Patient selection and treatment for multiple sclerosis

Abstract

The disclosure relates to methods of administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB1*11:04, HLA-DRB1*04:02, HLA-DRB1*04:04, HLA-DRB1*15:01, HLA-DRB1*15:02, or HLA-DRB1*15:03 wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB1*11:04 allele, an HLA-DRB1*04:02 allele, an HLA-DRB1*04:04 allele, HLA-DRB1*15:01 allele, or an HLA-DRB1*15:03 allele.

Description

PATIENT SELECTION AND TREATMENT FOR MULTIPLE SCLEROSIS CROSS-REFERECE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 552,941, filed February 13, 2024; and U.S. Provisional Application 63 / 685,474 filed August 21, 2024, and the contents of each are incorporated herein by reference in their entireties.REFERENCE TO SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (ABTH_008_02WO_SeqList_ST26.xml; Size: 122,750 bytes; and Date of Creation: February 7, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0003] While the nature of susceptibility to multiple sclerosis (MS) is complex and involves both environmental and genetic factors, certain alleles of the human leukocyte antigens inside the major histocompatibility complex are associated with MS prevalence. The human DRB 1*15:01 allele is strongly associated with MS, and there are estimated to be at least 45,000 patients in the U.S. with this allele who are affected with MS. Regulatory T cells have potential for the treatment of MS and other autoimmune diseases by selectively targeting diseased cell types and tissues, generating a local immune response via an antigen-specific mechanism.SUMMARY

[0004] Some aspects of the disclosure relate to a method comprising administering to a subject a regulatory T cell (Treg) comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an major histocompatibility complex (MHC) Class II comprising HLA-DRB1*11 :O4, wherein the subject has been diagnosed with multiple sclerosis (MS) and has been identified as having an HLA- DRB1*11 :O4 allele.

[0005] It should be understood that administration of a Treg to a subject encompasses administration of a composition (e.g., a pharmaceutical composition) comprising a Treg or a population of Tregs and can include (or not include) other cell types, unless stated otherwise.

[0006] Other aspects relate to a method comprising detecting an HLA-DRB 1 * 11 :04 allele in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

[0007] Yet other aspects relate to a method comprising human leukocyte antigen (HLA) haplotyping a subject diagnosed with MS, selecting the subject for treatment based on thepresence of an HLA-DRB1*11 :O4 allele in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1*11 :04.

[0008] Further aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subj ect for treatment based on (i) the presence of an HLA-DRB 1 * 11 : 04 allele in the subject and (ii) based on the presence of an HLA DRB 1 allele selected from HLA- DRB1*11:O4, HLA-DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB1* 15:03, HLA-DRBl*04:04, and HLA-DRB 1*04:02 in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1*11 :04.

[0009] Still other aspects provide a method comprising administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 02, wherein the subject has been diagnosed with MS and has been identified as having an HLA-DRB 1*04:02 allele.

[0010] Some aspects relate to a method comprising detecting an HLA-DRB 1*04: 02 allele in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 02.

[0011] Other aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on the presence of an HLA-DRB 1*04: 02 allele in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 02.

[0012] Yet other aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on (i) the presence of an HLA-DRB 1*04: 02 allele in the subject and (ii) the presence of an HLA DRB1 allele selected from HLA- DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB1* 15:03, HLA-DRBl*04:04, and HLA-DRB 1*04:02 in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 02.

[0013] Further aspects relate to a method comprising administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 04, wherein thesubject has been diagnosed with MS and has been identified as having an HLA-DRB 1*04:04 allele.

[0014] Still other aspects relate to a method comprising detecting an HLA-DRB 1*04: 04 allele in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 04.

[0015] Some aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on the presence of an HLA-DRB 1*04: 04 allele in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 04.

[0016] Other aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on (i) the presence of an HLA-DRB 1 *04:04 allele in the subject and (ii) the presence of an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA- DRBl*04:02 in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 04.

[0017] Yet other aspects relate to a method comprising administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl* 15:01, wherein the subject has been diagnosed with MS and has been identified as having an HLA-DRB 1*15:01 allele.

[0018] Further aspects relate to a method comprising detecting an HLA-DRBl*15:01 allele in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl*15:01.

[0019] Still other aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on the presence of an HLA-DRBl*15:01 allele in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl*15:01.

[0020] Some aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subj ect for treatment based on (i) the presence of an HLA-DRB 1 * 15 : 01 allelein the subject and (ii) the presence of an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HLA-DRB1*15:O1, HLA-DRB 1*15:02, HLA-DRB 1*15:03, HLA-DRBl*04:04, and HLA- DRBl*04:02 in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl*15:01.

[0021] Yet other aspects relate to a method comprising administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:02, wherein the subject has been diagnosed with MS and has been identified as having an HLA-DRB 1* 15:02 allele.

[0022] Further aspects relate to a method comprising detecting an HLA-DRB 1* 15:02 allele in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:02.

[0023] Still other aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on the presence of an HLA-DRB 1*15:02 allele in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1* 15:02.

[0024] Some aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subj ect for treatment based on (i) the presence of an HLA-DRB 1 * 15 :02 allele in the subject and (ii) the presence of an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB1* 15:02 HLA-DRB1* 15:03, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:02.

[0025] Yet other aspects relate to a method comprising administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:03, wherein the subject has been diagnosed with MS and has been identified as having an HLA-DRB 1* 15:03 allele.

[0026] Further aspects relate to a method comprising detecting an HLA-DRB 1* 15:03 allele in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineeredTCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:03.

[0027] Still other aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on the presence of an HLA-DRB 1*15:03 allele in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1* 15:03.

[0028] Some aspects relate to a method comprising HLA haplotyping a subject diagnosed with MS, selecting the subj ect for treatment based on (i) the presence of an HLA-DRB 1 * 15 :03 allele in the subject and (ii) the presence of an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:03.

[0029] In some embodiments of any of the above aspects, one or more of the HLA DRB1 alleles is identified by HLA haplotyping. In some embodiments, the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

[0030] In some embodiments of any of the above aspects, the MBP peptide is an MBP 83-99 peptide comprising the amino acid sequence of ENPVVHFFKNIVTPRTP (SEQ ID NO: 61).

[0031] In some embodiments, the engineered TCR is encoded as a single polypeptide, optionally comprising a self-cleaving peptide sequence.

[0032] In some embodiments, the administering comprises administering to the subject a cellular composition comprising a therapeutically effective amount of regulatory T cells comprising the engineered TCR. In some embodiments, the administering comprises one or more infusions of the cellular composition.

[0033] In some embodiments of any of the above aspects, the regulatory T cells are autologous relative to the subject. In some embodiments, the regulatory T cells are allogeneic relative to the subject. In some embodiments, the regulatory T cells are derived from stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells or hematopoietic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the regulatory T cells are derived from polarized T cells. In some embodiments, the regulatory T cells are derived from thymocytes. In some embodiments, the regulatory T cells are isolated fromperipheral blood or cord blood. In some embodiments, the regulatory T cells are induced regulatory T cells.

[0034] In some embodiments of any of the above aspects, the subject is diagnosed with multiple sclerosis. In some embodiments, the subject has Progression Independent of Relapse Activity (PIRA). In some embodiments, the subject is diagnosed with progressive multiple sclerosis, relapsing remitting multiple sclerosis, or multiple sclerosis. In some embodiments, the progressive multiple sclerosis is primary progressive multiple sclerosis (PPMS) or nonrelapsing progressive multiple sclerosis.

[0035] In some embodiments of any of the above aspects, the engineered TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and / or the beta chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the alpha chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and the beta chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, (i) the alpha chain comprises 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 (ii) the beta chain comprises 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 alpha chain comprises an amino acid sequence having at least 90%identity to SEQ ID NO: 4 or SEQ ID NO: 5; and the beta chain comprises an amino acid sequence having at least 90%identity to SEQ ID NO: 10 or SEQ ID NO: 11.

[0036] In some embodiments of any of the above aspects, an endogenous FOXP3 locus of the regulatory T cell is not engineered. In some embodiments the regulatory T cell comprising the engineered TCR has a hypomethylated Treg-specific demethylated region (TSDR) at an endogenous FOXP3 locus.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 provides a bar graph showing that Tregs expressing TCR-E are activated (as shown by CD69 expression) when co-cultured with myelin basic protein (MBP) 83-99 peptide antigen and antigen presenting cells expressing HLA-DRBl*15:01, HLA-DRBl*04:04, HLA- DRB1*11:O4, or HLA-DRBl*04:02.

[0038] FIG. 2 provides a graph showing ECso (half maximal effective concentration) curves for CD69 expression by Tregs expressing TCR-E when cocultured with antigen (MBP 83-99peptide or Selanginella moellendorffii) and antigen presenting cells expressing DRB1*15:O1, HLA-DRB 1 *04:04, HLA-DRB 1 * 11 :04, or HLA-DRB 1 *04:02.

[0039] FIG. 3 shows that activation of engineered Tregs of the disclosure by HLA- DRBl*15:01 PBMC is specific to tissues in which MBP 83-99 peptide is present (e.g., brain and spinal cord).

[0040] FIG. 4 shows activation of engineered Tregs expressing TCR-A when co-cultured with myelin basic protein (MBP) 83-99 peptide antigen and antigen presenting cells expressing HLA-DRBl*15:01 or HLA-DRB 1*15:03.

[0041] FIG. 5A-FIG. 5B shows EC50 antigen curves for TCR-E expressing Tregs in the context of HLA-DRB 1*15:02 (FIG. 5 A) and HLA-DRB 1*15:03 (FIG. 5B), compared to HLA-DRB 1*15:01. MBP83-99 was titrated 4-fold from 10 pM to 0 pM and co-cultured with PBMCs expressing HLA-DRB 1* 15:01 and HLA-DRB 1*15:02 (FIG. 5A) and HLA-DRB 1- 15:01 and HLA-DRB 1*15:03 (FIG. 5B).

[0042] FIG. 6A-FIG. 6B shows that activation of engineered Tregs of the disclosure by HLA- DRB 1*15:02 (FIG. 6A) and HLA-DRB 1*15:03 (FIG. 6B) PBMC is specific to tissues in which MBP 83-99 peptide is present (e.g., brain and spinal cord).DETAILED DESCRIPTIONOverview

[0043] The present disclosure provides methods related to the treatment of subjects diagnosed with multiple sclerosis (MS) who have an HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA- DRB1*15:O2, HLA-DRB 1*15:03, HLA-DRB 1*04:04, and / or HLA-DRB 1*04: 02 allele. The present disclosure also provides methods for the identification of subjects diagnosed with MS who would benefit from treatment with regulatory T cells (Tregs) comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide, e.g., MBP83-99, complexed with an MHC Class II comprising HLA-DRBl*15:01, HLA- DRB 1*15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, HLA-DRB1*11 :O4 or HLA- DRBl*04:02.

