 3 to 1×10 10 cells, 1×10 4 to 1×10 10 cells, 1×10 5 to 1×10 10 cells, 1×10 6 to 1×10 10 cells, 1×10 7 to 1×10 10 cells, 1×10 8 to 1×10 10 cells, 1×10 5 to 1×10 9 cells, 1×10 6 to 1×10 8 cells, 1×10 7 to 1×10 10 cells, or 1×10 4 to 1×106 a stable regulatory T cell. In some embodiments, the isolated cell population comprises 1×10 6 to 1×10 10 a stable regulatory T cell. In some embodiments, the isolated cell population comprises 1×10 7 cells, 2x10 7 cells, 3×10 7 cells, 4×10 7 cells, 5x10 7 cells, 6×10 7 cells, 7×10 7 cells, 8x10 7 cells or 9×10 7 a stable regulatory T cell. In some embodiments, the isolated cell population comprises 1×10 7 to 1×10 10 cells, 2×10 7 to 1×10 10 cells, 3×10 7 to 1×10 10 cells, 4×10 7 to 1×10 10 cells, 5×10 7 to 1×10 10 cells, 6×10 7 to 1×10 10 cells, 7×10 7 to 1×10 10 cells, 8×10 7 to 1×10 10 cells or 9×10 7 to 1×10 10 a stable regulatory T cell.

[0224] In some embodiments, the isolated cell population comprises stable T regulatory cells that comprise a hypomethylated TSDR at the FOXP3 locus, wherein the stable T regulatory cells retain markers of stability in the presence of pro-inflammatory conditions (i.e., maintain a hypomethylated TSDR at the FOXP3 locus and / or maintain CD25 + / 高 CD4 + CD127 - / 低 FOXP3 +protein expression profiles). Pro-inflammatory conditions refer 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 other cells 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). Pro-inflammatory conditions can be in vitro or in vivo. In some embodiments, stable regulatory T cells within an 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.

[0225] In some embodiments, an isolated cell population comprises stable T regulatory cells that comprise a hypomethylated TSDR at the FOXP3 locus, wherein the stable T regulatory cells retain markers of stability over time (i.e., maintain a hypomethylated TSDR at the FOXP3 locus and / or maintain CD25 + / 高 CD4 + CD127 - / 低 FOXP3 +protein expression profiles). For example, in some embodiments, stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after isolation from a biological sample 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. In some embodiments, stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after activation and / or expansion 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. In some embodiments, stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after transduction with an exogenous TCR 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. In some embodiments, stable T regulatory cells maintain hypomethylated TSDR at the FOXP3 locus ex vivo after cryopreservation-thaw cycles 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.

[0226] In some embodiments, stable T regulatory cells maintain CD25 ex vivo after isolation from a biological sample + / 高 CD4 + CD127 - / 低 FOXP3 + protein expression profiles 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. In some embodiments, stable T regulatory cells maintain CD25 ex vivo after activation and / or expansion +高 CD4 + CD127 - / 低 FOXP3 + protein expression profiles 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. In some embodiments, stable T regulatory cells maintain CD25 ex vivo after transduction with an exogenous TCR +高 CD4 + CD127 - / 低 FOXP3 +protein expression profiles 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. In some embodiments, stable T regulatory cells maintain CD25 ex vivo after cryopreservation freeze-thaw cycles +高 CD4 + CD127 - / 低 FOXP3 + protein expression profiles 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.

[0227] In some embodiments, at least 50% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 50% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 50% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 60% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 60% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 60% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 70% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 70% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 70% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 80% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 80% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 80% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 90% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 90% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 90% of the cells in the isolated cell population contain a hypomethylated TSDR at the FOXP3 locus for at least 15 days after isolation.

[0228] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated at multiple (e.g., two or more) time points during the isolation and generation process. In some embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 20% between these two or more time points.

[0229] For example, in some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after isolation from a 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 T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from the biological sample.

[0230] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after isolation from a biological sample and is evaluated again within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In some embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no 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 evaluated within 1 to 3 days after isolation from a biological sample and is evaluated again within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion.

[0231] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after isolation from a biological sample and re-evaluated within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after isolation from a biological sample and re-evaluated within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus is reduced by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus is reduced by no more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus is reduced by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR.

[0232] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after isolation from a biological sample and is evaluated again within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In some embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after isolation from a biological sample and is evaluated again within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles.

[0233] In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after transduction with an exogenous human TCR and is evaluated again within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In some embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In some embodiments, hypomethylation of the TSDR at the FOXP3 locus is evaluated within 1 to 3 days after isolation from a biological sample and is evaluated again within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles. In such embodiments, the percentage of stable T regulatory cells containing hypomethylated TSDR at the FOXP3 locus decreases by no more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after cryopreservation freeze-thaw cycles.

[0234] In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 5, 6, 7, 8, 9, 10, or 11 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percentage point. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 5, 6, 7, 8, 9, 10, or 11 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells increases by 1, 2, 3, 4, or 5 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 5 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 10 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 10 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 10 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 15 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 10 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 5 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 5 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 10 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 5 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 15 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 5 percentage points. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 5 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 1 percentage point. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 10 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 1 percentage point. In some embodiments, after transducing stable regulatory T cells with exogenous human TCR, during an amplification period of at least 15 days, the percentage of cells in the isolated cell population comprising the stable regulatory T cells decreases by no more than 1 percentage point.

[0235] The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells can be evaluated at about 1 hour, about 6 hours, about 12, about 24 hours, about 36, about 48, about 72 hours, about 96 hours, or about 120 hours after transducing cells with exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells can be evaluated at 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, 13, or 14 days after transducing cells with exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells can be evaluated at about 1 - 21 days, 1 - 7 days, 4 - 14 days, 4 - 7 days, 7 - 10 days, 7 - 14 days, 10 - 21 days, or 14 - 21 days after transducing cells with exogenous human TCR.

[0236] 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 in the isolated cell population express exogenous TCR (e.g., after transducing the isolated cell population with exogenous TCR). In some embodiments, 10% - 60% or 20% - 50% of the regulatory T cells in the isolated cell population express exogenous TCR. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the stable regulatory T cells in the isolated cell population comprising regulatory T cells express exogenous TCR (e.g., after transducing the isolated cell population with exogenous TCR). In some embodiments, 10% - 60% or 20% - 50% of the stable regulatory T cells in the isolated cell population express exogenous TCR.

[0237] In some embodiments, the TSDR at the endogenous FOXP3 locus of the stable regulatory T cells in the isolated cell population of regulatory T cells remains hypomethylated until the isolated cell population is administered to a subject. In some embodiments, the TSDR at the endogenous FOXP3 locus of the stable regulatory T cells in the isolated cell population of regulatory T cells remains hypomethylated after cryopreservation thaw - freeze cycles.

[0238] In some embodiments, regulatory T cells of a separated cell population (e.g., stable regulatory T cells) exhibit one or more cellular functions that are associated with regulatory T cells when activated by binding to pMHC. Non-limiting examples of such cellular functions include cytokine secretion activity, expression of certain activation markers, and inhibitory activity. Cytokine secretion activity refers only to the secretion of certain anti-inflammatory cytokines such as IL-10, TGFβ, and IL-35. Activation markers include, but are not limited to, CD69, 4-1BB, CD25, CD71, or CTLA-4. For example, when regulatory T cells are contacted with a target peptide complexed with MHC, they express one or more of these markers.

[0239] Inhibitory activity refers to inhibiting the activation, proliferation, and cytokine production of non-regulatory T cells (e.g., CD8+ T cells and CD4+ conventional T cells), in part to prevent the immune system from becoming overactive. Regulatory T cells exhibit inhibitory activity when contacted with a target peptide complexed with MHC that binds to the 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 inhibit the activation, proliferation, and cytokine production of conventional T cells specific for a shared target peptide complexed with MHC (e.g., a shared target peptide presented by an antigen-presenting cell (APC)). In some embodiments, relative to a control (e.g., non-regulatory T cells in the absence of regulatory T cells), regulatory T cells inhibit the proliferation and growth of non-regulatory T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%.

[0240] In some embodiments, relative to a control (e.g., conventional T cells in the absence of regulatory T cells), regulatory T cells inhibit IFN-γ production by conventional T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. In some embodiments, relative to a control, regulatory T cells inhibit IFN-γ production by conventional T cells by 50%-99%, 75%-99%, or 80%-100% (e.g., when present in a population containing a 1:1 to 1:8 ratio of regulatory T cells compared to conventional T cells). In some embodiments, relative to a control (e.g., conventional T cells in the absence of regulatory T cells), regulatory T cells inhibit CD71 production by conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%. In some embodiments, relative to a control, regulatory T cells inhibit CD71 production by conventional T cells by 20%-90% or 30%-80% (e.g., when present in a population containing a 1:1 to 1:8 ratio of regulatory T cells compared to conventional T cells). In some embodiments, relative to a control (e.g., conventional T cells in the absence of regulatory T cells), regulatory T cells inhibit CD25 production by conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%. In some embodiments, relative to a control, regulatory T cells inhibit CD25 production by conventional T cells by 40%-70% (e.g., when present in a population containing a 1:1 to 1:8 ratio of regulatory T cells compared to conventional T cells).

[0241] In some embodiments, when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by a regulatory T cell (e.g., a target peptide presented by an APC), the regulatory T cell exhibits cytokine secretion activity (e.g., secretion of IL-10). In some embodiments, when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by a regulatory T cell, the regulatory T cell exhibits expression of activation markers. For example, in some embodiments, when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by a regulatory T cell, the regulatory T cell exhibits expression of CD69, 4-1BB, CD25, CD71, and / or CTLA-4. In some embodiments, when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by a regulatory T cell, the regulatory T cell exhibits inhibitory activity. For example, in some embodiments, the regulatory T cell inhibits the activation of conventional T cells specific for a shared target peptide complexed with MHC.

[0242] The cellular function of stable regulatory T cells can be evaluated at about 1 hour, about 6 hours, about 12, about 24 hours, about 36, about 48, about 72 hours, about 96 hours or about 120 hours after transducing cells with exogenous human TCR. The cellular function of stable regulatory T cells can be evaluated at 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, 13 or 14 days after transducing cells with exogenous human TCR. The cellular function of stable regulatory T cells can be evaluated at about 1 - 21 days, 1 - 7 days, 4 - 14 days, 4 - 7 days, 7 - 10 days, 7 - 14 days, 10 - 21 days or 14 - 21 days after transducing cells with exogenous human TCR.

[0243] In some embodiments, transduced regulatory T cells (e.g., a population of isolated transduced regulatory T cells) retain their cellular functionality (e.g., the ability to be activated) after cryopreservation thaw cycles. In some embodiments, transduced regulatory T cells can be activated and / or expanded after cryopreservation thaw cycles. In some embodiments, regulatory T cells can exhibit cytokine secretion activity, expression of certain activation markers, and / or inhibitory activity after cryopreservation thaw cycles.

[0244] In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the cell population is CD25+ / high CD4+CD127− / lo before activation and / or transduction with exogenous human T cell receptor.

[0245] An isolated cell population comprising stable regulatory T cells may contain a small number of non-regulatory T cells (e.g., conventional T cells). Non-regulatory T cells can 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 the 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 the 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 the isolated cell population comprising regulatory T cells are non-regulatory T cells comprising exogenous human TCR. Conventional T cells typically 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 the isolated cell population comprising stable 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 the 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 the isolated cell population comprising regulatory T cells are conventional T cells comprising exogenous human TCR. In some embodiments, the isolated cell population comprising regulatory T cells contains an undetectable amount of conventional T cells. In some embodiments, the isolated cell population comprising regulatory T cells contains an undetectable amount of conventional CD4+ T cells. In some embodiments, the isolated cell population comprising regulatory T cells contains an undetectable amount of B cells. In some embodiments, the isolated cell population comprising regulatory T cells contains an undetectable amount of NK T cells. In some embodiments, the isolated cell population comprising regulatory T cells contains an undetectable amount of CD14+ cells. In some embodiments, the isolated cell population comprising regulatory T cells contains an undetectable number of eosinophils.In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of neutrophils. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of CD8+ T cells.

[0246] The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) can be evaluated at about 1 hour, about 6 hours, about 12, about 24 hours, about 36, about 48, about 72 hours, about 96 hours, or about 120 hours after transducing the cells with exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) can be evaluated at 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, 13, or 14 days after transducing the cells with exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) can be evaluated at about 1 - 21 days, 1 - 7 days, 4 - 14 days, 4 - 7 days, 7 - 10 days, 7 - 14 days, 10 - 21 days, or 14 - 21 days after transducing the cells with exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) can be evaluated at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after cryopreservation thaw cycles.

[0247] In some embodiments, in an isolated cell population comprising regulatory T cells, the ratio of regulatory T cells to conventional T cells is at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 100:1.

[0248] Methods for generating stable regulatory T cells

[0249] In some embodiments, the present disclosure provides methods for generating isolated cell populations that comprise stable CD4+ T regulatory cells derived from a subject with multiple sclerosis. Conventional methods for generating antigen-specific regulatory T cells are unable to produce clinical-scale and clinical-grade manufacturing of cell populations that comprise high concentrations of stable antigen-specific regulatory T cells. Although polyclonal T cells have been successfully generated and administered in a clinical setting, the use of stable thymic regulatory T cells engineered with exogenous TCRs poses increased safety risks that require increased cell purity and regulatory T cell stability. Without wishing to be bound by theory, stable thymic regulatory T cells represent a small fraction of white blood cells, and if the isolated regulatory T cell population is not highly pure, other types of white blood cells will be engineered with the exogenous TCR. If present in sufficient numbers, these engineered non-regulatory T cells can have deleterious effects by eliciting rather than suppressing immune responses at sites of autoimmune disease. By integrating and modifying several biomarker selection methods, including an unexpectedly effective dual selection method using CD25 and, in some cases, CD45RA, the methods devised herein provide the art with a tool for generating antigen-specific regulatory T cell populations with high yields and high relative concentrations of stable TCR-transduced regulatory T cells.

[0250] In some embodiments, the methods of the present disclosure involve removing conventional T cells and selecting cells with a CD25+ / high CD4+CD127− / lo phenotype to generate a cell population that comprises stable regulatory T cells. In some embodiments, the methods of the present disclosure involve removing conventional T cells, selecting cells with a CD25 + / 高 CD4 + CD127 - / 低 phenotype to generate a cell population that comprises stable regulatory T cells, and engineering the stable regulatory T cells to comprise an exogenous human TCR that specifically binds a target peptide complexed with MHC. In some embodiments, the method for generating a cell population of stable regulatory T cells comprises isolating a biological sample that comprises regulatory T cells from a human subject with multiple sclerosis.

[0251] Figure 1A - 1B Example methods of the present disclosure are provided in Figure 1AIn the provided embodiments, a biological sample (e.g., a blood sample) is first isolated from a human subject with multiple sclerosis using apheresis. The cells of the biological sample are labeled with anti-CD8 (targeting non-CD4 conventional T cells), anti-CD19 (targeting B cells), and anti-CD14 (targeting monocytes) antibodies; and the labeled cells are removed from the biological sample to produce a depleted biological sample. In some embodiments, the volume of the depleted biological sample is then reduced (e.g., by removing water / liquid) to facilitate 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., anti-biotin microbeads) 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 attached to a tandem conjugate of phycoerythrin (PE) and biotin. In some embodiments, this enrichment step can be repeated more than once (e.g., two or more times). Then, as Figure 1A and Figure 1B described, these CD25-enriched cultures are further processed by sorting the cells using a TCR gating strategy that utilizes fluorescence-activated cell sorting (FACS) or Tyto sorting (Miltenyi). The cells are first labeled with anti-CD45RA, anti-CD4, and anti-CD127 antibodies. The cells are then selected in multiple sorting steps. In each step, in some embodiments, the labeled CD25+ / hi CD4+ CD127− / lo cells are first identified by a gating strategy that includes first identifying singlets, then live cells, then CD4 + cells, then CD25 + / 高 CD127 - / 低 cells are identified from the CD4+ cells, and then CD25 高 CD45RA - and CD25 + / 高 CD45RA + cells. The identified cell populations (e.g., cells comprising the protein expression profile of CD4 + CD25 高 CD127 - / 低 CD45RA - and cells comprising the expression profile of CD4 + CD25 + CD127 - / 低 CD45RA + are collected). In some embodiments, the sorting procedure is performed twice to optimize the purity of the cells.

