Cell therapies for type 1 diabetes

Abstract

The present disclosure is directed to recombinant T cell receptors, isolated cell populations comprising the same, methods of producing the same and method of treating type 1 diabetes.

Description

CELL THERAPIES FOR TYPE 1 DIABETESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 614,065, filed December 22, 2023, the contents of which are herein incorporated in their entirety.REFERENCE TO THE ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (ABTH_002_05WO_SeqList_ST26.xml; Size: 150,962 bytes; and Date of Creation: December 17, 2024) are herein incorporated by reference in its entirety.BACKGROUND

[0003] Type 1 diabetes (T1 D) is a T cell-mediated autoimmune disease resulting in islet p cell destruction, hypoinsulinemia, and altered glucose homeostasis. The activity of effector T cells and regulatory T cells, which jointly function in immune homeostasis, are dysregulated in T1 D patients. It has been shown in the clinic that modulating T cells affects disease progression in these patients. There is evidence that therapeutic intervention in type 1 diabetes patients, who are early in autoimmune pathogenesis, can prevent the onset of symptomatic disease and insulin dependence. Currently, curative treatments for type 1 diabetes do not exist and available therapies rely on the treatment of symptoms often involving immunosuppressive reagents that can have severe side effects.SUMMARY

[0004] There is a need in the art for improved therapies for the treatment of Type 1 Diabetes (T1 D). Regulatory T cells have potential for the treatment of this disease and other autoimmune diseases because they can selectively target diseased cell types and tissues, generating a local immune response via an antigen-specific mechanism.

[0005] In some aspects disclosed herein is an isolated population of cells comprising stable CD4+ T regulatory cells (T regs) derived from a subject having type 1 diabetes (T1 D), wherein at least 80% of the cells are stable CD4+ T regs comprising a hypomethylated T cell specific demethylated region (TSDR) at the FOXP3 locus, and wherein the cells comprise an exogenous human T cell receptor (TCR), or a polynucleotide encoding the same, that binds specifically to a target peptide complexed with a major histocompatibility complex (MHC). In some embodiments, the target peptide is a peptide of GAD65. In some embodiments, the exogenous human TCR is a recombinant TCR comprising a TCR alpha (TCRa) chain polypeptide and a TCR beta (TCRP) chain polypeptide, wherein the TCR alpha chain comprises an alpha chain variable domain comprising an alpha CDR1 , and alpha CDR2, and an alpha CDR3 and wherein the TCR beta chain comprises a beta chain variable domaincomprising a beta CDR1 , a beta CDR2, and a beta CDR3; wherein (a) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 1 , the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 2, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3 and the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 7, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 8, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 9; (b) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 15, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 16, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 17; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 21 , the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 22, and beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 23; or (c) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 29, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 30, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 31 ; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 35, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 36, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 37; and wherein the TCR binds to a glutamic acid decarboxylase 65 (GAD65) peptide complexed with a major histocompatibility complex (MHC). In some embodiments, (a) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 4; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 10; (b) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 24; or (c) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 32; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, (a) the TCRa chain comprises or consists of SEQ ID NO: 4; and wherein the TCRp chain comprises or consists of SEQ ID NO: 10; (b) the TCRa chain comprises or consists of SEQ ID NO: 18; and wherein the TCRp chain comprises or consists of SEQ ID NO: 24; or (c) the TCRa chain comprises or consists of SEQ ID NO: 32; and wherein the TCRp chain comprises or consists of SEQ ID NO: 38. In some embodiments, the TCR binds to amino acids 339-352 or 555-567 of the GAD65 peptide when it is complexed with the MHC. In some embodiments, the target peptide is a peptide of proinsulin or preproinsulin. In some embodiments, the exogenous human TCR is a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chainpolypeptide and a TCR beta (TCRP) chain polypeptide, wherein the TCR alpha chain comprises an alpha chain variable domain comprising an alpha CDR1 , and alpha CDR2, and an alpha CDR3 and wherein the TCR beta chain comprises a beta chain variable domain comprising a beta CDR1 , a beta CDR2, and a beta CDR3; wherein (a) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 53, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 54, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 55; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 59, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 60, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 61 ; (b) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 66, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 67, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 68;the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 72, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 73, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 74; and wherein the TCR binds specifically to a proinsulin peptide complexed with a major histocompatibility complex (MHC). In some embodiments, (a) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 56; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 62; (b) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 69; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, (a) the TCRa chain comprises or consists of SEQ ID NO: 56; and wherein the TCRp chain comprises or consists of SEQ ID NO: 62; (b) the TCRa chain comprises or consists of SEQ ID NO: 69; and wherein TCRp chain comprises or consists of SEQ ID NO: 75. In some embodiments, the TCR binds specifically to amino acids 94-110 or 76-86 of the proinsulin peptide when it is complexed with the MHC. In some embodiments, the TCR binds specifically to amino acids 73-90 of preproinsulin. In some embodiments, the MHC molecule comprises an HLA-DRB1*04:01 molecule. In some embodiments, the exogenous human TCR is a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain polypeptide and a TCR beta (TCRP) chain polypeptide, wherein the TCR alpha chain comprises an alpha chain variable domain comprising an alpha CDR1 , and alpha CDR2, and an alpha CDR3 and wherein the TCR beta chain comprises a beta chain variable domain comprising a beta CDR1 , a beta CDR2, and a beta CDR3; wherein the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 125, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 126, and the alpha CDR3 sequence comprises the amino acid sequence ofSEQ ID NO: 127; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 133, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 134, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the TCRa variable domain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 128; and wherein the TCRp variable domain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the TCRa variable domain comprises or consists of sequence of SEQ ID NO: 128; and wherein the TCRp variable domain comprises or consists of SEQ ID NO: 136. In some embodiments, the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 130; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the TCRa chain comprises or consists of SEQ ID NO: 130; and wherein the TCRp chain comprises or consists of SEQ ID NO: 138. In some embodiments, the TCRa chain and the TCRp chain are encoded as two separate polypeptide chains. In some embodiments, the TCRa chain and the TCRp chain are encoded as a single polypeptide chain. In some embodiments, the polypeptide comprises an N-terminal TCRp chain and a C- terminal TCRa chain. In some embodiments, the polypeptide comprises a self-cleaving peptide sequence positioned between the TCRa chain and the TCRp 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. I some embodiments, the subject has residual beta cell function. In some embodiments, the stable CD4+ T regs do not express a FOXP3 protein from an engineered FOXP3 locus. In some embodiments, the TSDR is the CNS2 region of FOXP3. In some embodiments, the MHC is MHC Class I or MHC Class II. In some embodiments, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells are stable CD4+ T regs comprising a hypomethylated TSDR at an endogenous FOXP3 locus. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the CD4+ T regs cells are CD4+CD25+CD127' / |OW. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the CD4+ T regs cells are CD4+CD25+CD127' / |OWFOXP3+. In some embodiments, the isolated population comprises at least 4x107stable CD4+ T regs. In some embodiments, the isolated population comprises 4x107to 1x1010stable CD4+ T regs. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated population are conventional CD4+ T cells. In some embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1 % of the conventional T cells comprise the exogenous human TCR. In some embodiments,the ratio of stable CD4+ T regs to conventional T cells in the isolated population is at least 50: 1 , at least 60: 1 , at least 70: 1 , at least 80: 1 , at least 90: 1 , at least 100: 1 , at least 500: 1 , at least 1000:1 , or at least 10000:1. In some embodiments, less than 2%, less than 1%, less than 0.5%, less than 0.1 %, or less than 0.01% of the cells of the isolated population are CD8+T cells. In some embodiments, the isolated population does not comprise a detectable percentage of CD8+ T cells by fluorescence activated cell sorting (FACS). In some embodiments, at least 10% of the cells express the exogenous human TCR. In some embodiments, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells express the exogenous human TCR. In some embodiments, the MHC phenotype is HLA-DRB1*04:01 or HLA-DRB1*03:01.

[0006] In some aspects disclosed herein is a method of treating type 1 diabetes (T1 D) in a subject comprising administering to the subject the isolated population of any one of the above aspects or embodiments disclosed herein or a pharmaceutical composition thereof. In some embodiments, the subject is a human male subject. In some embodiments, the subject is a human female subject. In some embodiments, the administering comprises intravenous administration. In some embodiments, the administering comprises one or more infusions. In some embodiments, the administration of the isolated population, the pharmaceutical composition, or the cell or population of cells is administered in an effective amount to alleviate one or more symptom of type 1 diabetes. In some embodiments, the cells of the isolated population are autologous relative to the subject.

[0007] In some aspects, disclosed herein is a method of producing a population of cells comprising stable CD4+ T regulatory cells (T regs) comprising: (a) removing CD8+cells, CD19+cells, and optionally CD14+cells from a biological sample obtained from a subject having type 1 diabetes (T1 D) to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+ cells to produce an enriched population; (c) isolating CD4+CD25+CD127' / |OWcells from the enriched population; (d) expanding the enriched population to produce an expanded enriched population of cells; and (e) quantifying the methylation status of a T cell specific demethylated region (TSDR) at the FOXP3 locus in the population of cells, wherein at least 80% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus, thereby producing a population of cells 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 comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated population is selected for therapeutic use if the percentage of cells comprising a hypomethylated TSDR at the FOXP3 locus is 80% or greater. In some embodiments, step (c) is performed at least twice. In some embodiments, the method further comprises activating the population of cells of step (c). In some embodiments, the activating step comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28antibody. In some embodiments, the activating step is performed at least twice. In some embodiments, the second activating step is performed between 4 and 8 days after the first activating step. In some embodiments, the cells are expanded in a culture media comprising IL-2 and TNFa. In some embodiments, the activating and expanding steps comprise culturing the population of cells for at least 5, 6, 7, 8, 9, 10, 11 , or 12 days. In some embodiments, the activating and expanding steps comprise culturing the population of cells for no more than 15, 14, 13, or 12 days.

[0008] In some aspects disclosed herein is a method of producing a population of cells comprising engineered stable CD4+ T regs comprising: (a) removing CD8+cells, CD19+cells, and optionally CD14+cells from a biological sample obtained from a subject having type 1 diabetes (T1 D) to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+cells to produce an enriched population; (c) isolating CD4+CD25+CD127' / |OWcells from the enriched population; (d) delivering a vector comprising a nucleic acid encoding an exogenous human T cell receptor (TCR) to the isolated population of (c) to produce a population of engineered cells; (e) expanding the population of engineered cells to produce an expanded population of engineered cells; and (f) quantifying the methylation status of a T cell specific demethylated region (TSDR) at the FOXP3 locus in the expanded population of engineered cells, wherein at least 80% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, at least 85%, at least 90%, or at least 95% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, step (c) is performed at least twice. In some embodiments, the isolated population is selected for therapeutic use if the percentage of cells comprising a hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95%, or greater. In some embodiments, the method further comprises activating the population of engineered cells step (c). In some embodiments, the activating step comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the activating step is performed at least twice. In some embodiments, the second activating step is performed between 4 and 8 days after the first activating step. In some embodiments, the cells are expanded in a culture media comprising IL-2 and TNFa. In some embodiments, the expanded population comprises at least at least 1x107engineered stable CD4+ T regs. In some embodiments, the methylation status of the TSDR in step (f) is assessed at least 24 hours after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of CD4+CD25+ / highCD127' / l0Wregulatory T cells is assessed 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 comprise CD8+cells, CD19+cells, and / or CD14+cells. In some embodiments, 2% or less of the population of cells are CD8+ cells. In some embodiments, 20% or less of the population of cells are conventional T cells. Insome embodiments, the population of cells in step (c) comprises CD25highCD45RA_cells and CD25+CD45RA+cells and does not comprise CD25+CD45RA_cells. In some embodiments, the isolating of cells at step (c) comprises: (i) identifying a first subpopulation of CD4+cells from the enriched population of step (b); (ii) identifying a second subpopulation of CD25+ / highCD127' / l0Wcells from the first subpopulation; and (iii) selecting CD25highCD45RA_and CD25+ / highCD45RA+cells from the second subpopulation for isolation and excluding CD25+CD45RA_; thereby isolating the population of CD4+T regs. In some embodiments, the TSDR is the CNS2 region of FOXP3. In some embodiments, the subject is a human male subject. In some embodiments, the subject is a human female subject. In some embodiments, the subject has residual beta cell function. In some embodiments, the delivering the vector comprising a nucleic acid encoding the exogenous human TCR comprises transducing the cell population with the vector.

[0009] In some aspects the isolated cell population disclosed herein further comprises, the polynucleotide encoding the exogenous TCR comprises a promoter operably linked to a coding sequence encoding the recombinant TCR, optionally wherein the promoter is an EF-1 alpha promoter or an MND promoter. In some embodiments, the polynucleotide further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), further optionally WPRE-mut6. In some embodiments, the polynucleotide encoding the exogenous TCR is comprised in a vector. 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, 3rd generation lentiviral vector. In some embodiments, the coding sequence is codon-optimized.

[0010] In some aspects disclosed herein is a composition comprising the isolated cell population of any of the disclosed aspects or embodiments and a cryopreservative.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1A-FIG.1 B provide an exemplary method of producing a cell population comprising stable regulatory T cells. FIG. 1A shows a schematic of the overall process of isolating regulatory T cells having a CD4+CD45RA+CD25+ / highCD127l0W / _phenotype. FIG. 1B shows data depicting a fluorescence-activated cell sorting (FACS) strategy to enrich a cell population for cells having a CD45RA+CD4+CD25+CD127|OW / ' phenotype from the CD25- enriched sample to produce an isolated cell population comprising stable regulatory T cells. As is shown in FIG. 1A, this sorting strategy is employed in two consecutive steps to obtain high levels of purity.

[0012] FIG. 2 shows graphs depicting the recovery of regulatory T cells following depletion and CD25 enrichment steps in an exemplary method of producing an isolated cell population comprising stable regulatory T cells.

[0013] FIG. 3A-FIG. 3B show data depicting relative amount of various cell phenotypes following depletion and CD25 enrichment steps in an exemplary method of producing an isolated cell population comprising stable regulatory T cells.

[0014] FIG. 4 shows graphs depicting the growth of cells following transduction with an exogenous TOR. Cells that were not transduced (“Untransduced”) are included as a control.

[0015] FIG. 5 shows graphs depicting the phenotype and TCR expression of regulatory T cells in a cell population produced by an exemplary method of the disclosure.

[0016] FIG. 6 shows data demonstrating that regulatory T cells in a cell population produced by an exemplary method of the disclosure maintain a hypomethylated T cell-specific demethylation region (TSDR).

[0017] FIG. 7 shows a graph depicting the activation of lentivirus-transduced regulatory T cells of the disclosure following a cryopreservation freeze-thaw cycle.

[0018] FIG. 8A-FIG. 8D show graphs of an exemplary gating strategy for the purification of the CD4+CD45RA+CD127' / |OWCD25+cell population purification.

[0019] FIG. 9 shows the percent TSDR hypomethylation of different runs purified by gating strategy depicted in FIG. 8A-FIG. 8D.

[0020] FIG. 10A-FIG. 10D show expression of CTLA-4 (FIG. 10A), CD69 (FIG. 10B), TGF -1 (FIG. 10C), and IL-10 (FIG. 10D) after incubation of the cell populations with and without anti-CD3 and anti-CD28 beads following a cryopreservation freeze-thaw cycle.

[0021] FIG. 11 shows the change in the percent TSDR hypomethylation over time for the expansion of three different MS donors.

[0022] FIG. 12A-FIG. 12C show the population doubling level (FIG. 12A and FIG. 12B) and the percent TSDR hypomethylation (FIG. 12C) for CD45RA+and CD45RA' cells over eight days of expansion. The spike-in shows that CD45RA+cells can expand within the CD45RA' population.

[0023] FIG. 13A-FIG. 13C show the gating strategy for CD25+CD127' / |OWCD45' and CD25+ / highCD127' / l0WCD45‘ selection.

[0024] FIG. 14 shows the expansion of cells after restimulating on days 6, 8, or 10.

[0025] FIG. 15 shows the effect of the fold expansion with TNF-a, a second stimulation, or both.

[0026] FIG. 16A-FIG. 16B show the viability and purity of the cells expanding by both a TNF- a and second stimulation.

[0027] FIG. 17 shows the percent TSDR hypomethylation over the course of expansion for the same donor based on different sorting strategies.

[0028] FIG. 18A-FIG. 18D show the percent TSDR hypomethylation and percent FOXP3+ cells according to the proportion of Tregs in the initial population based on two trials.

[0029] FIG. 19 shows the proportion of FOXP3+and FOXP3' that expressed IL-2 and IFN-y post-activation with PMA and ionomycin.

[0030] FIG. 20A-FIG. 20D show graphs depicting the ability of GAD65-specific TCRs to be expressed in and induce activation of lentivirus-transduced Jurkat cells. FIG. 20A depicts the surface expression of each of the TCRs as expressed by the Jurkat cells. FIG. 20B depicts the responsiveness of the TCRs to GAD65 peptides complexed with MHC. FIG. 20C shows the relative expression of CD69, nuclear factor of activator T cells (NFAT)-driven luciferase expression from a NFAT luciferase reporter, and phospho-ERK cells as a percentage of all cells expressing GFP. FIG. 20D shows a dose response curve of a GAD65 peptide for activation of TCR-A (TCR2) and TCR-B (TCR1).

[0031] FIG. 21A-FIG. 21 C show graphs depicting the ability of GAD65-specific TCRs to be expressed in, and induce activation and proliferation of, lentivirus-transduced conventional CD4+T cells. FIG. 21A depicts the surface expression of each of the TCRs as expressed by the CD4+T cells. FIG. 21 B shows a graph depicting activation of the CD4+T cells in response to GAD65 peptides. FIG. 21 C shows a graph depicting the proliferation of the CD4+T cells in response to GAD65 peptides. The CLIP peptide is a control peptide.

[0032] FIG. 22A-FIG. 22B show graphs depicting the ability of proinsulin-specific TCRs to be expressed in (FIG. 22A) and induce activation of (FIG. 22B) lentivirus-transduced Jurkat cells.

[0033] FIG. 23 shows a graph depicting flow cytometry measurements of ABA-201 cells in blood, showing a decline in ABA-201 cells over the course of the study, with limited persistence of cells after day 11 . Data is shown as mean ± SEM. For each timepoint, dots on the left indicate PBMC control, dots in the middle indicate TCR-Tregs without peptide, and dots on the right indicate TCR-Tregs with PPI73-90.DETAILED DESCRIPTIONOverview

[0034] The present disclosure provides methods and compositions related to the treatment of type 1 diabetes through the production and utilization of stable regulatory T cells. The present disclosure also provides methods and compositions related to the treatment of type 1 diabetes through the production and utilization of engineered stable regulatory T cells comprising recombinant T cell receptors (TCRs) that bind to a target peptide associated with T1 D (e.g., a GAD65 or proinsulin peptide). Such engineered regulatory T cells are capable of specifically targeting discrete cell types and tissues associated with type 1 diabetes (T1 D), for example, in order to prevent immune-mediated destruction, restore homeostasis, and promote repair in affected tissues. Furthermore, the engineered regulatory T cells described herein are stable(e.g., committed to a Treg phenotype) thymically-derived regulatory T cells that are capable of persisting in vivo for extended periods and may provide therapeutic benefit for months or years following a single dose. These stable regulatory T cells are also, in some embodiments, resistant to pro-inflammatory triggers (e.g., pro-inflammatory cytokines). In some embodiments, the stable regulatory T cells of the disclosure are autologous cells, meaning they are obtained directly from a patient’s own cells, engineered to express an exogenous antigen-specific TCR, and subsequently administered back into the patient. The use of autologous cells minimizes the risk of a rejection (e.g., graft-versus-host disease) by the patient.