[0044] The human leukocyte antigen (HLA) locus encodes molecules of the human major histocompatibility complex (MHC) that are involved in the regulation of the immune system. The HLA locus is located on chromosome 6 (6p21), spanning approximately 3,600 kilobases of DNA. Within the HLA locus are nucleic acid sequences belonging to MHC class I, MHC class II, and MHC class III. The MHC class I sequences contain the HLA-A, HLA-B, andHLA-C genes that encode the heavy chains of MHC class I cell surface receptors. The MHC class II sequences contain a series of regions (HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR) that encode a and P chains of MHC class II cell surface receptors. The MHC class III sequences contain genes for complement components (C2, C4, factor B), 21 -hydroxylase, and tumor necrosis factors (TNFs), among others.

[0045] An HLA-DR protein (comprising a DR a chain and DR P chain) can form a complex with a peptide (generally between 9 and 30 amino acids in length); and that complex can function as a ligand for a T cell receptor (TCR) in order to modulate an immune response effectuated by T cells displaying the TCR. The HLA-DR region within the MHC class II sequences consists of a single DRA gene (encoding a DR a chain) and up to nine DRB genes (DRB1 to DRB9) (encoding DR P chains). There exists a large amount of allelic diversity within the genes of the HLA-DR region, particularly at DRB1. Much of the variation among distinct HLA-DRB1 alleles occurs at amino acid positions that contact peptides in HLA-DR complexes, allowing for different HLA-DRB1 alleles to specifically bind to different arrays of peptide antigens.

[0046] This diversity in peptide antigen recognition resulting from the allelic diversity in HLA- DRB1 means that not all human patients will respond to a particular Treg treatment. In order for a particular Treg treatment option to be viable for a particular patient, that patient must express the target antigen (e.g., MBP83-99) and the corresponding HL A allele (e.g., HLA- DRB 1 allele) that binds said target antigen. Thus, Treg treatments are generally restricted to patient populations expressing a particular HLA haplotype.

[0047] The inventors of the present disclosure have identified that HLA molecules encoded by each of HLA-DRB1*11 :04, HLA-DRBl*15:01, HLA-DRB 1*15:02, HLA-DRB1* 15:03, HLA-DRB 1*04:04, and HLA-DRB 1*04: 02 allele are capable of binding to MBP83-99 peptide and being recognized by the T cell receptors described herein. Thus, subjects expressing any one of these alleles (HLA-DRB1*11 :O4, HLA-DRB 1*15:01, HLA-DRB 1*15:02, HLA- DRB 1*15:03, HLA-DRB 1*04: 04, and HLA-DRB 1*04: 02 alleles) are candidates for treatment using regulatory T cells (Tregs) comprising an engineered T cell receptor (TCR) as described herein (e.g., Tregs comprising TCR-E). The HLA-DRB 1*04: 02 is reference no. HLA00687 of the IMGT database (https: / / www.ebi.ac.uk / ipd / imgt / hla / ). HLA-DRB 1*04: 04 is reference no. HLA00689 of the IMGT database. The HLA-DRB1*11 :O4 amino acid sequence is encoded by reference nos. HLA00756 and HLA00757 of the IMGT database. The HLA-DRBl*15:01 amino acid sequence is encoded by reference nos. HLA00865 and HLA00866 of the IMGT database. The HLA-DRB 1*15:02 amino acid sequence is encoded by reference nos.HLA00867, HLA00868, and HLA00869 of the IMGT database. The HLA-DRB 1*15:03 amino acid sequence is encoded by reference nos. HLA00870 and HLA03454 of the IMGT database.

[0048] Relapsing-remitting multiple sclerosis (RRMS) is the most common form of MS, characterized by episodes of new or worsening symptoms (relapses) followed by periods of partial or complete recovery (remissions). Approximately 80-85% of MS patients are initially diagnosed with this form of the disease. Progressive MS refers to the types of MS where the disease worsens more steadily over time, without the distinct relapses and remissions seen in RRMS. Progressive MS is categorized into two main types: primary progressive MS (PPMS) and secondary progressive MS (SPMS). PPMS is characterized by a gradual progression of the disease from its onset, without early relapses or remissions. Symptoms steadily worsen over time rather than appearing as sudden attacks. Approximately 10-15% of MS cases are diagnosed as PPMS. SPMS follows the initial course of RRMS. Some people who are diagnosed with RRMS eventually go on to have a secondary progressive course, in which neurologic function worsens over time and disability increases. According to estimates, in the United States, the prevalence of SPMS is 27-45 for every 100,000 members of the general population.

[0049] While Progressive MS affect over 250,000 patients, there are currently no therapies available for such patients. Moreover, although currently approved disease modifying therapies are effective at reducing relapse occurrence in RRMS, even RRMS subjects experience progression independent of relapse (PIRA), which has not been successfully treated.

[0050] With Progressive MS, compartmentalized inflammation, mainly localized in meningeal lymphoid aggregates, drives ongoing inflammatory demyelination behind an intact blood-brain barrier. Inflammatory T cells in a stable, long-lasting aggregate release inflammatory mediators that activate macrophages. This high density of meningeal lymphocytes and inflammation is associated with an increased level of subpial demyelination at the cortex and increased white matter lesions. Macrophages at the lesion edge continuously degrade myelin. PIRA in RRMS subjects has the same mechanistic basis as progression in progressive forms of MS.

[0051] Without wishing to be bound by theory, it is hypothesized that targeting Tregs to the meninges by engineering them to bind MBP, which is present at increased levels in the meninges of subjects with primary progressive MS (PPMS), will reduce the inflammatory response in the meninges to stop the progression of, or reverse the progression of, the disease. Meningeal targeting can be achieved by engineering the Tregs with TCRs that specifically bind MBP.

[0052] Surprisingly, the data described herein shows that the engineered Tregs of the disclosure are activated not only by MBP antigen, e.g., MBP83-99, complexed with an MHC Class II comprising HLA-DRBl*15:01, but are also activated by MBP antigen, e.g., MBPss- 99, complexed with an MHC Class II comprising HLA-DRB1*11 :O4, HLA-DRBl*04:04, HLA-DRB 1*15:02, HLA-DRB 1*15:03, and / or HLA-DRBl*04:02. Thus, the engineered Tregs described herein can be used to target diseased tissue in a newly identified, expanded patient population.Methods

[0053] In some embodiments, methods herein include administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g, MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1*11 :O4, HLA- DRB 1*04:04, HLA-DRB 1*04:02, HLA-DRB 1*15:02, HLA-DRB 1* 15:03, or HLA- DRB 1*15:01, wherein the subject has been diagnosed with MS and has been identified as having an HLA-DRB 1* 11 :04, HLA-DRB 1*04: 04, HLA-DRB 1*04: 02, HLA-DRB 1* 15:02, HLA-DRB 1*15:03, or HLA-DRBl*15:01 allele. Thus, some embodiments comprise detecting an HLA-DRB1*11 :O4, HLA-DRB 1*04: 04, HLA-DRB 1*04:02, HLA-DRB 1*15:02, HLA- DRBl*15:03, or HLA-DRBl*15:01 allele in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g, MBP 83-99 peptide) complexed with an MHC Class II comprising HLA- DRB1*11 :O4, HLA-DRB 1*04: 04, HLA-DRB 1*04: 02, HLA-DRB 1* 15:02, HLA- DRBl*15:03, or HLA-DRBl*15:01. Other embodiments comprise HLA haplotyping a subject diagnosed with MS, selecting the subject for treatment based on the presence of an HLA- DRB1*11 :O4, HLA-DRB 1*04: 04, HLA-DRB 1*04: 02, HLA-DRB 1* 15:02, HLA- DRBl*15:03, or HLA-DRBl*15:01 allele in the subject;, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1*11 :O4, HLA- DRB 1*04:04, HLA-DRB 1*04:02, HLA-DRB 1*15:02, HLA-DRB 1* 15:03, or HLA- DRBl*15:01.

[0054] Activation of a TCR can be assessed, for example, by measuring proliferation, upregulation of activation markers (such as CD69), and / or production of effector cytokines (such as IFN-y and / or TNF-a).

[0055] In some embodiments, an MBP peptide is myelin basic protein peptide 83-99 (MBP83- 99). Without wishing to be bound by theory, degraded MBP, including MBP 83-99 is present at increased levels at sites of disease in MS patients, and thus, is an ideal peptide for targeting TCR-based therapeutics for MS.

[0056] An engineered TCR of the Tregs described herein may be a TCR selected from the TCRs provided in Table 2.

[0057] A TCR can comprise the amino acid sequence of any alpha chain CDR1, CDR2, or CDR3 as provided in Table 2. In some embodiments, the alpha CDR1 of a TCR comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the alpha CDR2 of a TCR comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the alpha CDR3 of a TCR comprises the amino acid sequence of SEQ ID NO: 3. A TCR may comprise the amino acid sequence of any beta chain CDR1, CDR2, or CDR3 as provided in Table 2. In some embodiments, the beta CDR1 of a TCR comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the beta CDR2 of a TCR comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the beta CDR3 of a TCR comprises the amino acid sequence of SEQ ID NO: 9.

[0058] In some embodiments, an engineered TCR is TCR-A. In some embodiments, an engineered TCR is TCR-AA. In some embodiments, an engineered TCR is TCR-B. In some embodiments, an engineered TCR is TCR-C. In some embodiments, an engineered TCR is TCR-D. In some embodiments, an engineered TCR is TCR-E. In some embodiments, an engineered TCR is TCR-F. In preferred embodiments, an engineered TCR is TCR-E of Table 2.

[0059] In some embodiments, an engineered TCR binds to and is activated by MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1*11 :O4. In some embodiments, an engineered binds to and is activated by MBP peptide (e.g, MBP 83- 99 peptide) complexed with an MHC Class II comprising HLA-DRBl*04:02. In some embodiments, an engineered TCR binds to and is activated by MBP peptide (e.g, MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl *15:01. In some embodiments, an engineered TCR binds to and is activated by MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1 * 15:02. In some embodiments, an engineered TCR binds to and is activated by MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1 * 15:03. In some embodiments, an engineered TCR binds to and is activated by MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04:04.

[0060] In some embodiments, a subject has been diagnosed with MS and has been identified as having an HLA-DRB 1 * 11 : 04, HLA-DRB 1 * 04 : 02, HLA-DRB 1*15:01, HLA-DRB 1 * 15 : 02, HLA-DRB 1*15:03, and / or HLA-DRB 1*04: 04 allele. In some embodiments, a subject has been diagnosed with MS and has been identified as having an HLA-DRB1*11 :O4 allele. In some embodiments, a subject has been diagnosed with MS and has been identified as having an HLA-DRB 1*04:02 allele. In some embodiments, a subject has been diagnosed with MS and has been identified as having an HLA-DRB 1* 15:01 allele. In some embodiments, a subject has been diagnosed with MS and has been identified as having an HLA-DRB 1*15:02 allele. In some embodiments, a subject has been diagnosed with MS and has been identified as having an HLA-DRB 1 * 15 :03 allele. In some embodiments, a subject has been diagnosed with MS and has been identified as having an HLA-DRB 1*04: 04 allele.