[0252] In some embodiments, the present disclosure provides a method of generating a population of cells comprising stable CD4+ Method for isolating a population of T regulatory cells, wherein the method comprises (a) removing CD8 + cells and CD19 + cells from a biological sample obtained from a subject suffering from multiple sclerosis to produce a depleted biological sample; (b) enriching CD25 + cells in the depleted biological sample to produce an enriched population; and (c) isolating CD4 + CD25 + CD127 - / 低 cells from the enriched population. In some embodiments, the present disclosure provides a method for producing an isolated population of stable CD4 + T regulatory cells, wherein the method comprises (a) removing CD8 + cells and CD19 + cells from a biological sample obtained from a subject suffering from multiple sclerosis to produce a depleted biological sample; (b) enriching CD25 + cells in the depleted biological sample to produce an enriched population; (c) isolating CD4 + CD25 + CD127 - / 低 cells from the enriched population; and (d) engineering the cell population of (c) to express an exogenous human T cell receptor (TCR) that specifically binds a target peptide complexed with MHC. In some embodiments, step (a) further comprises removing CD14 + cells from the biological sample. In some embodiments, step (a) further comprises removing CD56 + cells from the biological sample.

[0253] In some embodiments, the method further comprises quantifying the methylation status of the T cell-specific demethylated region (TSDR) at the FOXP3 locus in the cell population, wherein at least 80% of the cells are stable CD4 + T regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the methylation status of the TSDR is quantified after the isolation step of (c). In some embodiments, the methylation status of the TSDR is quantified after the isolation step of (d).

[0254] In some embodiments, the step of isolating CD4 + CD25 + CD127 - / 低 cells from the enriched population comprises: (i) identifying a first subset of CD4 + cells from the enriched population of step (b); (ii) identifying CD25 + / 高 CD127 - / 低a second subset of cells; and (iii) selecting CD25 high CD45RA- and CD25 from the second subset + / 高 CD45RA + cells for isolation and excluding CD25 + CD45RA - thereby isolating a stable CD4 + T reg population.

[0255] In some embodiments, the methods provided herein further comprise isolating a biological sample comprising regulatory T cells from a human subject having multiple sclerosis. In some embodiments, the sample isolation is performed using apheresis techniques (e.g., leukapheresis techniques for isolating white blood cells). In some embodiments, isolating a biological sample comprising regulatory T cells from a human subject having multiple sclerosis is performed by taking blood from the subject and separating the blood into plasma and cells. In some embodiments, the apheresis technique involves taking whole blood from the subject and separating the whole blood to remove the desired cell type (e.g., T cells). In some embodiments, the separation step of the apheresis is performed using continuous flow centrifugation or intermittent flow centrifugation.

[0256] Cells expressing a specific biomarker (e.g., CD8 + cells, CD19 + cells, CD14 + cells and / or CD56 + cells) can be removed from a sample (e.g., a biological sample) using FACS, antibody pull-down assay techniques, or any other method known to those of ordinary skill in the art for removing cells expressing a specific biomarker. In some embodiments, those cells can be removed from the sample by labeling the cells expressing the specific biomarker with beads (e.g., anti-CD8, anti-CD14, anti-CD19, and / or anti-CD56 beads) and then subjecting the sample to FACS (to remove the cells expressing the specific biomarker).

[0257] A depleted biological sample is a biological sample (e.g., isolated from a human subject having multiple sclerosis) that has been processed to remove CD8 + cells, CD19 + cells, CD14 + cells and / or CD56 + cells. In some embodiments, removing CD8 + cells, CD19 + cells, CD14 + cells and / or CD56 +Cells generate a depleted biological sample in which less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, or less than 3% of the total cells contain CD8 + cells, CD19 + cells, CD14 + cells, and / or CD56 + cells. In some embodiments, CD8 + cells, CD19 + cells, CD14 + cells, and / or CD56 + cells generate a depleted biological sample in which less than 0.5% of the total cells contain CD8 + cells.

[0258] Cell selection techniques can be used to select cells with a specific phenotype (e.g., cells expressing a specific biomarker such as CD4, CD25, or CD45RA; or cells not expressing or low-expressing a specific biomarker such as CD127). The cell selection technique can be FACS technique, Tyto device, antibody pull-down assay technique, magnetic cell separation technique, or any other method known to those of ordinary skill in the art for selecting cells expressing a specific biomarker. In some embodiments, the cell selection technique includes labeling cells expressing a biomarker with a biotinylated antibody targeting the specific biomarker (e.g., CD25), and then performing a pull-down assay (e.g., with anti-biotin microbeads or streptavidin complex). In some embodiments, the cell selection technique includes labeling cells expressing a biomarker with an antibody targeting the specific biomarker (e.g., CD25, CD4, and / or CD45RA). Then cells labeled with an antibody targeting the specific biomarker can be selected from the sample (e.g., a CD25-enriched population) using FACS.

[0259] The CD25 enrichment step is followed by a multi-step sorting process for selecting CD25 + / 高 CD4 + CD127 - / 低 regulatory T cells. In some embodiments, the sorting step is performed on a Tyto device (Miltenyi Biotec). Those skilled in the art will understand that software for visualizing the sorted cell population enables data to be displayed in various ways, including dot plots and histograms. When identifying a population based on the presence or absence of a single protein, a dot plot or histogram can be used for identification. When identification of a population based on the presence or absence of two proteins (e.g., CD25 and CD45RA) is required, a dot plot should be used for identification.

[0260] In some embodiments, prior to the first sorting step, a CD25-rich cell population is separated into multiple cell populations, and the first sorting step is performed simultaneously and / or sequentially in multiple subsets. This provides an accelerated processing of a large number of cells that need to be processed in order to isolate a sufficient number of a small proportion of CD25 + / 高 CD4 + CD127 - / 低 cells. In some embodiments, the CD25-rich cell population is separated into 2, 3, 4, 5, or 6 or more subsets, each of which is sorted in the first sorting step. In some embodiments, after the first sorting step, the sorted subsets are combined prior to the second sorting step.

[0261] In some embodiments, the present disclosure provides a method of generating an isolated population of stable CD4 + T regulatory cells, wherein the method comprises (a) removing CD8 + cells and CD19 + cells from a biological sample obtained from a subject suffering from multiple sclerosis to produce a depleted biological sample; (b) enriching CD25 + cells in the depleted biological sample to produce an enriched population; and (c) isolating CD4 + CD25 + CD127 - / 低 cells from the enriched population, wherein the isolation comprises (i) identifying a first subset of CD4 + cells from the enriched population of step (b); (ii) identifying a second subset of CD25 + / 高 CD127 - / 低 cells from the first subset; and (iii) selecting CD25 高 CD45RA - and CD25 + / 高 CD45RA + cells for isolation and excluding CD25 + CD45RA - thereby isolating a stable CD4+ T reg population.

[0262] In some embodiments, selecting CD25 + / 高 CD4 + CD127 - / 低 cells from a CD25-rich population comprises identifying a first subset of CD4 + cells from the CD25-rich population. Then a second subset of CD25 + / 高 CD127 - / 低 cells is identified from the first subset. Finally, CD25 高CD45RA - and CD25 + / 高 CD45RA + populations of cells are separated.

[0263] In some embodiments, the first subset 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.

[0264] In some embodiments, the second subset 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 + / 高 CD4 + CD127 - / 低 regulatory T cells. In some embodiments, the second subset 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 + / 高 CD4 + CD127 - / 低 FoxP3 + regulatory T cells.

[0265] In some embodiments, the population selected for separation comprises CD25 高 CD4 + CD127 - / 低 CD45RA - (CD25高 CD45RA - ) and CD25 + / 高 CD4 + CD127 - / 低 CD45RA + (CD25 + / 高 CD45RA + ) regulatory T cells. In some embodiments, the population selected for isolation comprises at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% CD25 高 CD45RA - cells. In some embodiments, the population selected for isolation comprises at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% CD25 + / 高 CD45RA + cells. It is understood that the percentage of CD25 高 CD45RA - and CD25 + / 高 CD45RA + cells in the final population totals approximately 80%, 90% or 100% of the total cell population.

[0266] In some embodiments, the population selected for isolation comprises less than 10% CD25 + CD4 + CD127 - / 低 CD45RA cells. In some embodiments, the population selected for isolation comprises less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% CD25 + CD4 + CD127 - / 低 CD45RA cells.

[0267] In some embodiments, the methods described herein produce an isolated cell population wherein at least 80% of the cells are stable CD4 cells comprising a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus +Tregs. 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 cells containing a hypomethylated TSDR at the FOXP3 locus + Tregs. In some embodiments, the isolated cell population contains stable CD4 + T regulatory cells, wherein 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells are stable CD4 cells containing a hypomethylated TSDR at the FOXP3 locus + Tregs. In some embodiments, the cells between 80% and 90%, between 85% and 95%, between 80% and 85%, between 85% and 90%, between 90% and 95% are stable CD4 cells containing a hypomethylated TSDR at the FOXP3 locus + Tregs.

[0268] In some embodiments, if at least 80% of the cells are stable CD4 + Tregs that contain a hypomethylated T cell-specific demethylated region (TSDR) at the FOXP3 locus, then the isolated cell population generated by the methods described herein is selected for therapeutic use. In some embodiments, if at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells are stable CD4 + Tregs, then the isolated cell population is selected for therapeutic use. In some embodiments, if 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells are stable CD4 + Tregs, then the isolated cell population is selected for therapeutic use. In some embodiments, if the cells between 80% and 90%, between 85% and 95%, between 80% and 85%, between 85% and 90%, between 90% and 95% are stable CD4 + Tregs, then the isolated cell population is selected for therapeutic use.

[0269] In some embodiments, the methods provided herein further comprise introducing an exogenous TCR that specifically binds a target peptide complexed with MHC into a cell population comprising stable CD4 + T regulatory cells. In some embodiments, introducing the exogenous TCR comprises transfecting the cell population with a nucleic acid encoding the exogenous TCR. In some embodiments, introducing the exogenous TCR comprises transducing the cell population with a nucleic acid encoding the exogenous TCR. The nucleic acid encoding the exogenous TCR can be an RNA (e.g., mRNA) molecule or a DNA molecule. In some embodiments, the nucleic acid is a vector or plasmid. In some embodiments, a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a retroviral vector, or a baculovirus vector is used to deliver the nucleic acid to the cell population.

[0270] In some embodiments, the method or process of generating a cell population of regulatory T cells further comprises activating and / or expanding the regulatory T cells of the population (e.g., after the depletion, enrichment, and engineering steps described herein). In some embodiments, the regulatory T cells are activated prior to transfecting or transducing them with the exogenous TCR. In some embodiments, the regulatory T cells are activated with anti-CD28 and / or anti-CD8 antibodies. In some embodiments, activating the regulatory T cells comprises contacting the regulatory T cells with an antigen that specifically binds an exogenous TCR belonging to a separate cell population. In some embodiments, the antibody (e.g., anti-CD3, anti-CD28, and / or TCR-specific antibody) is conjugated or complexed with beads (e.g., magnetic beads or polymer beads). In some embodiments, the antibody is covalently attached to a polymeric matrix.

[0271] In some embodiments, the methods provided herein further comprise a second activation and / or expansion step. In some embodiments, the second activation and / or expansion step is performed 5, 6, or 7 days after the initial activation and / or expansion step.

[0272] In some embodiments, activating and / or expanding regulatory T cells comprises culturing a population of isolated cells in a cell culture medium. In some embodiments, the medium comprises IL-2. In some embodiments, the medium comprises about 100 IU / ml, 200 IU / ml, 300 IU / ml, 400 IU / ml, 500 IU / ml, 600 IU / ml, 700 IU / ml, 800 IU / ml, 900 IU / ml, 1,000 IU / ml, 1200 IU / ml, 1400 IU / ml, 1600 IU / ml, 1800 IU / ml, 2000 IU / ml, 3000 IU / ml or 4000 IU / ml IL-2. In some embodiments, the medium comprises 500 - 1500 IU / ml or 800 - 1200 IU / ml IL-2. In some embodiments, the medium comprises about 1,000 IU / ml IL-2. In some embodiments, the medium comprises 1,000 IU / ml IL-2. In some embodiments, the medium further comprises TNFα. In some embodiments, the medium comprises 500 IU / ml, 750 IU / ml, 1000 IU / ml, 1500 IU / ml, 1750 IU / ml, 2000 IU / ml, 2250 IU / ml, 2500 IU / ml or about 3000 IU / ml TNFα. In some embodiments, the medium comprises 2000 - 3000 IU / ml or 2250 - 2750 IU / ml TNFα. In some embodiments, the medium comprises about 2500 IU / ml TNFα. In some embodiments, the medium comprises 2500 IU / ml TNFα.

[0273] In some embodiments, activating and expanding regulatory T cells comprises culturing the population of cells for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some embodiments, activating and expanding regulatory T cells comprises culturing the population of cells for 2 - 14 days, 2 - 10 days, 2 - 5 days, 5 - 10 days or 5 - 14 days. In some embodiments, activating and expanding regulatory T cells comprises culturing the population of cells for no more than 15 days, 14 days, 13 days or 12 days. In some embodiments, after activation and / or expansion, the population of isolated cells comprises at least 1×10 2 cells, at least 1×10 3 cells, at least 1×10 4 cells, at least 1×10 5 cells, at least 1×10 6 cells, at least 1×10 7 cells, at least 1×10 8 cells, at least 1×10 9 cells or at least 1×10 10A stable CD25 + / 高 CD4 + CD127 - / 低 Regulatory T cells, which contain a hypomethylated TSDR at the endogenous FOXP3 locus.

[0274] In some embodiments, the method or process for generating a population of regulatory T cells may further comprise cryopreserving the population of cells. In some embodiments, the method or process for generating a population of regulatory T cells may further comprise thawing the cryopreserved population of cells. A cryopreservation-thaw cycle refers to the process of cryopreserving a population of cells and thawing the cryopreserved population of cells at a later time.

[0275] Pharmaceutical compositions

[0276] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated population of the regulatory T cells (e.g., stable regulatory T cells) described herein. In some embodiments, the pharmaceutical composition comprises a population of the regulatory T cells (e.g., stable regulatory T cells) described herein and a pharmaceutically acceptable excipient.

[0277] As used herein, a pharmaceutically acceptable excipient may also be referred to as a pharmaceutically acceptable carrier, pharmaceutically acceptable diluent, or pharmaceutically acceptable adjuvant. The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, and the development of suitable dosing and treatment regimens for using the specific compositions described herein in a variety of treatment regimens is also well known to those skilled in the art.

[0278] The pharmaceutical composition should generally be sterile and stable under the manufacturing and storage conditions. Sterile injectable formulations can be prepared using a non-toxic parenterally acceptable diluent or solvent. The pharmaceutical composition used according to the present invention may comprise a pharmaceutically acceptable dispersing agent, wetting agent, suspending agent, isotonic agent, coating, antibacterial agent, antifungal agent, carrier, excipient, salt, or stabilizer, which are non-toxic to the subject at the doses and concentrations used. In some embodiments, the pharmaceutical composition may comprise 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.

[0279] The pharmaceutical composition may comprise an effective amount of stable regulatory T cells sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of the stable regulatory T cells described herein may refer to the number of cells sufficient to ameliorate 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 formulation provided herein may vary depending on various factors, such as the desired biological response, e.g., the particular disease being treated, the particular symptoms to be alleviated, the cells or tissues being targeted, and the age, sex, and general health of the subject.

[0280] In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus) comprises at least 1×10 2 cells, at least 1×10 3 cells, at least 1×10 4 cells, at least 1×10 5 cells, at least 1×10 6 cells, at least 1×10 7 cells, at least 1×10 8 cells, at least 1×10 9 cells, or at least 1×10 10 stable regulatory T cells. 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 1×102 to 1×1010, 1×103 to 1×1010, 1×10 4 to 1×10 10 cells, 1×10 5 to 1×10 10 cells, 1×10 6 to 1×10 10 cells, 1×10 7 to 1×10 10 cells, 1×10 8 to 1×10 10 cells, 1×10 9 to 1×10 10 stable regulatory T cells. In some embodiments, an effective amount of stable regulatory T cells comprises 1×10 7 cells, 2x10 7 cells, 3×10 7 cells, 4×10 7 cells, 5x10 7 cells, 6×10 7 cells, 7×10 7 cells, 8x10 7 cells, or 9×10 7A stable regulatory T cell. In some embodiments, an effective amount of the stable regulatory T cell comprises 1×10 7 to 1×10 10 cells, 2×10 7 to 1×10 10 cells, 3×10 7 to 1×10 10 cells, 4×10 7 to 1×10 10 cells, 5×10 7 to 1×10 10 cells, 6×10 7 to 1×10 10 cells, 7×10 7 to 1×10 10 cells, 8×10 7 to 1×10 10 cells or 9×10 7 to 1×10 10 stable regulatory T cells.