[0035] Isolation of a highly stable and pure population of engineered regulatory T cells produced in the thymus and obtaining a sufficiently large and pure population of said engineered regulatory T cells to treat a patient has proved challenging, both because the population of stable thymic regulatory T cells is so small relative to the total population of lymphocytes, and because there are no cell surface markers that are solely associated with regulatory T cells. Although isolated polyclonal regulatory T cells have proved effective in clinical therapies, when engineering regulatory T cells with an exogenous TCR, a higher level of purity and stability are required because conventional T cells and non-stable regulatory T cells (i.e. , peripheral Tregs) engineered with the exogenous TCR, in sufficient numbers, could have a deleterious effect by causing, rather than suppressing, an immune response at the site of autoimmune disease.

[0036] These stable engineered regulatory T cells and methods of production provide advantages over several current methods of producing regulatory T cells for cellular therapies. Alternative approaches have been employed to generate populations of engineered regulatory T cells, or regulatory-like T cells, but these cells have disadvantages relative to the populations of cells described herein.

[0037] One possible approach is to increase the proportion of regulatory-like T cells by driving the expression of FOXP3 via engineering of conventional T cells or induced stem cells, either via engineering the FOXP3 promoter or introducing an ectopic FOXP3 gene into the cells. FOXP3 expression confers a regulatory-like phenotype. However, FOXP3 is only one of several genes that have upregulated expression in stable regulatory T cells, and it has been shown that these regulatory- 1 ike T cells differ from natural Treg. Indeed, when CD4+T cells were edited to exhibit a Treg-like phenotype by introducing an exogenous promoter upstream of FOXP3, the engineered cells were shown to have a reduced suppressive capacity relative to natural Treg (Buckner, Science Translational Medicine, 2022), suggesting that these cells will not exhibit the same level of efficacy as true stable regulatory T cells when administered in a clinical setting.

[0038] Conversion of conventional CD4+T cells has also been attempted, for example, by stimulation with TGF-p and IL-2, with retinoic acid, short-chain fatty acids, and TGF-p, or with rapamycin. However, these induced regulator T cells lack epigenomic changes associated with stable regulatory T cells, especially regulatory T cell-specific demethylation at the Conserved Non-coding Sequence 2 (CNS2) of the FOXP3 gene, and hence are functionally unstable and would not retain a stable phenotype when administered in a clinical setting. Moreover, proteomic analysis has shown that these induced Treg like cells have very little overlap in protein expression profile with stable, thymic Treg, and instead, share signaling and metabolic proteins with conventional T cells (Mensink et al., Sci Rep. 2022).

[0039] Contemplated herein is a population of cells and a strategy for isolating said population of cells that comprises highly pure and stable thymic regulatory T cells engineered with an exogenous TCR at sufficient purity and in sufficient numbers to be administered to a subject in a therapeutic amount. In some embodiments, the optimal population of said regulatory T cells are CD25+ / highCD4+CD127' / l0WCD45RA+following isolation, prior to introduction of the exogenous TCR, as this phenotype has been associated with thymic naive regulatory T cells. In some embodiments, the optimal population of said regulatory T cells further comprises CD25highCD4+CD127' / l0WCD45RA' following isolation, prior to introduction of the exogenous TCR, as this phenotype has been associated with thymic antigen-experienced regulatory T cells. These cells further exhibit a stable TSDR phenotype following introduction of the exogenous TCR, indicating that there is no outgrowth of non-regulatory T cell sub-populations or loss of regulatory T cell stability (e.g., no change in cell fate - cells remain terminally differentiated).

[0040] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0041] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, 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.

[0042] In the claims, as well as in the specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e. , to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0043] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0044] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.Regulatory T cells

[0045] In some embodiments, the present disclosure provides isolated populations of cells comprising stable CD4+T regulatory cells derived from a subject having type 1 diabetes, as well as compositions thereof. These isolated populations may be used, for example, to treat type 1 diabetes in a subject in need thereof.

[0046] The terms “Regulatory T cell”, “T regulatory cell” and “Treg” are used interchangeably herein and refer to T cells that suppress the effector functions of other cell populations of the immune system (e.g., conventional CD4+T cells, effector CD8+T cells, antigen presenting cells, and / or granulocytes). Regulatory T cells are defined by expression of the cell surface markers CD4 and CD25, as well as expression of the transcription factor FOXP3. Further, regulatory T cells do not express, or express at low levels, the cell surface marker CD127. Therefore, regulatory T cells are characterized by the following protein expression profile: CD4+CD25high / +CD127low / 'FOXP3+.

[0047] Throughout the present disclosure, the expression of an indicated protein by a cell or population of cells may include reference to various expression indicators such as “+” (e.g.,(e.g., CD127|OW), “High / +” (e.g., CD25High / +), or “low / -“ (e.g., CD127|OW / -). Herein, 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 as measured by a convention protein expression assay (e.g., flow cytometry, fluorescence active cell sorting (FACS), or Western blot). Unless otherwise indicated, protein expression as described throughout the present application is determined by FACS.(a) A positive (+) indicator refers to a detectable level of expression of the indicated protein by FACS. A population of cells that is positive (+) for a particular protein may be further divided into populations of “low” and / or “high” subpopulations.(b) A subpopulation of “low” cells expresses the indicated protein but at a lower level than the other cells in the population (e.g.sat least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the expression level of the other cells in the population) or at a lower level than expression in a control cell population (e.g., relative to a CD4+conventional T cell or a CD8+effector T cell).(c) A subpopulation of “high” cells expresses the indicated protein at a higher level than the other cells in the population (e.g.sat least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than the expression level of the other cells in the population) orat a higher level than expression in a control cell population (e.g., relative to a CD4+conventional T cell or a CD8+effector T cell).(d) A negative (') indicator refers to an absence of expression of the indicated protein, or an expression level of the indicated protein that is below the limit of detection for the particular detection assay (e.g., below the limit of detection for a particular fluorescent antibody and / or flow cytometer).(e) An indicator of “' / Low” refers to a cell population comprising cells that are (-) for the indicated protein and cells that are express “low” levels of the indicated protein.(f) An indicator of “+ / High” refers to a cell population comprising cells that are (+) for the indicated protein, including cells that express a high level of the indicated protein.

[0048] In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is CD25+. In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is CD25+ / high. In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is CD4+. In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is CD45RA+. In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is FOXP3+.

[0049] In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is a CD25+ / highCD4+CD127' / l0Wcell. That is, the regulatory T cell expresses, or expresses a high level of CD25, expresses CD4, and does not express, or expresses a low level of, CD127. In some embodiments, a regulatory T cell does not express CD127. In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is a CD25+ / highCD4+CD127' / l0WCD45RA+cell. Thus, the regulatory T cells expresses, or expresses a high level of CD25, expresses CD4 and CD45RA, and does not express, or expresses a low level of, CD127. In some embodiments, a regulatory T cell expresses FOXP3. In some embodiments, a regulatory T cell is a CD25+ / highCD4+CD127' / lowFOXP3+cell. Thus, the regulatory T cells expresses, or expresses a high level of CD25, expresses CD4 and FOXP3, and does not express, or expresses a low level of, CD127. In some embodiments, a regulatory T cell is a CD25+ / highCD4+CD45RA+CD127' / lowFOXP3+cell. Thus, the regulatory T cells expresses, or expresses a high level of CD25, expresses CD4, CD45RA and FOXP3, and do not express, or expresses a low level of, CD127.

[0050] In thymic regulatory T cell development, the genome organizer SATB1 (special AT- rich sequence-binding protein) binds to specific genomic sites from the CD4+CD8+thymocyte stage to open up the chromatin and activate super-enhancers associated with many regulatory T cell signature genes such as FOXP3, IL2RA, (CD25), CTLA4, IKZF2 (HELIOS), and IFZF4 (EOS). SATB1 and MLL4 (myeloid / lymphoid or mixed-lineage leukemia 4), an enzyme involved in enhancer priming, commonly occupy the newly identified conserved enhancer region, designated conserved noncoding sequence 0 (CNS0), at the FOXP3 locus, with subsequent activation of the enhancers at CNS3 and CNS2, and then the promoter. Thisresults in stable hypomethylation and expression of FOXP3 and other regulator T cell- associated genes, thereby resulting in a stable regulatory T cell phenotype (Piotrowska.et al.; Int J Mol Sci. 2021).

[0051] After hematopoietic development, populations of T cells are able to transition between one phenotype to another under various conditions (See e.g.sKitagawa et al, Nat Immunol. 2017 Feb; 18(2): 173-183). As used herein, a “stable regulatory T cell” is a regulatory T cell that comprises a hypomethylated T cell-specific demethylation region (TSDR) at the FOXP3 locus. In some embodiments, a stable regulatory T cell is further defined by expression of CD4, CD25, and / or FOXP3. Tregs that originate in the thymus (e.g., thymic T regs) are stable and a regulatory T cell that is no longer able to transition between T cell phenotypes by virtue of a change in the methylation status of one or more loci in the FOXP3 locus is also considered stable. In contrast, peripheral Tregs, which are not stable and can be induced in response to pro-inflammatory states, do not exhibit stable hypomethylation of the TSDR region of the FOXP3 locus. Hypomethylation of a TSDR, which is an evolutionary conserved CpG-rich regulatory element of the FOXP3 gene, is associated with expression of FOXP3. A TSDR of an endogenous FOXP3 locus is hypomethylated when the methyl group from one or more methylated cytosines in the TSDR have been removed to replace the methylated cytosine(s) with cytosine. Therefore, the stability of a T regulatory cell is determined by the presence of a hypomethylated T cell-specific demethylation region (TSDR) at the FOXP3 locus.

[0052] In some embodiments, measurement of the methylation status of the TSDR of a FOXP3 locus is as described in Kressler et. al. “Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Regulatory T Phenotype” Frontiers in Immunology, 07 January 2021.; or Schreiber, et. al. “The Regulatory T-Specific Demethylated Region Stabilizes Foxp3 Expression Independently of NF-KB Signaling” PLOS One, February 5, 2014. In some embodiments, the TSDR of the FOXP3 locus is selected from conserved noncoding sequence 0 (CNS0), CNS3, and CNS2. In some embodiments, the TSDR of the FOXP3 locus is CNS2.

[0053] In some embodiments, a stable regulatory T cell maintains a hypomethylated regulatory T cell-specific demethylation region (TSDR) at an endogenous FOXP3 locus in the presence of pro-inflammatory conditions (e.g., in presence of one or more pro-inflammatory cytokines). In some embodiments, a stable regulatory T cell comprises a hypomethylated regulatory T cell-specific demethylation region (TSDR) at an 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.

[0054] In some embodiments, a stable regulatory T cell further exhibits one or more of the following functions: (i) regulatory cytokine secretion activity (e.g., secretion of IL-10, TGFp, and IL-35); (ii) expression or activation markers associated with regulatory T cells (e.g.,expression of CD69, 4-1 BB, CD25, CD71 , and / or CTLA-4); and / or (iii) suppression activity (e.g., the ability of the stable regulatory T cell to suppress the activation and / or proliferation of other effector cells of the immune system).

[0055] Thus, in some embodiments, isolated cell populations provided herein comprise stable T regulatory cells comprising a hypomethylated TSDR of the FOXP3 locus, expression of CD4, CD25, and FOXP3, and / or exhibit cytokine secretion, activation, and / or suppression activity.

[0056] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus overtime after isolation from a biological sample. For example, in some embodiments, the stable regulatory T cells of the isolated cell populations maintain a hypomethylated TSDR at the FOXP3 locus for at least1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after isolation from a biological sample. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for 1-20 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.

[0057] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after transduction with a nucleic acid encoding an exogenous TCR. For example, in some embodiments, the stable regulatory T cells of the isolated cell populations maintain a hypomethylated TSDR at the FOXP3 locus for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or20 days after transduction with a nucleic acid encoding an exogenous TCR. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after transduction with a nucleic acid encoding an exogenous TCR. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the locus for 1-20 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.

[0058] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after cryopreservation. For example, in some embodiments, the isolated cell populations described herein are cryopreserved and later thawed for use and / or analysis, referred to herein as a cryopreservation freeze-thaw cycle. In such embodiments, the stable regulatory T cells of the isolated cell populations maintain a hypomethylated TSDR at the FOXP3 locus for at least 1 ,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after a cryopreservation freeze-thaw cycle. In some embodiments, the stable regulatory T cells maintain ahypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after a cryopreservation freeze-thaw cycle. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the locus for 1-20 days, 1-10 days, 1-5 days, 5-30 days, 5-20 days, 10-40 days, or 25-50 days after a cryopreservation freeze-thaw cycle.

[0059] In some embodiments, the stable regulatory T cells of the isolated cell populations provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after administration to a subject. For example, in some embodiments, the stable regulatory T cells of the isolated cell populations maintain a hypomethylated TSDR at the FOXP3 locus for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after administration to a subject. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after administration to a subject. In some embodiments, the stable regulatory T cells maintain a hypomethylated TSDR at the FOXP3 locus for 1-20 days, 1-10 days, 1-5 days, 5-30 days, 5-20 days, 10-40 days, or 25-50 days after administration to a subject.

[0060] In some embodiments, the isolated populations described herein are autologous cell populations. The term autologous in this context refers to cells that have been obtained from the same subject to which they are subsequently administered. For example, a population of cells may be obtained from a subject, subjected to the methods described herein, and then administered to the same subject (from which the population of cells was originally obtained) to treat type 1 diabetes. In such embodiments, the population of cells administered to the subject comprise autologous regulatory T cells.

[0061] In some embodiments, the isolated populations described herein are allogenic cell populations. The term allogenic in this context refers to cells that have been obtained from one subject and then administered to another subject. For example, a population of cells may be obtained from a subject, subjected to the methods described herein, and then administered to another subject in order to treat type 1 diabetes.

[0062] In some embodiments, the isolated populations of cells described herein (e.g., isolated populations comprising stable CD4+T regulatory cells) are isolated from a biological sample obtained from a subject diagnosed with, or suspected of having, type 1 diabetes.

[0063] In some embodiments, the stable CD4+T regulatory cells and isolated populations thereof are engineered to express a recombinant TCR described herein. In some embodiments, the recombinant TCR is an exogenous TCR.Recombinant TCRs

[0064] In some embodiments, the present disclosure provides recombinant TCRs that bind to a target peptide associated with type 1 diabetes.

[0065] A T cell receptor (TCR) is a transmembrane heterodimer that includes an alpha chain and beta chain linked by a disulfide bond. Within these chains are complementary determining regions (CDRs) that determine the target peptide to which the TCR will bind. TCRs activate T cells in which they reside leading to a plethora of immune responses. Antigen presenting cells digest certain proteins (antigens) and display their fragments (peptides) on major histocompatibility complexes (MHC). This peptide-MHC (pMHC) complex binds to the TCR while other co-stimulatory molecules are activated leading to T cell activation, proliferation, differentiation, apoptosis, or cytokine release.

[0066] The TCR binds specifically to a target peptide complexed with an MHC. A TCR is considered to bind “specifically” to a target peptide complexed with an MHC if the TCR has a higher binding affinity for the target peptide complexed with an MHC relative to a non-target peptide complexed with an MHC. A TCR may bind to a target peptide complexed with an MHC with a binding affinity of at least 10'4M, 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, or 10'10M (e.g., 10-4M to 10'10M). In some embodiments, a TCR is considered to bind “specifically” to a target peptide complexed with an MHC if a T cell expressing the TCR becomes activated (e.g., as assessed by increased CD69 expression) when contacted with the target peptide complexed with an MHC, or becomes more highly activated relative to a non-target peptide complexed with an MHC. In some embodiments, the peptide is presented by a cell expressing the MHC.

[0067] A target peptide may be a peptide associated with type 1 diabetes. In some embodiments, a target peptide associated with type 1 diabetes may be a peptide derived from the GAD65 protein, such as GAD65 (555-567) (SEQ ID NO: 46) or GAD65 (339-352) (SEQ ID NO: 47); or derived from proinsulin proteins, such as proinsulin (94-110) (SEQ ID NO: 118), proinsulin (94-108) (SEQ ID NO: 120), proinsulin (73-90) (also referred to herein as PPI73-90) (SEQ ID NO: 144), or proinsulin (76-86) (SEQ ID NO: 119). In some embodiments, a target peptide is derived from a protein that is overexpressed in a population of cells associated with type 1 diabetes relative to a control (e.g., relative to a population of cells that are not associated with type 1 diabetes).

[0068] In some embodiments, a target peptide is subject to an increased autoimmune reaction in a subject having type 1 diabetes relative to a control. In some embodiments, a target peptide is derived from a protein that is expressed at a site of T1 D inflammation within a subject relative to unaffected sites within the same subject (e.g., a peptide derived from a protein expressed in beta islet cells). In some embodiments, a target peptide is specifically presented by an MHC allele that is associated with T1 D in the subject.

[0069] In some embodiments, a target peptide is derived from a protein that is overexpressed in cells of a subject having type 1 diabetes relative to a control (e.g., relative to a healthy subject). A protein is considered to be overexpressed in cells (e.g., associated with type 1 diabetes) if expression of the protein 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 control cells (e.g., a population of cells that are not associated with the autoimmune disease). In some embodiments, a protein is overexpressed in cells of a subject having type 1 diabetes if expression of the protein in cells of the subject having type 1 diabetes 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 having type 1 diabetes relative to a healthy subject (e.g., a subject that does not have the autoimmune disease).

[0070] In some embodiments, a target peptide is derived from a protein that is highly expressed in cells at the site of disease in a subject having type 1 diabetes relative to a control (e.g., target peptide expression in an unaffected / non-disease site in the subject). A protein is considered to be highly expressed in cells (e.g., associated with type 1 diabetes) at the site of disease if expression of the protein in the cells the site of disease is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher in the cells relative to control cells (e.g., cells not at the site of disease).

[0071] In some embodiments, a target peptide is a peptide that is complexed with an MHC having an HLA haplotype associated with type 1 diabetes. In some embodiments, the HLA haplotype is HLA-DRB1*04:01. In some embodiments, the HLA haplotype is HLA- DRB1*04:02. In some embodiments, the HLA haplotype is HLA-DRB1*03:01.

[0072] In some embodiments, the recombinant TCR is (or is encoded as) a single polypeptide (e.g., comprising a beta chain and an alpha chain). In some embodiments, a TCR comprises an N-terminal beta chain and a C-terminal alpha chain. In other embodiments, a TCR comprises an N-terminal alpha chain and a C-terminal beta chain.

[0073] A recombinant TCR may comprise a linker domain positioned between an alpha chain and a beta chain. In some embodiments, a linker domain comprises a self-cleaving peptide sequence (e.g., a self-cleaving peptide sequence positioned between an alpha chain and a beta chain). A self-cleaving peptide sequence is a peptide sequence that induces a polypeptide to separate into two peptides using a non-classical mechanism. In some embodiments, a self-cleaving peptide sequence can induce ribosomal skipping during translation of a polypeptide. In some embodiments, a self-cleaving peptide sequence is 10- 30, 10-25, 15-30, 15-25, or 18-22 amino acids in length. In some embodiments, a self-cleaving peptide sequence may be a 2A peptide sequence. A 2A peptide sequence may comprise, forexample, a DXEXNPGP (SEQ ID NO: 48) amino acid motif, wherein X can be any amino acid. Exemplary 2A sequences are provided in Table 1.Table 1 : Exemplary 2A sequences

[0074] In some embodiments, the recombinant TCR comprises (or is encoded as) 2 or more polypeptides. For example, in some embodiments, the recombinant TCR comprises a first polypeptide comprising an alpha chain and a second polypeptide comprising a beta chain.