[0061] In some embodiments, a subject has been diagnosed with MS and has been identified as having (i) a first HLA DRB 1 allele selected from the group consisting of HLA-DRB 1 * 11 :04, HLA-DRB 1*04:02, HLA-DRBl*15:01, HLA-DRB 1*15:02, HLA-DRB1* 15:03, and HLA- DRB 1*04:04 allele; and (ii) a second HLA DRB1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA-DRB 1*04: 02, HLA-DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB1*15:O3, and HLA-DRB 1*04: 04 allele. In some embodiments, a subject has been diagnosed with MS and has been identified as having (i) a first HLA DRB 1 allele selected from the group consisting of HLA-DRB1*11:O4, HLA-DRB 1*04:02, HLA-DRBl*15:01, HLA- DRB 1 * 15 : 02, HLA-DRB 1 * 15 : 03 , and HLA-DRB 1 * 04 : 04 allele; and (ii) a second HLA DRB 1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA-DRB 1*04:02, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB1* 15:03, HLA-DRB 1*04: 04, HLA-DRBl*01 :01, HLA-DRBl*13:01, HLA-DRB1*14:O1, HLA-DRB 1*07:01, HLA-DRB1*12:O1, HLA-DRB 1*13:07, HLA-DRB 1*09:01, HLA-DRB 1*03:01, HLA-DRBl*08:01, HLA-DRB 1*01 :02, HLA-DRB 1*16:01, HLA-DRB 1*04: 03, and HLA- DRBl*04:05.

[0062] In some embodiments, a subject is homozygous for the HLA-DRB1*1 LO4 allele i.e., has two HLA-DRB1*1 LO4 alleles). In some embodiments, a subject is homozygous for the HLA-DRB 1*04:02 allele (i.e., has two HLA-DRB 1*04:02 alleles). In some embodiments, a subject is homozygous for the HLA-DRBl*15:01 allele i.e., has two HLA-DRBl*15:01 alleles). In some embodiments, a subject is homozygous for the HLA-DRB 1* 15:02 allele i.e., has two HLA-DRB1* 15:02 alleles). In some embodiments, a subject is homozygous for the HLA-DRB 1*15:03 allele i.e., has two HLA-DRB 1*15:03 alleles). In some embodiments, asubject is homozygous for the HLA-DRB 1*04: 04 allele (i.e., has two HLA-DRBl*04:04 alleles).

[0063] In some embodiments, a subject has an HLA-DRB1*11 :O4 allele and an HLA- DRBl*04:02 allele. In some embodiments, a subject has an HLA-DRB1*11 :O4 allele and an HLA-DRB 1*15:01 allele. In some embodiments, a subject has an HLA-DRB1*11 :O4 allele and an HLA-DRB 1*04: 04 allele. In some embodiments, a subject has an HLA-DRB 1*04: 02 allele and an HLA-DRB 1*15:01 allele. In some embodiments, a subject has an HLA- DRBl*04:02 allele and an HLA-DRB 1*04: 04 allele. In some embodiments, a subject has an HLA-DRBl*15:01 allele and an HLA-DRB 1*04: 04 allele. In some embodiments, a subject has an HLA-DRB 1*15:03 allele and an HLA-DRB 1*04: 02 allele. In some embodiments, a subject has an HLA-DRB 1* 15:03 allele and an HLA-DRB1*11 :O4 allele. In some embodiments, a subject has an HLA-DRB 1* 15:03 allele and an HLA-DRB 1*15:01 allele. In some embodiments, a subject has an HLA-DRB1* 15:03 allele and an HLA-DRB 1*04: 04 allele. In some embodiments, a subject has an HLA-DRB1* 15:02 allele and an HLA- DRBl*04:02 allele. In some embodiments, a subject has an HLA-DRB 1*15:02 allele and an HLA-DRB1*11 :O4 allele. In some embodiments, a subject has an HLA-DRB 1* 15:02 allele and an HLA-DRB 1*15:01 allele. In some embodiments, a subject has an HLA-DRB 1* 15:02 allele and an HLA-DRB 1*15:03 allele. In some embodiments, a subject has an HLA- DRB1* 15:02 allele and an HLA-DRB 1*04: 04 allele.

[0064] Accordingly, in some embodiments, a method comprises administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1*11 :O4, wherein the subject has been diagnosed with MS and has been identified as having an HLA- DRB1*11 :O4 allele. In some embodiments, a method comprises administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 04, wherein the subject has been diagnosed with MS and has been identified as having an HLA- DRB 1*04:04 allele. In some embodiments, a method comprises administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl*15:01, wherein the subject has been diagnosed with MS and has been identified as having an HLA- DRBl*15:01 allele. In some embodiments, a method comprises administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:02,wherein the subject has been diagnosed with MS and has been identified as having an HLA- DRB1*15:O2 allele. In some embodiments, a method comprises administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:03, wherein the subject has been diagnosed with MS and has been identified as having an HLA- DRBl*15:03 allele. In some embodiments, a method comprises administering to a subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 02, wherein the subject has been diagnosed with MS and has been identified as having an HLA- DRB 1*04:02 allele.

[0065] Some aspects of the disclosure provide a method comprising detecting an HLA DRB1 allele (e.g., an HLA-DRB1*11 :O4, HLA-DRB 1*04:02, HLA-DRBl*15:01, HLA- DRB1*15:O2, HLA-DRB1* 15:03, and / or HLA-DRB 1*04: 04 allele) in a subject diagnosed with MS, and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA DRB1 allele (e.g., an HLA-DRB1*11 :O4, HLA-DRB 1*04: 02, HLA- DRBl*15:01, DRB1*15:O2, HLA-DRB1* 15:03, and / or HLA-DRB 1*04: 04 allele).

[0066] In some embodiments, a method comprises detecting an HLA-DRB1*11 :O4 allele in a subject diagnosed with MS and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g, MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

[0067] In some embodiments, a method comprises detecting an HLA-DRB 1*04: 02 allele in a subject diagnosed with MS and administering to the subject a Treg comprising an engineered that binds to and is activated by an MBP peptide (e.g, MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 02.

[0068] In some embodiments, a method comprises detecting an HLA-DRBl*15:01 allele in a subject diagnosed with MS and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl*15:01.

[0069] In some embodiments, a method comprises detecting an HLA-DRB 1*15:02 allele in a subject diagnosed with MS and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:02.

[0070] In some embodiments, a method comprises detecting an HLA-DRB 1*15:03 allele in a subject diagnosed with MS and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*15:03.

[0071] In some embodiments, the method comprises detecting an HLA-DRB 1*04: 04 allele in a subject diagnosed with MS and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04:04.

[0072] Detecting an HLA allele in a subject can be accomplished by HLA haplotyping. HLA haplotyping can be used to identify the specific variations of genes within the HLA system in a subject. In some embodiments, detecting an HLA haplotype in a subject is performed by taking a sample (e.g., a blood sample) from the subject and undergoing HLA haplotyping. HLA haplotyping can be performed, for example, using next-generation sequencing (NGS), Sangerbased sequencing, and / or polymerase chain reaction (PCR) methods. In some embodiments, HLA haplotyping is performed in accordance with the methods set forth in US Patent Publication 20190002979; or Murphy N.M. et al. Scientific Reports 2016; 6 (30381). In some embodiments, HLA haplotyping comprises determining the sequence of the HLA DRB1 locus.

[0073] Some aspects of the disclosure provide a method comprising HLA haplotyping a subject diagnosed with MS and selecting the subject for treatment based on the presence of a specific HLA DRB1 allele (e.g., an HLA-DRB1*11 :O4, HLA-DRB 1*04: 02, HLA- DRB 1*15:01, HLA-DRB 1 * 15 : 02, HLA-DRB 1*15:03, and / or HLA-DRB 1 * 04 : 04 allele) in the subject. In some embodiments, a method comprising a patient selection step further comprises a step of administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising a specific HLA.

[0074] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on the presence of an HLA-DRB1*11 :O4 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II compri sing HLA-DRB 1 * 11 : 04.

[0075] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on the presence of an HLA-DRB 1*04:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds toand is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRBl*04:02.

[0076] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on the presence of an HLA-DRB 1*15:01 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II compri sing HLA-DRB 1*15:01.

[0077] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on the presence of an HLA-DRB 1*15:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II compri sing HLA-DRB 1 * 15 : 02.

[0078] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on the presence of an HLA-DRB 1*15:03 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II compri sing HLA-DRB 1*15:03.

[0079] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on the presence of an HLA-DRB 1*04:04 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB 1*04: 04.

[0080] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS and selecting the subject for treatment based on (i) the presence of a first HLA DRB 1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA- DRB1*15:O2, HLA-DRB1* 15:03, HLA-DRB 1*04:04, and HLA-DRB 1*04: 02 in the subject and (ii) the presence of a second HLA DRB 1 allele selected from the group consisting of HLA- DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB1* 15:03, HLA-DRB 1*04:04, and HLA-DRB 1*04: 02 in the subject. In some embodiments, a method comprising a patient selection step further comprises a step of administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA molecule selected from the group consisting of HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA-DRB 1*04: 02 molecule.

[0081] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on (i) the presence of a first HLA DRB1 allele selected from the group consisting of HLA-DRB 1 * 11 :04 in the subject and (ii) the presence of a second HLA DRB1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA-DRB 1 * 11 :04.

[0082] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on (i) the presence of a first HLA DRB1 allele selected from the group consisting of HLA-DRB 1*15:01 in the subject and (ii) the presence of a second HLA DRB1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA-DRBl*15:01.

[0083] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on (i) the presence of a first HLA DRB1 allele selected from the group consisting of HLA-DRB 1*15:02 in the subject and (ii) the presence of a second HLA DRB1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA-DRB 1* 15:02.

[0084] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on (i) the presence of a first HLA DRB1 allele selected from the group consisting of HLA-DRB 1*15:03 in the subject and (ii) the presence of a second HLA DRB1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA-DRB 1* 15:03.

[0085] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on (i) the presence of a first HLA DRB1 alleleselected from the group consisting of HLA-DRB 1*04: 04 in the subject and (ii) the presence of a second HLA DRB1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA- DRB1*15:O1, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRBl*04:04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA-DRB 1*04: 04.

[0086] In some embodiments, a method comprises HLA haplotyping a subject diagnosed with MS; selecting the subject for treatment based on (i) the presence of a first HLA DRB1 allele selected from the group consisting of HLA-DRB 1*04: 02 in the subject and (ii) the presence of a second HLA DRB1 allele selected from the group consisting of HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a Treg comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising an HLA-DRB 1*04: 02.