[0281] In some embodiments, an isolated cell population (e.g., intended for use in a pharmaceutical composition) is cryopreserved (e.g., by undergoing one or more freeze-thaw cycles). That is, the isolated cell populations generated herein can be combined with a cryoprotectant that lowers the melting temperature by forming chemical bonds with water and increasing the total concentration of solutes in the system. Non-limiting examples of cryoprotectants include glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, and propylene glycol. While traditional methods that typically involve the use of serum and DMSO can be used, the present disclosure also contemplates the use of a cryopreservation medium that is manufactured under cGMP conditions and formulated to be serum-free and non-animal sourced (e.g., using <10% DMSO in the cryomix). Other cryopreservation techniques are also provided herein, including more advanced cooling techniques, such as vitrifying the cells without the use of a cryoprotectant. See, e.g., Shinshu University. “A new way to 'freeze' cells promises to transform the common cell-freezing practice.” ScienceDaily.com April 2019. This ultra-rapid cooling process utilizes an inkjet unit to print at a rate of 10,000 degrees Celsius per second to cool the cells to near vitrification.

[0282] Treatment methods

[0283] In some embodiments, the present disclosure provides methods of treating a subject in need thereof for multiple sclerosis, the methods comprising administering a composition comprising a stable CD4 +An isolated cell population or composition thereof of regulatory T cells. In some embodiments, the present disclosure provides methods of administering an isolated cell population (and related pharmaceutical compositions) comprising regulatory T cells as described herein to a subject (e.g., a subject having multiple sclerosis).

[0284] Multiple sclerosis typically begins with relapses and symptom remissions in relapsing-remitting multiple sclerosis (RRMS). Approximately 80% to 85% of MS patients are initially diagnosed with this form of the disease. There are currently over 15 disease-modifying therapies for RRMS. In progressive MS, the disease progresses slowly and steadily. Progressive MS can be relapsing (sometimes called secondary progressive MS) or non-relapsing (sometimes called primary progressive MS). Progressive multiple sclerosis is characterized by a progressive increase in clinical disability that is not associated with acute relapsing pathology. There are currently no therapies available for progressive multiple sclerosis. Currently, over 250,000 patients are affected by progressive multiple sclerosis. In addition, although currently approved disease-modifying therapies are effective in reducing the relapsing occurrence of RRMS, even RRMS patients experience progression independent of relapse activity (PIRA), which has not been successfully treated (Ransohoff 2023).

[0285] In progressive MS, compartmentalized inflammation mainly localized in meningeal lymphoid aggregates drives persistent inflammatory demyelination during progressive multiple sclerosis behind an intact blood-brain barrier (Lassman, Frontiers in Immunology, 2019). Inflammatory T cells in stable, persistent aggregates release inflammatory mediators that activate macrophages. Studies have shown that this high density and inflammation of meningeal lymphocytes are associated with increased subpial demyelination levels in the cortex and an increase in white matter lesions (Ahmed et al., JCI Insight. 2022;7(5)). Macrophages at the lesion margins continuously degrade myelin. PIRA in RRMS subjects has the same mechanistic basis as the progression of the progressive form of MS (Ransohoff 2023).

[0286] Without wishing to be bound by theory, it is contemplated herein that by engineering stable engineered regulatory T cells to bind to MBP (which is presented by cells in the meninges of a subject having multiple sclerosis) and targeting them to the meninges, the progression of multiple sclerosis (and particularly progressive multiple sclerosis) will be reduced, halted, or reversed by reducing the inflammatory response in the meninges.

[0287] In some embodiments, the present disclosure provides methods of administering to a subject a cell population or pharmaceutical composition comprising regulatory T cells as described herein in an effective amount to alleviate one or more symptoms of multiple sclerosis. Non-limiting examples of symptoms of multiple sclerosis include fatigue, vision problems, numbness of muscle tissue, muscle spasms, muscle stiffness, muscle weakness, mobility problems, pain (e.g., muscle pain), depression, anxiety, sexual problems, bladder problems, bowel problems, speech problems, and dysphagia.

[0288] In some embodiments, the present disclosure provides methods of treating multiple sclerosis in a subject, the methods comprising administering to the subject a cell population or pharmaceutical composition comprising regulatory T cells as described herein. In some embodiments, at least a portion of the cell population is autologous cells (i.e., obtained from the same subject to be subsequently administered). In some embodiments, a cell population is isolated from a subject, subjected to a method of generating a cell population as described herein (e.g., to increase the relative concentration of regulatory T cells within the population), engineered to express an exogenous TCR, and then administered to the same subject to treat the disease.

[0289] In some embodiments, treating (or treatment) of a disease refers to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of multiple sclerosis or one or more of its symptoms. In some embodiments, treatment can be administered after the appearance of one or more symptoms and / or after the disease is diagnosed. In other embodiments, treatment can be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or to inhibit the onset or progression of the disease. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., due to knowledge of genetic factors). Treatment can also be continued after symptoms have subsided, e.g., to prevent or delay symptom recurrence.

[0290] A subject refers to an individual organism, e.g., an individual human. In some embodiments, the subject is a human subject, such as a male subject or a female subject. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cow, a cat, or a dog. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject can be male or female.

[0291] Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intravenous, subcutaneous, intravenous, intrathecal administration for 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. Routes of administration can be combined if desired. In some embodiments, the isolated population or a composition thereof described herein is administered intravenously.

[0292] A cell population or pharmaceutical composition comprising regulatory T cells can be administered as a bolus. In some embodiments, administration of a cell population or pharmaceutical composition comprising regulatory T cells includes infusing the isolated cell population or pharmaceutical composition into a subject one or more times (e.g., infusing cells through a central line, similar to a blood transfusion).

[0293] Additional numbered embodiments

[0294] Additional numbered embodiments of the present disclosure are as follows:

[0295] Embodiment 1. An isolated cell population comprising regulatory T cells, wherein: the regulatory T cells of the isolated cell population comprise stable regulatory T cells, and these stable regulatory T cells (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 a hypomethylated regulatory T cell-specific demethylated region (TSDR) at the endogenous FOXP3 locus. At least 80% of the cells of the isolated cell population are CD25+ / high CD4+CD127− / lo regulatory T cells, and fewer than 10% of the cells of the isolated cell population express FOXP3 protein from an engineered FOXP3 locus.

[0296] Embodiment 2. The isolated population according to Embodiment 1, wherein the MBP peptide is the MBP 83-99 peptide comprising the amino acid sequence of SEQ ID NO: 61.

[0297] Embodiment 3. The isolated population according to Embodiment 1 or 2, wherein the MHC comprises HLA-DRB1*15:01.

[0298] Embodiment 4. The isolated population according to Embodiment 3, wherein the MHC further comprises HLA-DRA*.

[0299] Embodiment 5. The isolated population according to any one of the foregoing embodiments, wherein the exogenous TCR comprises a TCR α-chain, and the TCR α-chain comprises (a) a CDR1-α sequence comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR2-α sequence comprising the amino acid sequence of SEQ ID NO: 2, and / or (c) a CDR3-α sequence comprising the amino acid sequence of SEQ ID NO: 3.

[0300] Embodiment 6. The isolated population according to Embodiment 5, wherein the exogenous TCR comprises a variable region of the TCR α-chain, and the variable region comprises an amino acid sequence having at least 90%, 95% or 100% identity with the amino acid sequence of SEQ ID NO: 4.

[0301] Embodiment 7. The isolated population according to Embodiment 5, wherein the exogenous TCR comprises a TCR α-chain, and the TCR α-chain comprises an amino acid sequence having at least 90%, 95% or 100% identity with the amino acid sequence of SEQ ID NO: 5 or 73.

[0302] Embodiment 8. The isolated population according to any one of the foregoing embodiments, wherein the exogenous TCR comprises a TCR β-chain, and the TCR β-chain comprises: 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.

[0303] Embodiment 9. The isolated population according to Embodiment 8, wherein the exogenous TCR comprises a variable region of the TCR β-chain, and the variable region comprises an amino acid sequence having at least 90%, 95% or 100% identity with the amino acid sequence of SEQ ID NO: 10.

[0304] Embodiment 10. The isolated population according to Embodiment 8, wherein the exogenous TCR comprises a TCR β-chain, and the TCR β-chain comprises an amino acid sequence having at least 90%, 95% or 100% identity with the amino acid sequence of SEQ ID NO: 11 or 75.

[0305] Embodiment 11. The isolated population according to any one of the foregoing embodiments, wherein the exogenous TCR comprises a TCR α-chain, and the TCR α-chain comprises (a) a CDR1-α sequence comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR2-α sequence comprising the amino acid sequence of SEQ ID NO: 19, and / or (c) a CDR3-α sequence comprising the amino acid sequence of SEQ ID NO: 20.

[0306] Embodiment 12. The isolated population according to Embodiment 11, wherein the exogenous TCR comprises a variable region of the TCR α-chain, and the variable region comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 21.

[0307] Embodiment 13. The isolated population according to Embodiment 11, wherein the exogenous TCR comprises a TCR α-chain, and the TCR α-chain comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 22 or 80.

[0308] Embodiment 14. The isolated population according to any one of the foregoing embodiments, wherein the exogenous TCR comprises a TCR β-chain, and the TCR β-chain comprises: 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.

[0309] Embodiment 15. The isolated population according to Embodiment 14, wherein the exogenous TCR comprises a variable region of the TCR β-chain, and the variable region comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 27.

[0310] Embodiment 16. The isolated population according to Embodiment 14, wherein the exogenous TCR comprises a TCR β-chain, and the TCR β-chain comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 28 or 82.

[0311] Embodiment 17. The isolated population according to any one of the foregoing embodiments, wherein the exogenous TCR comprises a TCR α-chain, and the TCR α-chain comprises (a) a CDR1-α sequence comprising the amino acid sequence of SEQ ID NO: 32, (b) a CDR2-α sequence comprising the amino acid sequence of SEQ ID NO: 33, and / or (c) a CDR3-α sequence comprising the amino acid sequence of SEQ ID NO: 34.

[0312] Embodiment 18. The isolated population according to Embodiment 17, wherein the exogenous TCR comprises a variable region of the TCR α-chain, and the variable region comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 35.

[0313] Embodiment 19. The isolated population according to Embodiment 17, wherein the exogenous TCR comprises a TCR α chain, and the TCR α chain comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 36.

[0314] Embodiment 20. The isolated population according to any one of the foregoing embodiments, wherein the exogenous TCR comprises a TCR β chain, and the TCR β chain comprises: 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.

[0315] Embodiment 21. The isolated population according to Embodiment 20, wherein the exogenous TCR comprises a TCR β chain variable region, and the variable region comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 41.

[0316] Embodiment 22. The isolated population according to Embodiment 20, wherein the exogenous TCR comprises a TCR β chain, and the TCR β chain comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 42.

[0317] Embodiment 23. The isolated population according to any one of the foregoing embodiments, 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.

[0318] Embodiment 24. The isolated population according to Embodiment 23, wherein the single polypeptide comprises a TCR α chain and a TCR β chain, and wherein the polypeptide comprises a self-cleaving peptide sequence positioned between the TCR α chain and the TCR β chain.

[0319] Embodiment 25. The isolated population according to Embodiment 24, 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.

[0320] Embodiment 26. The isolated population according to any one of the foregoing embodiments, wherein the exogenous TCR comprises a TCR α chain containing a constant region, and the constant region comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 58.

[0321] Embodiment 27. The isolated population according to any one of the preceding embodiments, wherein the exogenous TCR comprises a TCRβ chain containing a constant region, and the constant region comprises an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 59 or 60.

[0322] Embodiment 28. The isolated population according to any one of the preceding embodiments, wherein the exogenous human TCR comprises one or more amino acid substitutions for cysteine residues in the constant regions of the TCRα chain and the TCRβ chain, and wherein these cysteine residues are capable of forming one or more disulfide bridges.

[0323] Embodiment 29. The isolated population according to any one of the preceding embodiments, wherein the TCRα chain constant region comprises a T48C amino acid substitution relative to the TCRα chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and wherein the TCRβ chain constant region comprises an S57C amino acid substitution relative to the TCRβ chain constant region comprising the amino acid sequence of SEQ ID NO: 60.

[0324] Embodiment 30. The isolated cell population according to any one of the preceding embodiments, wherein at least 70% of the cells of the isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

[0325] Embodiment 31. The isolated population according to 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.

[0326] Embodiment 32. The isolated cell population according to 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+ / high CD4+CD127- / lo regulatory T cells.

[0327] Embodiment 33. The isolated cell population according to any one of the preceding embodiments, wherein at least 70% of the cells of the isolated cell population are CD25 + / 高 CD4 + CD127 - / 低 FOXP3 + regulatory T cells.

[0328] Embodiment 34. The isolated cell population according to Embodiment 33, 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 CD25 + / 高 CD4 + CD127 - / 低 FOXP3 + Regulatory T cells.

[0329] Embodiment 35. The isolated cell population according to any one of the preceding embodiments, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an expansion period of at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days, the percentage of cells of the isolated cell population comprising the stable regulatory T cells decreases by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 percentage point.

[0330] Embodiment 36. The isolated cell population according to Embodiment 35, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an expansion period of at least 5 days, the percentage of cells of the isolated cell population comprising the stable regulatory T cells decreases by no more than 10 percentage points, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an expansion period of at least 10 days, the percentage of cells of the isolated cell population comprising the stable regulatory T cells decreases by no more than 10 percentage points.

[0331] Embodiment 37. The isolated cell population according to Embodiment 35, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an expansion period of at least 5 days, the percentage of cells of the isolated cell population comprising the stable regulatory T cells decreases by no more than 5 percentage points, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an expansion period of at least 10 days, the percentage of cells of the isolated cell population comprising the stable regulatory T cells decreases by no more than 5 percentage points.

[0332] Embodiment 38. The isolated cell population according to Embodiment 35, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an expansion period of at least 5 days, the percentage of cells of the isolated cell population comprising the stable regulatory T cells decreases by no more than 1 percentage point, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an expansion period of at least 10 days, the percentage of cells of the isolated cell population comprising the stable regulatory T cells decreases by no more than 1 percentage point.

[0333] Embodiment 39. The isolated cell population according to any one of the foregoing embodiments, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an amplification period of at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days, the percentage of cells in the isolated cell population comprising stable regulatory T cells is reduced by no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0334] Embodiment 40. The isolated cell population according to Embodiment 39, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an amplification period of at least 5 days, the percentage of cells in the isolated cell population comprising stable regulatory T cells is reduced by no more than 10%, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an amplification period of at least 10 days, the percentage of cells in the isolated cell population comprising stable regulatory T cells is reduced by no more than 10%.

[0335] Embodiment 41. The isolated cell population according to Embodiment 39, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an amplification period of at least 5 days, the percentage of cells in the isolated cell population comprising stable regulatory T cells is reduced by no more than 5%, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an amplification period of at least 10 days, the percentage of cells in the isolated cell population comprising stable regulatory T cells is reduced by no more than 5%.

[0336] Embodiment 42. The isolated cell population according to Embodiment 39, wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an amplification period of at least 5 days, the percentage of cells in the isolated cell population comprising stable regulatory T cells is reduced by no more than 1%, or wherein after transducing the stable regulatory T cells with the exogenous human TCR, during an amplification period of at least 10 days, the percentage of cells in the isolated cell population comprising stable regulatory T cells is reduced by no more than 1%.

[0337] Embodiment 43. The isolated cell population according to any one of the foregoing embodiments, which comprises at least 1×10 7 stable regulatory T cells.

[0338] Embodiment 44. The isolated cell population according to Embodiment 43, which comprises from 1×107 to 1×109 stable regulatory T cells.

[0339] Embodiment 45. The isolated cell population according to any one of the foregoing embodiments, wherein less than 5%, less than 2%, or less than 1% of the cells of the isolated cell population express FOXP3 protein from an engineered FOXP3 locus.

[0340] Embodiment 46. The isolated cell population according to any one of the foregoing 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.