[0075] In some embodiments, the recombinant TCR comprises one or more cysteine residues present in the alpha chain of the TCR that are capable of forming one or more disulfide bonds with one or more cysteine residues in the beta chain of the TCR. In some embodiments, the recombinant TCR comprises one or more cysteine residues present in the alpha chain constant region of the TCR that are capable of forming one or more disulfide bonds with one or more cysteine residues in the beta chain constant region of the TCR. TCR constant region sequences are provided in Table 2.Table 2: Human TCR Constant Region Sequences

[0076] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43, and the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein the cysteine residue at position 48 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 57 of the beta chain.

[0077] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 146. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 146, and the TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein the cysteine residue at position 48 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 57 of the beta chain.

[0078] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43. In someembodiments, the TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43, and the TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein the cysteine residue at position 45 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 77 of the beta chain.

[0079] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43, and the TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., SVC) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein the cysteine residue at position 10 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 17 of the beta chain.

[0080] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43, and the TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein the cysteine residue at position 45 of the alphachain is capable of forming a disulfide bond with the cysteine residue at position 59 of the beta chain.

[0081] In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 43, and the TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein the cysteine residue at position 15 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 15 of the beta chain.

[0082] In some embodiments, the TCR comprises one or more amino acid sequences as described in Table 3 (e.g., one or more amino acid sequences belonging to any one of TCR- A, TCR-B, TCR-C, TCR-D, TCR-E, TCR-F, TCR-G, TCR-H, TCR-I). A TCR may comprise the amino acid sequence of any alpha chain CDR1 , CDR2, or CDR3 as provided in Table 3. In some embodiments, the alpha chain CDR1 of the TCR is any one of SEQ ID NOs: 1 , 15, 29, 53, 66, 79, 92, 105, or 125. In some embodiments, the alpha chain CDR2 of the TCR is any one of SEQ ID NOs: 2, 16, 30, 54, 67, 80, 93, 106, or 126. In some embodiments, the alpha chain CDR3 of the TCR is any one of SEQ ID NOs: 3, 17, 31 , 55, 68, 81 , 94, 107, or 127. A TCR may comprise the amino acid sequence of any beta chain CDR1 , CDR2, or CDR3 as provided in Table 3. In some embodiments, the beta chain CDR1 of the TCR is any one of SEQ ID NOs: 7, 21 , 35, 59, 72, 85, 98, 111 , or 133. In some embodiments, the beta chain CDR2 of the TCR is any one of SEQ ID NOs: 8, 22, 36, 60, 73, 86, 99, 112, or 134. In some embodiments, the beta chain CDR3 of the TCR is any one of SEQ ID NOs: 9, 23, 37, 61 , 74, 87, 100, 113, or 135.

[0083] In some embodiments, the alpha chain variable region of the TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 4, 18, 32, 56, 69, 82, 95, 108, or 128. In some embodiments, the beta chain variable region of the TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 10, 24, 38, 62, 75, 88, 101 , 114, or 136. In some embodiments, the alpha chain of the TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequenceof any one of SEQ ID NOs: 4, 18, 32, 56, 69, 82, 95, 108, or 128. In some embodiments, the beta chain of the TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 10, 24, 38, 62, 75, 88, 101 , 114, or 136.

[0084] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 , a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 1 , a CDR2 consisting of SEQ ID NO: 2, and a CDR3 consisting of SEQ ID NO: 3; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 7, a CDR2 consisting of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0085] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 21 , a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 15, a CDR2 consisting of SEQ ID NO: 16, and a CDR3 consisting of SEQ ID NO: 17; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 21 , a CDR2 consisting of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0086] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31 ; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO:29, a CDR2 consisting of SEQ ID NO: 30, and a CDR3 consisting of SEQ ID NO: 31 ; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 35, a CDR2 consisting of SEQ ID NO: 36, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 37.

[0087] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 55; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 53, a CDR2 consisting of SEQ ID NO: 54, and a CDR3 consisting of SEQ ID NO: 55; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 59, a CDR2 consisting of SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 61.

[0088] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 68; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 66, a CDR2 consisting of SEQ ID NO: 67, and a CDR3 consisting of SEQ ID NO: 68; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 72, a CDR2 consisting of SEQ ID NO: 73, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0089] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 81; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 79, a CDR2 consisting of SEQ ID NO: 80, and a CDR3 consisting of SEQ ID NO: 81; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 85, a CDR2 consisting of SEQ ID NO: 86, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87.

[0090] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 92, a CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 94; and (b) a TCRp chain variable region comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 98, a CDR2 comprising the amino acid sequence of SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 100. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 92, a CDR2 consisting of SEQ ID NO: 93, and a CDR3 consisting of SEQ ID NO: 94; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 98, a CDR2 consisting of SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 100.

[0091] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 105, a CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 107; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 111 , a CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 105, a CDR2 consisting of SEQ ID NO: 106, and a CDR3 consisting of SEQ ID NO: 107; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 111 , a CDR2 consisting of SEQ ID NO: 112, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 113.

[0092] In some embodiments, the recombinant TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 125, a CDR2 comprising the amino acid sequence of SEQ ID NO: 126, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127; and (b) a TCRp chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 133, a CDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 135. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 125, a CDR2 consisting of SEQ ID NO: 126, and a CDR3 consisting of SEQ ID NO: 127; and (b) a TCRp chain variable region comprising a CDR1 consisting of SEQ ID NO: 133, a CDR2 consisting of SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 135.

[0093] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 4. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 4. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 10. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 10.

[0094] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 4, and b) a TCRp chain variable region comprising SEQ ID NO: 10. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 4, and b) a TCRp chain variable region consisting of SEQ ID NO: 10.

[0095] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 5, and b) a TCRp chain comprising SEQ ID NO: 11. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 5, and b) a TCRp chain consisting of SEQ ID NO: 11.

[0096] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 14. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 14.

[0097] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 18. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 18. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 24. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 24.

[0098] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 18, and b) a TCRp chain variable region comprising SEQ ID NO: 24. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 18, and b) a TCRp chain variable region consisting of SEQ ID NO: 24.

[0099] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 25. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 19, and b) a TCRp chain comprising SEQ ID NO: 25. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 19, and b) a TCRp chain consisting of SEQ ID NO: 25.

[0100] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 28. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 28. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 28.

[0101] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 32. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 32. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 32. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 38. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 38. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 38.

[0102] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 32, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 38. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 32, and b) a TCRp chain variable region comprising SEQ ID NO: 38. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 32, and b) a TCRp chain variable region consisting of SEQ ID NO: 38.

[0103] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 33, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 39. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 33, and b) a TCRp chain comprising SEQ ID NO: 39. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 33, and b) a TCRp chain consisting of SEQ ID NO: 39.

[0104] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 42. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 42. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 42.

[0105] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 56. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 56. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 56. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 62. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 62. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 62.

[0106] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 56, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 62. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 56, and b) a TCRp chain variable region comprising SEQ ID NO: 62. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 56, and b) a TCRp chain variable region consisting of SEQ ID NO: 62.

[0107] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 57, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 63. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 57, and b) a TCRp chain comprising SEQ ID NO: 63. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 57, and b) a TCRp chain consisting of SEQ ID NO: 63.

[0108] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 65. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 65. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 65.

[0109] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 69. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 69. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 69. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 75. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 75. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 75.

[0110] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 69, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 75. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 69, and b) a TCRp chain variable region comprising SEQ ID NO: 75. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 69, and b) a TCRp chain variable region consisting of SEQ ID NO: 75.

[0111] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 70, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 76. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 70, and b) a TCRp chain comprising SEQ ID NO: 76. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 70, and b) a TCRp chain consisting of SEQ ID NO: 76.

[0112] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 78. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 78. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 78.

[0113] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 82. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 82. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 82. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 88. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 88. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 88.

[0114] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 82, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 88. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 82, and b) a TCRp chain variable region comprising SEQ ID NO: 88. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 82, and b) a TCRp chain variable region consisting of SEQ ID NO: 88.

[0115] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 83, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 89. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 83, and b) a TCRp chain comprising SEQ ID NO: 89. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 83, and b) a TCRp chain consisting of SEQ ID NO: 89.

[0116] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 91. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 91.

[0117] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 95. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 95. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 95. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 101. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 101.

[0118] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 95, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 95, and b) a TCRp chain variable region comprising SEQ ID NO: 101. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 95, and b) a TCRp chain variable region consisting of SEQ ID NO: 101.

[0119] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 96, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 102. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 96, and b) a TCRp chain comprising SEQ ID NO: 102. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 96, and b) a TCRp chain consisting of SEQ ID NO: 102.

[0120] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 104. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 104. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 104.

[0121] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 108. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 108. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 114. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 114.

[0122] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 108, and b) a TCRp chain variable region comprising SEQ ID NO: 114. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 108, and b) a TCRp chain variable region consisting of SEQ ID NO: 114.

[0123] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 109, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 115. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 109, and b) a TCRp chain comprising SEQ ID NO: 115. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 109, and b) a TCRp chain consisting of SEQ ID NO: 115.

[0124] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 117. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 117. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 117.

[0125] In some embodiments, the TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128. In some embodiments, the TCRa chain variable region comprises SEQ ID NO: 128. In some embodiments, the TCRa chain variable region consists of SEQ ID NO: 128. In some embodiments, the TCRp chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 136. In some embodiments, the TCRpchain variable region comprises SEQ ID NO: 136. In some embodiments, the TCRp chain variable region consists of SEQ ID NO: 136.

[0126] In some embodiments, the recombinant human TOR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128, and b) a TCRp chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 136. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 128, and b) a TCRp chain variable region comprising SEQ ID NO: 136. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 128, and b) a TCRp chain variable region consisting of SEQ ID NO: 136.

[0127] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 129, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 137. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 129, and b) a TCRp chain comprising SEQ ID NO: 137. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 129, and b) a TCRp chain consisting of SEQ ID NO: 137.

[0128] In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130, and b) a TCRp chain comprising an amino acid sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 138. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain comprising SEQ ID NO: 130, and b) a TCRp chain comprising SEQ ID NO: 138. In some embodiments, the recombinant human TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 130, and b) a TCRp chain consisting of SEQ ID NO: 138.

[0129] In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 141 . In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprisingSEQ ID NO: 141. In some embodiments, the recombinant human TOR is expressed as a single polypeptide consisting of SEQ ID NO: 141.

[0130] In some embodiments, the recombinant human TOR is expressed as a single polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 142. In some embodiments, the recombinant human TCR is expressed as a single polypeptide comprising SEQ ID NO: 142. In some embodiments, the recombinant human TCR is expressed as a single polypeptide consisting of SEQ ID NO: 142.Table 3: TCR Sequences

[0131] In some embodiments, the amino acid sequence of the polypeptide further comprises an amino terminal leader sequence, optionally METLLGVSLVILWLQLARVN (SEQ ID NO: 123). In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 123.

[0132] In some embodiments, the amino acid sequence of the polypeptide further comprises a carboxy terminal P2A overhang sequence, optionally ATNFSLLKQAGDVEENPG (SEQ ID NO: 124).Nucleic Acids Encoding a TCR

[0133] In some embodiments, the disclosure provides nucleic acids encoding a TCR (e.g., an exogenous TCR). Nucleic acids may be or may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., messenger RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras.

[0134] The nucleic acids used herein are generally engineered nucleic acids. An engineered nucleic acid is a polynucleotide (e.g., at least two nucleotides covalently linked together, and in some instances, containing phosphodiester bonds, referred to as a phosphodiester backbone) that does not occur in nature. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. A recombinant nucleic acid is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) from two different organisms (e.g., human and mouse). A synthetic nucleic acid is a molecule that is amplified or chemically, or by other means, synthesized. A synthetic nucleic acid includes those that are chemically modified, or otherwise modified, butcan base pair with (bind to) naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.

[0135] Engineered nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, nucleic acids are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343- 345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5' exonuclease, the 3' extension activity of a DNA polymerase and DNA ligase activity. The 5' exonuclease activity chews back the 5' end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed domains. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. The MegaGate molecular cloning method may also be used. MegaGate is a toxin-less Gateway technology that eliminates the ccdb toxin used in Gateway recombinase cloning and instead utilizes meganuclease-mediated digestion to eliminate background vectors during cloning (see, e.g., Kramme C. et al. STAR Protoc. 2021 Oct 22;2(4): 100907, incorporated herein by reference). Other methods of producing engineered polynucleotides may be used in accordance with the present disclosure.

[0136] In some embodiments, the present disclosure provides an expression cassette comprising an open reading frame comprising a nucleic acid encoding the recombinant TOR operably linked to a promoter. A promoter is a nucleotide sequence to which RNA polymerase binds to initial transcription (e.g., ATG). Promoters are typically located directly upstream from (at the 5' end of) a transcription initiation site. In some embodiments, a promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which is it operably linked. In some embodiments, a promoter is an inducible promoter. An inducible promoter may be regulated in vivo by a chemical agent, temperature, or light, for example.

[0137] 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, for example, a protein. An open reading frame is operably linked to a promoter if that promoter regulates transcription of the open reading frame.

[0138] In some embodiments, the present disclosure provides a vector comprising the nucleic acid encoding the recombinant TCR or an expression vector comprising the same. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. Forexample, the vector may be a lentiviral vector, an adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, or a baculoviral vector. A viral vector provides efficient delivery of the recombinant TCR into regulatory T cells of the disclosure. Exemplary viral vectors may be derived from lentivirus, retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication deficient herpes virus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV- BLV group, alpharetrovirus, gammaretrovirus, spumavirus, murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.

[0139] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vectors of the present disclosure comprise a lentiviral gag, pol and rev genes and two long terminal repeats (LTRs) which flank the expression cassette comprising the nucleic acid encoding the recombinant TCR. For safety, 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.

[0140] In some embodiments, the lentiviral vector is a self-inactivating vector. Self-inactivating vectors are vectors where the production of full-length vector RNA in transduced cells in greatly reduced or abolished altogether. This feature greatly minimizes the risk that replication- competent recombinants (RCRs) will emerge. Furthermore, it reduces the risk that that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed. Furthermore, an SIN design reduces the possibility of interference between the LTR and the promoter that is driving the expression of the transgene.

[0141] Self-inactivation is preferably achieved through the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, i.e. , the DNA used to produce the vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA. However, the elements of the LTR that are involved with polyadenylation of the viral RNA arenot modified. Together this diminishes or abolishes the production of full-length vector RNA in transduced cells.

[0142] In some embodiments, a nucleic acid encoding a recombinant TCR is an RNA (e.g., a messenger RNA (mRNA)). In some embodiments, an mRNA comprises a 5' cap, a 5' untranslated region (UTR), an open reading frame (ORF), a 3' UTR, and / or a poly(A) tail.

[0143] In some embodiments, a nucleic acid is codon-optimized. Codon optimization methods are known in the art. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase RNA (e.g., mRNA) stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add posttranslation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide

[0144] In some embodiments, a nucleic acid encoding a TCR comprises a promoter operably linked to a coding sequence encoding the recombinant human TCR. A promoter may be a viral promoter or a natural TCR promoter. In some embodiments, a promoter is a constitutively active promoter or an inducible promoter. In some embodiments, a promoter is the eukaryotic translation elongation factor 1 alpha (EF-1 alpha) promoter and the MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primerbinding site substituted) (see, e.g., Gill, DR. et al. Gene Ther. 2001 ; 8: 1539-46 and Astrakhan, A. et al. Blood 2012; 119: 4395-4407).

[0145] A vector may also include a termination codon and / or expression enhancer elements. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used. In some embodiments, an enhancer element is an optimized post-transcriptional regulatory element (oPRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). A WPRE may be a wild-type WPRE or a WPRE mutant sequence (e.g., WPRE-mut6). In some embodiments, a WPRE is as described in Zanta-Boussif, M.A. et al., Gene Therapy volume 16, pages 605-619 (2009).

[0146] In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to a sequence selected from SEQ ID NO: 4, 10, 18, 24, 32, 38, 56, 62, 69, 75, 82, 88, 95, 101 , 108, 114, 128, and 136. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding apolypeptide comprising 100% identity to a sequence selected from SEQ ID NO: 4, 10, 18, 24, 32, 38, 56, 62, 69, 75, 82, 88, 95, 101, 108, 114, 128, and 136. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide sequence selected from SEQ ID NOs: 4, 10, 18, 24, 32, 38, 56, 62, 69, 75, 82, 88, 95, 101, 108, 114, 128, and 136. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide consisting of a sequence selected from SEQ ID NOs: 4, 10, 18, 24, 32, 38, 56, 62, 69, 75, 82, 88, 95, 101, 108, 114, 128, and 136.

[0147] In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to a sequence selected from SEQ ID NO: 5- 6, 11-13, 19-20, 25-27, 33-34, 39-41, 57-58, 63-64, 70-71, 76-77, 83-84, 89-90, 96-97, 102- 103, 109-110, 115-116, 129-132, and 137-140. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide comprising 100% identity to a sequence selected from SEQ ID NO: 5-6, 11-13, 19-20, 25-27, 33-34, 39-41, 57- 58, 63-64, 70-71, 76-77, 83-84, 89-90, 96-97, 102-103, 109-110, 115-116, 129-132, and 137- 140. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide sequence selected from SEQ ID NOs: 5-6, 11-13, 19-20, 25-27, 33- 34, 39-41, 57-58, 63-64, 70-71 , 76-77, 83-84, 89-90, 96-97, 102-103, 109-110, 115-116, 129- 132, and 137-140. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide consisting of a sequence selected from SEQ ID NOs: 5-6, 11-13, 19-20, 25-27, 33-34, 39-41, 57-58, 63-64, 70-71 , 76-77, 83-84, 89-90, 96-97, 102- 103, 109-110, 115-116, 129-132, and 137-140.

[0148] In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to a sequence selected from SEQ ID NO: 14, 28, 42, 65, 78, 91, 104, 117, 141, and 142. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide comprising 100% identity to a sequence selected from SEQ ID NO: 14, 28, 42, 65, 78, 91, 104, 117, 141, and 142. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide sequence selected from SEQ ID NOs: 14, 28, 42, 65, 78, 91 , 104, 117, 141, and 142. In some embodiments, an engineered polynucleotide comprises an open reading frame encoding a polypeptide consisting of a sequence selected from SEQ ID NOs: 14, 28, 42, 65, 78, 91, 104, 117, 141, and 142.Engineered Regulatory T cells and Isolated Cell Populations

[0149] In some embodiments, the present disclosure provides an isolated population of cells comprising regulatory T cells (e.g., stable regulatory T cells) comprising 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”.

[0150] An exogenous TCR may be any TCR that is introduced to a regulatory T cell, wherein the TCR is not endogenous to (i.e. , naturally occurring in) that regulatory T cell. For example, in some embodiments, an exogenous TCR is encoded by a nucleic acid that is not endogenous to a regulatory T cell (i.e., not naturally occurring in the genome of the regulatory T cell). In some embodiments, the nucleic acid is an engineered nucleic acid, for example, a recombinant or synthetic nucleic acid. The exogenous TCR may be a human TCR. In some embodiments, the TCR is a TCR from a monkey, mouse, rat, or any other animal. The exogenous TCR may be a recombinant TCR as disclosed herein.

[0151] In some embodiments, the present disclosure provides isolated populations of cells comprising stable CD4+T regulatory cells derived from a subject having type 1 diabetes, as well as compositions thereof. In some embodiments, the present disclosure provides isolated populations of cells comprising stable CD4+T regulatory cells derived from a subject having type 1 diabetes, an engineered to express an exogenous human TCR, as well as compositions thereof. 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.

[0152] The methylation status of the TSDR at the FOXP3 locus can be evaluated by means known in the art. For example, in some embodiments, 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, methylation status of the TSDR at the FOXP3 locus is evaluated by single-cell sequencing methods.