[0087] Any one of the methods of administering a Treg described herein can be modified to administer more than one Treg to a subject (e.g., a population of Tregs). In some embodiments, administering a Treg to a subject comprises administering a cellular composition comprising a therapeutically effective amount of Tregs comprising an engineered TCR that binds to and is activated by an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC Class II comprising HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB1* 15:03, HLA-DRB 1*04:04, and / or HLA-DRB 1*04: 02. A “therapeutically effective amount,” also referred to as an “effective amount,” of Tregs includes an amount or concentration of Tregs that is sufficient to elicit a desired biological response. For example, an effective amount of Tregs can include a number of Tregs sufficient to improve a symptom associated with MS (e.g., Progressive MS).

[0088] In some embodiments, methods of administering involve administering Tregs to a subject in an effective amount to alleviate one or more symptoms of MS. Non-limiting examples of symptoms of MS include fatigue, vision problems, numbness in muscle tissues, muscle spasms, muscle stiffness, muscle weakness, mobility problems, pain (e.g., muscle pain), depression, anxiety, sexual problems, bladder problems, bowel problems, speech problems, and difficulty with swallowing.

[0089] In some embodiments, Tregs of the disclosure are autologous. Autologous cells include cells obtained from the same subject to which they are subsequently administered. In some embodiments, engineered Tregs are isolated from a subject, engineered to express a TCR asdescribed herein, and then administered to the same subject in order to treat a disease. The use of autologous cells minimizes the risk of a rejection (e.g., graft-versus-host disease) by the patient.

[0090] In other embodiments, Tregs of the disclosure are allogeneic. Allogenic cells include cells obtained from one subject and then administered to another subject. In some embodiments, engineered Tregs are isolated from a subject, engineered to express a TCR as described herein, and then administered to another subject in order to treat a disease.

[0091] In some embodiments, treating (or treatment of) a disease includes a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progression of MS, or one or more symptoms thereof. In some embodiments, treatment can be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment can 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 can be administered to a susceptible individual prior to the onset of symptoms (e.g., because of knowledge of genetic factors). Treatment can also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0092] A subject includes an individual organism, for example, an individual human. In some embodiments, a subject is a human subject, such as a male subject or a female subject. In some embodiments, a subject is a non-human mammal. In some embodiments, a subject is a nonhuman primate. In some embodiments, a subject is a rodent. In some embodiments, a subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, a subject is a research animal. In some embodiments, a subject is genetically engineered, e.g., a genetically engineered non- human subject. A subject can be male or female.

[0093] Conventional and pharmaceutically acceptable routes of administration of the include, but are not limited to, intravenous, subcutaneous, intravenous, intrathecal administration, direct delivery to a selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intramuscular, intradermal, intratumoral, and other parental routes of administration. Routes of administration can be combined, if desired. In some embodiments, engineered Tregs are administered intravenously.

[0094] Tregs can be administered as a bolus administration. In some embodiments, administration of Tregs comprises one or more infusion(s) of Tregs (e.g., cells are infused through a central line, similar to a blood transfusion).

[0095] In some embodiments, Tregs are derived from stem cells, which can divide and selfrenewal as well as divide to form cells that mature into cells that make up every type of tissueand organ in the body. In some embodiments, the stem cells are pluripotent stem cells, which have the potential to differentiate into all the more than 200 different cell types. In some embodiments, a pluripotent stem cell is capable of differentiating into a muscle, vascular, dermal, nerve, or brain cell. In some embodiments, a pluripotent stem cell is an embryonic stem cell or hematopoietic stem cell. In some embodiments, a pluripotent stem cell is an induced pluripotent stem cell (iPSC), which include cells reprogrammed into pluripotent stem cells from adult tissues. Thus, an iPSC can be derived from a non-pluripotent cell and has been induced into a pluripotent state through induced expression of certain genes and transcription factors.

[0096] In some embodiments, Tregs are derived from polarized T cells. T cells can ‘polarize’ into T1 and T2 effector cell types in response to distinct cytokines. In some embodiments, Tregs are derived from thymocytes (z.e., thymic-derived Tregs). In some embodiments, Tregs are isolated from blood (e.g., whole blood). In some embodiments, Tregs are isolated from peripheral blood (from peripheral blood mononuclear cells (PBMCs)). In some embodiments, Tregs are isolated from cord blood. In some embodiments, Tregs are induced Tregs (iTregs), which can arise in the periphery from CD4+FOXP3 conventional T cells and can be generated in vitro.

[0097] The present disclosure also provides methods and compositions related to the treatment of MS through the production and utilization of Tregs comprising an engineered TCR that binds to MBP peptide. Such engineered Tregs are capable of specifically targeting discrete cell types and tissues associated with degraded myelin, for example, to prevent immune-mediated destruction, restore homeostasis, and promote repair in affected tissues. Additionally, the engineered Tregs of the disclosure suppress local inflammation at the target and do not cause systemic immune suppression. Furthermore, engineered Tregs described herein, in some embodiments, are stable (e.g., committed to a Treg phenotype) thymically-derived Tregs that are capable of persisting in vivo for extended periods and can provide therapeutic benefit for months or years following a single dose. These Tregs are also, in some embodiments, resistant to pro-inflammatory triggers (e.g., pro-inflammatory cytokines).Engineered Regulatory T cells

[0098] In some embodiments, the present disclosure provides regulatory T cells (Tregs) derived from a subject having MS. These Tregs can be used, for example, to treat MS in a subject in need thereof.

[0099] The terms “regulatory T cell” and “Treg” are used interchangeably herein and include 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 express the cell surface markers CD4 and CD25 as well as the transcription factor F0XP3. Further, Tregs do not express, or express at low levels, the cell surface marker CD127. Thus, Tregs are characterized by the following protein expression profile: CD4+CD25hlgh / +CD127low / 'FOAP3+. That is, a Treg expresses CD4, expresses or expresses a high level of CD25, does not express or expresses a low level of CD127, and expresses F0XP3.

[0100] An “engineered Treg” is a Treg that comprises an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II .

[0101] Throughout the present disclosure, the expression of an indicated protein by a cell or population of cells can include reference to various expression indicators such as “+” (e.g., CD4+), “-“ (e.g., CD127-), “high” (e.g., CD25High), “int” or “intermediate” (e.g., CD25int), “low” (e.g., CD127low), “High / +” (e.g., CD25High / +), or “low / -“ (e.g., CD127low / -). 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.

[0102] A “stable Treg” includes a Treg that comprises a hypomethylated T cell-specific demethylation region (TSDR) at the F0XP3 locus. Any one or more of the engineered Tregs of the disclosure may be a stable engineered Treg.

[0103] 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 can be further divided into populations of “low” and / or “high” subpopulations.

[0104] A subpopulation of “low” cells expresses the indicated protein but at a lower level than the other cells in the population (e.g.4at 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).

[0105] A subpopulation of “high” cells expresses the indicated protein at a higher level than the other cells in the population (e.g.4at 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) or at a higherlevel than expression in a control cell population (e.g., relative to a CD4+conventional T cell or a CD8+ effector T cell).

[0106] 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).

[0107] 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.

[0108] An indicator of “+ / H1gh” refers to a cell population comprising cells that are (+) for the indicated protein, including cells that express a high level of the indicated protein.

[0109] In thymic Treg development, the genome organizer SATB1 (special AT-rich sequencebinding protein) binds to specific genomic sites from the CD4+CD8+thymocyte stage to open up the chromatin and activate super-enhancers associated with many Treg signature genes such as F0XP3, 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 F0XP3 locus, with subsequent activation of the enhancers at CNS3 and CNS2, and then the promoter. This results in stable hypomethylation and expression of F0XP3 and other Treg-associated genes, thereby resulting in a stable Treg phenotype (Piotrowska, et al.; Int J Mol Sci. 2021).

[0110] After hematopoietic development, populations of T cells can transition between one phenotype to another under various conditions (see e.g. , Kitagawa et al. Nat Immunol. 2017 Feb; 18(2): 173-183). Tregs that originate in the thymus (e.g., thymic Tregs) are stable, and a Treg 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 F0XP3 locus. Hypomethylation of a TSDR, which is an evolutionary conserved CpG-rich regulatory element of the F0XP3 gene, is associated with expression of F0XP3. A TSDR of an endogenous F0XP3 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 Treg is determined by the presence of a hypomethylated TSDR at the FOXP3 locus.[OHl] In some embodiments, measurement of the methylation status of the TSDR of & FOXP3 locus is as described in Kressler et. al. Frontiers in Immunology, 07 January 2021; Schreiber et. al. PLOS One, February 5, 2014. In some embodiments, the TSDR of the FOXP3 locus is selected from conserved noncoding sequence 0 (CNSO), CNS3, and CNS2. In some embodiments, the TSDR of the FOXP3 locus is CNS2.

[0112] In some embodiments, an engineered Treg maintains a hypomethylated 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, an engineered Treg comprises a hypomethylated 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.

[0113] In some embodiments, an engineered Treg 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 Tregs (e.g., expression of CD69, 4- IBB, CD25, CD71, and / or CTLA-4); and / or (iii) suppression activity (e.g., the ability of a Treg to suppress the activation and / or proliferation of other effector cells of the immune system).

[0114] In some embodiments, engineered Tregs maintain a hypomethylated TSDR at the FOXP3 locus over time after isolation from a biological sample. For example, engineered Tregs can maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after isolation from a biological sample. In some embodiments, engineered Tregs 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, engineered Tregs 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.

[0115] In some embodiments, engineered Tregs provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after transduction with a nucleic acid encoding a TCR. For example, Tregs can maintain a hypomethylated TSDR at the FOXP3 locus for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after transduction with a nucleic acid encoding an engineered TCR. In some embodiments, engineered Tregs 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 engineered TCR. In some embodiments, engineered Tregs maintain a hypomethylated TSDRat 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 engineered TCR.

[0116] In some embodiments, engineered Tregs provided herein can maintain a hypom ethylated TSDR at the FOXP3 locus over time after cry opreservation. For example, Tregs can be cryopreserved and later thawed for use and / or analysis, referred to herein as a cryopreservation freeze-thaw cycle. In such embodiments, Tregs maintain a hypomethylated TSDR at the F0XP3 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 cry opreservation freeze-thaw cycle. In some embodiments, engineered Tregs maintain a hypomethylated TSDR at the F0XP3 locus for more than 5 days, more than 10 days, more than 15 days, or more than 20 days after a cry opreservation freeze-thaw cycle. In some embodiments, engineered Tregs 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.

[0117] In some embodiments, engineered Tregs provided herein maintain a hypomethylated TSDR at the FOXP3 locus over time after administration to a subject. For example, Tregs can 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, engineered Tregs 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, engineered Tregs 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.

[0118] In some embodiments, the isolated populations of cells described herein (e.g., isolated populations comprising Tregs) are isolated from a biological sample obtained from a subject diagnosed with, or suspected of having, MS.Engineered T Cell Receptors

[0119] Engineered Tregs provided herein comprise an engineered TCR that binds to MBP peptide. This binding occurs when the MBP peptide is complexed with a major histocompatibility complex (MHC) (e.g., MHC Class I or MHC Class II).