[0341] Embodiment 47. The isolated cell population according to any one of the foregoing embodiments, wherein at least 90%, at least 95%, or 100% of the cells of the isolated cell population comprise an unmodified endogenous FOXP3 locus.

[0342] Embodiment 48. The isolated cell population according to any one of the foregoing 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 an engineered FOXP3 protein.

[0343] Embodiment 49. The isolated cell population according to any one of the foregoing 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 comprise an exogenous FOXP3 gene or an exogenous FOXP3 protein.

[0344] Embodiment 50. The isolated cell population according to any one of the foregoing embodiments, wherein at least 90%, at least 95%, or 100% of the cells of the isolated cell population do not exhibit ectopic FOXP3 expression.

[0345] Embodiment 51. The isolated cell population according to any one of the foregoing embodiments, wherein at least 90%, at least 95%, or 100% of the cells of the isolated cell population express a physiological level of functional FOXP3 protein.

[0346] Embodiment 52. The isolated cell population according to any one of the foregoing 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.

[0347] Embodiment 53. The isolated cell population according to any one of the foregoing 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 in the isolated cell population are conventional T cells.

[0348] Embodiment 54. The isolated cell population according to any one of the foregoing embodiments, wherein about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 5%, or about 5% to about 10% of the cells in the isolated cell population are conventional T cells.

[0349] Embodiment 55. The isolated cell population according to Embodiment 53 or 54, wherein the conventional T cells in the isolated population comprise the exogenous human TCR.

[0350] Embodiment 56. The isolated cell population according to Embodiment 55, wherein about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, or about 60% of the conventional T cells comprise the exogenous TCR.

[0351] Embodiment 57. The isolated cell population according to any one of the foregoing 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.

[0352] Embodiment 58. The isolated cell population according to any one of the foregoing embodiments, wherein less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated cell population are CD8+ T cells.

[0353] Embodiment 59. The isolated cell population according to Embodiment 58, wherein these CD8+ T cells are undetectable, optionally undetectable by fluorescence-activated cell sorting (FACS).

[0354] Embodiment 60. The isolated cell population according to any one of the foregoing embodiments, wherein at least 10% of the regulatory T cells in the isolated cell population express the exogenous human TCR.

[0355] Embodiment 61. The isolated cell population according to 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 in the isolated cell population express the exogenous human TCR.

[0356] Embodiment 62. The isolated cell population according to any one of the preceding embodiments, wherein CD25 is evaluated from 1 hour to 120 days after transduction + / 高 CD4 + CD127 - / 低 The percentage of regulatory T cells.

[0357] Embodiment 63. The isolated cell population according to any one of Embodiments 1 to 62, wherein CD25 is evaluated at least 1 hour, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours or 120 hours after transduction + / 高 CD4 + CD127 - / 低 The percentage of regulatory T cells.

[0358] Embodiment 64. The isolated cell population according to any one of Embodiments 1 to 62, wherein CD25 is evaluated from 7 days to 14 days after transduction + / 高 CD4 + CD127 - / 低 The percentage of regulatory T cells.

[0359] Embodiment 65. The isolated cell population according to any one of Embodiments 1 to 62, wherein CD25 is evaluated at least 10 days, 15 days, 30 days, 60 days or 120 days after transduction + / 高 CD4 + CD127 - / 低 The percentage of regulatory T cells.

[0360] Embodiment 66. The isolated cell population according to 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.

[0361] Embodiment 67. The isolated cell population according to any one of the preceding embodiments, wherein the stable regulatory T cells of the isolated cell population exhibit one or more functions selected from the following: (a) cytokine secretion activity; (b) expression of activation markers associated with regulatory T cells; and (c) inhibitory activity.

[0362] Embodiment 68. The isolated cell population according to Embodiment 67, wherein the one or more functions are evaluated from 1 hour to 14 days after transduction.

[0363] Embodiment 69. The isolated cell population according to Embodiment 67, wherein the one or more functions are evaluated at least 12 hours, 24 hours, 48 hours, 72 hours, 96 hours or 120 hours after transduction.

[0364] Embodiment 70. The isolated cell population according to Embodiment 67, wherein the one or more functions are evaluated at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after transduction.

[0365] Embodiment 71. The isolated cell population according to any one of Embodiments 67 to 70, wherein the one or more functions are evaluated after cryopreservation of the isolated cell population, optionally at least 24 hours after cryopreservation.

[0366] Embodiment 72. The isolated cell population according to any one of the foregoing embodiments, wherein at least 10% of the stable regulatory T cells are CD45RA+.

[0367] Embodiment 73. The isolated cell population according to Embodiment 72, wherein at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the stable regulatory T cells are CD45RA + 。

[0368] Embodiment 74. The isolated cell population according to any one of the foregoing embodiments, wherein at least 10% of the cells of the isolated cell population are CD25 + / 高 CD4 + CD127 - / 低 CD45RA + 。

[0369] Embodiment 75. The isolated cell population according to any one of the foregoing embodiments, wherein at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the cells of the isolated cell population are CD25 + / 高 CD4 + CD127 - / 低 CD45RA + 。

[0370] Embodiment 76. The isolated population according to any one of the foregoing embodiments, wherein the CD25 + / 高 CD4 + CD127 - / 低 regulatory T cells have the human leukocyte antigen (HLA)-DR15 haplotype.

[0371] Embodiment 77. The isolated population according to Embodiment 76, wherein the CD25 + / 高 CD4 + CD127 - / 低 regulatory T cells comprise the HLA-DRB1*15:01 allele.

[0372] Embodiment 78. The isolated population according to Embodiment 77, wherein the CD25 + / 高 CD4 + CD127 - / 低 regulatory T cells further comprise the HLA-DRB5*01:01 allele.

[0373] Embodiment 79. A method of generating an isolated cell population according to any one of the foregoing embodiments, the method comprising: (a) isolating a biological sample comprising regulatory T cells from a human subject having multiple sclerosis, optionally progressive multiple sclerosis; (b) removing CD8+ cells, CD19+ cells, and optionally CD14 + cells from the biological sample to produce a depleted biological sample; (c) selecting CD25 + / 高 cells from the depleted biological sample to produce a CD25-enriched cell population; (d) selecting CD25 + / 高 CD4 + CD127 - / 低 CD45RA + cells from the CD25-enriched cell population to produce one or more positive fractions; (e) selecting CD25 + / 高 CD4 + CD127 - / 低 CD45RA + cells from the one or more positive fractions to produce a cell population comprising stable regulatory T cells; and (f) engineering the cell population comprising stable regulatory T cells such that the stable regulatory T cells comprise the exogenous human TCR, thereby producing the isolated cell population.

[0374] Embodiment 80. The method according to 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.

[0375] Embodiment 81. The method according to any one of Embodiments 79 to 80, wherein less than 0.5% of the cells of the depleted sample comprise CD8 + cells.

[0376] Embodiment 82. The method according to any one of embodiments 79 to 81, wherein the selection in (d) comprises identifying CD4 + CD45RA + cells, and then identifying CD25 + CD45RA + from the identified CD4 + / 高 CD127 - / 低 cells to identify CD25 + / 高 CD4 + CD127 - / 低 CD45RA + cells of the first population, and then selecting the CD25 + / 高 CD4 + CD127 - / 低 CD45RA + from the first subset of cells of the first population to produce the one or more positive fractions.

[0377] Embodiment 83. The method according to any one of embodiments 79 to 82, wherein the selection in (e) comprises identifying CD4 + CD45RA + cells, and then identifying CD25 + CD45RA + from the identified CD4 + / 高 CD127 - / 低 cells to identify CD25 + / 高 CD4 + CD127 - / 低 CD45RA + cells of the second population, and then selecting the CD25 + / 高 CD4 + CD127 - / 低 CD45RA + from the second subset of cells of the second population to produce a cell population comprising stable regulatory T cells.

[0378] Embodiment 84. The method according to any one of embodiments 79 to 83, wherein the subject has a human leukocyte antigen (HLA)-DR15 haplotype.

[0379] Embodiment 85. The method according to embodiment 84, wherein the regulatory T cells comprise the HLA-DRB1*15:01 allele.

[0380] Embodiment 86. The method according to any one of embodiments 79 to 85, wherein the subject is a male subject.

[0381] Embodiment 87. The method according to any one of Embodiments 79 to 85, wherein the subject is a female subject.

[0382] Embodiment 88. The method according to any one of Embodiments 79 to 87, wherein engineering the isolated cell population comprises transducing the cell population with a nucleic acid encoding the exogenous human TCR.

[0383] Embodiment 89. The method according to any one of Embodiments 79 to 88, further comprising activating and expanding the cells of the isolated cell population to produce a cell population comprising at least 1×107 stable CD25 + / 高 CD4 + CD127 - / 低 regulatory T cells, which regulatory T cells comprise a hypomethylated TSDR at the endogenous FOXP3 locus.

[0384] Embodiment 90. The method according to Embodiment 89, wherein the activation and expansion comprises culturing the cells of the isolated cell population for at least 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

[0385] Embodiment 91. The method according to Embodiment 89, wherein the activation and expansion comprises culturing the cells of the isolated cell population for no more than 15 days, 14 days, 13 days or 12 days.

[0386] Embodiment 92. The method according to any one of Embodiments 88 to 91, wherein the nucleic acid is a vector.

[0387] Embodiment 93. The method according to Embodiment 92, wherein the vector is a viral vector.

[0388] Embodiment 94. The method according to Embodiment 93, wherein the viral vector is a lentiviral vector.

[0389] Embodiment 95. The method according to any one of Embodiments 88 to 94, wherein the nucleic acid comprises a promoter operably linked to the coding sequence encoding the exogenous human TCR, optionally wherein the promoter is an EF-1α promoter or an MND promoter.

[0390] Embodiment 96. The method according to any one of Embodiments 88 to 95, wherein the nucleic acid further comprises an enhancer element, an optionally optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and also optionally WPRE-mut6.

[0391] Embodiment 97. The method according to any one of Embodiments 88 to 96, wherein the coding sequence is codon-optimized.

[0392] Embodiment 98. The vector according to any one of Embodiments 88 to 97.

[0393] Embodiment 99. A pharmaceutical composition comprising a separated cell population according to any one of Embodiments 1 to 78 and a pharmaceutically acceptable excipient.

[0394] Embodiment 100. A composition comprising a separated cell population according to any one of Embodiments 1 to 78 and a cryopreservative.

[0395] Embodiment 101. A method comprising administering to a subject the pharmaceutical composition according to Embodiment 99, wherein the subject has multiple sclerosis.

[0396] Embodiment 102. The method according to Embodiment 101, wherein the subject has progressive multiple sclerosis.

[0397] Embodiment 103. The method according to Embodiment 102, wherein the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

[0398] Embodiment 104. The method according to any one of Embodiments 101 to 103, wherein the pharmaceutical composition is administered in an effective amount to alleviate one or more symptoms of multiple sclerosis.

[0399] Embodiment 105. The method according to any one of Embodiments 101 to 104, wherein the separated population is obtained from the subject.

[0400] Embodiment 106. The method according to any one of Embodiments 101 to 105, wherein the subject has the HLA-DR15 haplotype.

[0401] Embodiment 107. The method according to Embodiment 106, wherein the subject has the HLA-DRB1*15:01 allele.

[0402] Embodiment 108. The method according to Embodiment 106 or 107, wherein the subject has the HLA-DRB5*01:01 allele.

[0403] Embodiment 109. A method comprising administering to a subject a separated population according to any one of Embodiments 1 to 78.

[0404] Embodiment 110. The method according to Embodiment 109, wherein the subject has multiple sclerosis.

[0405] Embodiment 111. The method according to Embodiment 110, wherein the subject has progressive multiple sclerosis.

[0406] Embodiment 112. The method according to Embodiment 111, wherein the subject has primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

[0407] Embodiment 113. The method according to any one of Embodiments 101 to 112, wherein the isolated population is obtained from the subject.

[0408] Embodiment 114. The method according to any one of Embodiments 101 to 113, wherein the subject has an HLA-DR15 haplotype.

[0409] Embodiment 115. The method according to Embodiment 114, wherein the subject has an HLA-DRB1*15:01 allele.

[0410] Embodiment 116. The method according to Embodiment 114 or 115, wherein the subject has an HLA-DRB5*01:01 allele.

[0411] Embodiment 117. The method according to any one of Embodiments 101 to 116, wherein the administration comprises intravenous administration.

[0412] Embodiment 118. The method according to any one of Embodiments 101 to 117, wherein the administration comprises one or more infusions.

[0413] Embodiment 133. An engineered polynucleotide comprising an open reading frame that comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 107.

[0414] Embodiment 134. The engineered polynucleotide according to 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.

[0415] Embodiment 135. The engineered polynucleotide according to Embodiment 134, wherein the nucleotide sequence of the open reading frame has 100% identity to the nucleotide sequence of SEQ ID NO: 107.

[0416] 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.

[0417] Embodiment 137. The engineered polynucleotide according to 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.

[0418] Embodiment 138. The engineered polynucleotide according to 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.

[0419] Embodiment 139. The engineered polynucleotide according to Embodiment 138, wherein the amino acid sequence of the polypeptide has 100% identity to the amino acid sequence of SEQ ID NO: 108.

[0420] Embodiment 140. The engineered polynucleotide according to any one of Embodiments 133 to 139, wherein the open reading frame encodes a human / mouse chimeric T cell receptor (TCR), for example, wherein the variable region is a human variable region and the constant domain is a mouse constant domain.

[0421] Embodiment 141. The engineered polynucleotide according to Embodiment 140, wherein the human / mouse chimeric TCR comprises an α chain containing the amino acid sequences of SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111.

[0422] Embodiment 142. The engineered polynucleotide according to Embodiment 141, wherein the human / mouse chimeric TCR comprises an α chain containing the amino acid sequence of SEQ ID NO: 112.

[0423] Embodiment 143. The engineered polynucleotide according to Embodiment 141, wherein the human / mouse chimeric TCR comprises an α chain containing the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 114.

[0424] Embodiment 144. The engineered polynucleotide according to Embodiment 141, wherein the human / mouse chimeric TCR comprises an α constant domain containing the amino acid sequence of SEQ ID NO: 123.

[0425] Embodiment 145. The engineered polynucleotide according to any one of embodiments 140 to 144, wherein the human / mouse chimeric TCR comprises a β-chain having an amino acid sequence containing SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0426] Embodiment 146. The engineered polynucleotide according to embodiment 145, wherein the human / mouse chimeric TCR comprises a β-chain having an amino acid sequence containing SEQ ID NO: 118.

[0427] Embodiment 147. The engineered polynucleotide according to embodiment 145, wherein the human / mouse chimeric TCR comprises a β-chain having an amino acid sequence containing SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.

[0428] Embodiment 148. The engineered polynucleotide according to embodiment 145, wherein the human / mouse chimeric TCR comprises a β constant domain having an amino acid sequence containing SEQ ID NO: 124.

[0429] Embodiment 149. The engineered polynucleotide according to 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).

[0430] Embodiment 150. An engineered polynucleotide comprising an open reading frame encoding a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 113.

[0431] Embodiment 151. The engineered polynucleotide according to 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.

[0432] Embodiment 152. The engineered polynucleotide according to embodiment 151, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.

[0433] Embodiment 153. The engineered polynucleotide according to 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).

[0434] Embodiment 154. The engineered polynucleotide according to Embodiment 153, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 114.

[0435] 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.

[0436] Embodiment 156. The engineered polynucleotide according to 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.

[0437] Embodiment 157. The engineered polynucleotide according to Embodiment 156, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 119.

[0438] Embodiment 158. The engineered polynucleotide according to 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).

[0439] Embodiment 159. The engineered polynucleotide according to Embodiment 158, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 121.

[0440] Embodiment 160. The engineered polynucleotide according to any one of Embodiments 155 to 159, wherein the amino acid sequence of the polypeptide further comprises a carboxy-terminal self-cleaving peptide sequence, which is optionally selected from P2A, E2A, F2A and T2A, and optionally comprises the sequence GSG ATNFSLLKQA GDVEENPG (SEQ ID NO: 104).

[0441] Embodiment 161. The engineered polynucleotide according to 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.

[0442] Embodiment 162. The engineered polynucleotide according to 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.

[0443] Embodiment 163. The engineered polynucleotide according to any one of the foregoing embodiments, wherein the engineered polynucleotide is messenger ribonucleic acid (mRNA).