[0153] An isolated cell population is a cell population that is removed from a human body, or removed from a sample obtained from a human body. Thus, it is considered “isolated” from the human body. A population of cells may be isolated (e.g., obtained from) a subject, or from a biological sample obtained from the subject, for example, using any known cell collection method, such as apheresis. An isolated cell population of the disclosure may be subjected to the methods described herein to produce an isolated cell population a higher number of regulatory T cells (e.g., stable regulatory T cells) relative to a population of cells obtained directly from a subject, or from a biological sample obtained from the subject, by apheresis, for example.

[0154] In some embodiments, the isolated cell population comprises stable CD4+T regulatory cells, wherein at least 80% of the cells are stable CD4+T regs comprising a 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 regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated cell population comprises stable CD4+ T regulatory cells, wherein 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+T regs comprising 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+T regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells of an isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at an endogenous FOXP3 locus.

[0155] In some embodiments, the isolated population comprises CD25+ / highCD4+CD127_ / l0Wregulatory T cells. In some embodiments, the isolated cell population comprises CD25+ / highCD4+CD127' / lowFOXP3+regulatory T cells. In some embodiments, the isolated cell population comprises CD25+ / highCD4+CD127' / lowFOXP3+CD45RA+regulatory T cells.

[0156] In some embodiments, the isolated cell population comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25+ / highCD4+CD127' / l0Wregulatory T cells. In some embodiments, the isolated cell population comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25+ / highCD4+CD127- / lowFOXP3+regulatory T cells.

[0157] 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 CD45RAT In some embodiments, the isolated cell population comprises at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD25+ / highCD4+CD127- / l0WCD45RA+regulatory T cells.

[0158] As described herein, the present disclosure provides isolated cell populations comprising stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus. While the isolated populations described herein are, in some embodiments, engineered to express an exogenous TCR, the FOXP3 locus remains unmodified. Therefore, the cells of the isolated populations described herein do not comprise an engineered FOXP3 locus. An“engineered F0XP3 locus” refers to any engineered modification (e.g., a modification by the hand of man) intended to alter 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., gene editing system, small molecule, or peptide) intended to activate expression of FOXP3.

[0159] In some embodiments, the isolated cell population comprises at least 1x102, at least 1x103, at least 1x104, at least 1x105, at least 1x106, at least 1x107, at least 1x108, at least 1x109, or at least 1x101° stable regulatory T cells. In some embodiments, the isolated cell population comprises 1x102to 1x101°, 1x103to 1x101°, 1x104to 1x101°, 1x105to 1x101°, 1x106to 1x1010, 1x107to 1x101°, 1x108to 1x101°, 1x105to 1x109, 1x106to 1x108, 1x107to 1x101°, or 1x104to 1x106stable regulatory T cells. In some embodiments, the isolated cell population comprises 1x106to 1x101° stable regulatory T cells. In some embodiments, the isolated cell population comprises 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, or 9x107stable regulatory T cells. In some embodiments, the isolated cell population comprises 1x107to 1x1010, 2x107to 1x1010, 3x107to 1x1010, 4x107to 1x1010, 5x107to 1x1010, 6x107to 1x1010, 7x107to 1x1010, 8x107to 1x1010, or 9x107to 1x1010stable regulatory T cells.

[0160] In some embodiments, the isolated cell population comprises stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus, wherein the stable T regulatory cells retain markers of stability (i.e., maintain the hypomethylated TSDR at the FOXP3 locus and / or maintain the protein expression profile of CD25+ / highCD4+CD127_ / lowFOXP3+) in the presence of proinflammatory conditions. Proinflammatory conditions refer to cells and factors known to drive inflammatory immune responses and can include pro-inflammatory cytokines (e.g., IL- 17, IL-22, IL-21 , IFNy, IL-12, TNFa, IL-1 p, IL-6, IL-1 , GM-CSF, and others known in the art), immune effector cells (e.g., conventional CD4+ T cells, CD8+ effector T cells, granulocytes, etc.), and other proinflammatory mediators (e.g., prostaglandins, thrombin, histamine, and matrix proteases). The proinflammatory conditions may be in vitro or in vivo. In some embodiments, the stable regulatory T cells within an isolated cell population maintain a hypomethylated TSDR at an endogenous FOXP3 locus in the presence of pro-inflammatory conditions for at Ieast 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 21 , 22, 23, 24, or 25 days.

[0161] In some embodiments, the isolated cell population comprises stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus, wherein the stable T regulatory cells retain markers of stability (i.e., maintain the hypomethylated TSDR at the FOXP3 locus and / or maintain the protein expression profile of CD25+ / highCD4+CD127' / lowFOXP3+) over time. For example, in some embodiments, the stable T regulatory cells maintain the hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from the biological sample. In some embodiments, the stable T regulatory cells maintain the hypomethylated TSDR at the FOXP3 locus ex vivofor at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after activation and / or expansion. In some embodiments, the stable T regulatory cells maintain the hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with an exogenous TCR. In some embodiments, the stable T regulatory cells maintain the hypomethylated TSDR at the FOXP3 locus ex vivo for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after a cryopreservation freeze-thaw cycle.

[0162] In some embodiments, the stable T regulatory cells maintain the protein expression profile of CD25+ / highCD4+CD127- / lowFOXP3+ex vivo for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from the biological sample. In some embodiments, the stable T regulatory cells maintain the protein expression profile of CD25+ / highCD4+CD127' / lowFOXP3+ex vivo for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after activation and / or expansion. In some embodiments, the stable T regulatory cells maintain the protein expression profile of CD25+ / highCD4+CD127' / |OWFOXP3+ex vivo for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with an exogenous TCR. In some embodiments, the stable T regulatory cells maintain the protein expression profile of CD25+ / highCD4+CD127_ / lowFOXP3+ex vivo for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after a cryopreservation freeze-thaw cycle.

[0163] In some embodiments, at least 50% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 50% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 50% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 60% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 60% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 60% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 70% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 70% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 70% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 15 days after isolation. In someembodiments, at least 80% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 80% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 80% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 15 days after isolation. In some embodiments, at least 90% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 5 days after isolation. In some embodiments, at least 90% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 10 days after isolation. In some embodiments, at least 90% of the cells of an isolated cell population comprises a hypomethylated TSDR at a FOXP3 locus for at least 15 days after isolation.

[0164] In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed at multiple (e.g., 2 or more) timepoints during the isolation and production process. In some embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% between these 2 or more timepoints.

[0165] For example, in some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after isolation from a biological sample. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11 %, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1 % within 5, 6, 7, 8, 9, 10, or 11 days after isolation from a biological sample. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from a biological sample. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from a biological sample.

[0166] In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In some embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days afteractivation and / or expansion. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11 %, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after activation and / or expansion.

[0167] In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11 %, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after transduction with an exogenous human TCR.

[0168] In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freezethaw cycle. In some embodiments, hypomethylation of a TSDR at the FOXP3 locus isassessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11%, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freezethaw cycle. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0169] In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after transduction with an exogenous human TCR and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In some embodiments, hypomethylation of a TSDR at the FOXP3 locus is assessed within 1-3 days after isolation from a biological sample and again within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 19%, more than 18%, more than 17%, more than 16%, more than 15%, more than 14%, more than 13%, more than 12%, more than 11 %, more than 10%, more than 9%, more than 8%, more than 7%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 10% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle. In such embodiments, the percentage of stable T regulatory cells comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after a cryopreservation freeze-thaw cycle.

[0170] In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percentage point(s) during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stableregulatory T cells increases by 1 , 2, 3, 4, or 5 percentage point(s) during at least 5, 6, 7, 8, 9, 10, or 11 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 10 percentage points during at least 5 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 10 percentage points during at least 10 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 10 percentage points during at least 15 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 5 percentage points during at least 5 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 5 percentage points during at least 10 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 5 percentage points during at least 15 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 1 percentage point during at least 5 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 1 percentage point during at least 10 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR. In some embodiments, the percentage of cells of the isolated cell population comprising the stable regulatory T cells does not decrease by more than 1 percentage point during at least 15 days of expansion following transduction of the stable regulatory T cells with the exogenous human TCR.

[0171] The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed about 1 , about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of cells with an exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed 4, 5, 6, 7, 8, 9,10, 11 , 12, 13, or 14 days after transduction of cells with an exogenous human TCR. The percentage of stable regulatory T cells relative to total cells in an isolated cell population comprising regulatory T cells may be assessed about 1-21 , 1-7, 4-14, 4-7, 7-10, 7-14, 10-21 , or 14-21 days after transduction of cells with an exogenous human TCR.

[0172] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the regulatory T cells of an isolated cell population express an exogenous TCR (e.g., following transduction of the isolated cell population with an exogenous TCR). In some embodiments, 10%-60% or 20%-50% of the regulatory T cells of an isolated cell population express an exogenous TCR. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the stable regulatory T cells of an isolated cell population express an exogenous TCR (e.g., following transduction of the isolated cell population with an exogenous TCR). In some embodiments, 10%-60% or 20%-50% of the stable regulatory T cells of an isolated cell population express an exogenous TCR.

[0173] In some embodiments, the TSDR at the endogenous FOXP3 locus of stable regulatory T cells of an isolated cell population of regulatory T cells remains hypomethylated until administration of the isolated cell population to a subject. In some embodiments, the TSDR at the endogenous FOXP3 locus of stable regulatory T cells of an isolated cell population of regulatory T cells remains hypomethylated following a cryopreservation freeze-thaw cycle.

[0174] In some embodiments, regulatory T cells (e.g., stable regulatory T cells) of an isolated cell population exhibit one or more cellular functions that are associated with regulatory T cells when activated by binding the pMHC. Non-limiting examples of such cellular functions include cytokine secretion activity, expression of certain activation markers, and suppression activity. Cytokine secretion activity simply refers to the secretion of certain anti-inflammatory cytokines, such as IL-10, TGFp, and IL-35. Activation markers include, but are not limited to CD69, 4- 1 BB, CD25, CD71 , or CTLA-4. A regulatory T cell expresses one or more of these markers when it comes into contact with a target peptide complexed with MHC, for example.

[0175] Suppression activity refers to the suppression of activation, proliferation and cytokine production of non-regulatory T cells (e.g., CD8+ T cells and CD4+ conventional T cells) in part to suppress the immune system from becoming overactive. Regulatory T cells exhibit suppression activity when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. In some embodiments, the target peptide is presented by a cell expressing the MHC. For example, in some embodiments, regulatory T cells suppress the activation, proliferation and cytokine production of conventional T cells having specificity towards a shared target peptide complexed with MHC, e.g., a shared target peptide presented by an Antigen Presenting Cell (APC). In some embodiments,regulatory T cells suppress 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%, relative to a control (e.g., non-regulatory T cells in the absence of regulatory T cells).

[0176] In some embodiments, regulatory T cells suppress the production of IFN-gamma from conventional T cells by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, relative to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress the production of IFN-gamma from conventional T cells by 50%-99%, 75%-99%, or 80%-100% relative to a control (e.g., when present in a population comprising a ratio of 1 :1 to 1 :8 regulatory T cells compared to conventional T cells). In some embodiments, regulatory T cells suppress the production of CD71 from conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, relative to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress the production of CD71 of conventional T cells by 20%-90% or 30%-80% relative to a control (e.g., when present in a population comprising a ratio of 1 :1 to 1 :8 regulatory T cells compared to conventional T cells). In some embodiments, regulatory T cells suppress the production of CD25 from conventional T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, relative to a control (e.g., conventional T cells in the absence of regulatory T cells). In some embodiments, regulatory T cells suppress the production of CD25 of conventional T cells by 40%-70% relative to a control (e.g., when present in a population comprising a ratio of 1 :1 to 1 :8 regulatory T cells compared to conventional T cells).

[0177] In some embodiments, regulatory T cells exhibit cytokine secretion activity (e.g., secretion of IL-10) when contacted with a target peptide complexed with MHC, e.g., a target peptide presented by an APC, that binds to an exogenous TCR expressed by the regulatory T cells. In some embodiments, regulatory ? cells exhibit expression of activation markers when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. For example, in some embodiments, regulatory T cells exhibit expression of CD69, 4-1 BB, CD25, CD71 , and / or CTLA-4 when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. In some embodiments, regulatory T cells exhibit suppression activity when contacted with a target peptide complexed with MHC that binds to an exogenous TCR expressed by the regulatory T cells. For example, in some embodiments, regulatory T cells suppress the activation of conventional T cells having specificity towards a shared target peptide complexed with MHC.

[0178] The cellular functions of stable regulatory T cells may be assessed about 1 , about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of cells with an exogenous human TCR. The cellular functions of stable regulatoryT cells may be assessed 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days after transduction of cells with an exogenous human TCR. The cellular functions of stable regulatory T cells may be assessed about 1-21 , 1-7, 4-14, 4-7, 7-10, 7-14, 10-21 , or 14-21 days after transduction of cells with an exogenous human TCR.

[0179] In some embodiments, transduced regulatory T cells (e.g., transduced regulatory T cells of an isolated population) retain their cellular functionality (e.g., ability to be activated) following a cryopreservation freeze-thaw cycle. In some embodiments, transduced regulatory T cells can be activated and / or expanded following a cryopreservation freeze-thaw cycle. In some embodiments, regulatory T cells can exhibit cytokine secretion activity, expression of certain activation markers, and / or suppression activity following a cryopreservation freezethaw cycle.

[0180] 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 a population of cells are CD25+ / highCD4+CD127' / l0Wprior to activation and / or transduction with an exogenous human T cell receptor.

[0181] An isolated cell population comprising stable regulatory T cells may comprise a minority amount of non-regulatory T cells (e.g., conventional T cells). Non-regulatory T cells may be NK T cells, B cells, CD8+ T cells, neutrophils, eosinophils, CD14+ cells, or conventional (CD4+) T cells that are 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 of an isolated cell population comprising stable regulatory T cells are non-regulatory T cells. In some embodiments, about 0.01% to about 0.1 %, about 0.1% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 10%, about 2% to about 5%, or about 5% to about 10% of the cells of an isolated cell population comprising regulatory T cells are non-regulatory T cells. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1 %, or less than 0.01 % of the cells of an isolated cell population comprising regulatory T cells are non-regulatory T cells that comprise an exogenous human TCR. A conventional T cell commonly produces IL-2 and other interleukin factors. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1 %, less than 0.5%, less than 0.1%, or less than 0.01% of the cells of an isolated cell population comprising regulatory T cells are conventional T cells. In some embodiments, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 10%, about 2% to about 5%, or about 5% to about 10% of the cells of an isolated cell population comprising regulatory T cells are conventional T cells. In some embodiments, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, orless than 0.01% of the cells of an isolated cell population comprising regulatory T cells are conventional T cells that comprise an exogenous human TCR. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of conventional T cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of conventional CD4+ T cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of B cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of NK T cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable amount of CD14+ cells. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable number of eosinophils. In some embodiments, an isolated cell population comprising regulatory T cells comprises an undetectable number of neutrophils. In some embodiments, an isolated population of cells comprising regulatory T cells comprises an undetectable amount of CD8+ T cells.

[0182] The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) may be assessed about 1 , about 6, about 12, about 24, about 36, about 48, about 72, about 96, or about 120 hours after transduction of cells with an exogenous human TCR. The percentage of conventional T cells or other non- regulatory T cells relative to total cells (or relative to stable regulatory T cells) may be assessed 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days after transduction of cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) may be assessed about 1-21 , 1-7, 4-14, 4-7, 7- 10, 7-14, 10-21 , or 14-21 days after transduction of cells with an exogenous human TCR. The percentage of conventional T cells or other non-regulatory T cells relative to total cells (or relative to stable regulatory T cells) may be assessed 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days after a cryopreservation freeze-thaw cycle.

[0183] In some embodiments, the ratio of regulatory T cells to conventional T cells in an isolate population of cells comprising regulatory T cells is at least 5:1 , at least 10:1 , at least 15:1 , at least 20: 1 , at least 25: 1 , at least 30: 1 , at least 35: 1 , at least 40: 1 , at least 45: 1 , at least 50: 1 , at least 60: 1 , at least 70: 1 , at least 80: 1 , at least 90: 1 , or at least 100: 1.Methods of Producing Stable Regulatory T cells

[0184] In some embodiments, the present disclosure provides methods of producing isolated populations of cells comprising stable CD4+ T regulatory cells derived from a subject having type 1 diabetes. Conventional methods of producing antigen-specific regulatory T cells have been unable to produce clinical scale and clinical grade manufacturing of cell populations comprising a high concentration of stable antigen-specific regulatory T cells. Althoughpolyclonal T cells have successfully been produced and administered in clinical settings, the use of stable, thymic regulatory T cells engineered with an exogenous TCR poses increased safety risks that require increased cellular purity and regulatory T cell stability. Without wishing to be bound by theory, stable thymic regulatory T cells represent a small percentage of leukocytes, and if the isolated regulatory T cell population is not highly pure, other types of leukocytes will be engineered with the exogenous TCR. In sufficient numbers, these engineered non- regulatory T cells could have a deleterious effect by causing, rather than suppressing, an immune response at the site of autoimmune disease. Through integration and modification of several biomarker selection processes, including an unexpectedly effective double selection process using CD25, and in some instances CD45RA, the methods designed herein provide the field with the tools to produce populations of antigen-specific regulatory T cells at high yield and with a high relative concentration of stable TCR-transduced regulatory T cells.

[0185] In some embodiments, the method of the present disclosure involves removal of conventional T cells and selection for cells having a CD25+ / highCD4+CD127_ / l0Wphenotype to produce cell populations comprising stable regulatory T cells. In some embodiments, the method of the present disclosure involves removal of conventional T cells, selection for cells having a CD25+ / highCD4+CD127_ / l0Wphenotype to produce cell populations comprising stable regulatory T cells, and engineering of the stable regulatory T cells to comprise an exogenous human TCR that binds specifically to a target peptide complexed with an MHC. In some embodiments, a method of producing a cell population of stable regulatory T cells comprises isolating a biological sample comprising regulatory T cells from a human subject having type 1 diabetes.

[0186] An example method of the disclosure is provided in FIGs. 1A-1B. In the embodiment provided in FIG. 1A, a biological sample (e.g., blood sample) is first isolated from a human subject having type 1 diabetes 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 antiCDF (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 removal of water / liquid) to assist with downstream processing of the sample. The sample is then labeled with an anti-CD25 antibody (e.g., a CD25-PE-Biotin antibody) and a secondary molecule (e.g., an anti-biotin microbead) to produce a CD25-enriched cell population. In some embodiments, a CD25-PE-Biotin antibody is used. The CD25-PE-Biotin antibody comprises an anti-CD25 antibody attached to a tandem conjugate of phycoerythrin (PE) and Biotin. In some embodiments, this enrichment step may be repeated more than once (e.g., two or more times). These CD25-enriched cultures are then further processed by sorting cells using a TCR gating strategy that utilizesfluorescence-activated cell sorting (FACS) or Tyto sorting (Miltenyi) as described in FIG. 1A and 1 B. Cells are first labeled with anti-CD45RA, anti-CD4, and anti-CD127 antibodies. Cells are then selected in multiple sorting steps. In each step, in some embodiments, labeled CD25+ / highCD4+CD127' / l0Wcells are first identified by a gating strategy comprising first identifying singlets, then identifying living cells, then identifying CD4+cells, then identifying CD25+ / highCD127' / l0Wcells from the CD4+ cells, and then identifying CD25highCD45RA' and CD25+ / highCD45RA+cells. The identified population of cells (e.g., cells comprising the protein expression profile of CD4+CD25highCD127' / l0WCD45RA' and cells comprising the expression profile of CD4+CD25+CD127' / |OWCD45RA+) is the collected. In some embodiments, the sorting procedure is performed twice to optimize purity of the cells.