[0120] A 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 digestcertain proteins (antigens) and display their fragments (peptides) on 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.

[0121] TCRs expressed by the Tregs of the disclosure are exogenous TCRs that are introduced to a Treg - that is, the TCRs are not endogenous to (z.e., naturally occurring in) a Treg of the disclosure. For example, an engineered TCR can be encoded by a nucleic acid that is not endogenous to a Treg (z.e., not naturally occurring in the genome of the Treg). In some embodiments, a nucleic acid is an engineered nucleic acid, for example, a recombinant or synthetic nucleic acid.

[0122] A TCR described herein binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA- DRB 1*15:02, HLA-DRB 1* 15:03, HLA-DRB 1*04:04, and HLA-DRBl*04:02. A TCR is considered to bind “specifically” to an MBP peptide (e.g., such as MBP 83-99) complexed with an MHC if the TCR has a higher binding affinity for the MBP peptide complexed with an MHC relative to a non-target peptide complexed with an MHC. A TCR can bind to an MBP 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 an MBP peptide complexed with an MHC if a Treg expressing the TCR becomes activated (e.g., as assessed by increased CD69 expression) when contacted with the MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA- DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB1* 15:03, HLA-DRB 1*04:04, and HLA-DRB 1*04: 02, or becomes more highly activated relative to a nontarget peptide complexed with an MHC comprising HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1 * 15 : 02, HLA-DRB 1*15:03, HLA-DRB 1 * 04 : 04, and HLA-DRB 1 * 04 : 02.

[0123] A TCR, in some embodiments, 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.

[0124] A TCR can 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 selfcleaving 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 someembodiments, 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 can be a 2A peptide sequence. A 2A peptide sequence can comprise, for example, a DXEXNPGP (SEQ ID NO: 102) amino acid motif, wherein X can be any amino acid. In some embodiments, a 2 A peptide sequence is a P2A (derived from porcine teschovirus-1 2 A), E2A (derived from equine rhinitis A virus), F2A (derived from foot-and-mouth disease virus), or T2A (derived from Thosea asigna virus 2A) peptide sequence. A T2A peptide sequence can comprise, for example, the amino acid sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 103). A P2A peptide sequence can comprise, for example, the amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 104). An E2A peptide sequence can comprise, for example, the amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 105). A F2A peptide sequence can comprise, for example, the amino acid sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 106).

[0125] In some embodiments, an engineered TCR comprises two or more polypeptides. For example, an engineered TCR can comprise a first polypeptide comprising an alpha chain and a second polypeptide comprising a beta chain.

[0126] In some embodiments, an engineered 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, an engineered 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.

[0127] In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, a TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 48 to introduce a cysteine (e.g., T48C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and a TCR beta chain constant region comprises an amino acid substitution at position 57 to introduce a cysteine (e.g., S57C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60,wherein the cysteine residue at position 48 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 57 of the beta chain.

[0128] In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, a TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and a TCR beta chain constant region comprises an amino acid substitution at position 77 to introduce a cysteine (e.g., S77C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 45 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 77 of the beta chain.

[0129] In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, a TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 10 to introduce a cysteine (e.g., Y10C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and a TCR beta chain constant region comprises an amino acid substitution at position 17 to introduce a cysteine (e.g., S17C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 10 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 17 of the beta chain.

[0130] In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, a TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, a TCRalpha chain constant region comprises an amino acid substitution at position 45 to introduce a cysteine (e.g., T45C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and a TCR beta chain constant region comprises an amino acid substitution at position 59 to introduce a cysteine (e.g., D59C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 45 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 59 of the beta chain.

[0131] In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, a TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, a TCR alpha chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., S15C) relative to a TCR alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 58, and a TCR beta chain constant region comprises an amino acid substitution at position 15 to introduce a cysteine (e.g., E15C) amino acid substitution relative to a TCR beta chain constant region comprising the amino acid sequence of SEQ ID NO: 60, wherein the cysteine residue at position 15 of the alpha chain is capable of forming a disulfide bond with the cysteine residue at position 15 of the beta chain.Table 1. TCR Constant Domains

[0132] In some embodiments, a TCR comprises one or more amino acid sequences as described in Table 2 or Table 3 (e.g., one or more amino acid sequences belonging to any one of TCR- A, TCR-AA, TCR-B, TCR-C, TCR-D, TCR-E, or TCR-F). A TCR can comprise the amino acid sequence of any alpha chain CDR1, CDR2, or CDR3 as provided in Table 2 or Table 3. In some embodiments, the alpha chain CDR1 of a TCR is any one of SEQ ID NOs: 1, 18, 32, 46, or 109. In some embodiments, the alpha chain CDR2 of a TCR is any one of SEQ ID NOs: 2, 19, 33, 47, or 110. In some embodiments, the alpha chain CDR3 of a TCR is any one of SEQ ID NOs: 3, 20, 34, 48, or 111. A TCR can comprise the amino acid sequence of any beta chain CDR1, CDR2, or CDR3 as provided in Table 2. In some embodiments, the beta chain CDR1 of a TCR is any one of SEQ ID NOs: 7, 24, 38, 52, or 115. In some embodiments, the beta chain CDR2 of a TCR is any one of SEQ ID NOs: 8, 25, 39, 53, 116. In some embodiments, the beta chain CDR3 of a TCR is any one of SEQ ID NOs: 9, 26, 40, 54, 117.

[0133] In some embodiments, the alpha chain variable region of a TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4, 21, 35, 49, or 112. In some embodiments, the beta chain variable region of a TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 10, 14, 27, 41, 55, or 118. In some embodiments, the alpha chain of a TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 5, 22, 36, 50, 73, 80 or 113. In some embodiments, the beta chain of a TCR comprises an amino acid sequence having at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11, 15, 28, 43, 56, 75, 82, or 119.

[0134] In some embodiments, an engineered 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, an engineered 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.

[0135] In some embodiments, a TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4. In someembodiments, a TCRa chain variable region comprises SEQ ID NO: 4. In some embodiments, a TCRa chain variable region consists of SEQ ID NO: 4. In some embodiments, a TCRP chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 10. In some embodiments, a TCRP chain variable region comprises SEQ ID NO: 10. In some embodiments, a TCRP chain variable region consists of SEQ ID NO: 10.

[0136] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4, and (b) a TCRP chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 10. In some embodiments, an engineered 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, an engineered 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.

[0137] In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 5, and (b) a TCRP chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 11. In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising SEQ ID NO: 5, and (b) a TCRP chain comprising SEQ ID NO: 11. In some embodiments, an engineered TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 5, and (b) a TCRP chain consisting of SEQ ID NO: 11.

[0138] In some embodiments, an engineered TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 17. In some embodiments, an engineered TCR is expressed as a single polypeptide comprising SEQ ID NO: 17. In some embodiments, an engineered TCR is expressed as a single polypeptide consisting of SEQ ID NO: 17.

[0139] In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 73, and (b) a TCRP chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 75. In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising SEQ ID NO: 73, and (b) a TCRP chain comprising SEQ ID NO: 75. In some embodiments, an engineered TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 73, and (b) a TCRP chain consisting of SEQ ID NO: 75.

[0140] In some embodiments, an engineered TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 79. In some embodiments, an engineered TCR is expressed as a single polypeptide comprising SEQ ID NO: 79. In some embodiments, an engineered TCR is expressed as a single polypeptide consisting of SEQ ID NO: 79.

[0141] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and (b) a TCRP chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 18, a CDR2 consisting of SEQ ID NO: 19, and a CDR3 consisting of SEQ ID NO: 20; and (b) a TCRP chain variable region comprising a CDR1 consisting of SEQ ID NO: 24, a CDR2 consisting of SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0142] In some embodiments, a TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 21. In some embodiments, a TCRa chain variable region comprises SEQ ID NO: 21. In some embodiments, a TCRa chain variable region consists of SEQ ID NO: 21. In some embodiments, a TCRP chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 27. In some embodiments, a TCRP chain variable region comprises SEQ ID NO: 27. In some embodiments, a TCRP chain variable region consists of SEQ ID NO: 27.

[0143] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 21, and (b) a TCRP chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 27. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 21, and (b) a TCRP chain variable region comprising SEQ ID NO: 27. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 21, and (b) a TCRP chain variable region consisting of SEQ ID NO: 27.

[0144] In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:22, and (b) a TCRP chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 28. In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising SEQ ID NO: 22, and (b) a TCRP chain comprising SEQ ID NO: 28. In some embodiments, an engineered TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 22, and (b) a TCRP chain consisting of SEQ ID NO: 28.

[0145] In some embodiments, an engineered TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, an engineered TCR is expressed as a single polypeptide comprising SEQ ID NO: 31. In some embodiments, an engineered TCR is expressed as a single polypeptide consisting of SEQ ID NO: 31.

[0146] In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 80, and (b) a TCRP chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 82. In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising SEQ ID NO: 80, and (b) a TCRP chain comprising SEQ ID NO: 82. In some embodiments, an engineered TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 80, and (b) a TCRP chain consisting of SEQ ID NO: 82.

[0147] In some embodiments, an engineered TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 86. In some embodiments, an engineered TCR is expressed as a single polypeptide comprising SEQ ID NO: 86. In some embodiments, an engineered TCR is expressed as a single polypeptide consisting of SEQ ID NO: 86.

[0148] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34; and (b) a TCRP chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 32, a CDR2 consisting of SEQ ID NO: 33, and a CDR3 consisting of SEQ ID NO: 34; and (b) a TCRP chain variable region comprising a CDR1 consisting of SEQ ID NO: 38, a CDR2 consisting of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 40.

[0149] In some embodiments, a TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 35. In some embodiments, a TCRa chain variable region comprises SEQ ID NO: 35. In some embodiments, a TCRa chain variable region consists of SEQ ID NO: 35. In some embodiments, a TCRP chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 41. In some embodiments, a TCRP chain variable region comprises SEQ ID NO: 41. In some embodiments, a TCRP chain variable region consists of SEQ ID NO: 41.

[0150] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 35, and (b) a TCRP chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 41. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 35, and (b) a TCRP chain variable region comprising SEQ ID NO: 41. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 35, and (b) a TCRP chain variable region consisting of SEQ ID NO: 41.

[0151] In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 36, and (b) a TCRP chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 43. In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising SEQ ID NO: 36, and (b) a TCRP chain comprising SEQ ID NO: 42. In some embodiments, an engineered TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 36, and (b) a TCRP chain consisting of SEQ ID NO: 42.

[0152] In some embodiments, an engineered TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 45. In some embodiments, an engineered TCR is expressed as a single polypeptide comprising SEQ ID NO: 45. In some embodiments, an engineered TCR is expressed as a single polypeptide consisting of SEQ ID NO: 45.