[0444] Embodiment 164. The engineered polynucleotide according to any one of Embodiments 133 to 162, wherein the engineered polynucleotide is deoxyribonucleic acid (DNA).

[0445] Embodiment 165. The engineered polynucleotide according to Embodiment 164, which further comprises a promoter operably linked to the open reading frame.

[0446] Embodiment 166. The engineered polynucleotide according to Embodiment 165, wherein the promoter is an EF-1α promoter or an MND promoter.

[0447] 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.

[0448] Embodiment 180. The engineered polypeptide according to 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.

[0449] Embodiment 181. The engineered polypeptide according to Embodiment 180, wherein the amino acid sequence of the polypeptide has 100% identity to the amino acid sequence of SEQ ID NO: 108.

[0450] 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.

[0451] Embodiment 183. The engineered polypeptide according to 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.

[0452] Embodiment 184. The engineered polypeptide according to Embodiment 183, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.

[0453] Embodiment 185. The engineered polypeptide according to 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).

[0454] Embodiment 186. The engineered polypeptide according to embodiment 185, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 114.

[0455] Embodiment 187. An engineered polypeptide encoding a polypeptide having an amino acid sequence with 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.

[0456] Embodiment 188. The engineered polypeptide according to embodiment 187, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence with 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.

[0457] Embodiment 189. The engineered polypeptide according to embodiment 188, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 119.

[0458] Embodiment 190. The engineered polypeptide according to 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).

[0459] Embodiment 191. The engineered polypeptide according to embodiment 190, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 121.

[0460] Embodiment 192. The engineered polypeptide according to any one of embodiments 187 to 191, wherein the amino acid sequence of the polypeptide further comprises a carboxyl-terminal P2A hanging sequence, optionally GSG ATNFSLLKQA GDVEENPG (SEQ ID NO: 104).

[0461] Embodiment 193. The engineered polypeptide according to 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.

[0462] Embodiment 194. The engineered polypeptide according to 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.

[0463] Embodiment 195. A T cell receptor comprising (a) an amino acid sequence of an α chain 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 or SEQ ID NO: 114; and / or (b) an amino acid sequence of a β chain 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, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122.

[0464] Embodiment 196. The T cell receptor according to Embodiment 195, wherein (a) the amino acid sequence of the α 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 β 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.

[0465] 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.

[0466] Embodiment 198. The T cell receptor according to 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.

[0467] Embodiment 199. The T cell receptor according to 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.

[0468] Embodiment 200. A T cell receptor encoded by an engineered polynucleotide according to any one of Embodiments 123 to 166.

[0469] Embodiment 201. A T cell receptor comprising an engineered polypeptide according to any one of Embodiments 173 to 194.

[0470] Example 202. The isolated population according to any one of Examples 1 to 74, comprising an engineered polypeptide according to any one of Examples 173 to 194.

[0471] Examples

[0472] Example 1. Manufacturing process for generating autologous stable regulatory T cells expressing exogenous TCR

[0473] The generation of a cell population comprising stable thymus-derived regulatory T cells expressing an exogenous TCR is shown in this example. The overall enrichment and sorting strategy is shown in Figures 1A to 1B .

[0474] Leukapheresis, depletion, and enrichment

[0475] First, leukapheresis is collected from a human subject, transported to a central processing facility, and maintained at 4 °C until processing approximately 24 hours after collection. The incoming leukapheresis product is washed in phosphate-buffered saline containing 0.5% human serum albumin (HSA) to reduce the platelet count, and then labeled with a mixture of magnetic microbeads (CD8, CD14, and CD19 microbeads; Miltenyi Biotec) using an automated cell processing system.

[0476] Using magnetic cell separation technology, cytotoxic T cells (CD8 + ) are depleted before enriching for CD25 + cells, B cells (CD19 + ) and monocytes (CD14 + ). The volume of the depleted biological sample is reduced by centrifugation. The depleted biological sample is then labeled with a CD25-PE-biotin antibody (Miltenyi Biotec) according to the manufacturer's recommendations. The cells are then washed with phosphate-buffered saline containing 0.5% HSA and labeled with anti-biotin microbeads (Miltenyi Biotec). CD25 + cells are then isolated using magnetic cell separation technology to produce a CD25-enriched sample.

[0477] Figure 2 Shows the viable cells (VC) of all cells after running 1 using the leukapheresis, removing CD8 + , CD19 + and CD14 + cells (depleted sample) and selecting CD25 + (CD25-enriched sample). After the CD25 selection step to produce the CD25-enriched sample, approximately 98% of the total cells in the population are CD3 + cells, and approximately 95% of the cells are CD4 + T cells.

[0478] Table 1 shows the apheresis cell population and the CD4 + cell and naïve Treg cell composition after depletion and enrichment steps in 5 additional manufacturing runs.

[0479] Table 1: Composition of cell populations during depletion and enrichment

[0480] Cells Run 2 Run 3 Run 4 Run 5 Run 6 Donor gender 33 59 29 37 26 Donor age M M M M M Donor health status (MS / non - MS) MS Healthy donor Healthy donor Healthy donor Healthy donor Apheresis VC 1.1E+10 1.1E+10 8.7E+09 1.5E+10 1.1E+10 Apheresis CD4 T cell percentage 38％ 30％ 22％ 36％ 49％ Apheresis naive Treg percentage 1％ 1％ 1％ 1％ 1％ Post - depletion VC 3.3E+09 2.6E+09 1.9E+09 4.6E+09 4.0E+09 Post - depletion CD4 T cell percentage 83％ 59％ 65％ 89％ 88％ Post - depletion naive Treg percentage 1％ 3％ 4％ 3％ 2％ Post - enrichment VC 3.5E+09 2.4E+08 3.0E+08 4.6E+08 2.0E+08 Post - enrichment CD4 T cell percentage 95％ 96％ 93％ 96％ 97％ Post - enrichment naive Treg percentage 10％ 19％ 21％ 18％ 19％

[0481] Figure 3A shows the viable cell count (VC) of naïve regulatory T cells (specifically, CD4 + CD25 + CD25 + / high CD127- / lo CD45RA+ cells) after apheresis, depletion of CD8 Figure 3B shows the recovery of naïve regulatory T cells (CD4+CD25+ / high CD127- / lo CD45RA+ cells) after each step, where approximately 80% of CD4+CD25+ / high CD127- / lo CD45RA cells were recovered after the depletion step and 75% of CD4+CD25+ / high CD127- / lo CD45RA+ cells were recovered after the CD25 enrichment step.

[0482] Table 2 shows the VC of naïve regulatory T cells after apheresis, depletion, and enrichment, as well as the recovery of naïve regulatory T cells from the previous step after depletion and enrichment.

[0483] Table 2: Total viable cell count (TVC) of naive regulatory T cells and recovery percentage after each step of the manufacturing process

[0484]

[0485] Gating strategy

[0486] The CD25-enriched sample was then stained with fluorescently labeled anti-CD45RA, anti-CD4, and anti-CD127 antibodies and sorted by fluorescence-activated cell sorting (FACS) for CD4 + CD25 + / 高 CD127 - / lo CD45RA +Sort the phenotypes to generate a cell population containing stable regulatory T cells. FACS is performed in two rounds. The first round is subtractive hierarchical sorting, and the second round is hierarchical purity sorting. For subtractive sorting, the CD25-rich sample is divided into multiple fractions, and these multiple fractions are sorted through multiple cell sorters. After subtractive sorting, the fractions are combined for the second-round purity sorting. The purpose of hierarchical sorting of cells in subtractive and / or purity sorting is to accelerate the sorting process by sorting on multiple machines, thereby ensuring that the cells remain viable. The gating strategies for subtractive and purity sorting are the same. For the gating strategy, a dot plot-based method is used, in which CD45RA + CD4 + cells are selected first, and then CD25 + CD4 + cells are selected from the CD45RA + / 高 CD127 - / lo cell population to optimize the purity of stable regulatory T cells in the population as determined by the TSDR phenotype. As Figure 1B shown, this FACS gating strategy first plots CD45RA + cells against CD4 + cells on a dot plot and selects these cells using a box gate based on the population distribution. From the selected cell population, CD25 + / 高 cells are plotted against CD127 - / lo cells on a dot plot, and a polygon gate is used for gating to select the population of CD4 + CD25 + / 高 CD127 - / lo CD45RA + cells for collection. This FACS strategy was developed to optimize the selection of stable naive regulatory T cells based on the TSDR hypomethylation phenotype. The composition of the cell population before sorting, after subtraction, and after generating the CD4 + CD25 + / 高 CD127 - / lo CD45RA + cell population for Run 1 is provided in Table 3.

[0487] Table 3. Composition of cell populations after FACS

[0488]

[0489] The purity and recovery of naive Tregs during the sorting process for 5 additional manufacturing runs are shown in Table 4.

[0490] Table 4: Purity and recovery of naive Tregs after FACS sorting

[0491]

[0492] After FACS, regulatory T cells were activated with T cell TransAct activation reagent (Miltenyi Biotec) for approximately 48 hours. Then, the activated regulatory T cells were 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 TCRβ chain and a C-terminal TCRα chain with a linker domain that contains the GSG amino acid sequence, followed by a P2A self-cleaving peptide. The TCRβ-GSG-P2A-TCRα fusion construct is expressed under the EF1α promoter. Figure 5 The proportion of transduced cells measured by FACS analysis with an anti-Vβ antibody is shown.

[0493] The transduced regulatory T cells were then expanded in T cell expansion medium supplemented with 1,000 IU / ml IL-2 until harvest (day 9, day 10, or day 11). The cell count, size, population doubling number, and viability of untransduced and transduced regulatory T cells were evaluated, and the results for run 1 are shown ( Figure 4 ). Notably, the transduced regulatory T cells shared a similar growth curve with untransduced regulatory T cells, indicating that the exogenous TCR does not impair cell growth and proliferation.

[0494] The population doubling levels and percentage viability for 5 additional manufacturing runs are shown in Table 5. Also shown is the percentage of the target dose achieved over the entire course of all 6 manufacturing runs. The percentage of the target dose was calculated as follows: the total final number of primary Tregs was determined based on the initial number and population doubling level of primary Tregs before amplification after the sorting process, assuming a transduction efficiency of 25%. The percentage of the target dose was calculated based on a target dose of 100 million transduced cells. As shown, the manufacturing process in this example yielded a Treg product with high purity and sufficient cells for administration to a patient from a single leukopak. As used herein, PDL is the population doubling level used throughout. TDN denotes transduced cells.

[0495] Table 5: Properties of the manufactured cells

[0496] Days PDL (TDN) Viability percentage Percentage of target dose Run 2 9 6.8 94％ 370％ Run 3 9 7.8 97％ 420％ Run 4 9 6.4 94％ 450％ Run 5 9 6.6 96％ 1400％ Run 6 9 6.5 Not applicable 482％

[0497] Transduced cells were collected on days 0 to 11 of the activation and expansion process, and TSDR hypomethylation at the FOXP3 CNS2 locus was measured by ddPCR. Genomic DNA (gDNA) was obtained from enriched regulatory T cells and subjected to bisulfite treatment. Then, digital droplet PCR (ddPCR) assays using methylation-specific primers and probes were employed to quantify unmethylated and methylated sequences. Figure 6 The TSDR hypomethylation status of the collected cells from all 6 post-transduction manufacturing runs is shown in Figure 6 . During expansion, for any manufacturing run, the TSDR hypomethylation level decreased by no more than 10%, and remained above 80% for all manufacturing runs. Table 6 below shows the TSDR hypomethylation status of all 6 donor samples processed using the method described in this example.

[0498] These data demonstrate that the obtained regulatory T cells are stable, thymus-derived regulatory T cells.

[0499] Table 6: TSDR status of cells generated using the method of this example

[0500] TSDR hypomethylation % Donor gender Day 0 Day 3 Day 6 Day 9 Run 1 M Not applicable Not applicable 93％ 93％ Run 2 M 96％ Not applicable 94％ 90％ Run 3 M 94％ Not applicable 90％ 86％ Run 4 M 90％ Not applicable 89％ 88％ Run 5 M 95％ 95％ 94％ Not applicable Run 6 M Not applicable 98％ 99％ 99％

[0501] Activity of Tregs after cryopreservation freeze - thaw cycles

[0502] Untransduced or lentiviral vector-transduced regulatory T cells generated according to the method in this example were cryopreserved for storage. The cells were frozen and stored in a vapor-phase nitrogen storage freezer.

[0503] After refrigeration for a period of time, 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)), while the second experimental group was not activated (untransduced (UNT)). Both groups were expanded in medium containing IL-2. After incubation, IL-10 levels were measured at 1 day, 2 days, and 3 days.

[0504] Figure 7 The increase in IL-10 levels per run over a three-day period for cells activated with anti-CD3 and anti-CD28 antibodies is shown in Figure 7 . The cells showed an increase in the secretion of the IL-10 cytokine over a three-day period, which demonstrates that the cell population can be activated and retains its cellular functionality after cryopreservation freeze-thaw cycles ( Figure 7 ). The unactivated cells showed no increase in IL-10 levels. This data further demonstrates that the regulatory T cells retained their function.

[0505] Example 2. Gating strategy optimized for GMP conditions

[0506] A second gating strategy was developed using a 1-D histogram-based method instead of a 2-D point-based method to reduce user error in the GMP manufacturing environment. Cells were prepared as in Example 1 up to the FACS sorting step. In the FACS sorting step, the gating scheme used, as Figure 8 shown, was a one-dimensional histogram-based method in which cells enriched for CD25 were gated successively by CD4+, CD45RA+, CD127− / lo, and CD25+ / hi (CD4+ > CD45RA+ (histogram) > CD127− / lo (histogram) 40% > CD25Hi (histogram) 70%). For runs with the same number of runs as in Example 1, the two gating strategies were run in parallel on the same donor samples. Cells were transduced with TCR-A or a codon-optimized version of disulfide-modified (referred to as 'TCR-E v2').

[0507] The properties of the cells before sorting are shown in Table 7, and the viability of the cells after each step of the manufacturing process is shown in Table 8.

[0508] Table 7: Composition of cell populations during depletion and enrichment

[0509]

[0510] Table 8: VC and percent recovery of naive regulatory T cells after each step of the manufacturing process

[0511]

[0512] The purity and recovery of naive Tregs during the sorting process for each run are shown in Table 9.

[0513] Table 9: Purity and recovery of naive Tregs after FACS sorting

[0514]

[0515] The properties of the manufactured cells are shown in Table 10 below.

[0516] Table 10: Properties of the manufactured cells

[0517]

[0518] The TSDR status of the cells is shown in Tables 11 below and Figure 9 . The TSDR level exceeded 100% in one run because the donor was female. Since 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 of female subjects is doubled to obtain the proportion of cells with a hypomethylated FoxP3 locus.

[0519] Table 11: TSDR status of cells generated using the method of this example

[0520]

[0521] Activity of Tregs after cryopreservation freeze-thaw cycles

[0522] Untransduced or lentiviral vector-transduced regulatory T cells generated according to the method in Example 5-7 are cryopreserved for storage. The cells are frozen and stored in a vapor phase nitrogen storage freezer.

[0523] After refrigeration for a period of time, the frozen cell population is thawed. The cell population is divided into two experimental groups. The first experimental group is activated with anti-CD3 and anti-CD28 antibodies, while the second experimental group is not activated. Both groups are expanded in a medium containing IL-2. After incubation, the regulatory T cell activation markers and cytokine levels (IL-10, CTLA-4, TGF-β-1, and CD69) are measured at 1 day, 2 days, and 3 days.

[0524] Figure 10A to Figure 10B Shown is the increase in the level of each of IL-10, CTLA-4, TGF-β-1, and CD69 for all runs over a three-day period for cells activated with anti-CD3 and anti-CD28 beads. The activated cells showed an increase in the levels of IL-10 ( Figure 10D ), CTLA-4 ( Figure 10A ), TGF-β-1 ( Figure 10C ), and CD69 ( Figure 10B ) during this period, which demonstrated that the cell population could be activated and retained its cellular functionality after cryopreservation freeze-thaw cycles ( Figure 10A to Figure 10D ). The non-activated cells showed no increase in levels. This data further demonstrated that the regulatory T cells retained their function.