[0187] In some embodiments, the present disclosure provides a method of producing an isolated population comprising stable CD4+T regulatory cells, wherein the method comprises (a) removing CD8+cells and CD19+cells from a biological sample obtained from a subject having type 1 diabetes to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+cells to produce an enriched population; and (c) isolating CD4+CD25+CD127_ / |OWcells from the enriched population. In some embodiments, the present disclosure provides a method of producing an isolated population comprising stable CD4+T regulatory cells, wherein the method comprises (a) removing CD8+cells and CD19+cells from a biological sample obtained from a subject having type 1 diabetes to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+cells to produce an enriched population; (c) isolating CD4+CD25+CD127' / |OWcells from the enriched population; and (d) engineering the population of cells of (c) to express an exogenous human T cell receptor (TCR) that binds specifically to a target peptide complexed with an MHC. In some embodiments, a step (a) further comprises removing CD14+cells from the biological sample. In some embodiments, a step (a) further comprises removing CD56+cells from the biological sample.

[0188] In some embodiments, the method further comprises quantifying the methylation status of a T cell specific demethylated region (TSDR) at the FOXP3 locus in the population of cells, wherein at least 80% of the cells are stable CD4+T 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).

[0189] In some embodiments, the step of isolating CD4+CD25+CD127' / |OWcells from the enriched population comprises (i) identifying a first subpopulation of CD4+cells from the enriched population of step (b); (ii) identifying a second subpopulation of CD25+ / highCD127' / l0Wcells from the first subpopulation; and (iii) selecting CD25highCD45RA- andCD25+ / highCD45RA+cells from the second subpopulation for isolation and excluding CD25+CD45RA thereby isolating the population of stable CD4+T regs.

[0190] In some embodiments, the methods provided herein further comprise isolating a biological sample comprising regulatory T cells from a human subject having type 1 diabetes In some embodiments, the sample isolation is performed using an apheresis technique (e.g., a leukapheresis technique to isolate white blood cells). In some embodiments, isolating a biological sample comprising regulatory T cells from a human subject having type 1 diabetes is performed by removing blood from the subject and separating the blood into plasma and cells. In some embodiments, an apheresis technique involves the removal of whole blood from a subject and separation of the whole blood to remove desired cell types (e.g., T cells). In some embodiments, the separation step of apheresis is performed using continuous flow centrifugation or intermittent flow centrifugation.

[0191] Cells expressing a specific biomarker (e.g., CD8+cells, CD19+cells, CD14+cells, and / or CD56+cells) may be removed from a sample (e.g., a biological sample) using FACS, an antibody pulldown assay technique, or any other method of removing cells expressing a specific biomarker that is known to a person of ordinary skill in the art. In some embodiments, cells expressing a specific biomarker may be removed from a sample by labeling those cells with a microbead (e.g., anti-CD8, anti-CD14, anti-CD19 and / or anti-CD56 microbeads) and then subjected the sample to FACS (to remove the cells expressing the specific biomarker).

[0192] A depleted biological sample is a biological sample (e.g., isolated from a human subject having type 1 diabetes) that has been processed to remove CD8+cells, CD19+cells, CD14+cells and / or CD56+cells. In some embodiments, removing CD8+cells, CD19+cells, CD14+cells and / or CD56+cells from a biological sample generates a depleted biological sample having less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, or less than 3% of its total cells comprising CD8+cells, CD19+cells, CD14+cells, and / or CD56+cells. In some embodiments, removing CD8+cells, CD19+cells, CD14+cells and / or CD56+cells from a biological sample generates a depleted biological sample having less than 0.5% of its total cells comprising CD8+cells.

[0193] A cell selection technique may be used to select for cells having a specific phenotype (e.g., cells expressing a specific biomarker such as CD4, CD25, or CD45RA; or cells that have no to low expression of a specific biomarker such as CD127). A cell selection technique may be a FACS technique, a Tyto device, an antibody pulldown assay technique, magnetic cell separation technique, or any other method of selecting cells expressing a specific biomarker that is known to a person of ordinary skill in the art. In some embodiments, a cell selection technique comprises labeling cells expressing a specific biomarker (e.g., CD25) with a biotinylated antibody that targets that biomarker and then performing a pulldown assay (e.g., with anti-biotin microbeads or a streptavidin complex). In some embodiments, a cell selectiontechnique comprises labeling cells expressing a specific biomarker (e.g., CD25, CD4, and / or CD45RA) with antibodies that target that biomarker. Cells labeled with an antibody that targets a specific biomarker can then be selected from a sample (e.g., a CD25-enriched population) using FACS.

[0194] The CD25 enrichment step is then followed by a multi-step sorting process to select for CD25+ / highCD4+CD127' / l0Wregulatory T cells. In some embodiments, the sorting steps are performed on a Tyto device (Miltenyi). One of skill in the art will appreciate that software for visualizing sorted cell populations enable display of the data in various ways including dot plots and histograms. When a population is being identified based on the presence or absence of single protein, identification can be made using a dot plot or histogram. When it is necessary to identify a population based on the presence or absence of two proteins (e.g., CD25 and CD45RA), identification should be made using a dot plot.

[0195] In some embodiments, prior to the first sorting step, the CD25-enriched cell population is separated into multiple populations of cells and the first sorting step is performed simultaneously and / or sequentially in the multiple sub-populations. This provides for expedited processing of the large number of cells that need to be processed in order to isolate sufficient numbers of the small proportion of cells that are CD25+ / highCD4+CD127' / l0W. In some embodiments, the CD25-enriched cell population is separated into 2, 3, 4, 5, or 6 or more subpopulations that are each sorted in the first sorting step. In some embodiments, after the first sorting step, the sorted sub-populations are combined prior to the second sorting step.

[0196] In some embodiments, the present disclosure provides a method of producing an isolated population comprising stable CD4+T regulatory cells, wherein the method comprises (a) removing CD8+cells and CD19+cells from a biological sample obtained from a subject having type 1 diabetes to produce a depleted biological sample; (b) enriching the depleted biological sample for CD25+cells to produce an enriched population; and (c) isolating CD4+CD25+CD127' / |OWcells from the enriched population, wherein the isolating comprises (i) identifying a first subpopulation of CD4+cells from the enriched population of step (b); (ii) identifying a second subpopulation of CD25+ / highCD127' / l0Wcells from the first subpopulation; and (iii) selecting CD25highCD45RA' and CD25+ / highCD45RA+cells from the second subpopulation for isolation and excluding CD25+CD45RA; thereby isolating the population of stable CD4+ T regs

[0197] In some embodiments, selecting CD25+ / highCD4+CD127' / l0Wcells from a CD25-enriched population comprises identifying a first subpopulation CD4+cells from the CD25-enriched population. A second subpopulation of CD25+ / highCD127_ / l0Wcells is then identified from the first subpopulation. Finally, populations of CD25highCD45RA' and CD25+ / highCD45RA+cells are selected from the second subpopulation for isolation.

[0198] In some embodiments, the first subpopulation comprises at least 60%, at least 70%, at least 71%, at least 72% at least 73% at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD4+ cells.

[0199] In some embodiments, the second subpopulation comprises at least 60%, at least 70%, at least 71%, at least 72% at least 73% at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD25+ / highCD4+CD127- / l0Wregulatory T cells. In some embodiments, the second subpopulation comprises at least 60%, at least 70%, at least 71 %, at least 72% at least 73% at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD25+ / highCD4+CD127' / lowFoxP3+regulatory T cells.

[0200] In some embodiments, the population selected for isolation comprises CD25highCD4+CD127' / l0WCD45RA' (CD25highCD45RA’) and CD25+ / highCD4+CD127- / |OWCD45RA+(CD25+ / highCD45RA+) 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% CD25highCD45RA' 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+ / highCD45RA+cells. It is understood that percentages of CD25highCD45RA' and CD25+ / highCD45RA+cells in the final population add together to equal approximately 80%, 90%, or 100% of the total cell population.

[0201] In some embodiments, the population selected for isolation comprises less than 10% CD25+CD4+CD127' / |OWCD45RA' 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' / |OWCD45RA' cells.

[0202] In some embodiments, the methods described herein result in an isolated population of cells, wherein at least 80% of the cells are stable CD4+T regs comprising a hypomethylated T cell specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated cell population comprises stable CD4+T regulatory cells, wherein 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+T regs comprising 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+T regs comprising a hypomethylated TSDR at the FOXP3 locus.

[0203] In some embodiments, the isolated population of cells produced by the methods described herein is selected for therapeutic use if at least 80% of the cells are stable CD4+T regs comprising a hypomethylated T cell specific demethylated region (TSDR) at the FOXP3 locus. In some embodiments, the isolated population of cells is selected for therapeutic use if at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated population of cells is selected for therapeutic use if 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus. In some embodiments, the isolated population of cells is selected for therapeutic use if between 80% and 90%, between 85% and 95%, between 80% and 85%, between 85% and 90%, between 90% and 95% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus.

[0204] In some embodiments, the methods provided herein further comprise introducing an exogenous TCR that binds specifically to a target peptide complexed with an MHC to the population of cells comprising stable CD4+T regulator cells. In some embodiments, introducing the exogenous TCR comprises transfecting the population of cells with a nucleic acid encoding the exogenous TCR. In some embodiments, introducing the exogenous TCR comprises transducing the population of cells with a nucleic acid encoding the exogenous TCR. The nucleic acid encoding the exogenous TCR may be an RNA (e.g., mRNA) molecule or a DNA molecule. In some embodiments, the nucleic acid is a vector or plasmid. In some embodiments, the nucleic acid is delivered to the population of cells using a lentiviral vector, an adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, or a baculoviral vector.

[0205] In some embodiments, a method or process of producing a cell population of regulatory T cells further comprises activating and / or expanding regulatory T cells of the population (e.g., following the deletion, enrichment, and engineering steps described herein). In some embodiments, the regulatory T cells are activated prior to transfection or transduction of theregulatory T cells 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 regulatory T cells comprises contacting the regulatory T cells with the antigen that specifically binds to the exogenous TCR belonging to the isolated cell population. In some embodiments, the antibodies (e.g., the anti-CD3, anti-CD28, and / or TCR-specific antibodies) are conjugated or complexed with a bead, e.g., a magnetic bead or a polymeric bead. In some embodiments, the antibodies are covalently attached to a polymeric matrix.

[0206] 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.

[0207] In some embodiments, activating and / or expanding the regulatory T cells comprises culturing the isolated cell population in a cell media. In some embodiments, the media comprises IL-2. In some embodiments, the media comprises about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, 1200, 1400, 1600, 1800, 2000, 3000, or 4000 lU / ml IL-2. In some embodiments, the media comprises 500-1500 or 800-1200 lU / ml IL-2. In some embodiments, the media comprises about 1,000 lU / ml IL-2. In some embodiments, the media comprises 1,000 lU / ml IL-2. In some embodiments, the media comprises further comprises TNFa. In some embodiments, the media comprises about 500, 750, 1000, 1500, 1750, 2000, 2250, 2500, or about 3000 lU / ml TNFa. In some embodiments, the media comprises 2000-3000 or 2250-2750 lU / ml TNFa. In some embodiments, the media comprises about 2500 lU / ml TNFa. In some embodiments, the media comprises 2500 lU / ml TNFa.

[0208] In some embodiments, activating and expanding the regulatory T cells comprises culturing cells of the population for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, activating and expanding the regulatory T cells comprises culturing cells of the population for 2-14, 2-10, 2-5, 5-10, or 5-14 days. In some embodiments, activating and expanding the regulatory T cells comprises culturing cells of the population for no more than 15, 14, 13, or 12 days. In some embodiments, following activation and / or expansion, isolated the isolated cell population comprises at least 1x102, at least 1x103, at least 1x104, at least 1x105, at least 1x106, at least 1x107, at least 1x108, at least 1x109, or at least 1x101° stable CD25+ / highCD4+CD127' / l0Wregulatory T cells comprising a hypomethylated TSDR at an endogenous FOXP3 locus.

[0209] In some embodiments, a method or process of producing a cell population of regulatory T cells may further comprise cryopreserving the cell population. In some embodiments, a method or process of producing a cell population of regulatory T cells may further comprise thawing the cryopreserved cell population. A cryopreservation freeze-thaw cycle refers to the process of cryopreserving a cell population, and thawing the cryopreserved cell population at a later time.Pharmaceutical Compositions

[0210] In some embodiments, the disclosure provides pharmaceutical compositions comprising the isolated cell populations of regulatory T cells (e.g., stable regulatory T cells) described herein. In some embodiments, a pharmaceutical composition comprises a cell population of regulatory T cells (e.g., stable regulatory T cells) described herein and a pharmaceutically acceptable excipient.

[0211] As used herein, a pharmaceutically acceptable excipient may also be referred to as a pharmaceutically acceptable carrier, pharmaceutically acceptable diluent, or pharmaceutically acceptable adjuvant. Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

[0212] The pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. Sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent. A pharmaceutical composition for use in accordance with the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers which are non-toxic to the subjects at the dosages and concentrations employed. 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.

[0213] A pharmaceutical composition may comprise an effective amount of stable regulatory T cells that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of stable regulatory T cells as described herein may refer to number of cells that is sufficient to improve a symptom associated with type 1 diabetes (e.g., reduced or no production of insulin, build-up of glucose / sugar in the bloodstream, feeling more thirsty than usual, excess urination, bed-wetting in children who have never wet the bed during the night, hunger, weight loss, irritability or other mood changes, fatigue, and blurred vision). As will be appreciated by the skilled artisan, the effective amount of a solution or preparation provided herein may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific disease being treated, the specific symptom to be alleviated, on the cell or tissue being targeted, and on the subject’s age, gender, and general health status.

[0214] In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising a hypomethylated TSDR at an endogenous FOXP3 locus) comprises at least 1x102, at least 1x103, at least 1x104, at least 1x105, at least 1x106, at least 1x107, at least1x108, at least 1x109, or at least 1x1O10stable regulatory T cells. In some embodiments, an effective amount of stable regulatory T cells (e.g., regulatory T cells comprising a hypomethylated TSDR at an endogenous FOXP3 locus) is 1x102to 1x1010, 1x103to 1x1010, 1x104to 1x1010, 1x105to 1x1010, 1x106to 1x1010, 1x107to 1x1010, 1x108to 1x1010, 1x109to 1x1010stable regulatory T cells. In some embodiments, an effective amount of stable regulatory ? cells comprises 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, or 9x107stable regulatory T cells. In some embodiments, an effective amount of stable regulatory T cells comprises 1x107to 1x1O10, 2x107to 1x1O10, 3x107to 1x1O10, 4x107to 1x1O10, 5x107to 1x1010, 6x107to 1x1O10, 7x107to 1x1O10, 8x107to 1x1O10, or 9x107to 1x1O10stable regulatory T cells.

[0215] In some embodiments, the isolated cell populations (e.g., intended to be used in a pharmaceutical composition) are cryopreserved (e.g., subjected to one or more cryopreservation freeze-thaw cycles). That is, the isolated cell populations produced herein may be combined with a cryoprotecting agent, which lowers the melting temperature by forming chemical bonds with water and increasing the total concentration of solutes in the system. Non-limiting examples of cryoprotecting agents include glycerol, dimethyl sulfoxide (DMSO), ethanediol, and propanediol. While traditional methods that often include the use of serum and DMSO may be used, the disclosure also contemplates the use of freezing medium manufactured under cGMP conditions and formulated serum-free and of non-animal origin (e.g., using <10% DMSO in the freeze cocktail). Other cryopreservation techniques are also provided herein, including more advanced techniques of cooling, for example, vitrifying cells without the use of cryoprotecting agents. See, e.g., Shinshu University. “A new way to 'freeze' cells promises to transform the common cell-freezing practice.” ScienceDaily.com April 2019. This process of ultrarapid cooling, utilizes inkjet cell printing to cool at a rate of 10,000 degrees Celsius / second, causing near-vitrification of the cells.Methods of Treatment

[0216] In some embodiments, the present disclosure provides methods of treating type 1 diabetes in a subject in need thereof comprising administering an isolated population of cells comprising stable CD4+T regulatory cells or a composition thereof. In some embodiments, the disclosure provides methods of administration of isolated cell populations comprising regulatory T cells as described herein (and related pharmaceutical compositions) to a subject (e.g., a subject having type 1 diabetes). In some embodiments, the stable CD4+T regulatory cells are engineered to express an exogenous TCR that binds to a target peptide associated with T 1 D.

[0217] In some embodiments, the disclosure provides methods of administering a cell population comprising regulatory T cells or a pharmaceutical composition as described hereinto a subject in an effective amount to alleviate one or more symptoms of type 1 diabetes. Nonlimiting examples of symptoms of type 1 diabetes include reduced or no production of insulin, build-up of glucose / sugar in the bloodstream, feeling more thirsty than usual, excess urination, bed-wetting in children who have never wet the bed during the night, hunger, weight loss, irritability or other mood changes, fatigue, and blurred vision. In some embodiments, the subject has T1 D but retains residual beta cell function.

[0218] In some embodiments, the disclosure provides methods of treating type 1 diabetes in a subject comprising administering a cell population comprising regulatory T cells or a pharmaceutical composition as described herein to the subject. In some embodiments, at least a portion of the population of cells are autologous cells (i.e. , obtained from the same subject to which they are subsequently administered). In some embodiments, a population of cells is isolated from a subject, subjected to a method of producing a cell population (e.g., to increase the relative concentration of regulatory T cells within the population) as described herein, engineered to express an exogenous TCR, and then administered to the same subject in order to treat a disease.

[0219] In some embodiments, treating (or treatment of) a disease refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of type 1 diabetes, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., because of knowledge of genetic factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0220] A subject refers to an individual organism, for example, 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 cattle, 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 may be male or female.

[0221] Conventional and pharmaceutically acceptable routes of administration of the include, but are not limited to, intravenous, subcutaneous, intravenous, intrathecal administration direct delivery to the selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intramuscular, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.In some embodiments, the isolated populations or compositions thereof described herein are administered intravenously.

[0222] A cell population comprising regulatory T cells or a pharmaceutical composition may be administered as a bolus administration. In some embodiments, administration of a cell population comprising regulatory T cells or a pharmaceutical composition comprises one or more infusion of the isolated cell population or pharmaceutical composition to the subject (e.g., cells are infused through a central line, similar to a blood transfusion).INCORPORATION BY REFERENCE

[0223] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.EXAMPLESExample 1. Manufacturing process for production of autologous stable regulatory T cells expressing an exogenous TCR

[0224] The generation of a population of cells comprising stable thymically-derived regulatory T cells that express an exogenous TCR is shown in this Example. The overall enrichment and sorting strategy is illustrated in FIGs. 1A-1B.Leukapheresis, Depletion, and Enrichment

[0225] First, leukapheresis from a human subject was collected, shipped to a central processing facility, and held at 4 °C until processing at about 24 hours following collection. Incoming leukapheresis product was washed in phosphate-buffered saline containing 0.5% human serum albumin (HSA) to reduce platelet count and then labeled with a cocktail of magnetic microbeads (CD8, CD14, and CD19 microbeads; Miltenyi) using an automated cell processing system.

[0226] Using magnetic cell separation technology, cytotoxic T cells (CD8+), B cells (CD19+), and monocytes (CD14+) were depleted prior to enrichment for CD25+cells. The volume of the depleted biological sample was reduced by centrifugation. The depleted biological sample was then labeled with CD25-PE-Biotin antibody (Miltenyi) according to the manufacturer’s recommendations. The cells were then washed with phosphate-buffered saline containing 0.5% HSA and labeled with anti-biotin microbeads (Miltenyi). CD25+cells were then isolated using magnetic cell separation technology to produce a CD25-enriched sample.

[0227] FIG. 2 shows the Viable Cells (VC)s of all cells following apheresis, removal of the CD8+, CD19+, and CD14+cells (depleted sample), and selection for CD25+(CD25-enriched sample) for Run 1. Approximately 98% of the total cells of the population are CD3+cellsfollowing the CD25 selection step to produce the CD25-enriched sample and approximately 95% of the cells are CD4+T cells.