[0153] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and (b) a TCRP chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 46, a CDR2 consisting of SEQ ID NO: 47, and a CDR3 consisting of SEQ ID NO: 48; and (b) a TCRP chain variable region comprising a CDR1 consisting of SEQ ID NO: 52, a CDR2 consisting of SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 54.

[0154] In some embodiments, a TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 49. In some embodiments, a TCRa chain variable region comprises SEQ ID NO: 49. In some embodiments, a TCRa chain variable region consists of SEQ ID NO: 49. In some embodiments, a TCRP chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 55. In some embodiments, a TCRP chain variable region comprises SEQ ID NO: 55. In some embodiments, a TCRP chain variable region consists of SEQ ID NO: 55.

[0155] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 49, and (b) a TCRP chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 55. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 49, and (b) a TCRP chain variable region comprising SEQ ID NO: 55. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 49, and (b) a TCRP chain variable region consisting of SEQ ID NO: 55.

[0156] In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 50, and (b) a TCRP chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 56. In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising SEQ ID NO: 50, and (b) a TCRP chain comprising SEQ ID NO: 56. In some embodiments, an engineered TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 50, and (b) a TCRP chain consisting of SEQ ID NO: 56.

[0157] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 109, a CDR2 comprising the amino acid sequence of SEQ ID NO: 110, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 111; and (b) a TCRP chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 115, a CDR2 comprising the amino acid sequence of SEQ ID NO: 116, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 117. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising a CDR1 consisting of SEQ ID NO: 109, a CDR2 consisting of SEQ ID NO: 110, and a CDR3 consisting of SEQ ID NO: 111; and (b) a TCRP chain variable region comprising a CDR1 consisting of SEQ ID NO: 115, a CDR2 consisting of SEQ ID NO: 116, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 117.

[0158] In some embodiments, a TCRa chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 112. In some embodiments, a TCRa chain variable region comprises SEQ ID NO: 112. In some embodiments, a TCRa chain variable region consists of SEQ ID NO: 112. In some embodiments, a TCRP chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 118. In some embodiments, a TCRP chain variable region comprises SEQ ID NO: 118. In some embodiments, a TCRP chain variable region consists of SEQ ID NO: 118.

[0159] In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 112, and (b) a TCRP chain variable region comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 118. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region comprising SEQ ID NO: 112, and (b) a TCRP chain variable region comprising SEQ ID NO: 118. In some embodiments, an engineered TCR comprises (a) a TCRa chain variable region consisting of SEQ ID NO: 112, and (b) a TCRP chain variable region consisting of SEQ ID NO: 118.

[0160] In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 113, and (b) a TCRP chain comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 119. In some embodiments, an engineered TCR comprises (a) a TCRa chain comprising SEQ ID NO: 113, and (b) a TCRP chain comprising SEQ ID NO: 119. In some embodiments, an engineered TCR comprises (a) a TCRa chain consisting of SEQ ID NO: 113, and (b) a TCRP chain consisting of SEQ ID NO: 119.

[0161] In some embodiments, an engineered TCR is expressed as a single polypeptide comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 108. In some embodiments, an engineered TCR is expressed as a single polypeptide comprising SEQ ID NO: 108. In some embodiments, an engineered TCR is expressed as a single polypeptide consisting of SEQ ID NO: 108.Table 2: TCR SequencesTable 3: Additional Exemplary TCR Sequences

[0162] In some embodiments, an engineered TCR comprises two or more polypeptides. For example, an engineered TCR can comprise a first polypeptide comprising an alpha chain and a second polypeptide comprising a beta chain.

[0163] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g.,MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRB1*15:O1, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRBl*04:04, and / or HLA- DRB 1*04:02.

[0164] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and / or HLA- DRB 1*04:02.

[0165] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:73; and / or (b) a beta chain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and / or HLA- DRB 1*04:02.

[0166] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and / or HLA- DRB 1*04:02.

[0167] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and / or HLA- DRB 1*04:02.

[0168] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 97% identity to the aminoacid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRB1*15:O1, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRBl*04:04, and / or HLA- DRB 1*04:02.

[0169] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and / or HLA- DRB 1*04:02.

[0170] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and / or HLA- DRB 1*04:02.

[0171] In some embodiments, a TCR comprises (a) an alpha chain comprising an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 73; and / or (b) a beta chain comprising an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 75, wherein the TCR binds specifically to an MBP peptide (e.g., MBP 83-99 peptide) complexed with an MHC comprising HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRB1* 15:02, HLA-DRB 1*15:03, HLA-DRB 1*04: 04, and / or HLA- DRB 1*04:02.

[0172] In some embodiments, the amino acid sequence of the alpha chain has at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the alpha chain has at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the alpha chain has at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the alpha chain has at least 96% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the alpha chain has at least 97% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the alpha chain has at least 98% identity to the amino acid sequence of SEQ IDNO: 73. In some embodiments, the amino acid sequence of the alpha chain has at least 99% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the alpha chain has 100% identity to the amino acid sequence of SEQ ID NO: 73.

[0173] In some embodiments, the amino acid sequence of the alpha chain has at least 85% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the alpha chain has at least 90% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the alpha chain has at least 95% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the alpha chain has at least 96% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the alpha chain has at least 97% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the alpha chain has at least 98% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the alpha chain has at least 99% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the alpha chain has 100% identity to the amino acid sequence of SEQ ID NO: 74.

[0174] In some embodiments, the amino acid sequence of the beta chain has at least 85% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the beta chain has at least 90% identity to the amino acid sequence of SEQ ID NO:75. In some embodiments, the amino acid sequence of the beta chain has at least 95% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the beta chain has at least 96% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the beta chain has at least 97% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the beta chain has at least 98% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the beta chain has at least 99% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the beta chain has 100% identity to the amino acid sequence of SEQ ID NO: 75.

[0175] In some embodiments, the amino acid sequence of the beta chain has at least 85% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the beta chain has at least 90% identity to the amino acid sequence of SEQ ID NO:76. In some embodiments, the amino acid sequence of the beta chain has at least 95% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the beta chain has at least 96% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the beta chain has at least 97% identity to theamino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the beta chain has at least 98% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the beta chain has at least 99% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the beta chain has 100% identity to the amino acid sequence of SEQ ID NO: 76.

[0176] In some embodiments, the amino acid sequence of the beta chain has at least 85% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the beta chain has at least 90% identity to the amino acid sequence of SEQ ID NO:77. In some embodiments, the amino acid sequence of the beta chain has at least 95% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the beta chain has at least 96% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the beta chain has at least 97% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the beta chain has at least 98% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the beta chain has at least 99% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the beta chain has 100% identity to the amino acid sequence of SEQ ID NO: 77.

[0177] In some embodiments, the amino acid sequence of the beta chain has at least 85% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the beta chain has at least 90% identity to the amino acid sequence of SEQ ID NO:78. In some embodiments, the amino acid sequence of the beta chain has at least 95% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the beta chain has at least 96% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the beta chain has at least 97% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the beta chain has at least 98% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the beta chain has at least 99% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the beta chain has 100% identity to the amino acid sequence of SEQ ID NO: 78.

[0178] In some embodiments, a TCR comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, a TCR comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, a TCR comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, aTCR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, a TCR comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, a TCR comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, a TCR comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, a TCR comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, a TCR comprises an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 79.

[0179] In some embodiments, the alpha chain variable region of a TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the beta chain variable region of a TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 10.

[0180] In some embodiments, the alpha chain constant region of a TCR is a murine constant region. In some embodiments, the beta chain constant region of a TCR is a murine constant region.

[0181] In some embodiments, the alpha chain constant region of a TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the beta chain constant region of a TCR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 124.

[0182] In some embodiments, an engineered TCR comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having at least 96% identity to the aminoacid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, an engineered TCR comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 79.

[0183] In some embodiments, the present disclosure provides engineered TCR comprising a TCR sequences described herein.Nucleic Acids Encoding a TCR

[0184] In some embodiments, the disclosure provides nucleic acids encoding a TCR (e.g., an engineered TCR). Nucleic acids can be or can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., messenger RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras.

[0185] 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, but can 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.

[0186] Engineered nucleic acids of the present disclosure can be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, nucleic acids are produced using GIBSON 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' endsequences 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 can 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 can be used in accordance with the present disclosure.

[0187] In some embodiments, the present disclosure provides an expression cassette comprising an open reading frame comprising a nucleic acid encoding an engineered TCR operably linked to a promoter. A promoter includes 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 can be regulated in vivo by a chemical agent, temperature, or light, for example.

[0188] An open reading frame includes 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.

[0189] In some embodiments, the present disclosure provides a vector comprising the nucleic acid encoding an engineered TCR or an expression vector comprising the same. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. For example, the vector can 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 an engineered TCR into Tregs of the disclosure. Exemplary viral vectors can 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 doublestranded 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 (MV A), 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.

[0190] 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 an engineered 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.

[0191] In some embodiments, the lentiviral vector is a self-inactivating vector. Selfinactivating 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.

[0192] Self-inactivation can be 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 are not be modified. Together this diminishes or abolishes the production of full-length vector RNA in transduced cells.

[0193] In some embodiments, a nucleic acid encoding an engineered 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.

[0194] In some embodiments, a nucleic acid is codon optimized. Codon optimization methods are known in the art. Codon optimization, in some embodiments, can 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 can 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

[0195] In some embodiments, a nucleic acid encoding a TCR comprises a promoter operably linked to a coding sequence encoding an engineered human TCR. A promoter can 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).

[0196] A vector can also include a termination codon and / or expression enhancer elements. Any suitable vectors, promoters, enhancers and termination codons known in the art can 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 can 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).Regulatory T Cell Populations

[0197] In some embodiments, the present disclosure provides cell populations comprising engineered Tregs obtained from a subject having MS. In some embodiments, at least 80% of the cells are Tregs comprising a hypomethylated T cell specific demethylated region (TSDR) at the FOXP3 locus.

[0198] The methylation status of the TSDR at the FOXP3 locus can be evaluated by means known in the art. For example, methylation status of a TSDR at the FOXP3 locus can beevaluated by bisulfite treatment and digital droplet PCR (ddPCR) using methylation-specific primers and probes. In some embodiments, methylation status of the TSDR at the F0XP3 locus is evaluated by single-cell sequencing methods.

[0199] Cell populations comprising Tregs herein are considered “isolated” cell populations. An isolated cell population is a cell population that is removed from (isolated from) a body (e.g., a human body), or removed from a sample obtained from a body. Cell populations comprising Tregs can be isolated from a subject, or from a biological sample obtained from the subject, using for example, apheresis or other methods used in the art for a similar purpose.