[0525] Example 3: Increasing the stringency of the manufacturing strategy for selecting stable Tregs

[0526] Challenges in manufacturing stable Tregs from patient populations

[0527] After establishing the manufacturing process with healthy donor samples, the manufacturing process is evaluated with samples from MS donors. For some MS donors, lower initial TSDR hypomethylation levels and / or more unstable TSDR hypomethylation levels during expansion (from day 0 to harvest) were observed compared to the results using healthy donor cells. Figure 11 Shows the change of TSDR hypomethylation with amplification for three MS donors. For two of the three MS donors, the initial TSDR was below 90%, and the TSDR decreased by more than 10% during amplification. This may be because there are differences in the Treg populations between healthy donors and autoimmune donors (including MS donors), making the boundary between Treg and T conventional cells less clear when sorting cells. These results suggest that for some donors, increasing the stringency of the gating strategy will likely improve the purity of the isolated Treg population and thus increase the proportion of stable Tregs obtained in this process. However, increasing the stringency may lead to a decrease in yield. Described in this example is a more stringent gating method in combination with an improved amplification protocol. The improved amplification protocol enhances the proliferation of Tregs, which is required to overcome the decreased yield due to the increased stringency of selecting stable Tregs.

[0528] Evaluation of Treg subsets included in the process

[0529] The sorting strategy described in the above example focuses on the selection of naive Tregs. Considering that a more stringent sorting strategy is required for some donors and that such a strategy may exclude an increasing proportion of naive Tregs, we evaluated whether the CD45RA+ selection step for selecting naive Tregs is necessary in the context of a more stringent sorting strategy, or whether antigen-experienced Tregs with more stringent selection can be used. Naive Tregs are CD4 + CD25 + CD127 - / 低 CD45RA + . Antigen-experienced Tregs are CD4+CD25hiCD127− / loCD45−. Both of these populations are stable Tregs, which will have a stable TSDR hypomethylated phenotype. To evaluate whether CD45RA-based selection is required, antigen-experienced (CD45RA−) and naive (CD45RA + ) Tregs were isolated and their amplification ability and stability were compared.

[0530] Prepare cells as described in Example 1 and then perform the FACS sorting step. Then perform two rounds of FACS sorting on the cells for depletion and purity. The CD45RA - population and the CD45RA + population were sorted by CD4, CD25, and CD127 during depletion sorting. First, the cells were identified as CD4 + , and then a polygon gate was used to identify CD25 + / 高 CD127 - / 低 Tregs, which was set at 70% of the standard polygon gate for Treg identification and selection. The sorting was repeated, and for CD45RA- Cells, in identifying CD25 + / 高 CD127 - / 低 Treg population, identify and select CD45RA - cells. For CD45RA + cells, in identifying CD25 + / 高 CD127 - / 低 Treg population, identify and select CD45RA + cells. The percentage of naive (CD45RA + ) Tregs in each population is shown in Table 12 below.

[0531] At the end of sorting, 3 groups were established:

[0532] (a) CD45RA + Treg–enriched CD45RA + Treg fraction

[0533] (b) CD45RA - Treg–enriched CD45RA - Treg fraction

[0534] (c) “Spiked” Treg–80% enriched CD45RA - Treg fraction + 20% enriched CD45RA + Treg fraction

[0535] Table 12: Percent naive Tregs under each condition before expansion

[0536] Condition Day 0 nTreg% CD45RA+ 94％ Spiked 20％ CD45RA- 2％

[0537] The cells were then expanded in culture for 8 days as described in Example 1. Figure 12A to Figure 12C The population doubling level (PDL) and TSDR hypomethylation after cell expansion are shown. As Figure 12A and Figure 12C shown, relative to the CD45RA - population, the CD45RA + population showed a significant increase in expansion, as well as increased TSDR hypomethylation. This indicates that CD45RA + naive Tregs are required for the expansion process, as compared to CD45RA - Tregs, CD45RA + naive Tregs have a relative expansion capacity. Importantly, the “spiked” samples indicate that in the presence of a large excess of CD45RA - antigen-experienced Tregs, CD45RA + naive Tregs are still able to expand effectively.

[0538] Developing a more stringent gating strategy for isolating stable Tregs

[0539] Based on the above findings, an improved gating strategy was developed to increase the stringency of sorting while still capturing CD45RA + Treg. In particular, the strategy identifies antigen-experienced (CD4 + CD25 高 CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + ) Treg, while specifically excluding CD4 + CD25 + CD127 - / lo CD45RA - non-Treg. By separately identifying antigen-experienced Treg and naive Treg, a sufficient number of Treg can be obtained to generate a cell therapy product from a single leukapheresis, and the antigen-experienced Treg have high inhibitory capacity, inhibiting any non-Treg ex vivo and in vivo. In this method, CD4 + cells are identified, followed by identifying CD127 - / lo and CD25 + (sequentially or simultaneously), as the final step of the gating protocol, to select antigen-experienced (CD4 + CD25 高 CD127 - / lo CD45RA - ) and naive (CD4 + CD25 + CD127 - / lo CD45RA + ) Treg, while specifically excluding unwanted CD4 + CD25 + CD127 - / lo CD45RA - cells. This gating method is shown in Figure 13A to Figure 13C and Figure 14A to Figure 14E , where Figure 13A to Figure 13C shows a dot plot method for the CD25 / CD127 selection step, and Figure 14A to Figure 14E shows a histogram method for selecting CD127 - / lo followed by selecting CD25 + .

[0540] To further describe the gating strategy in detail, cells enriched for CD25 were stained with fluorescently labeled antibodies that recognize the cell surface markers CD4, CD127, and CD45RA. The cells had already been labeled with CD25 antibody during the enrichment step of this process. The cells were then sorted twice using a FACS instrument with the gating protocol described below (using the same strategy at each step). For dot plot-based methods, as Figure 13A to Figure 13C shown, in the first step of the gating process, CD4 + T cells ( Figure 13A ) were first identified from all live cell events. In the second step, CD25 + CD127 - / 低 Treg cells were identified based on a two-parameter flow plot, thereby allowing the spatial context of the Treg cell population to be visualized relative to another cell population (i.e., Tconv cells), visualizing the CD25 and CD127 levels, and drawing a polygon gate in the upper left quadrant to capture CD25 + CD127 - / 低 cells. The polygon gate was then shifted upward to reduce the selected cell population to 70% of the original population, thereby increasing the stringency of the selection ( Figure 13B ). In the final step, a "not" gate or L-shaped gate was applied, thereby visualizing the CD45RA and CD25 levels to select naive Tregs (CD4 + CD45RA - ) for expansion by excluding the CD25 + CD25 + CD127 - / lo CD45RA + ) and antigen-experienced Treg (CD4 + CD25 高 CD127 - / 低 CD45RA - ) cell populations ( Figure 13C ). Although both the antigen-experienced Treg population and the unstable Treg population are CD45RA - , they can be distinguished by their CD25 levels, where the antigen-experienced Tregs are CD25 高 , while the unstable Treg population is only CD25 + but not highly. Since naive Tregs are CD45RA + and CD25 + (but not highly), the CD45RA - cells were used to determine whether they are CD25 + or CD25 高The threshold is determined based on the CD25 level in the naive Treg population, where CD25 高 is defined as having a CD25 level higher than that expressed in the naive Treg population. The L-shaped gate is crucial as it allows the selection of two important cell populations. Selecting CD25 高 CD45RA - antigen-experienced Tregs and CD25 + CD45RA + naive Tregs, while excluding CD25 + CD45RA - contaminating cells.

[0541] The histogram-based method was implemented as an operational alternative for certain manufacturing scenarios and produced results similar to the dot plot strategy ( Figure 14A to Figure 14E ). Using a sequential histogram gating strategy, CD4 - / lo T cells with the lowest CD127 expression (i.e., 40 ± 2% CD127 + ) were selected, followed by those with the highest CD25 expression (i.e., 70 ± 2% CD25 + ). The cell population was further refined by selecting 70 ± 2% of Tregs in a CD25 versus CD127 biparametric dot plot ( Figure 14A to Figure 14C ). Then, naive Tregs (CD4 Figure 14D ) and antigen-experienced Tregs (CD4 + CD25 + CD127 - / lo CD45RA + ) were identified using the same "not" gate or L-shaped gate as in the dot plot-based method ( + CD25 高 CD127 - / lo CD45RA - ). ( Figure 14E )

[0542] The MS donor for Run 10 donated a second apheresis (Run 12), and this apheresis was processed using the new sorting strategy (histogram + L-shaped gate). All other elements of the manufacturing process were the same as described in Example 1, except for the FACS sorting step. Table 13 shows the properties of the cells before enrichment and sorting, demonstrating a high degree of similarity in cell properties between the two runs prior to the sorting step. In the context of Table 13 and all tables in this article, N / A means the data is not applicable as it was not collected.

[0543] Table 13: Properties of MS donor cells before FACS sorting

[0544]

[0545]

[0546] Table 14 shows the properties of the cells after isolation and before amplification. As expected, although the purity of Tregs remains high after sorting with a strict sorting method, the proportion of naive Tregs is reduced relative to the original process described in Example 1 due to the inclusion of antigen-experienced Tregs in the sorting strategy. The recovery of naive Tregs is also reduced due to the increased strictness of the gating protocol.

[0547] Table 14: Properties of MS donor cells after FACS sorting

[0548]

[0549] The population doubling level and percentage viability are shown in Table 15. As in Example 1, the percentage of the target dose achieved over the entire process is also shown and calculated, but for the new sorting strategy, the target dose calculation is based on the total number of Tregs rather than the total number of naive Tregs, because two populations were selected.

[0550] Table 15: Properties of the manufactured cells

[0551]

[0552]

[0553] Figure 15 And Table 16 below shows the percentage of TSDR hypomethylation after sorting and during the amplification process. For the L-shaped gating method, greater than 95% initial TSDR hypomethylation was achieved (compared to 87.79% for the sorting method from Example 1). In addition, for the L-gate-based method, the TSDR hypomethylation level decreased by less than 10% during the amplification process.

[0554] Table 16 :

[0555] Run 10 (Example 1 process) Run 12 (histogram + L-shaped gate) Day 0 88％ 97％ Day 3 86％ Not applicable Day 6 85％ 95％ Day 9 75％ Not applicable Day 10 Not applicable 87％

[0556] New expansion process

[0557] Since the newly developed gating strategy selects a smaller number of naive Tregs compared to the original process, the amplification process was modified to increase the amplification of Tregs.

[0558] The amplification protocol was changed to add TNF-α to the amplification medium and add a second anti-CD28 anti-CD3 stimulation. First, the effect of restimulation was evaluated at different time points after transduction. For this experiment, cells were prepared according to the method described in Example 1, except that gating was performed only on CD4, CD25, and CD127 and not on CD45RA. Then the cells were activated and transduced as in Example 1, where a second activation step was performed with anti-CD3 and anti-CD28 on days 6, 8, and 10 after the first activation step. It was found that this restimulation on day 6 produced a similar amplification to that on day 7 or day 8, but advantageously, when the cells were harvested on day 10, restimulation on day 6 of the amplification process gave the greatest increase in amplification, and crucially this did not require an extension of the amplification process( Figure 16 ). Next, the effect of adding TNF-α to the amplification protocol was evaluated. Cells prepared according to the method of this example (including the L-shaped gate) were amplified as described in Example 1, where a second stimulation was performed or not on day six, and 2500 IU / ml TNF-α was added or not added, to evaluate the effect of TNF-α alone or in combination with the second stimulation on Treg amplification. As Figure 17 shown, adding TNF-α throughout the amplification process and performing a second stimulation with anti-CD3 and anti-CD28 on day 6 of the amplification process gave the greatest increase in amplification. However, each individual change to the process also increased the amplification. As shown in Figure 18, cells amplified in TNF-α and combined with the second stimulation showed the highest viability and purity at harvest compared to the individual process changes or the original amplification protocol.

[0559] Tregs generated with the new gating and expansion process

[0560] Then the manufacturing process described in this example was applied to a second repeated MS donor. The MS donor from Run 11 donated additional apheresis. This apheresis was processed using a strict sorting strategy based on dot plots and histograms and improvements to the above amplification conditions. In the CD127 / CD25 selection step( Figure 19C ), a minor modification was made to the L-shaped gate method based on histograms. CD4 - / lo T cells with the lowest CD127 expression (i.e., 30 ± 2% CD127 + ) and then the highest CD25 expression (i.e., 85 ± 2% CD25 + ) were selected. The cell population was further refined by selecting 85 ± 2% of Treg cells in a CD25 vs CD127 bivariate flow plot( Figure 19D ). The L-shaped gate itself was not changed( Figure 19E ). Before the sorting step, the cells were processed as in Example 1( Figure 19A to Figure 19E )。Table 17 shows the properties of the cells after enrichment and before sorting, thus demonstrating that the properties of the cells from each run are highly similar before the sorting step.

[0561] Table 17: Properties of MS donor cells before FACS sorting

[0562]

[0563] The properties of the sorted cells are shown in Table 18 below:

[0564] Table 18: Properties of MS donor cells after FACS sorting

[0565]

[0566]

[0567] Table 19 below shows the properties of the manufactured cells. The target dose percentage is calculated as described above.

[0568] Table 19: Properties of the manufactured cells

[0569]

[0570] Figure 20 And Table 20 below shows the relationship between the low methylation of the TSDR and the amplification of this donor using the procedure described in Example 2 and the dot plot and histogram-based L-shaped gating strategy described in this example.

[0571] Table 20: TSDR of MS donors using the original process and L-gate

[0572]

[0573] The comparative data presented in this example show that the combination of a strict gating strategy and an updated amplification process results in a highly stable Treg population and generates sufficient transduced Tregs to achieve the target dose.

[0574] This process was repeated on another MS donor and two other healthy donors using two L-shaped gating methods, and similar results were obtained. Table 21 shows the properties of the cells after enrichment and before sorting.

[0575] Table 21: Composition of cell populations during depletion and enrichment

[0576] Cell Run 14 Run 15 Run 16 Donor sex 25 33 50 Donor age M F F Donor health status (MS / non-MS) Healthy donor Healthy donor MS Apheresis VC 7.4E+09 1.4E+10 3.7E+09 Apheresis CD4 T cell percentage 50％ 39％ 34％ Apheresis naive Treg percentage Not applicable 2％ 1％ Apheresis Treg percentage Not applicable 3％ 4％ VC after depletion 2.6E+09 4.8E+09 1.4E+09 CD4 T cell percentage after depletion 89％ 60％ 71％ Naive Treg percentage after depletion 3％ 3％ 3％ Treg percentage after depletion 5％ 6％ 8％ VC after enrichment 2.3+08 4.4E+08 1.7E+08 CD4 T cell percentage after enrichment 82％ 88％ 97％ Naive Treg percentage after enrichment 26％ 23％ 7％ Treg percentage after enrichment 41％ 45％ 29％

[0577] The properties of the sorted cells are shown in Table 22 below:

[0578] Table 22: VC and percent recovery of regulatory T cells after sorting

[0579]

[0580]

[0581] The properties of the manufactured cells are shown in Table 23 below. Calculate the target dose as described earlier in this example.

[0582] Table 23: Properties of the manufactured cells

[0583]

[0584] The TSDR status of the cells is shown in Table 24 below and Figure 21 in.

[0585] Table 24: TSDR status of cells generated using the method of this example

[0586]

[0587] For all donors processed using the sorting and expansion strategies described in this example, a TSDR hypomethylated state of more than 90% was achieved after sorting, which decreased by no more than 10% during the expansion process. This indicates that this process generates a highly pure and stable Treg population from multiple donors. In addition, this process was also able to obtain a sufficient number of transduced Tregs to achieve the target dose.