[0228] Table 4 shows CD4+cell and naive Treg cell composition of the apheresis cell population and after the depletion and enrichment steps for 5 additional manufacturing runs.Table 4: Composition of cell population during depletion and enrichment process

[0229] FIG. 3A shows the viable counts (VC) of naive regulatory T cells (specifically, CD4+CD25+ / highCD127' / l0WCD45RA+cells) following apheresis, removal of the CD8+, CD19+, and CD14+cells (depleted sample), and selection for CD25+(CD25-enriched sample) for Run 1 . FIG. 3B shows the recovery of naive regulatory T cells (CD4+CD25+ / highCD127' / l0WCD45RA+cells) after each step and that approximately 80% of the CD4+CD25+ / highCD127' / l0WCD45RA+cells are recovered after the depletion step and 75% of the CD4+CD25+ / highCD127' / l0WCD45RA+cells are recovered after the CD25-enrichement step.

[0230] Table 5 shows VCs of naive regulatory T cells following apheresis, depletion, and enrichment, and recovery from the previous step of naive regulatory T cells after the depletion, and enrichment.Table 5: Total viable counts (TVC) of naive regulatory T cells and % recovery following steps of the manufacturing processGating Strategy

[0231] 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 the CD4+CD25+ / highCD127' / l0WCD45RA+phenotype to produce a population of cells comprising stable regulatory T cells. FACS was performed in two rounds, with a first debulking fractional sort and a second fractional purity sort. For the debulking sort, the CD25-enriched sample was divided into multiple fractions that were sorted across multiple cell sorters. Following the debulking sort, the fractions were combined for the second round of purity sort. The purpose of fractionally sorting the cells in the debulking and / or purity sort is to expedite the sorting process by spreading the sorts over multiple machines, ensuring that the cells remain viable. The gating strategy used for the debulk and purity sorts were identical. For the gating strategy, a dot-plot based approach was used in which CD45RA+CD4+cells were first selected, and then CD25+ / highCD127_ / l0Wcells were selected from the CD45RA+CD4+cell population to optimize the purity of stable regulatory T cells in the population as determined by TSDR phenotype. As shown in FIG. 1 B, this FACS gating strategy first plotted CD45RA+versus CD4+cells on a dot plot and selected them based on population distribution using a box gate. From the selected population of cells, CD25+ / highversus CD127 ' / |OWcells were plotted on a dot plot and gated using a polygon gate to select a population of CD4+CD25+ / highCD127' / l0WCD45RA+cells for collection. This FACS strategy was developed to optimize selection of stable naive regulatory T cells based on TSDR hypomethylation phenotype. The composition of the cell population before sorting, after debulking and after production of the population of CD4+CD25+ / highCD127' / l0WCD45RA+cells for Run 1 is provided in Table 6.Table 6. Composition of Cell Population following FACS

[0232] The purity and recovery of naive Tregs during the sorting process for 5 additional manufacturing runs is shown in Table 7.Table 7: Purity and recovery of naive Tregs post-FACS sorting

[0233] The regulatory T cells were activated after the FACS using T cell TransAct activation reagent (Miltenyi) for about 48 hours. The activated regulatory T cells were then transduced with a third generation, VSV-pseudotyped, self-inactivating lentiviral vector encoding an exogenous TCR. The vector construct comprised an N-terminal TCR beta chain and a C- terminal TCR alpha chain with a linker domain comprising a GSG amino acid sequence followed by a P2A self-cleaving peptide. The TCR beta-GSG-P2A-TCR alpha fusion constructs were expressed under the EF1 alpha promoter. The proportion of transduced cells, as measured by FACS analysis with an anti-Vbeta antibody, is shown in FIG. 5.

[0234] The transduced regulatory T cells were then expanded in T cell expansion media supplemented with 1 ,000 lll / ml IL-2 until harvest (Day 9, 10, or 11). Cell count, size, population doubling, and viability of the untransduced and transduced regulatory T cells were assessed and are shown for Run 1 (FIG. 4). Notably, the transduced regulatory T cells shared a similar growth profile with the untransduced regulatory T cells, suggesting that the exogenous TCR does not impair the growth and proliferation of the cells.

[0235] The population doubling level and % viability for 5 additional manufacturing runs is shown in Table 8. The % target dose achieved for the full process for all 6 manufacturing runs is also shown. The % target dose is calculated by determining the total final number of naive Tregs based on the initial number of naive Tregs prior to expansion following the sorting process and the population doubling level and assumes a transduction efficiency of 25%. The % target dose is calculated based on a target dose of 100 million transduced cells. As is shown, the manufacturing process in this example achieves a Treg product with high purity and sufficient cells to dose a patient from a single leukopak. As is used herein, PDL is population doubling level as used throughout. TDN indicates transduced cells.Table 8: Properties of manufactured cells

[0236] Transduced cells were collected at days 0-11 of the activation and expansion process and TSDR hypomethylation at the FOXP3 CNS2 locus was measured by a ddPCR assay. Genomic DNA (gDNA) was obtained from enriched regulatory T cells and bisulfite treated. Then a digital droplet PCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. TSDR hypomethylation status of the collected cells for all 6 transduced manufacturing runs is shown in FIG. 6. During expansion, TSDR hypomethylation levels did not decrease by more than 10% for any manufacturing run and remained above 80% for all manufacturing runs. Table 9 below shows the TSDR hypomethylation status for all 6 donor samples processed using the method described in this Example.

[0237] These data demonstrate that the regulatory T cells obtained are stable, thymically derived regulatory T cells.Table 9: TSDR status of cells produced using the methods of this ExampleActivity of Tregs following a cryopreservation freeze-thaw cycle

[0238] Regulatory T cells produced according to the methods in this example that were untransduced or transduced with the lentiviral vector encoding the TCR described herein were cryopreserved for storage. The cells were frozen and stored in a vapor-phase nitrogen storage freezer.

[0239] After a period of time in cold storage, the frozen cell population was thawed. The cell population was split into two experimental groups. The first experimental group was activated with anti-CD3 and anti-CD28 antibodies (transduced (TDN)) while the second experimentalgroup was not (untransduced (UNT)). Both groups were expanded in media containing IL-2. Following incubation, IL-10 levels were measured after 1 , 2, and 3 days.

[0240] The increase in IL-10 levels for each of the runs over the three-day period for cells activated with anti-CD3 and anti-CD28 antibodies is shown in FIG. 7. The cells exhibited increasing secretion of the IL-10 cytokine over the period of three days, demonstrating that cell populations can be activated and retain their cellular functionality following a cryopreservation freeze-thaw cycle (FIG. 7). The cells that were not activated showed no increase in IL-10 levels. This data further demonstrates that the regulatory T cells retained function.Example 2. Altered gating strategy for GMP conditions

[0241] A second gating strategy was developed using a 1-D histogram-based approach rather than a 2-D dot-based approach to reduce user error in a GMP manufacturing setting. Cells were prepared as in Example 1 up to the FACS sorting step. In the FACS sorting step, the gating scheme used, shown in FIG. 8, was a one-dimensional, histogram plot-based approach in which the CD25-enriched cells were sequentially gated on CD4+, CD45RA+, CD127' / |OWand CD25+ / highusing histograms (CD4+> CD45RA+(histo)> CD127' / Iow(histo) 40% > CD25Hi(histo) 70%). For runs with the same run number as in Example 1 , both gating strategies were run in parallel on the same donor sample.

[0242] The properties of the cells prior to sorting are shown in Table 10 and the viability of the cells after each step of the manufacturing process is Table 11.Table 10: Composition of cell population during depletion and enrichment processTable 11 : VC of naive regulatory T cells and % recovery following steps of the manufacturing process

[0243] The purity and recovery of naive Tregs during the sorting process for each run is shown in Table 12.Table 12: Purity and recovery of naive Tregs post-FACS sorting

[0244] The properties of the manufactured cells are shown below in Table 13.Table 13: Properties of manufactured cells

[0245] The TSDR status of the cells is shown in Table 14 below and in Fig. 9. The one run having TSDR levels above 100% is due to the donor being female. Because the TSDR assays measures the FoxP3 locus on the X chromosome, and females have one silenced (methylated) copy of the X chromosome, TSDR demethylation levels are doubled for female subjects to obtain the proportion of cells that have a hypomethylated FoxP3 locus. The data presented in this example shows that the populations of cells produced are similar when the methods of Example 1 or Example 2 are used.Table 14: TSDR status of cells produced using the methods of this ExampleActivity of Tregs following a cryopreservation freeze-thaw cycle

[0246] Regulatory T cells produced according to the methods in this example for runs 5-7 that were untransduced or transduced with the lentiviral vectors shown were cryopreserved for storage. The cells were frozen and stored in a vapor-phase nitrogen storage freezer.

[0247] After a period of time in cold storage, the frozen cell population was thawed. The cell population was split into two experimental groups. The first experimental group was activated with anti-CD3 and anti-CD28 antibodies while the second experimental group was not. Both groups were expanded in media containing IL-2. Following incubation, regulatory T cell activation markers and cytokine levels were measured after 1 , 2, and 3 days (IL-10, CTLA-4, TGF-beta-1 , and CD69).

[0248] The increase in levels for each of IL-10, CTLA-4, TGF-beta-1 , and CD69 for all of the runs over the three day period for cells activated with anti-CD3 and anti-CD28 beads is shown in FIG. 10A-FIG. 10D. The activated cells showed increasing levels of IL-10 (FIG. 10D), CTLA- 4 (FIG. 10A), TGF-beta-1 (FIG. 10C), and CD69 (FIG. 10B) over the period, demonstrating that cell populations can be activated and retain their cellular functionality following a cryopreservation freeze-thaw cycle (FIG. 10A-FIG.10D). The cells that were not activated showed no increase in levels. This data further demonstrates that the regulatory T cells retained function.Example 3: Manufacturing Strategy with Increased Stringency of Stable Treg SelectionChallenges manufacturing stable Tregs from patient populations

[0249] Following establishment of a manufacturing process with healthy donor samples, the manufacturing process was evaluated with samples from autoimmune donors. In this Example, manufacturing with cells from MS donors is described. For certain MS donors, a lower initial TSDR hypomethylation level and / or less stable TSDR hypomethylation levels over expansion (Day 0 to harvest) were observed compared to the results using healthy donor cells. FIG. 11 shows the change in TSDR hypomethylation over expansion for the three MS donors. For all three donors, cells were isolated according to the healthy donor process described in Examples 1 or 2. For two of the three MS donors tested, an initial TSDR was below 90% was observed and TSDR decreased by more than 10% over the course of expansion. This is likely because of differences in Treg populations between healthy and autoimmune donors, including MS donors, making the boundary between Treg and T conventional cells less clear when sorting the cells. These results suggested that, for some donors, increasing the stringency of the gating strategy would likely improve the purity of the isolated Treg population, and consequently, the proportion of stable Tregs obtained in the process. However, an increase in stringency would likely result in a decrease in yield. Described in this example is a stricter gating approach coupled with a modified expansion protocol. The modified expansion protocol enhances the proliferation of Tregs that is needed to overcome the reduced yield due to the increased stringency of stable Treg selection.Evaluation of Treg subpopulations for inclusion in process

[0250] The sorting strategies described in the above examples focus on the selection of naive Tregs. Given the need for a more stringent sorting strategy for certain donors, and that this strategy would likely exclude an increased proportion of naive Tregs, we evaluated whether the CD45RA+selection step to select naive Tregs was necessary in the context of a stricter sorting strategy, or whether more stringently selected antigen-experienced Tregs could be used. Naive Tregs are CD4+CD25+CD127' / |OWCD45RA+. Antigen-experienced Tregs are CD4+CD25highCD127' / l0WCD45'. Both of these populations are stable Tregs that will have a stable TSDR hypomethylation phenotype. To assess whether selection based on CD45RA is needed, antigen experienced (CD45RA-) and naive (CD45RA+) Tregs were isolated and compared for expansion capacity and stability.

[0251] Cells were prepared as described in Example 1 prior to the FACS sorting step. Cells then underwent two rounds of FACS sorting for debulk and purity. Both the CD45RA' and CD45RA+population were sorted on CD4, CD25, and CD127 at the debulk sort. Cells were first identified as CD4+and then a polygon gate was used to identify CD25+ / highCD127_ / l0WTreg set at 70% of a standard polygon gate for Treg identification and selection. The sort was therepeated and for CD45RA' cells, after the identification of the CD25+ / highCD127_ / l0WTreg population, CD45RA' cells were identified and selected. For CD45RA+cells, after the identification of the CD25+ / highCD127_ / l0WTreg population, CD45RA+cells were identified and selected. The percent naive (CD45RA+) Tregs in each population is shown below in Table 15.

[0252] At the conclusion of the sort, 3 groups were set up:(a) CD45RA+Tregs - Enriched CD45RA+Treg Fraction(b) CD45RA" Tregs - Enriched CD45RA' Treg Fraction(c) “Spike-In” Tregs - 80% Enriched CD45RA' Treg Fraction + 20% Enriched CD45RA+Treg FractionTable 15: % naive Treg in each condition prior to expansion

[0253] Cells were then expanded for 8 days in culture as described in Example 1. Fig. 12A- 12C show the population doubling level (PDL) and the TSDR hypomethylation following the expansion of the cells. As shown in FIG. 12A and FIG. 12C, the CD45RA+population showed a dramatic increase in expansion relative to the CD45RA' population along with increased TSDR hypomethylation. This suggests that CD45RA+naive Tregs are necessary for the expansion process because of the relative expansion capacity of CD45RA+naive Tregs compared to CD45RA' Tregs. Importantly, the “spike-in” sample shows that CD45RA+naive T regs are still able to expand effectively in the presence of a large excess of CD45RA' antigen- experienced Tregs.Development of a stricter gating strategy for isolation of stable Tregs

[0254] In light of the findings above, an improved gating strategy was developed for increasing the stringency of the sort while still capturing CD45RA+Tregs. In particular, the strategy identifies both antigen-experienced (CD4+CD25highCD127' / l0WCD45RA‘) and naive (CD4+CD25+CD127- / |OWCD45RA+) Tregs, while specifically excluding CD4+CD25+CD127’ / |OWCD45RA' non-Tregs. By separately identifying both antigen-experienced and naive Tregs, sufficient numbers of Tregs are obtained to produce a cell therapy product from a single leukopak, and the antigen experienced Tregs have high suppressive capacity, which suppresses any non-Treg both ex vivo and in vivo. In this approach, CD4+cells are identified, followed by identification of CD127' / |OWand CD25+(either sequentially or concurrently) combined with a critical “not” or L-shaped gate as the last step of the gating scheme, to select antigen-experienced (CD4+CD25highCD127' / l0WCD45RA‘) and naive (CD4+CD25+CD127‘ / |OWCD45RA+) Tregs, while specifically excluding the undesired CD4+CD25+CD127' / |OWCD45RA_cells based on CD25 and CD45RA levels. This gating approach is shown in FIGs 13A-FIG.13C.

[0255] To describe the gating strategy in further detail, CD25 enriched cells were stained with fluorescently labeled antibodies that recognize human CD4, CD127, CD45RA cell surface markers. Cells were already labeled with CD25 antibodies in the enrichment step of the process. The cells were then sorted twice using the gating scheme described below (using the same strategy in each step) using a FACS instrument. For the dot plot-based approach, shown in FIG. 13A-FIG.13C, in the first step of the gating process, CD4+T cells were first identified from all the live cellular events (FIG. 13A). In the second step, CD25+CD127' / l0WTreg cells were identified based on two-parameter flow plots, whereby spatial context of the Treg cell population could be visualized relative to another cell population (i.e. , Tconv cells), CD25 and CD127 levels were visualized and a polygon gate was drawn in the upper left quadrant to capture CD25+CD127' / |OWcells. That polygon gate was then shifted upwards to reduce the selected cell population to 70% of the original population, thereby increasing the stringency of selection (FIG. 13B). In the final step, the “not” or L-shaped gate was applied visualizing CD45RA and CD25 levels to select the naive Treg (CD4+CD25+CD127' / |OWCD45RA+) and antigen experienced Treg (CD4+CD25highCD127' / l0WCD45RA‘) cell population for expansion by excluding the CD25+CD45RA_cell population from the sorting gate (FIG. 13C). While both the antigen experienced and non-stable Treg populations are CD45RA they are distinguishable by CD25 levels, with the antigen-experienced Tregs being CD25highand non-stable Treg population being only CD25+but not high. Because naive Tregs are CD45RA+and CD25+(but not high), the threshold for determining whether CD45RA' cells are CD25+or CD25highis determined based on CD25 levels in the naive Treg population, with CD25highbeing defined as CD25 levels being above that expressed in the naive Treg population. The L-shaped gate is critical because it allows for the selection of two important populations of cells. CD25highCD45RA- antigen-experienced Tregs and CD25+CD45RA+naive Tregs are selected, while CD25+CD45RA' contaminating cells are excluded.New Expansion Process

[0256] Because the newly developed gating strategy is expected to select a smaller number of naive Tregs than the original process, the expansion process was modified to increase expansion of the Tregs.

[0257] The expansion protocol was altered to add TNF-alpha to the expansion media and to add a second anti-CD28 anti-CD3 stimulation. First, the effect of restimulation was evaluated at various timepoints following transduction. For this experiment, cells were preparedaccording to the methods described in Example 1 , except that cells were gated on CD4, CD25, and CD127 only and not CD45RA. Cells were then activated and transduced as in Example 1 , with a second activation step with anti-CD3 and anti-CD28 at days 6, 8, and 10 following the first activation step. It was found that this restimulation at Day 6 gave similar expansion to restimulation at days 7 or 8 but advantageously, restimulation at day 6 of the expansion process gave the greatest increase in expansion when cells were harvested at day 10, which, critically, does not require lengthening of the expansion process (FIG. 14) Next, the effect of adding TNF-alpha to the expansion protocol was assessed. Cells prepared according to the methods described in this example (including the L-shaped gate) were expanded as described in Example 1 with or without the second stimulation at day six and with or without 2500 lll / ml TNF-alpha to assess the effect of TNF-alpha on expansion of Tregs, either alone or in combination with a second stimulation. As is shown in FIG. 15, the addition of TNF-alpha throughout the expansion process in combination with a second stimulation with anti-CD3 and anti-CD28 at day 6 of the expansion process gave the largest increase in expansion. Although, each individual change to the process also increased expansion. As is shown in FIG. 16A- 16B, cells expanded in TNF-alpha in combination with a second stimulation showed the highest viability and purity at harvest relative to either process change alone or the original expansion protocol.Tregs generated with new gating and expansion process

[0258] The manufacturing process described in this Example was then applied to a repeat MS donor. The MS donor from Run 11 donated an additional leukopak. This leukopak was processed using the strict sorting strategy and with the improvements to the expansion conditions described above. Table 16 shows the properties of the cells following enrichment and prior to sorting, demonstrating that the properties of the cells from each run are highly similar prior to the sorting step.Table 16: Properties of MS donor cells prior to FACS sorting

[0259] The properties of the cells following sorting are shown in Table 17 below:Table 17: Properties of MS donor cells following FACS sorting

[0260] Table 18 below shows the properties of the manufactured cells. The % target dose was calculated as described above.Table 18: Properties of manufactured cells

[0261] FIG. 17 and Table 19 below shows TSDR hypomethylation over expansion for this donor using the healthy donor process and the L-shaped gate strategies described in this example.Table 19: TSDR of MS donor using original process and L-gate

[0262] The comparative data presented in this example shows that the strict gating strategy combined with the updated expansion process results in a highly stable Treg population and generates sufficient transduced Tregs to achieve the target dose.Example 4: Assessment of TSDR threshold levels

[0263] The threshold level of TSDR hypomethylation, representing the level of stable Tregs, necessary for a stable Treg cell product was investigated. We first asked what proportion of stable T reg at Day 0 to maintain a stable level of T reg cells throughout the expansion process, indicating that there was low to no outgrowth of contaminating conventional T cells or other cellular impurities. To do this we spiked conventional T cells into Tregs, each isolated from the same healthy donor, at various percentages on Day 0 and tracked the TSDR levels throughout the expansion process. TSDR hypomethylation levels and FOXP3+levels correlated with initial Treg purity and remained stable with no more than a 10% drop in TSDR hypomethylation throughout expansion when Treg purity was greater than 60% (FIG. 18A-FIG.18D). Next, the Treg purity of the expanded cell product was evaluated for the presence of conventional T cells and production of pro-inflammatory cytokines when activated with PMA and ionomycin. Cryopreserved cells from the harvest were thawed into fresh media and then stimulated with PMA and ionomycin for 4 hours at 37°C and 5% CO2. Intracellular cytokine (ICC) for IFN-y and IL-2 production was measured by flow cytometry. FIG. 19 shows the proportion of FoxP3+and FoxP3' cells expressing IL-2 and IFN-y post-activation with PMA and ionomycin. In the absence of conventional T cells, the 100% Treg product shows little to no IFN-y or IL-2 cytokines secreted, indicating that there are insignificant numbers of unstable Treg cells that expanded from the initial culture. As the initial Treg purity decreased, there was an increase in the proportion of T conventional cells secreting IFN-y and IL-2 cytokines, with the largest increases observed when the starting Treg purity was less than 90%. This increase is likely due to the increased Tcon:Treg ratio. Upon activation, some conventional T cells upregulate FoxP3 which is why IFN-gamma and IL-2 are produced from some FoxP3+cells. This suggests that a high TSDR level is critical to maintain a stable population of cells that are able to suppress contaminating conventional T cells.Example 5. Properties of T cells expressing an exogenous TCR

[0264] T cells transduced with lentiviral vectors encoding an exogenous TCR were tested in functional assays for their ability to express the exogenous TCR and to activate T cells in response to a GAD65 peptide when presented by HLA. T cells of this Example were transduced with one of three unique TCRs (termed ‘TCR-A’, ‘TCR-B,’ and TCR-C) that specifically bind to a GAD65 peptide when complexed with an MHC. Each of the TCRs were encoded by vector constructs that comprised an N-terminal TCR beta chain and a C-terminal TCR alpha chain with a linker domain comprising a GSG amino acid sequence followed by aP2A self-cleaving peptide. The TCR beta-GSG-P2A-TCR alpha fusion constructs were expressed under the EF-1a promoter.