[0200] In some embodiments, at least 80% of the cells of a cell population are Tregs comprising a hypomethylated TSDR at the endogenous F0XP3 locus. In some embodiments, at least 85%, 90%, 95%, or 99% of the cells are Tregs comprising a hypomethylated TSDR at the endogenous FOXP3 locus. In some embodiments, a cell population comprises Tregs, wherein 80%, 85%, 90%, 95%, or 99% of the cells are Tregs comprising a hypomethylated TSDR at the endogenous FOXP3 locus.

[0201] In some embodiments, a cell population comprises CD25+ / hlghCD4+CD127' / lowTregs. In some embodiments, a cell population comprises CD25lllgllCD4 CD I 27‘oviFOXP3+Tregs. In some embodiments, a cell population comprises CD25+ / hlghCD4+CD127‘lowRW7J3 CD45RA Tregs.

[0202] In some embodiments, a 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+ / hlghCD4+CD127' / lowTregs. In some embodiments, a 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+CD 12TliovFOXP3+Tregs.

[0203] 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 Tregs in the isolated population are CD45RA+. In some embodiments, a 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 / 10WCD45RA+Tregs.

[0204] As described herein, the present disclosure provides cell populations comprising Tregs comprising a hypomethylated TSDR at the FOXP3 locus. While the isolated cell populations described herein are, in some embodiments, engineered to express an engineered TCR, the FOXP3 locus remains unmodified, in some embodiments. Therefore, in some embodiments,Tregs do not comprise an engineered F0XP3 locus. An “engineered F0XP3 locus” includes any engineered modification (e.g., a modification by the hand of man) intended to alter expression of F0XP3. Such engineered modifications include, but are not limited to, introduction of a F0XP3 transgene, introduction of a modified promoter, and / or use of an engineered agent (e.g., gene editing system, small molecule, or peptide) intended to activate expression of F0XP3.

[0205] In some embodiments, a cell population comprises at least IxlO2, at least IxlO3, at least IxlO4, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, or at least IxlO10Tregs. In some embodiments, a cell population comprises IxlO2to IxlO10, IxlO3to IxlO10, IxlO4to IxlO10, IxlO5to IxlO10, IxlO6to IxlO10, IxlO7to IxlO10, IxlO8to IxlO10, IxlO5to IxlO9, IxlO6to IxlO8, IxlO7to IxlO10, or IxlO4to IxlO6Tregs. In some embodiments, a cell population comprises IxlO6to IxlO10Tregs. In some embodiments, a cell population comprises IxlO7, 2xl07, 3xl07, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, or 9xl07Tregs. In some embodiments, a cell population comprises IxlO7to IxlO10, 2xl07to IxlO10, 3xl07to IxlO10, 4xl07to IxlO10, 5xl07to IxlO10, 6xl07to IxlO10, 7xl07to IxlO10, 8xl07to IxlO10, or 9xl07to IxlO10Tregs.

[0206] In some embodiments, a cell population comprises Tregs comprising a hypomethylated TSDR at the FOXP3 locus, wherein Tregs retain markers of stability (z.e., maintain the hypomethylated TSDR at the FOXP3 locus and / or maintain the protein expression profile of CD25+ / hlghCD4+CD127' / lowFOAP3+) in the presence of proinflammatory conditions. Proinflammatory conditions include 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-ip, 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 can be in vitro or in vivo. In some embodiments, engineered Tregs within a cell population maintain a hypomethylated 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.

[0207] In some embodiments, a cell population comprises Tregs comprising a hypomethylated TSDR at the FOXP3 locus, wherein Tregs retain markers of stability (z.e., maintain the hypomethylated TSDR at the FOXP3 locus and / or maintain the protein expression profile of CD25+ / hlghCD4+CD127' / lowFOAP3+) over time. For example, Tregs can 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, engineered Tregs 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 activation and / or expansion. In some embodiments, engineered Tregs 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 engineered TCR. In some embodiments, engineered Tregs 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.

[0208] In some embodiments, engineered Tregs maintain the protein expression profile of CD25+ / highCD4+CD127' / lowFCL¥P3+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, engineered Tregs maintain the protein expression profile of CD25+ / highCD4+CD127' / lowFCL¥P3+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, engineered Tregs maintain the protein expression profile of CD25+ / hlghCD4+CD127' / lowFO^P3+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 engineered TCR. In some embodiments, engineered Tregs maintain the protein expression profile of CD25+ / hlghCD4+CD127' / lowFO P3+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 freezethaw cycle.

[0209] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells of a cell population comprises a hypomethylated TSDR at Z.FOXP3 locus for at least 5 days, at least 10 days, or at least 15 days after isolation.

[0210] 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 Tregs comprising a hypomethylated TSDR at theFOXP3 locus does not decrease by more than 20% between these 2 or more timepoints.

[0211] For example, hypomethylation of a TSDR at the FOXP3 locus can be 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 Tregs comprising a hypomethylated TSDR at the FOXP3 locus does not decrease by more than 20%, more than 15%, more than 10%, more than 5%, 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 Tregscomprising 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 Tregs comprising a hypomethylated TSDR at 3\Q OXP3 locus does not decrease by more than 5% within 5, 6, 7, 8, 9, 10, or 11 days after isolation from a biological sample.

[0212] 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 Tregs of a cell population express an engineered TCR (e.g., following transduction of a cell population with an engineered TCR). In some embodiments, 10%-60% or 20%-50% of Tregs of a cell population express an engineered 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 Tregs of a cell population express an engineered TCR (e.g., following transduction of a cell population with an engineered TCR). In some embodiments, 10%-60% or 20%-50% of Tregs of a cell population express an engineered TCR.

[0213] In some embodiments, the TSDR at the endogenous FOXP3 locus of Tregs of a cell population of Tregs remains hypomethylated until administration of a cell population to a subject. In some embodiments, the TSDR at the endogenous FOXP3 locus of Tregs of a cell population of Tregs remains hypomethylated following a cryopreservation freeze-thaw cycle.

[0214] In some embodiments, engineered Tregs (e.g., Tregs) of a cell population exhibit one or more cellular functions that are associated with Tregs 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 includes 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- IBB, CD25, CD71, or CTLA-4. A Treg as provided herein expresses one or more of these markers when it comes into contact with an MBP peptide complexed with MHC.

[0215] Suppression activity includes the suppression of activation, proliferation and cytokine production of non-Tregs (e.g., CD8+ T cells and CD4+ conventional T cells) in part to suppress the immune system from becoming overactive. Tregs provided herein exhibit suppression activity when contacted with an MBP peptide complexed with MHC that binds to an engineered TCR expressed by Tregs. In some embodiments, an MBP peptide is presented by a cell expressing the MHC. For example, Tregs can suppress the activation, proliferation and cytokine production of conventional T cells having specificity towards a shared target peptidecomplexed with MHC, e.g., a shared target peptide presented by an Antigen Presenting Cell (APC). In some embodiments, engineered Tregs suppress proliferation and growth of non- Tregs 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-Tregs in the absence of Tregs).

[0216] In some embodiments, engineered Tregs 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 Tregs). In some embodiments, engineered Tregs 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 Tregs compared to conventional T cells). In some embodiments, engineered Tregs 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 Tregs). In some embodiments, engineered Tregs 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 Tregs compared to conventional T cells). In some embodiments, engineered Tregs 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 Tregs). In some embodiments, engineered Tregs 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 Tregs compared to conventional T cells).

[0217] In some embodiments, engineered Tregs exhibit cytokine secretion activity (e.g., secretion of IL- 10) when contacted with an MBP peptide complexed with MHC, e.g., an MBP peptide presented by an APC, that binds to an engineered TCR expressed by Tregs. In some embodiments, engineered Tregs exhibit expression of activation markers when contacted with an MBP peptide complexed with MHC that binds to an engineered TCR expressed by Tregs. For example, Tregs can exhibit expression of CD69, 4- IBB, CD25, CD71, and / or CTLA-4 when contacted with an MBP peptide complexed with MHC that binds to an engineered TCR expressed by Tregs. In some embodiments, engineered Tregs exhibit suppression activity when contacted with an MBP peptide complexed with MHC that binds to an engineered TCR expressed by Tregs. For example, Tregs can suppress the activation of conventional T cells having specificity towards a shared MBP peptide complexed with MHC.

[0218] The cellular functions of Tregs can 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 anengineered human TCR. The cellular functions of Tregs can be assessed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after transduction of cells with an engineered human TCR. The cellular functions of Tregs can 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 engineered human TCR.

[0219] In some embodiments, transduced Tregs (e.g., transduced Tregs of an isolated population) retain their cellular functionality (e.g., ability to be activated) following a cryopreservation freeze-thaw cycle. In some embodiments, transduced Tregs can be activated and / or expanded following a cryopreservation freeze-thaw cycle. In some embodiments, engineered Tregs can exhibit cytokine secretion activity, expression of certain activation markers, and / or suppression activity following a cryopreservation freeze-thaw cycle.

[0220] 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+ / hlghCD4+CD127' / lowprior to activation and / or transduction with an engineered TCR.

[0221] A cell population comprising Tregs can comprise a minority amount of non-Tregs (e.g., conventional T cells). Non-Tregs can 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 a cell population comprising Tregs are non-Tregs.

[0222] 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 a cell population comprising Tregs are conventional T cells. In some embodiments, a cell population comprising Tregs comprises an undetectable amount of conventional T cells. In some embodiments, a cell population comprising Tregs comprises an undetectable amount of conventional CD4+ T cells. In some embodiments, a cell population comprising Tregs comprises an undetectable amount of B cells. In some embodiments, a cell population comprising Tregs comprises an undetectable amount of NK T cells. In some embodiments, a cell population comprising Tregs comprises an undetectable amount of CD14+ cells. In some embodiments, a cell population comprising Tregs comprises an undetectable number of eosinophils. In some embodiments, a cell population comprising Tregs comprises an undetectable number of neutrophils. In some embodiments, an isolated population of cells comprising Tregs comprises an undetectable amount of CD8+ T cells.

[0223] In some embodiments, the ratio of Tregs to conventional T cells in an isolate population of cells comprising Tregs 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.Pharmaceutical Compositions

[0224] In some embodiments, the disclosure provides pharmaceutical compositions comprising cell populations of Tregs (e.g., comprising an engineered TCR) described herein. In some embodiments, a pharmaceutical composition comprises a cell population of engineered Tregs (e.g., comprising an engineered TCR) described herein and a pharmaceutically acceptable excipient.

[0225] Pharmaceutically acceptable excipients include pharmaceutically acceptable carriers, pharmaceutically acceptable diluents, and / or pharmaceutically acceptable adjuvants.

[0226] Pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. Sterile injectable formulations can be prepared using a non-toxic parenterally acceptable diluent or solvent. A pharmaceutical composition for use in accordance with the present invention can 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 can 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.

[0227] A pharmaceutical composition can comprise an effective amount of Tregs that is sufficient to elicit a desired biological response. For example, an effective amount of Tregs as described herein can include a number of cells that is sufficient to improve a symptom associated with multiple sclerosis (e.g., progressive Multiple Sclerosis). As will be appreciated by the skilled artisan, an effective amount of a solution or preparation provided herein can 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.