[0588] Example 4: Evaluation of TSDR threshold levels

[0589] The threshold level of TSDR hypomethylation required for a stable Treg cell product was investigated, which represents the level of stable Tregs. We first asked what proportion of stable Tregs on day 0 would be required to maintain a stable level of Treg cells throughout the expansion process, indicating a low or no growth of contaminating conventional T cells or other cellular impurities. To this end, we incorporated conventional T cells into Tregs at various percentages on day 0, each Treg being isolated from the same healthy donor, and tracked the TSDR level throughout the expansion process. The TSDR hypomethylation level and the FOXP3+ level were correlated with the initial Treg purity and remained stable. When the Treg purity was greater than 60%, the TSDR hypomethylation decreased by no more than 10% throughout the expansion process ( Figure 22A to Figure 22D ). Next, the Treg purity of the expanded cell product was evaluated to determine the presence of conventional T cells and whether pro-inflammatory cytokines were produced upon activation with PMA and ionomycin. Cryopreserved cells from the harvest were thawed into fresh medium and then stimulated with PMA and ionomycin at 37 °C and 5% CO2 for 4 hours. Intracellular cytokines (ICC) for IFN-γ and IL-2 production were measured by flow cytometry. Figure 23 Shows the proportions of FoxP3+ and FoxP3− cells expressing IL-2 and IFN-γ after activation with PMA and ionomycin. In the absence of conventional T cells, 100% of the Treg product secreted little or no IFN-γ or IL-2 cytokine, indicating that the number of unstable Treg cells expanded from the initial culture was not significant. As the purity of the naive Treg decreased, the proportion of T conventional cells secreting IFN-γ and IL-2 cytokines increased, with the largest increase observed when the starting Treg purity was below 90%. This increase may be due to an increase in the Tcon:Treg ratio. Upon activation, some conventional T cells upregulate FoxP3, which is why IFN-γ and IL-2 are produced from some FoxP3+ cells. This indicates that high TSDR levels are crucial for maintaining a stable cell population capable of suppressing contaminating conventional T cells.

[0590] Example 5: MBP 83-99 Characterization of the Reactive TCR Construct

[0591] 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. TCRs targeting the MBP peptide were selected as therapeutic agents for multiple sclerosis because MBP is localized to the meninges where inflammatory processes associated with the pathogenesis of MS are known to occur.

[0592] The TCR with the α and β chains of TCR A, described in further detail below, was cloned into a lentiviral vector in the α-P2A-β and β-P2A-α orientations to determine the optimal configuration of the TCR components in the vector. Each of the α and β chains was labeled to visualize the expression level of each component of the TCR. The construct was 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 hypothesized that if the second subunit was expressed, the first encoded subunit was also expressed. The second encoded subunit was followed by an IRES and a GFP tag to confirm the expression of the full ORF (EF1αTCRβ_P2A_cMyc_TCRα_IRES_EGFP or EF1αTCRα_P2A_cMyc_TCRβ_IRES_EGFP).

[0593] The constructs were transduced into TCR-null Jurkat cells at an MOI of 10, 20, or 30. The cells were then incubated overnight and fresh medium was added prior to staining for flow cytometry. The cells were stained with DAPI, anti-cMYC AX647, and CD3 BV786. Cell surface staining for CD3 indicated that the TCR assembled and was expressed on the cell surface. Detection of Myc indicated the expression of the second expressed TCR component. GFP+ cells indicated expression of the full construct.

[0594] As Figure 24 shown, for the TCRβ_P2A_cMyc_TCRα_IRES_EGFP construct, flow cytometry analysis showed cell surface localization of CD3 in GFP-positive cells, indicating assembly of the TCR complex on the cell surface. cMyc-TCRβ was also detected on the cell surface, indicating expression and cell surface localization of TCRα and TCRβ. However, as Figure 25A to Figure 25C shown, less CD3 ( Figure 25A ) and cMyc ( Figure 25C ) were detected on the cell surface of the TCRα_P2A_cMyc_TCRβ_IRES_EGFP construct relative to the construct with the opposite orientation of TCRα and TCRβ, indicating poor TCRα expression or cell surface localization. The proportion of GFP-positive cells was also reduced ( Figure 25B ), indicating reduced TCR expression of the TCRα_P2A_cMyc_TCRβ_IRES_EGFP construct relative to the TCRβ_P2A_cMyc_TCRα_IRES_EGFP construct. Based on this data, constructs with the TCRβ shifted upstream of TCRα were generated.

[0595] Each TCR (TCR-A, TCR-B, and TCR-C) was then cloned into a vector containing an N-terminal TCRβ chain with a linker domain and a C-terminal TCRα chain, the linker domain containing the GSG amino acid sequence followed by the P2A self-cleaving peptide. The TCRβ-GSG-P2A-TCRα fusion construct was expressed under the EF1α promoter. The constructs used in Examples 5-10 also included a GFP tag at the C-terminus of TCRα to allow in vitro detection.

[0596] TCR-A contains a TCRα variable domain having the amino acid sequence of SEQ ID NO:4 (which contains a CDR1-α sequence having the amino acid sequence of SEQ ID NO:1, a CDR2-α sequence having the amino acid sequence of SEQ ID NO:2, and a CDR3-α sequence having the amino acid sequence of SEQ ID NO:3) and a TCRβ variable domain having the amino acid sequence of SEQ ID NO:10 (which contains a CDR1-β sequence having the amino acid sequence of SEQ ID NO:7, a CDR2-β sequence having the amino acid sequence of SEQ ID NO:8, and a CDR3-β sequence having the amino acid sequence of SEQ ID NO:9).

[0597] TCR-B contains a TCRα variable domain having the amino acid sequence of SEQ ID NO:21 (which contains a CDR1-α sequence having the amino acid sequence of SEQ ID NO:18, a CDR2-α sequence having the amino acid sequence of SEQ ID NO:19, and a CDR3-α sequence having the amino acid sequence of SEQ ID NO:20) and a TCRβ variable domain having the amino acid sequence of SEQ ID NO:27 (which contains a CDR1-β sequence having the amino acid sequence of SEQ ID NO:24, a CDR2-β sequence having the amino acid sequence of SEQ ID NO:25, and a CDR3-β sequence having the amino acid sequence of SEQ ID NO:26).

[0598] TCR-C contains a TCRα variable domain having the amino acid sequence of SEQ ID NO:35 (which contains a CDR1-α sequence having the amino acid sequence of SEQ ID NO:32, a CDR2-α sequence having the amino acid sequence of SEQ ID NO:33, and a CDR3-α sequence having the amino acid sequence of SEQ ID NO:34) and a TCRβ variable domain having the amino acid sequence of SEQ ID NO:41 (which contains a CDR1-β sequence having the amino acid sequence of SEQ ID NO:38, a CDR2-β sequence having the amino acid sequence of SEQ ID NO:39, and a CDR3-β sequence having the amino acid sequence of SEQ ID NO:40).

[0599] TCRs were expressed in TCR-null Jurkat cells to evaluate the ability of the TCRs to be expressed on the cell surface and to activate T cells in the absence of endogenous TCRs.

[0600] TCR-null Jurkat cells were plated at 1×105 cells / well in 96-well plates and transduced with lentiviral vectors encoding one of the three TCRs. The cells were then expanded, TCR expression was evaluated, and the cells were collected for downstream assays to assess function. Surface expression of each of these TCRs was verified by flow cytometry for CD3 expression and the lentiviral GFP reporter gene ( Figure 26 ). Surface expression was shown for all three tested TCRs in TCR-null Jurkat cells.

[0601] To test the responsiveness of the TCRs to MBP complexed with MHC, the transduced Jurkat cells were co-cultured overnight with BLS2b cells (B cells that specifically express HLA-DRA*01:01 and HLA-DRB1*15:01) and the MBP83-99 peptide. The peptide was titrated 4-fold at the indicated concentrations. Upregulation of CD69 surface expression was measured using flow cytometry ( Figure 27A to Figure 27C ). This data demonstrated that all three TCRs were expressed on the cell surface and were able to activate TCR-null Jurkat cells.

[0602] TCR-A and TCR-B were expressed independently in conventional CD4 T cells to evaluate the ability of the TCRs to be expressed on the cell surface and to activate T cells in the presence of the endogenous TCR.

[0603] For each of these TCRs, CD4 T cells transduced with the TCR were plated at 100,000 cells / well in a row (12 wells) of a 96-well round-bottom plate. 50,000 BLS2b cells (B cells that specifically express only HLA-DRA*01:01 and HLA-DRB1*15:01) were added to each well containing the transduced T cells, and the MBP83-99 peptide was serially titrated onto the plate. The cells were cultured with the peptide overnight for 20 hours in a humidified 37°C, 5% CO2 incubator. The next day, the cells were prepared for flow cytometry analysis by staining with fluorescently labeled antibodies (anti-CD4 BV605, anti-CD69 BV421, anti-CD20 PE). Data was obtained on a BioRad ZE5 cell counter and analyzed in FlowJo v10. Transduced T cells were identified as CD20-, CD4+, GFP+ singlet events. T cell activation was determined by the level of CD69 expression ( Figure 28A to Figure 28C ). This data demonstrated that both TCR-A and TCR-B were able to activate conventional T cells.

[0604] Example 6: Properties of regulatory T cells expressing exogenous TCR

[0605] In a functional assay, the ability of regulatory T cells transduced with a lentiviral vector encoding the TCR from Example 5 to be activated was tested, and when presented by HLA, the regulatory T cells had inhibitory function in response to the MBP83-99 peptide. Using EasySep TM Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (Stemcell Technologies), Tregs were isolated from PBMCs and transduced and expanded as described in Example 1.

[0606] To test whether the transduced regulatory T cells could be activated, the transduced regulatory T cells or untransduced controls were incubated with mitomycin C-treated peripheral blood mononuclear cells (PBMCs) isolated from an HLA DRB1*15:01 donor, MBP83-99 peptide at a concentration of 0 μM to 100 μM (0 μM, 0.000001 μM, 0.00001 μM, 0.0001 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM), and 200 U / ml IL-2 in T cell medium at 37°C. The ratio of PBMCs to regulatory T cells was 4:1. The transduced regulatory T cells were differentiated from the untransduced regulatory T cells by GFP integrated in the lentiviral vector. After incubation, regulatory T activation markers were measured by FACS after 1 day, and cytokine levels were measured after 3 days.

[0607] Representative plots of CD69 expression levels on populations of regulatory T cells transduced with each of two TCRs (TCR-A and TCR-B) and untransduced regulatory T cells within the MBP83-99 peptide range are provided in Figure 29A . TCR-A provided an EC50 of 7.0 nM for CD69; and TCR-B provided an EC50 of 7.9 nM for CD69.

[0608] Representative plots of 41-BB expression levels on populations of regulatory T cells transduced with each of two TCRs (TCR-A and TCR-B) and untransduced regulatory T cells within the MBP83-99 peptide range are provided in Figure 29B . TCR-A provided an EC50 of 11.0 nM for 4-1BB; and TCR-B provided an EC50 of 18.0 nM for 4-1BB.

[0609] Representative plots of IL-10 expression levels on populations of regulatory T cells transduced with each of two TCRs (TCR-A and TCR-B) and untransduced regulatory T cells within the MBP83-99 peptide range are provided in Figure 29CAmong them, TCR-A provides an EC50 of 16.1 nM for IL-10; and TCR-B provides an EC50 of 13.7 nM for IL-10.

[0610] As shown, regulatory T cells expressing TCR-A or TCR-B show increased levels of CD69, 4-1BB, and IL-10 in response to MBP83-99 presented by PBMCs compared to regulatory T cells that do not express exogenous TCR. These data demonstrate that the transduced regulatory T cells are activated in response to MBP83-99 and exhibit the desired characteristics.

[0611] To test the ability of the transduced regulatory T cells to inhibit conventional T cell function, the activation of conventional T cells expressing TCR-A or TCR-B was stimulated with MBP83-99 peptide presented by PBMCs isolated from an HLA DRB1*15:01 donor in the presence of regulatory T cells expressing TCR-A or TCR-B. The activation of conventional T cells was evaluated by the presence of activation markers and cytokine release.

[0612] Regulatory T cells were incubated with mitomycin C-treated PBMCs isolated from an HLA DRB1*15:01 donor, MBP83-99 peptide at a concentration of 1 μM or 0.1 μM, conventional CD4 T cells expressing TCR-A or TCR-B, and 200 U / ml IL-2. The ratio of PBMCs to conventional T cells was 4:1. Inhibition of conventional T cells was measured at different ratios of regulatory T cells to conventional T cells (1:1, 1:2, 1:4, and 1:8). The cells were cultured for 4 days. After incubation, T cell activation markers were measured by FACS and cytokine levels were measured.

[0613] Figure 30A Inhibition of conventional T cells expressing TCR-A by regulatory T cells expressing TCR-A is shown. The percentage of inhibition of CD71, CD25, and IFN-γ is provided at 1 μM and 0.1 μM MBP83-99 peptides. Figure 30B Inhibition of conventional T cells expressing TCR-B by regulatory T cells expressing TCR-A is shown. The percentage of inhibition of CD71, CD25, and IFN-γ is provided at 1 μM and 0.1 μM MBP83-99 peptides. Figure 30C Inhibition of conventional T cells expressing TCR-A by regulatory T cells expressing TCR-B is shown. The percentage of inhibition of CD71, CD25, and IFN-γ is provided at 1 μM and 0.1 μM MBP83-99 peptides. Figure 30DShows the inhibition of conventional T cells expressing TCR-β by regulatory T cells expressing TCR-β. The percentage of inhibition of CD71, CD25, and IFN-γ is provided at 1 μM and 0.1 μM MBP83-99 peptide.

[0614] The ability of regulatory T cells expressing TCR-α to inhibit polyclonal (untransfected) conventional T cells was measured using the same method. For polyclonal conventional T cells, the inhibition of proliferation ( Figure 31A ), CD25 ( Figure 31B ), and interferon γ ( Figure 31C ) was measured at the indicated Treg:Tcon ratios.

[0615] These data demonstrate that stable regulatory T cells expressing exogenous TCRs are able to inhibit conventional T cells. Specifically, regulatory T cells expressing an exogenous TCR targeting MBP83-99 are able to inhibit the activation and cytokine release of conventional T cells. Collectively, these data suggest that a cell population comprising regulatory T cells is able to inhibit the immune response through an MBP-specific mechanism and should therefore be useful for treating subjects suffering from multiple sclerosis (e.g., progressive multiple sclerosis).

[0616] Example 7: Characterization of T cells expressing engineered T cell receptors

[0617] Cysteine substitutions were introduced into the TCRα and TCRβ sequences of TCR-α and TCR-β to introduce disulfide bonds and increase TCR pairing at the cell surface. For each TCR, a T48C mutation was introduced relative to the TRAC constant region (SEQ ID NO. 58), and an S57C mutation was introduced relative to the TRBC2 constant region (SEQ ID NO. 60). Engineered TCR-E v1 (middle row) is an engineered version of TCR-α with additional engineered disulfide bonds (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-F v1 (middle row) is an engineered version of TCR-β with additional engineered disulfide bonds (the amino acid sequence of v1 corresponds to SEQ ID NO: 86); and engineered TCR-F v2 (top row) 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.

[0618] TCRs were expressed in TCR-null Jurkat cells to evaluate the ability of the engineered TCRs to be expressed at the cell surface and to activate T cells in the absence of endogenous TCRs.

[0619] TCR-null Jurkat cells were plated at 1×105 cells / well in 96-well plates and transduced with lentiviral vectors encoding one of the TCRs. The cells were then expanded and collected for downstream assays to evaluate function.

[0620] To test the responsiveness of TCR-expressing cells to MBP complexed with MHC, the transduced Jurkat cells were co-cultured overnight with BLS2b cells (B cells that specifically express HLA-DRA*01:01 and HLA-DRB1*15:01) and the MBP83-99 peptide. The peptide was titrated 4-fold at the concentrations shown in Figures 32A to 32B . Upregulation of CD69 surface expression was measured using flow cytometry ( Figures 32A to 32B and Table 25). These data demonstrate that Jurkat expressing each of these TCRs responds to increasing concentrations of antigen.

[0621] Table 25. CD69 expression in TCR-transduced Jurkat cells

[0622] Maximum response EC-50 (nM) TCR-A 1.00 0.0042 TCR-A v2 1.03 0.0039 TCR-E engineered v1 0.95 0.0040 TCR-E engineered v2 0.97 0.0037 TCR-B 1.00 0.0030 TCR-B v2 1.03 0.0026 TCR-F engineered v1 1.03 0.0030 TCR-F engineered v2 1.04 0.0029

[0623] The TCRs were also expressed in conventional CD4+ T cells to evaluate cell surface expression of the TCR and T cell activation in the presence of the endogenous TCR.

[0624] CD4+ T cells transduced with each of these TCRs were plated at 100,000 cells / well in a row (12 wells) of a 96-well round-bottom plate. 50,000 BLS2b cells (B cells that specifically express only HLA-DRA*01:01 and HLA-DRB1*15:01) were added to each well containing the transduced T cells, and the MBP83-99 peptide was serially titrated onto the plate. The cells were cultured with the peptide overnight for 20 hours in a humidified 37 °C, 5% CO2 incubator. The next day, the cells were prepared for flow cytometry analysis by staining with fluorescently labeled antibodies (anti-CD4 BV605, anti-CD69 BV421, anti-CD20 PE). Data were acquired on a BioRad ZE5 cell counter and analyzed in FlowJo v10. The transduced T cells were identified as CD20 - , CD4 + , GFP + singlet events. T cell activation was determined by the level of CD69 expression ( Figures 33A to 33C and Table 26).