[0265] TCR-A comprised a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 4 (which includes a CDR1 -alpha sequence comprising the amino acid sequence of SEQ ID NO: 1 , a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 3) and a TCR beta variable domain having the amino acid sequence of SEQ ID NO: 10 (which includes a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 7, a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3- beta sequence comprising the amino acid sequence of SEQ ID NO: 9).

[0266] TCR-B comprised a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 18 (which includes a CDR1 -alpha sequence comprising the amino acid sequence of SEQ ID NO: 15, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 17) and a TCR beta variable domain having the amino acid sequence of SEQ I D NO: 24 (which includes a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 21 , a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3- beta sequence comprising the amino acid sequence of SEQ ID NO: 23).

[0267] TCR-C comprised a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 32 (which includes a CDR1 -alpha sequence comprising the amino acid sequence of SEQ ID NO: 29, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 31 ) and a TCR beta variable domain having the amino acid sequence of SEQ I D NO: 38 (which includes a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 35, a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 36, and a CDR3- beta sequence comprising the amino acid sequence of SEQ ID NO: 37).

[0268] TCR-A and TCR-B specifically bind to a GAD65 (555-567) peptide comprising the amino acid sequence of NFFRMVISNPAAT (SEQ ID NO: 46) when complexed with an MHC (e.g., comprising HLA-DRB1*04:01). TCR-C specifically binds to a GAD65 (339-352) peptide comprising the amino acid sequence of TVYGAFDPLLAVAD (SEQ ID NO: 47) when complexed with an MHC (e.g., comprising HLA-DRB1*03:01).

[0269] TCR-null Jurkat cells were plated at 1 x105per well in a 96-well plate and transduced with lentiviral vectors encoding one of the three TCRs. Cells were then expanded, assessed for TCR expression, and collected for downstream assays to assess function. Surface expression of each of the TCRs was verified by flow cytometric detection of CD3 expression and a lentiviral GFP-reporter (FIG. 20A). All three of the tested TCRs demonstrated surface expression in the TCR-null Jurkat cells.

[0270] To test responsiveness of the TCRs to GAD65 peptides complexed with MHC, the transduced Jurkat cells were co-cultured with BLS cells and GAD65 peptide. Jurkat cells expressing either TCR-A or TCR-B were incubated with BLS DR4 cells from HLA-DRB1*04:01 donors and GAD65 (555-567) peptide. Jurkat cells expressing TCR-C were incubated with BLS DR3 cells from HLA-DRB1*03:01 donors and GAD65 (339-352) peptide. Peptide was titrated 4-fold at the concentrations shown. Flow cytometry was used to measure upregulation of CD69 surface expression (FIG. 20B). As shown in FIG. 20C, for experiments performed using 10 pM GAD65 peptide concentration, activation was measured after 18 hours by determining the percentage of cells that expressed CD69 (relative to total cells that expressed GFP) and nuclear factor of activator T cells (NFAT)-driven luciferase expression from a NFAT luciferase reporter in the Jurkat cells. The percentage of cells that expressed phospho-ERK cells (relative to total cells that expressed GFP) was measured after 15 minutes. For all three indicators, activation in response to a control CLIP (87-101) peptide ( / .e., a peptide that does not specifically bind to the GAD65-specific TCR-A, TCR-B, or TCR-C) was also determined. A dose-responsive experiment was also performed for TCR-A and TCR-B to show that increasing concentrations of antigen leads to an increase in expression of CD69 (FIG. 20D). In Figure 20D, TCR1 is TCR-B and TCR2 is TCR-A. This data demonstrates that all three TCRs are expressed at the cell surface and are capable of activating TCR-null Jurkat cells. Jurkat cells in this example were also transduced with 6 additional TCRs specific for either GAD65 or proinsulin in the same configuration that did not show activity in the activation assays shown.

[0271] TCR-A, TCR-B, and TCR-C were independently expressed in conventional CD4+ T cells to assess the ability of the TCRs to be expressed at the cell surface and to activate T cells in the presence of endogenous TCRs.

[0272] Conventional CD4+T cells and CD14+monocytes were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors. 5 x105 CD4 conventional T cells were seeded in a 24-well plate, activated 3:1 with anti-CD3 / CD28 beads (Dyna beads; ThermoFisher) and transduced with lentivirus expressing GAD65-specific TCRs (TCR-A, TCR-B, or TCR-C) in the presence of IL-7 / IL-2 and lentiboost (Sirion Biotech) after 24 hours. Four days after transduction, the Dyna beads were removed and the T cells were further expanded for 8 days.

[0273] Expression of each of the GAD65-specific TCRs was measured by FACs as determined by CD3 cell surface expression and expression of a GFP tag included in the lentiviral vectors (FIG. 21A).

[0274] To test activation of the conventional T cells in response to GAD65 peptides complexed with MHC, CD69 expression from the conventional T cells expressing TCR-B or TCR-C was assessed by FACS (FIG. 21 B). T cells were incubated with donor-matched monocytes and 10 pM GAD65 peptide for 18 hours prior to measurement of CD69 levels.Cells expressing TCR-B were incubated with GAD65 (555-567) peptide; cells expressing TCR-C were incubated with GAD65 (339-352) peptide. Activation in response to the control CLIP (87-101) peptide was also measured.

[0275] To test proliferative capacity, conventional T cells were incubated with donor-matched monocytes and 10 pM GAD65 for 5 days. The percentage of cells that expressed CD71 and had low CTV expression (relative to total cells that expressed GFP) was measured by FACS (FIG. 21 C). Cells expressing TCR-B were incubated with GAD65 (555-567) peptide; cells expressing TCR-C were incubated with GAD65 (339-352) peptide. Proliferation in response to the control CLIP (87-101) peptide was also measured.

[0276] These data demonstrate that all three of the GAD65-specific TCRs (TCR-A, TCR-B, and TCR-C) are expressed and show activation in response to stimulation with H LA- complexed GAD65 peptide. Moreover, these data demonstrate that the GAD65-specific TCRs show increased proliferative capacity in CD4+T cells in response to with HLA-complexed GAD65 peptide.Example 6: Range of responsiveness of T cells expressing an exogenous TCR

[0277] T cells transduced with lentiviral vectors encoding an exogenous TCR were tested in functional assays for their ability to express the exogenous TCR and to activate T cells in response to a proinsulin peptide when presented by HLA. T cells of this Example were transduced with one of two TCRs (termed ‘TCR-D’ and ‘TCR-E) that specifically bind to a proinsulin peptide when complexed with an MHC. Each of the TCRs were encoded by vector constructs that comprised an N-terminal TCR beta chain and a C-terminal TCR alpha chain with a linker domain comprising a GSG amino acid sequence followed by a P2A self-cleaving peptide. The TCR beta-GSG-P2A-TCR alpha fusion constructs were expressed under the EF-1a promoter.

[0278] TCR-D comprised a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 56 (which includes a CDR1 -alpha sequence comprising the amino acid sequence of SEQ ID NO: 53, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 54, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 55) and a TCR beta variable domain having the amino acid sequence of SEQ ID NO: 62 (which includes a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 59, a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 60, and a CDR3- beta sequence comprising the amino acid sequence of SEQ ID NO: 61).

[0279] TCR-E comprised a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 69 (which includes a CDR1 -alpha sequence comprising the amino acid sequence of SEQ ID NO: 66, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 67, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO:68) and a TCR beta variable domain having the amino acid sequence of SEQ I D NO: 75 (which includes a CDR1-beta sequence comprising the amino acid sequence of SEQ ID NO: 72, a CDR2-beta sequence comprising the amino acid sequence of SEQ ID NO: 73, and a CDR3- beta sequence comprising the amino acid sequence of SEQ ID NO: 74).

[0280] TCR-D specifically binds to a proinsulin (94-110) peptide comprising the amino acid sequence of QCCTSICSLYQLENYCN (SEQ ID NO: 118) when complexed with an MHC (e.g., comprising HLA-DRB1*04:01). TCR-E specifically binds to a proinsulin (76-86) peptide comprising the amino acid sequence of SLQPLALEGSL (SEQ ID NO: 119) when complexed with an MHC (e.g., comprising HLA-DRB1*04:01).

[0281] TCR-null Jurkat cells were plated at 1 x105per well in a 96-well plate and transduced with lentiviral vectors encoding TCR-D or TCR-E. Cells were then expanded and collected for downstream assays to assess function.

[0282] To test responsiveness of the TCRs to proinsulin peptides complexed with MHC, the transduced Jurkat cells were co-cultured with BLS cells and proinsulin peptides. Jurkat cells expressing either TCR-D or TCR-E were incubated with BLS DR4 cells from HLA-DRB1*04:01 donors and proinsulin peptide (proinsulin (94-110) for cells expressing TCR-D or proinsulin (76-86) for cells expressing TCR-E). Peptide was titrated 4-fold at the concentrations shown. Flow cytometry was used to measure upregulation of CD69 surface expression (FIG. 22A). In FIG. 22A, TCR3 is TCR-D and TCR4 is TCR-E. As shown in FIG. 22B, for experiments performed using 10 pM proinsulin peptide concentration, activation was measured after 18 hours by determining the percentage of cells that expressed CD69 (relative to total cells that expressed GFP). Activation in response to a control CLIP (87-101) peptide (i.e. , a peptide that does not specifically bind to the proinsulin specific TCR-D or TCR-E) was also determined. These data demonstrate that the proinsulin-specific TCRs are expressed at the cell surface and are capable of activating TCR-null Jurkat cells.Example 7.- TCRs binding preproinsulin (73-90) - Manufacturing Data

[0283] TCRs targeting preproinsulin 73-90 (SEQ ID NO: 144) were identified and characterized as described in PCT publication PCT / US23 / 84859, which is incorporated herein by reference in its entirety. TCR-I comprises a TCR alpha variable domain having the amino acid sequence of SEQ ID NO: 128 (which includes a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 125, a CDR2-alpha sequence comprising the amino acid sequence of SEQ ID NO: 126, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 127), a disulfide-modified constant region in the TCRa chain (SEQ ID NO: 130), a TCR beta variable domain comprising SEQ ID NO: 136 (which includes which includes a CDR1-alpha sequence comprising the amino acid sequence of SEQ ID NO: 133, a CDR2-alpha sequence comprising the amino acid sequence of SEQ I D NO: 134, and a CDR3-alpha sequence comprising the amino acid sequence of SEQ ID NO: 135), and a disulfide- modified constant region in the TCRp chain (SEQ ID NO: 138). TCR-I is identified as TCR-A DS in PCT publication PCT / US23 / 84859, and was identified as a TCR with maximum activity towards preproinsulin 73-90 with minimal cross-reactivity. The remainder of the examples characterize Tregs generated from subjects having T1 D and engineered TCR-I targeting preproinsulin 73-90. Tregs from three T1 D donors were isolated, transduced, and expanded using the optimized process described in Example 3.

[0284] Table 20 shows the viability and purity (Treg and naive Treg) of the cells at input, following the first FACS (debulk) sort (post-primary) and following the second FACS (purity) sort (post-secondary). Table 21 shows the percentage step recovery (Treg and naive Treg) following the first FACS (debulk) sort (post-primary) and following the second FACS (purity) sort (post-secondary). Treg are identified as being CD4+CD25+ / hiCD127' / l0Wby FACS and naive Tregs are identified as being CD4+CD25+ / hiCD127' / l0WCD45RA+Table 20: Summary of Viability and Purity Data for T1D Donor CellsTable 21 : Percent Recovery of Treg and nTreg

[0285] The population doubling level and % viability during expansion is shown for each T1 D donor in Table 22.Table 22: Cell Expansion

[0286] As shown in Table 23, the average Treg purity of cells seeded on Day 0 was 96.15%. Drug product purity was stable through culture, as indicated by an average Treg purity post expansion of 96.84% and an average Day 10 TSDR of 106.8%. These data highlight the process’s capability in producing highly pure and stable Treg with T1 D donor cells.Table 23. Properties of manufactured cells from T1D donors - Summary TableExample 8: Characterization of TCR-engineered Tregs from T1D donorsAntigen Stimulated Activation of TCR-engineered healthy donor and T1D donor cells

[0287] The capability of TCR-Tregs to be stimulated in an antigen specific manner was compared between healthy and T1 D donor cells engineered with TCR I targeting PPI73-90. The TCR used in this example was GFP labeled to allow detection of TCR expression by flow cytometry.

[0288] Three representative batches of Tregs from 2 healthy and 2 T1 D donors, engineered with TCR-I (disulfide modified) and manufactured using the process described in Example 3, were cultured with PBMCs that were CTV-labeled and Mitomycin C-treated in the presence of titrated concentration of PPI73-90 peptide (1 E-06 - 100 pM) and recombinant human IL-2 (1000 lll / mL). After 1 day of co-culture, cells were collected for immunostaining for activation markers by FACS (CD69, CD71 , and 4-1 BB). After 3 days of co-culture, culture supernatants were collected to measure the production of anti-inflammatory cytokine IL-10.

[0289] TCR-Tregs were activated by antigen PPI73-90 stimulation presented by PBMCs in vitro. In response to antigen stimulation, TCR-Tregs upregulated expression of activation markers CD69, CD71 , and 4-1 BB and increased IL- 10 secretion in an antigen-dose dependent manner. The average EC50 of the antigen dose response curves is shown in Table 24 below and was 0.06 pM, 0.02 pM, and 0.02 pM for the expression of activation markers CD69, CD71 , and 4- 1 BB, respectively, and 0.28 pM for IL- 10 production. No significant difference was seen between TCR-Treg generated from healthy and T1 D donors.Table 24: Summary of Antigen Dose-Dependent Activation

[0290] TCR VP13.6 was used to gate cells into TCR-Tregs (contained in the TCR Vpi3.6+subset) and non-TCR-engineered Tregs (contained in the TCR Vpi3.6_). In comparison to the TCR Vpi3.6_subset, antigen PPI 73-90 stimulation specifically upregulated the expression of cell activation marker CD69, CD71 , and 4-1 BB in the TCR Vpi3.6+subset (Table 25).Table 25: Summary of Expression of CD69, CD71, 4-1 BB in Treg SubpopulationsSuppressive Capacity of Engineered Treg from T1 D Donors

[0291] The suppressive capacity of human Tregs from healthy and T1 D donors engineered with TCR-I (disulfide modified) and manufactured using the process described in Example 3 was evaluated.

[0292] TCR-engineered human Tregs and TCR-engineered Tconv cells were produced from healthy and T1 D donors. TCR-Tconv cells were used as responder cells in this study and engineered with a distinct TCR also targeting PPI73-90. CTV labeled TCR-Tconv cells were cocultured with TCR-Tregs at different Treg:Tconv ratios (2:1 to 1 :8) and stimulated by antigen peptide PPI73-90 presented by autologous HLA-DRB1*04:01 complex expressing PBMCs as antigen presenting cells (APCs). After four days of culture, cell culture supernatants were collected for cytokine analysis, and the suppressive ability of TCR-Tregs was evaluated by measuring the inhibition of proliferation dye dilution and the expression of activation markers (CD25 and CD71) on TCR-Tconv cells, using multicolor flow cytometry.

[0293] Treg suppression of Tconv cell proliferation, Tconv cell activation, and IFN-y secretion were calculated as follows:

[0294] Treg suppression of Tconv cell proliferation was calculated as follows: 100

[0295] Treg suppression of Tconv cell activation was calculated as follows:100 x[geoMFI of activated Tconv cells without Tregs] — [geoMFI of activated Tconv cells with Tregs] [geoMFI of activated Tconv cells without Tregs]

[0296] Treg suppression of IFN-y release was calculated as follows:100

[0297] TCR-Tregs suppressed the proliferation and activation of PPI 73-90- reactive Tconv cells in vitro in response to peptide PPI73-90 stimulation presented by HLA-DRB1*04:01 complexexpressing APCs within PBMCs by >72.8 % at ratio of 2:1 Tregs:Tconv. TCR-Tregs decreased the levels of proinflammatory cytokine IFN-y secreted by antigen-activated responder Tconv cells in a Treg dose-dependent manner at different Treg:Tconv ratios with a maximuminhibition of 88.8 % and 84.4% at a 2:1 and 1 :1 ratio, respectively, of Tregs to Tconv. TCR- Tregs produced from both healthy and T1 D donors showed comparable suppression capabilities. They inhibited the proliferation, activation, and proinflammatory cytokine production by responder TCR-Tconv, regardless of whether these cells were produced from healthy or T1 D donors (Table 26).Table 26: Average Percentages of Responder Tconv Activity Supressed by TCR-TregsData is presented as Mean±SEM; n=3Bystander Suppressive Capacity of Engineered Treg from T1 D Donors

[0298] The bystander suppressive ability of the TCR-Treg described above from T1 D donors was evaluated by measuring the activation status (CD71 and CD25) and proinflammatory cytokine (IFN-y) production of TCR-Tconv cells labeled with CTV, transduced with a different islet antigen reactive TCR specific for GAD6555s-567. Autologous TCR-Tconv cells were cultured with the TCR-Tregs at different Treg:Tconv ratios (2:1 to 1 :16) under conditions for maximal stimulation by their respective antigen peptides, GAD65555-567 (1 pM) and PPl73-9o (1 pM), both presented by autologous HLA-DRB1* 04:01 complex expressing PBMCs.

[0299] Activation of TCR-Tconv cells (Tconv cells responsive to an alternative islet peptide, GAD65s55-567, in the context of HLA-DRB1*04:01 peptide) and secretion of IFN-y weresuppressed in an TCR-Treg dose-dependent manner at different Treg:Tconv ratios. At 2:1 Treg to Tconv ratio, antigen-stimulated TCR-Tregs suppressed the expression of activation markers CD71 and CD25 in TCR-Tconv cells by 28.8 ± 6.9 % and 38.9 ± 4.0 %, respectively, and decreased the production of inflammatory cytokine IFN-y by 62.0 ± 5.1 % from TCR-Tconv cells (Table 27).

[0300] This data shows that TCR-Tregs from a T1 D donor Tregs functionally suppressed the activation and inflammatory cytokine production of engineered Tconv cells, which responded to a potent human islet antigen GAD65s55-567 in a bystander manner.Table 27: Average percentage of TCR-Tcon suppression by TCR-TregData is presented as Mean±SD: n=2In vivo suppression of inflammation by TCR-Treg from T1D donors

[0301] In vivo models of T1 D are extremely limited, especially in the context of TCR cell therapies due to the need for MHC compatibility. In light of this, a GvHD model was used to test the ability of PPI targeting TCR-Treg to suppress inflammation generally. The TCR-Treg used were those used throughout this example (Treg from T1 D donors produced according to Example 3 and engineered with TCR I disulfide modified).

[0302] Xenoreactivity of human donor PBMCs in immune deficient NOD.Cg-Pr cfcscid / L2rstm1Wjl / SzJ (NSG) mice was used to induce an inflammatory environment. Exposure and impact of TCR-Treg on weight loss in the in vivo inflammatory GvHD model was measured.

[0303] Whole body irradiation at 50R was administered to NSG mice 1 day prior to study initiation. Donor HLA-DRB1*04:01+, HLA-A2' PBMCs (5E06 cells / animal) were administered on Day 0 to establish inflammation due to CD4+and CD8+T cell recognition of murine pMHC complexes. HLA-DRB1*04:01+, HLA-A2+TCR-Treg (5E06 cells / animal) were delivered intravenously (IV) into the mice (n = 14 / group) and a subcutaneous (SC) administration of PPI73-90 peptide (250 pg / animal) in incomplete Freund’s adjuvant (I FA). All animals received an intraperitoneal (IP) administration of human recombinant IL-2 (hulL-2) at 10,000 III every other day (Q2D) from Day 0 - 2 to enable persistence of the TCR-Treg. The study was terminated at day 19. The set-up of the study is shown in Table 28.Table 28: xGvHD Study Design and Treatment Groups

[0304] This model was not run as a conventional survival model, as the intent of this study was to evaluate exposure and pharmacodynamics of TCR-Treg in an inflammatory mouse model. For GvHD scoring (on hunching posture, fur ruffling, activity, anemia, and weight loss), any individual parameter score of 2 or sum clinical score >5 constituted a humane endpoint resulting in euthanasia of the mouse. All groups were scored 3x / week starting on Day 10 by blinded scorer. At study termination, cells were counted and stained for flow cytometric analysis and plasma sample was analyzed for human cytokines IL-10, IFN-y, IL-2 and TNF- a, using the MSD multiplexing platform.

[0305] TCR-Treg persisted in the mice 7-10 days but declined thereafter (FIG. 23). The pharmacodynamic effect of TCR-Treg was durable through day 19. NSG mice treated with TCR-Treg showed reduced body weight loss, maintained lower GvHD score, and demonstrated a reduction in engraftment in the colon, spleen and liver as well as a reduction in the level of activated CD4 and CD8 T cells compared to the negative control PBMC treated animals (Table 29).Table 29: Summary of NSG Mice Treated with TCR-Treg

[0306] On Day 19 post-cell administration the level of the proinflammatory cytokine IFN-y was significantly lower in the TCR-Treg treated animals compared to the PBMC negative control animals, with a more significant reduction in IFNy in the TCR-Treg + peptide treated group. Noother measured cytokines were detected. Flow cytometry analysis of human T cells, cytotoxic T cells, and helper T cells in the blood, spleen, colon, and liver showed significantly lower numbers of hCD45+ cells in the TCR-Treg treated animals compared to the PBMC control animals. Moreover, animals dosed with TCR-Treg + PPI 73-90 demonstrated a further reduction in CD8+ T cell numbers in the blood, colon, and liver as well as a reduction in activated HLA- DR+CD4+T cells in the colon and liver.

[0307] This study showed that TCR-Treg were present for 7-10 days in the blood. Inflammation-induced weight loss was abrogated by the administration of TCR-Treg in this model, suggesting that TCR-Treg were broadly activated via its transgenic TCR interacting with both human and mouse peptide:MHC complexes expressed on donor and mouse cells, leading to immune suppressive function. In response to peptide activation, TCR-Treg demonstrated a significantly greater impact on reducing human CD45+ engraftment in the colon at the terminal day 19 timepoint, as well as significantly lower concentrations of IFNy in the plasma.

[0308] This study demonstrates that TCR-Treg exhibit robust functional suppression in an inflammatory environment with no contribution to exacerbation disease. Moreover, we observed durable pharmacodynamic effects of TCR-Treg beyond the window of exposure.

Claims

CLAIMS1. An isolated population of cells comprising stable CD4+ T regulatory cells (T regs) derived from a subject having type 1 diabetes (T1 D), wherein at least 80% of the cells are stable CD4+ T regs comprising a hypomethylated T cell specific demethylated region (TSDR) at the FOXP3 locus, and wherein the cells comprise an exogenous human T cell receptor (TCR), or a polynucleotide encoding the same, that binds specifically to a target peptide complexed with a major histocompatibility complex (MHC).

2. The isolated population of claim 1 , wherein the target peptide is a peptide of GAD65.

3. The isolated population of claim 2, wherein the exogenous human TCR is a recombinant TCR comprising a TCR alpha (TCRa) chain polypeptide and a TCR beta (TCRP) chain polypeptide, wherein the TCR alpha chain comprises an alpha chain variable domain comprising an alpha CDR1 , and alpha CDR2, and an alpha CDR3 and wherein the TCR beta chain comprises a beta chain variable domain comprising a beta CDR1 , a beta CDR2, and a beta CDR3; wherein a) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 1 , the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 2, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3 and the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 7, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 8, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 9; b) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 15, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 16, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 17; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 21 , the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 22, and beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 23; or c) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 29, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 30, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 31 ; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 35, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 36, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 37;and wherein the TCR binds to a glutamic acid decarboxylase 65 (GAD65) peptide complexed with a major histocompatibility complex (MHC).

4. The isolated population of claims 2-3, wherein a) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 4; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 10; b) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 24; or c) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 32; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 38.

5. The isolated population of claims 2-4, wherein a) the TCRa chain comprises or consists of SEQ ID NO: 4; and wherein the TCRp chain comprises or consists of SEQ ID NO: 10; b) the TCRa chain comprises or consists of SEQ ID NO: 18; and wherein the TCRp chain comprises or consists of SEQ ID NO: 24; or c) the TCRa chain comprises or consists of SEQ ID NO: 32; and wherein the TCRp chain comprises or consists of SEQ ID NO: 38.

6. The isolated population of any of claims 2-5, wherein the TCR binds to amino acids 339-352 or 555-567 of the GAD65 peptide when it is complexed with the MHC.

7. The isolated population of claim 1 , wherein the target peptide is a peptide of proinsulin or preproinsulin.

8. The isolated population of claim 7, wherein the exogenous human TCR is a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain polypeptide and a TCR beta (TCRP) chain polypeptide, wherein the TCR alpha chain comprises an alpha chain variable domain comprising an alpha CDR1 , and alpha CDR2, and an alpha CDR3 and wherein the TCR beta chaincomprises a beta chain variable domain comprising a beta CDR1 , a beta CDR2, and a beta CDR3; wherein a) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 53, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 54, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 55; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 59, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 60, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 61 ; b) the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 66, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 67, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 68; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 72, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 73, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 74; and wherein the TCR binds specifically to a proinsulin peptide complexed with a major histocompatibility complex (MHC).

9. The isolated population of claims 7-8, wherein a) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 56; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 62; b) the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 69; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 75.

10. The isolated population of any of claims 7-9, wherein a) the TCRa chain comprises or consists of SEQ ID NO: 56; and wherein the TCRp chain comprises or consists of SEQ ID NO: 62; b) the TCRa chain comprises or consists of SEQ ID NO: 69; and wherein TCRp chain comprises or consists of SEQ ID NO: 75.

11. The isolated population of any of claims 7-10, wherein the TCR binds specifically to amino acids 94-110 or 76-86 of the proinsulin peptide when it is complexed with the MHC.

12. The isolated population of claim 7, wherein the TCR binds specifically to amino acids 73-90 of preproinsulin.

13. The isolated population of claim 12, wherein the MHC molecule comprises an HLA- DRB1*04:01 molecule.

14. The isolated population of claim 12 or 13, wherein the exogenous human TCR is a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain polypeptide and a TCR beta (TCRP) chain polypeptide, wherein the TCR alpha chain comprises an alpha chain variable domain comprising an alpha CDR1 , and alpha CDR2, and an alpha CDR3 and wherein the TCR beta chain comprises a beta chain variable domain comprising a beta CDR1 , a beta CDR2, and a beta CDR3; wherein the alpha CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 125, the alpha CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 126, and the alpha CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 127; the beta CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 133, the beta CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 134, and the beta CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 135.

15. The isolated population of claim 14, wherein the TCRa variable domain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 128; and wherein the TCRp variable domain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 136.

16. The isolated population of claim 12-15, wherein the TCRa variable domain comprises or consists of sequence of SEQ ID NO: 128; and wherein the TCRp variable domain comprises or consists of SEQ ID NO: 136.

17. The isolated population of claims 12-16, wherein the TCRa chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 130; and wherein the TCRp chain comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 138.

18. The isolated population of any of claims 12-16, wherein the TCRa chain comprises or consists of SEQ ID NO: 130; and wherein the TCRp chain comprises or consists of SEQ ID NO: 138.

19. The isolated population of claims 1-18, wherein the TCRa chain and the TCRp chain are encoded as two separate polypeptide chains.

20. The isolated population of claims 1-18, wherein the TCRa chain and the TCRp chain are encoded as a single polypeptide chain.

21. The isolated population of claim 20, wherein the polypeptide comprises an N-terminal TCRp chain and a C-terminal TCRa chain.

22. The isolated population of claims 20 or 21 , wherein the polypeptide comprises a selfcleaving peptide sequence positioned between the TCRa chain and the TCRp chain.

23. The isolated population of claim 22, wherein the self-cleaving peptide sequence is a 2A peptide sequence.

24. The isolated population of claim 23, wherein the 2A peptide sequence is a P2A, E2A, F2A, or T2A peptide sequence.

25. The isolated population of any of claims 1-24, wherein the subject has residual beta cell function.

26. The isolated population of any one of claims 1-25, wherein the stable CD4+ T regs do not express a FOXP3 protein from an engineered FOXP3 locus.

27. The isolated population of any one of claims 1-26, wherein the TSDR is the CNS2 region of FOXP3.

28. The isolated population of any one of claims 1-27, wherein the MHC is MHC Class I or MHC Class II.

29. The isolated population of any one of claims 1-28, 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 an endogenous F0XP3 locus.

30. The isolated population of any one of claims 1-29, wherein at least 80%, at least 85%, at least 90%, or at least 95% of the CD4+ T regs cells are CD4+CD25+CD127' / |OW.31 . The isolated population of any one of claims 1-30, wherein at least 80%, at least 85%, at least 90%, or at least 95% of the CD4+ T regs cells are CD4+CD25+CD127' / |OWFOXP3+.

32. The isolated population of any one of claims 1-31 , comprising at least 4x107stable CD4+ T regs.

33. The isolated population of claim 32, comprising 4x107to 1x1010stable CD4+ T regs.

34. The isolated population of any one of claims 1-33, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the cells in the isolated population are conventional CD4+ T cells.

35. The isolated population of claim 34, 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.

36. The isolated population of any one of claims 1-35, wherein the ratio of stable CD4+ T regs to conventional T cells in the isolated population is at least 50:1 , at least 60:1 , at least 70:1 , at least 80:1 , at least 90:1 , at least 100:1 , at least 500:1 , at least 1000:1 , or at least 10000:1.

37. The isolated population of any one of claims 1-36, wherein less than 2%, less than 1 %, less than 0.5%, less than 0.1%, or less than 0.01% of the cells of the isolated population are CD8+T cells.

38. The isolated population of claim 37, wherein the isolated population does not comprise a detectable percentage of CD8+ T cells by fluorescence activated cell sorting (FACS).

39. The isolated population of any one of claims 1-38, wherein at least 10% of the cells express the exogenous human TCR.

40. The isolated population of claim 39, 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.

41. The isolated population of claims 1-40, wherein the MHC phenotype is HLA- DRB1*04:01 or HLA-DRB1*03:01.

42. A method of treating type 1 diabetes (T1 D) in a subject comprising administering to the subject the isolated population of any one of claims 1-41 or a pharmaceutical composition thereof.

43. The method of claim 42, wherein the subject is a human male subject.

44. The method of claim 42, wherein the subject is a human female subject.

45. The method of any one of claims 42-44, wherein the administering comprises intravenous administration.

46. The method of claim 45, wherein the administering comprises one or more infusions.

47. The method of any of claims 42-46, wherein the administration of the isolated population, the pharmaceutical composition, or the cell or population of cells is administered in an effective amount to alleviate one or more symptom of type 1 diabetes.

48. The method of any one of claims 42-47, wherein the cells of the isolated population are autologous relative to the subject.

49. A method of producing a population of cells comprising stable CD4+ T regulatory cells (T regs) comprising: a. removing CD8+cells, CD19+cells, and optionally CD14+cells from a biological sample obtained from a subject having type 1 diabetes (T1 D) to produce a depleted biological sample; b. enriching the depleted biological sample for CD25+ cells to produce an enriched population; c. isolating CD4+CD25+CD127_ / |OWcells from the enriched population; d. expanding the enriched population to produce an expanded enriched population of cells; and e. quantifying the methylation status of a T cell specific demethylated region (TSDR) at the FOXP3 locus in the population of cells, wherein at least 80% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus, thereby producing a population of cells comprising stable CD4+T regs.

50. The method of claim 49, 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.

51. The method of claim 49 or 50, wherein the isolated population is selected for therapeutic use if the percentage of cells comprising a hypomethylated TSDR at the FOXP3 locus is 80% or greater.

52. The method of any one of claims 49-51 , wherein step (c) is performed at least twice.

53. The method of any one of claims 49-52, further comprising activating the population of cells of step (c).

54. The method of claim 53, wherein the activating step comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28 antibody.

55. The method of claim 53 or 54, wherein the activating step is performed at least twice.

56. The method of claim 55, wherein the second activating step is performed between 4 and 8 days after the first activating step.

57. The method of claim 54 or 55, wherein the cells are expanded in a culture media comprising IL-2 and TNFa.

58. The method of any one of claims 49-57, wherein the activating and expanding steps comprise culturing the population of cells for at least 5, 6, 7, 8, 9, 10, 11 , or 12 days.

59. The method of any one of claims 49-58, wherein the activating and expanding steps comprise culturing the population of cells for no more than 15, 14, 13, or 12 days.

60. A method of producing a population of cells comprising engineered stable CD4+ T regs comprising: a. removing CD8+cells, CD19+cells, and optionally CD14+cells from a biological sample obtained from a subject having type 1 diabetes (T1 D) to produce a depleted biological sample; b. enriching the depleted biological sample for CD25+cells to produce an enriched population; c. isolating CD4+CD25+CD127_ / |OWcells from the enriched population; d. delivering a vector comprising a nucleic acid encoding an exogenous human T cell receptor (TCR) to the isolated population of (c) to produce a population of engineered cells; e. expanding the population of engineered cells to produce an expanded population of engineered cells; and f. quantifying the methylation status of a T cell specific demethylated region (TSDR) at the FOXP3 locus in the expanded population of engineered cells, wherein at least 80% of the cells are stable CD4+T regs comprising a hypomethylated TSDR at the FOXP3 locus.

61. The method of claim 60, 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.

62. The method of claim 60 or 61 , wherein step (c) is performed at least twice.

63. The method of any one of claims 60-62, wherein the isolated population is selected for therapeutic use if the percentage of cells comprising a hypomethylated TSDR at the FOXP3 locus is 80%, 85%, 90%, 95%, or greater.

64. The method of any one of claims 60-63, further comprising activating the population of engineered cells step (c).

65. The method of claim 64, wherein the activating step comprises culturing the population of cells with an anti-CD3 antibody and an anti-CD28 antibody.

66. The method of claim 64 or 65, wherein the activating step is performed at least twice.

67. The method of claim 66, wherein the second activating step is performed between 4 and 8 days after the first activating step.

68. The method of any one of claims 60-66, wherein the cells are expanded in a culture media comprising IL-2 and TNFa.

69. The method of any one of claims 60-68, wherein the expanded population comprises at least at least 1x107engineered stable CD4+ T regs.

70. The method of any one of claims 60-69, wherein the methylation status of the TSDR in step (f) is assessed at least 24 hours after a cryopreservation freeze-thaw cycle.

71. The method of any one of claims 60-70, wherein the percentage of CD4+CD25+ / highCD127' / l0Wregulatory T cells is assessed at least 24 hours after a cryopreservation freeze-thaw cycle.

72. The method of any one of claims 60-71 , 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.

73. The method of any one of claims 60-72, wherein 2% or less of the population of cells are CD8+ cells.

74. The method of any one of claims 60-73, wherein 20% or less of the population of cells are conventional T cells.

75. The method of any one of claims 60-74, wherein the population of cells in step (c) comprises CD25highCD45RA' cells and CD25+CD45RA+cells and does not comprise CD25+CD45RA- cells.

76. The method of claim 60-75, wherein the isolating of (c) comprises:i. identifying a first subpopulation of CD4+cells from the enriched population of step (b); ii. identifying a second subpopulation of CD25+ / highCD127_ / l0Wcells from the first subpopulation; and iii. selecting CD25highCD45RA' and CD25+ / highCD45RA+cells from the second subpopulation for isolation and excluding CD25+CD45RA; iv. thereby isolating the population of CD4+T regs.

77. The method of any one of claims 60-76, wherein the TSDR is the CNS2 region of FOXP3.

78. The method of any one of claims 60-77, wherein the subject is a human male subject.

79. The method of any one of claims 60-77, wherein the subject is a human female subject.

80. The method of any one of claims 60-79, wherein the subject has residual beta cell function.81 . The method of any one of claims 60-80, wherein the delivering the vector comprising a nucleic acid encoding the exogenous human TCR comprises transducing the cell population with the vector.

82. The isolated population of any one of claims 1-41 , wherein the polynucleotide encoding the exogenous TCR comprises a promoter operably linked to a coding sequence encoding the recombinant TCR, optionally wherein the promoter is an EF-1 alpha promoter or an MND promoter.

83. The isolated population of claim 82, wherein the polynucleotide further comprises an enhancer element, optionally an optimized post-transcriptional regulatory element (oPRE) or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), further optionally WPRE-mut6.

84. The isolated population of claim 82 or 83, wherein the polynucleotide encoding the exogenous TCR is comprised in a vector.

85. The isolated population of claim 84, wherein the vector is a viral vector.

86. The isolated population of claim 85, wherein the viral vector is a lentiviral vector.

87. The isolated population of claim 86, wherein the lentiviral vector is a VSVg pseudotyped, self-inactivating, 3rd generation lentiviral vector.

88. The isolated population of any one of claims 82-87, wherein the coding sequence is codon-optimized.

89. A composition comprising the isolated population of claim 88 and a cryopreservative.

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