[0625] Table 26. CD4 Transduced by TCR + CD69 Expression in T Cells

[0626] Maximum response EC-50 (μM) TCR-A 1.00 0.0076 TCR-A v2 1.05 0.0059 TCR-E engineered v1 1.36 0.0050 TCR-E engineered v2 1.44 0.0056 TCR-B 1.00 0.0075 TCR-B v2 1.04 0.0055 TCR-F engineered v1 1.57 0.0052 TCR-F engineered v2 1.59 0.0045

[0627] These data demonstrate that, relative to cells expressing the parental versions (TCR-A and TCR-B), the expression levels of cell activation markers (e.g., CD69) are higher in CD4 + T cells expressing engineered TCRs (e.g., TCR-E engineered v2 and TCR-F engineered v2).

[0628] The surface expression of each of these TCRs was verified by flow cytometry for Vβ2 expression and CD69 expression ( Figure 34A ). The expression of Vβ2 correlates with the surface level expression of the TCR ( Figures 34B to 34C ). These data demonstrate that, relative to the parental versions (TCR-A and TCR-B), the engineered TCRs (e.g., TCR-E engineered v2 and TCR-F engineered v2) do not significantly affect the surface expression of these TCRs in conventional T cells.

[0629] Example 8. Activation of regulatory T cells expressing engineered T cell receptors

[0630] 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-F v2). The Treg cells were then contacted with BLS2b cells (which are B cells expressing only HLA-DRA*01:01 and HLA-DRB1*15:01) and the MBP83-99 peptide to measure Treg activation as demonstrated by the expression of CD69, as described in Example 6. Figures 35A to 35B A graph depicting Treg activation in response to increasing doses of MBP peptide is shown.

[0631] As Figure 35A shown, the activation of Tregs expressing TCR-E engineered v2 was 1.53-fold that of Tregs expressing the parental version (TCR-A), while the activation of Tregs expressing TCR-E engineered v1 was 1.37-fold that of Tregs expressing the parental version (TCR-A).

[0632] As Figure 35B shown, the activation of Tregs expressing TCR-F engineered v2 was 1.89-fold that of Tregs expressing the parental version (TCR-B), while the activation of Tregs expressing TCR-F engineered v1 was 1.56-fold that of Tregs expressing the parental version (TCR-B).

[0633] The surface expression of each of these TCRs was verified by flow cytometry for Vβ2 expression. The proportion of cells transduced with TCR-E engineered v2 or TCR-F engineered v2 expressed higher levels of Vβ2 relative to parental controls and engineered v1 versions, respectively ( Figure 35C ). The expression of Vβ2 correlated with the surface level expression of the TCR.

[0634] These data demonstrate that Tregs transduced with the engineered TCRs of the present disclosure (e.g., TCRs encoded by nucleotide sequences having 100% identity to the nucleotide sequences of SEQ ID NO: 87 or 88) have a higher level of activation in response to the target antigen MBP83-99 peptide relative to the corresponding parental (non-engineered) and engineered v1 TCRs. Additionally, these data demonstrate that Tregs transduced with the engineered v2 versions of TCR-E and TCR-F have higher levels of TCR expression on their cell surface.

[0635] Example 9: Inhibitory ability of engineered TCR constructs in regulatory T cells

[0636] As described in Example 6, regulatory T cells were transduced with a lentiviral vector encoding one of TCR-A, TCR-B, TCR-E v1, TCR-E v2, TCR-F v1, and TCR-F v2. The transduced regulatory T cells were then tested for their affinity and response to the MBP83-99 peptide in a functional assay.

[0637] To test which TCR has the highest affinity and greatest response, a lentivirally transduced cell population containing regulatory T cells was incubated with mitomycin C-treated peripheral blood mononuclear cells (PBMCs) isolated from an HLA DRB1*15:01 donor, Figures 36A to 36B the MBP83-99 peptide at the concentrations shown, and 200 U / ml IL-2 in T cell medium at 37°C. The ratio of PBMCs to regulatory T cells was 4:1. After incubation, regulatory T activation markers were measured by FACS after 1 day, and cytokine levels were measured after 3 days.

[0638] Representative plots of the 4-1BB expression levels on regulatory T cell populations transduced with TCRs within the MBP83-99 peptide range are provided in Figures 36A to 36B and Table 27. These data demonstrate that regulatory T cells expressing the engineered TCRs of the present disclosu...

Claims

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

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

3. The isolated population according to claim 2, wherein said MBP peptide is the MBP 83-99 peptide comprising the amino acid sequence of SEQ ID NO:

61.

4. The isolated population according to claim 2 or claim 3, wherein said MHC comprises HLA-DRB1*15:

01.

5. The isolated population according to claim 4, wherein said MHC further comprises HLA-DRA*01:

01.

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

9.

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

4.

8. The isolated population according to claim 6 or claim 7, wherein said variable region of the TCR α chain comprises SEQ ID NO:4 or consists of the same.

9. The isolated population according to claim 6, wherein said TCR α chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:

5.

10. The isolated population according to claim 6 or claim 9, wherein said TCR α chain comprises SEQ ID NO:5 or consists of the same.

11. The isolated population according to claim 6, wherein said TCR α chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:

73.

12. The isolated population according to claim 6 or claim 11, wherein said TCR α chain comprises SEQ ID NO:73 or consists of the same.

13. The isolated population according to claim 6, wherein said TCR α chain comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:

91.

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

91.

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

10.

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

10.

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

11.

19. The isolated population according to 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:

75.

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

75.

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

93.

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

17.

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

17.

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

79.

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

79.

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

97.

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

97.

29. The isolated population according to claim 25 or claim 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 with the nucleotide sequence of SEQ ID NO:

87.

30. The isolated population according to any one of claims 2 to 5, wherein the exogenous TCR comprises: a. A variable region of the TCR α chain, which comprises a CDR1 containing the amino acid sequence of SEQ ID NO:18, a CDR2 containing the amino acid sequence of SEQ ID NO:19, and a CDR3 containing the amino acid sequence of SEQ ID NO:20; and b. A variable region of the TCR β chain, which comprises a CDR1 containing the amino acid sequence of SEQ ID NO:24, a CDR2 containing the amino acid sequence of SEQ ID NO:25, and a CDR3 containing the amino acid sequence of SEQ ID NO:

26.

31. The isolated population according to claim 30, wherein the variable region of the TCR α chain comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO:

21.

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

21.

33. The isolated population according to claim 30, wherein the TCR α chain comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO:

22.

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

22.

35. The isolated population according to claim 30, 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:

80.

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

80.

37. The isolated population according to any one of claims 30 to 36, wherein the variable region of the TCR β chain comprises an amino acid sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO:

27.

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

27.

39. The isolated population according to any one of claims 30 to 38, 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:

28.

40. An isolated population according to any one of claims 30 to 39, wherein the TCRβ chain comprises or consists of SEQ ID NO:

28.

41. An isolated population according to any one of claims 30 to 36, wherein the TCRβ 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. An isolated population according to any one of claims 30 to 36 or claim 41, wherein the TCRβ chain comprises or consists of SEQ ID NO:

82.

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

31.

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

31.

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

86.

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

86.

47. An isolated population according to 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. An isolated population according to any one of claims 1 to 47, wherein the stable CD4+ T regs do not express FOXP3 protein from the engineered FOXP3 locus.

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

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

51. An isolated population according to any one of claims 1 to 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+ T regs comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

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

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

54. The isolated population according to any one of the preceding claims, which comprises at least 4×10 7 stable CD4+ Tregs.

55. The isolated population according to claim 54, comprising from 4×10 7 to 1×10 10 stable CD4+ Tregs.

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

57. The isolated population according to 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 the conventional T cells comprise the exogenous human TCR.

58. The isolated population according to any one of the preceding claims, wherein 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.

59. The isolated population according to any one of the preceding claims, wherein fewer than 2%, fewer than 1%, fewer than 0.5%, fewer than 0.1% or fewer than 0.01% of the cells of the isolated population are CD8 + T cells.

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

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

62. The isolated population according to 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. The isolated population according to any one of the preceding claims, wherein after cryopreservation freeze-thaw cycles, the percentage of stable CD4+ Tregs comprising the hypomethylated TSDR at the FOXP3 locus is reduced by 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

64. The isolated population according to any one of the preceding claims, wherein the stable CD4+ 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. Inhibitory activity.

65. The isolated population according to 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. The isolated population according to claim 65, wherein the single polypeptide comprises a TCR α chain and a TCR β chain, and wherein the polypeptide comprises a self-cleaving peptide sequence positioned between the TCR α chain and the TCR β chain.

67. The isolated population according to 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. The isolated population according to any one of the preceding claims, wherein the exogenous TCR comprises a TCR α chain containing a constant region, the 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. A separated population according to any one of the preceding claims, wherein the exogenous TCR comprises a TCRβ chain containing a constant region, and the constant region comprises an amino acid sequence having at least 90%, 95% or 100% identity with the amino acid sequence of SEQ ID NO:

60.

70. A separated population according to any one of the preceding claims, wherein the exogenous human TCR comprises one or more amino acid substitutions for cysteine residues in the constant region of the TCRα chain and the constant region of the TCRβ chain, and wherein the cysteine residues are capable of forming one or more disulfide bonds.

71. The separated population according to claim 65, wherein the TCRα chain constant region comprises a T48C amino acid substitution relative to the TCRα chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and wherein the TCRβ chain constant region comprises an S57C amino acid substitution relative to the TCRβ chain constant region comprising the amino acid sequence of SEQ ID NO: 59 or 60.

72. A separated population according to any one of the preceding claims, wherein the stable CD4+ T reg has a human leukocyte antigen (HLA)-DR15 haplotype.

73. The separated population according to claim 72, wherein the stable CD4+ T reg comprises the HLA-DRB1*15:01 allele.

74. The separated population according to claim 73, wherein the stable CD4+ T reg further comprises the HLA-DRB5*01:01 allele.

75. A method of generating a cell population comprising stable CD4+ T regulatory cells (T regs), which comprises: a. Remove CD8 + cells, CD19 + cells and optionally CD14 + cells from a biological sample obtained from a subject suffering from multiple sclerosis to produce a depleted biological sample; b. enriching CD25+ cells in the depleted biological sample to generate an enriched population; c. Isolate CD4+CD25+CD127 - / lo cells from the enriched population; d. amplifying the enriched population to generate an amplified enriched cell population; and e. quantifying the methylation status of the T cell-specific demethylated region (TSDR) at the FOXP3 locus in the cell population, wherein at least 80% of the cells are stable CD4+ Tregs comprising a hypomethylated TSDR at the FOXP3 locus, thereby generating a cell population comprising stable CD4+ T regs.

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

77. The method according to claim 75 or 76, wherein if the percentage of cells comprising a hypomethylated TSDR at the FOXP3 locus is 80% or greater, the separated population is selected for therapeutic use.

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

79. The method according to any one of claims 75 to 78, which further comprises activating the cell population of step (c).

80. The method according to claim 79, wherein said activation comprises culturing said cell population with an anti-CD3 antibody and an anti-CD28 antibody.

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

82. The method according to claim 80 or 81, wherein said cells are expanded in a medium comprising IL-2 and TNFα.

83. The method according to any one of claims 75 to 82, wherein said activation and expansion comprise culturing said cell population for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days.

84. The method according to any one of claims 75 to 83, wherein said activation and expansion comprise culturing said cell population for no more than 15 days, 14 days, 13 days or 12 days.

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

86. The method according to 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. The method according to claim 85 or 86, wherein step (c) is carried out at least twice.

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

89. The method according to any one of claims 85 to 88, further comprising an activation step of the engineered cell population of (c).

90. The method according to claim 89, wherein said activation comprises culturing said cell population with an anti-CD3 antibody and an anti-CD28 antibody.

91. The method according to claim 89 or 90, wherein said activation step is carried out at least twice.

92. The method according to claim 91, wherein the second activation step is carried out between 4 and 8 days after the first activation step.

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

94. The method according to any one of claims 85 to 93, wherein the expanded population comprises at least 1×10 7 engineered stable CD4+ Tregs.

95. The method according to any one of claims 85 to 94, wherein the methylation status of the TSDR in step (f) is evaluated at least 24 hours after cryopreservation and thawing cycles.

96. The method according to any one of claims 85 to 95, wherein CD4 is evaluated at least 24 hours after cryopreservation and freeze-thaw cycles + CD25 + / 高 CD127 - / lo The percentage of regulatory T cells.

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

98. The method according to any one of claims 75 to 97, wherein 2% or less of the cell population is CD8+ cells.

99. The method according to any one of claims 75 to 98, wherein 20% or less of the cell population is conventional T cells.

100. The method according to any one of claims 75 to 99, wherein the cell population in step (c) comprises CD25 高 CD45RA - cells and CD25+CD45RA+ cells, and does not comprise CD25 + CD45RA - cells.

101. The method according to claims 75 to 100, wherein the isolation in (c) comprises: i. Identify a first subset of CD4 + cells from the enriched population of step (b); ii. Identify CD25 from the first subset + / 高 CD127 - / lo a second subset of cells; and iii. Select CD25 from the second subpopulation 高 CD45RA - and CD25 + / 高 CD45RA + cells for isolation and exclude CD25 + CD45RA - , iv. thereby isolating the CD4+ T reg population.

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

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

104. The method according to claim 103, wherein the regulatory T cells comprise the HLA-DRB1*15:01 allele.

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

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

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

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

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

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

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

112. The method according to claims 85 to 111, wherein the exogenous human TCR is encoded as a single polypeptide comprising a TCRα chain and a TCRβ chain.

113. The method according to claim 110, wherein the polypeptide comprises an N-terminal TCRβ chain and a C-terminal TCRα chain.

114. The method according to claim 110 or 113, wherein the polypeptide comprises a self-cleaving peptide sequence positioned between the TCRα chain and the TCRβ chain.

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

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

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

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

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

120. The method according to any one of claims 85 to 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-1α promoter or an MND promoter.

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

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

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

9.

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

10.

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

75.

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

87.

128. An engineered polynucleotide encoding a polypeptide comprising a TCRα chain and a TCRβ chain, wherein a. The TCR α chain comprises a variable region, which contains a CDR1 having the amino acid sequence of SEQ ID NO:18, a CDR2 having the amino acid sequence of SEQ ID NO:19, and a CDR3 having the amino acid sequence of SEQ ID NO:20; and b. The TCR β chain comprises a variable region, which contains a CDR1 having the amino acid sequence of SEQ ID NO:24, a CDR2 having the amino acid sequence of SEQ ID NO:25, and a CDR3 having the amino acid sequence of SEQ ID NO:

26.

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

27.

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

82.

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

88.

133. An expression cassette, which comprises the 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. The expression cassette according to claim 133, which further comprises an enhancer element, an optionally optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and also optionally WPRE-mut6.

135. A vector, which comprises the expression cassette according to claim 133 or 134.

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

137. The vector according to claim 136, wherein the viral vector is a lentiviral vector.

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

139. An engineered polypeptide, which comprises a human TCR α chain and a human TCR β chain, wherein a. the TCR α chain is at least 95% identical to SEQ ID NO:73 and the TCR β chain is at least 95% identical to SEQ ID NO:75; or b. the TCR α chain is at least 95% identical to SEQ ID NO:80 and the TCR β chain is at least 95% identical to SEQ ID NO:

82.

140. The engineered polypeptide according to claim 139, wherein a. the TCR α chain comprises or consists of SEQ ID NO:73 and the TCR β chain comprises or consists of SEQ ID NO:75; or b. the TCR α chain comprises or consists of SEQ ID NO:80 and the TCR β chain comprises or consists of SEQ ID NO:

82.

141. The engineered polypeptide according to claim 139 or 140, which comprises, from the N-terminus to the C-terminus: a TCR β chain, a self-cleaving peptide sequence, and a TCR α chain.

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

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

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

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

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

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

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

149. The method according to claim 147 or 148, wherein the administration comprises intravenous administration.

150. The method according to any one of claims 147 to 149, wherein the administration comprises one or more infusions.

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

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

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

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