[0228] In some embodiments, cell populations (e.g., intended to be used in a pharmaceutical composition) are cryopreserved (e.g., subjected to one or more cry opreservation freeze-thaw cycles). That is, cell populations produced herein can 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 can 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.Additional Aspects

[0229] Paragraph 1. A method comprising: administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB1*11 :O4, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1 * 11 :04 allele.

[0230] Paragraph 2. The method of Paragraph 1, wherein the HLA-DRB1*11 :O4 allele is identified by HLA haplotyping.

[0231] Paragraph s. A method comprising: detecting an HLA-DRB1*11 :04 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

[0232] Paragraph 4. The method of Paragraph 3, wherein the HLA-DRB1*11 :O4 allele is detected by HLA haplotyping.

[0233] Paragraph 5. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on the presence of an HLA-DRB1*11 :O4 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

[0234] Paragraph 6. The method of any one of the preceding Paragraphs, wherein the subject has been identified as also having an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HL A-DRB 1*15:01, HL A-DRB 1 * 04 : 04, and HL A-DRB 1 * 04 : 02.

[0235] Paragraph 7. The method of Paragraph 5 or 6, wherein one or more HLA DRB1 allele is identified by HLA haplotyping.

[0236] Paragraph 8. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB1*11 :O4 allele in the subject and (ii) based on the presence of an HLA DRB 1 allele selected from HLA-DRB 1 * 11 : 04, HLA-DRB 1*15:01, HL A-DRB 1*04: 04, and HLA-DRB 1*04: 02 in the subject; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

[0237] Paragraph 9. The method of any one of Paragraphs 2 or 4-7, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

[0238] Paragraph 10. The method of any one of the preceding Paragraphs, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRBl*15:01, HLA-DRB 1*04:04, and HLA- DRB 1*04:02.

[0239] Paragraph I L A method comprising: administering to a subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1*04:02, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1*04:02 allele.

[0240] Paragraph 12. The method of Paragraph 11, wherein the HLA-DRB 1*04: 02 allele is identified by HLA haplotyping.

[0241] Paragraph 13. A method comprising: detecting an HLA-DRB 1*04: 02 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1*04:02.

[0242] Paragraph 14. The method of Paragraph 13, wherein the HLA-DRB 1*04: 02 allele is detected by HLA haplotyping.

[0243] Paragraph 15. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on the presence of an HLA-DRB 1*04: 02 allele in the subject; and administering to the subject aregulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRBl*04:02.

[0244] Paragraph 16. The method of any one of Paragraphs 11-15, wherein the subject has been identified as having an HLA DRB1 allele selected from HLA-DRBl*04:02, HLA- DRB 1*15:01, HL A-DRB 1 * 04 : 04, and HL A-DRB 1 * 11 : 04.

[0245] Paragraph 17. The method of Paragraph 15 or 16, wherein the HLA-DRBl*04:02 allele is identified by HLA haplotyping.

[0246] Paragraph 18. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRBl*04:02 allele in the subject and (ii) the presence of an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRBl*04:04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRBl*04:02.

[0247] Paragraph 19. The method of any one of Paragraphs 12 or 14-18, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods .

[0248] Paragraph 20. The method of any one of Paragraphs 11-19, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRBl*15:01, HLA-DRBl*04:04, and HLA- DRB1*11 :O4.

[0249] Paragraph 21. A method comprising: administering to a subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1*04:04, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1*04:04 allele.

[0250] Paragraph 22. The method of Paragraph 21, wherein the HLA-DRB 1*04: 04 allele is identified by HLA haplotyping.

[0251] Paragraph 23. A method comprising: detecting an HLA-DRB 1*04: 04 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1*04:04.

[0252] Paragraph 24. The method of Paragraph 23, wherein the HLA-DRB 1*04: 04 allele is detected by HLA haplotyping.

[0253] Paragraph 25. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on the presence of an HLA-DRBl*04:04 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRBl*04:04.

[0254] Paragraph 26. The method of any one of Paragraphs 21-25, wherein the subject has been identified as having an HLA DRB1 allele selected from HLA-DRBl*04:02, HLA- DRB 1*15:01, HL A-DRB 1 * 04 : 04, and HL A-DRB 1 * 11 : 04.

[0255] Paragraph 27. The method of Paragraph 25 or 26, wherein the HLA-DRB 1 *04:04 allele is identified by HLA haplotyping.

[0256] Paragraph 28. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB 1*04: 04 allele in the subject and (ii) the presence of an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*04: 04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB 1*04: 04.

[0257] Paragraph 29. The method of any one of Paragraphs 22 or 24-28, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

[0258] Paragraph 30. The method of any one of Paragraphs 21-29, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRBl*15:01, HLA-DRB 1*04: 02, and HLA- DRB1*11 :O4.

[0259] Paragraph 31. A method comprising: administering to a subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRBl*15:01, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRBl*15:01 allele.

[0260] Paragraph 32. The method of Paragraph 31, wherein the HLA-DRBl*15:01 allele is identified by HLA haplotyping.

[0261] Paragraph 33. A method comprising: detecting an HLA-DRBl*15:01 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRBl*15:01.

[0262] Paragraph 34. The method of Paragraph 33, wherein the HLA-DRBl*15:01 allele is detected by HLA haplotyping.

[0263] Paragraph 35. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on the presence of an HLA-DRBl*15:01 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRBl*15:01.

[0264] Paragraph 36. The method of any one of Paragraphs 31-35, wherein the subject has been identified as having an HLA DRB1 allele selected from HLA-DRBl*04:02, HLA- DRB 1*15:01, HL A-DRB 1 * 04 : 04, and HL A-DRB 1 * 11 : 04.

[0265] Paragraph 37. The method of Paragraph 35 or 36, wherein the HL A-DRB 1*15:01 allele is identified by HLA haplotyping.

[0266] Paragraph 38. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB1* 15:01 allele in the subject and (ii) the presence of an HLA DRB1 allele selected from HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRBl*04:04, and HLA- DRB 1*04:02 in the subject; and administering to the subject a regulatory T cell comprising an engineered TCR that binds to and is activated by an MBP peptide complexed with an MHC Class II comprising HLA-DRB1* 15:01.

[0267] Paragraph 39. The method of any one of Paragraphs 32 or 34-38, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

[0268] Paragraph 40. The method of any one of Paragraphs 31-39, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRBl*04:02, HLA-DRBl*04:04, and HLA- DRB1*11 :O4.

[0269] Paragraph 41. The method of any one of the preceding Paragraphs, wherein the MBP peptide is an MBP 83-99 peptide comprising the amino acid sequence of ENPVVHFFKNIVTPRTP (SEQ ID NO: 61).

[0270] Paragraph 42. The method of any one of the preceding Paragraphs, wherein the engineered TCR is encoded as a single polypeptide, optionally comprising a self-cleaving peptide sequence.

[0271] Paragraph 43. The method of any one of the preceding Paragraphs, wherein the administering comprises administering to the subject a cellular composition comprising a therapeutically effective amount of regulatory T cells comprising the engineered TCR.

[0272] Paragraph 44. The method of Paragraph 43, wherein the administering comprises one or more infusions of the cellular composition.

[0273] Paragraph 45. The method of Paragraph 43 or 44, wherein the regulatory T cells are autologous relative to the subject.

[0274] Paragraph 46. The method of Paragraph 43 or 44, wherein the regulatory T cells are allogeneic relative to the subject.

[0275] Paragraph 47. The method of any one of Paragraphs 43-46, wherein the regulatory T cells are derived from stem cells.

[0276] Paragraph 48. The method of Paragraph 47, wherein the stem cells are pluripotent stem cells

[0277] Paragraph 49. The method of Paragraph 48, wherein the pluripotent stem cells are embryonic stem cells or hematopoietic stem cells.

[0278] Paragraph 50. The method of Paragraph 48, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0279] Paragraph 51. The method of any one of Paragraphs 43-46, wherein the regulatory T cells are derived from polarized T cells.

[0280] Paragraph 52. The method of any one of Paragraphs 43-46, wherein the regulatory T cells are derived from thymocytes.

[0281] Paragraph 53. The method of Paragraph 52, wherein the regulatory T cells are isolated from peripheral blood or cord blood.

[0282] Paragraph 54. The method of any one of Paragraphs 43-46, wherein the regulatory T cells are induced regulatory T cells.

[0283] Paragraph 55. The method of any one of the preceding Paragraphs, wherein the subject is diagnosed with multiple sclerosis.

[0284] Paragraph 56. The method of any one of the preceding Paragraphs, wherein the subject has Progression Independent of Relapse Activity (PIRA).

[0285] Paragraph 57. The method of Paragraphs 55 or 56, wherein the subject is diagnosed with progressive multiple sclerosis, relapsing remitting multiple sclerosis, or multiple sclerosis.

[0286] Paragraph 58. The method of Paragraph 57, wherein the progressive multiple sclerosis is primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

[0287] Paragraph 59. The method of any one of the preceding Paragraphs, wherein the engineered TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and / or the beta chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0288] Paragraph 60. The method of Paragraph 59, wherein the alpha chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and the beta chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0289] Paragraph 61. The method of Paragraph 60, wherein: (i) the alpha chain comprises 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 (ii) the beta chain comprises 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.

[0290] Paragraph 62. The method of Paragraph 61, wherein: the alpha chain comprises an amino acid sequence having at least 90%identity to SEQ ID NO: 4 or SEQ ID NO: 5; and the beta chain comprises an amino acid sequence having at least 90%identity to SEQ ID NO: 10 or SEQ ID NO: 11.

[0291] Paragraph 63. The method of any one of the preceding Paragraphs, wherein an endogenous FOXP3 locus of the regulatory T cell is not engineered.

[0292] Paragraph 64. The method of any one of the preceding Paragraphs, wherein the regulatory T cell comprising the engineered TCR has a hypomethylated Treg-specific demethylated region (TSDR) at an endogenous FOXP3 locus.EXAMPLESExample 1. An engineered T cell receptor is capable of binding to selected HLA-MBP peptide complexes.

[0293] This Example involved the determination of the ability of a panel of cell lines or PBMCs expressing MHC Class II with different human leukocyte antigen (HLA) DRB1 alleles, when complexed with a myelin basic protein (MBP) 83-99 peptide, to bind to and activate a Treg comprising TCR-E (an engineered T cell receptor (TCR) that binds to and is activated by MBP 83-99 peptide, having SEQ ID NO. 79). TCR-E was previously disclosed in PCT / US2023 / 072045, incorporated herein by reference in its entirety.

[0294] More than thirty different cell lines or PBMCs (as shown in FIG. 1) having different combinations of HLA-DRB1 alleles were cultured with a population of Tregs comprising anengineered TCR (TCR-E) that binds to and is activated by MBP 83-99 peptide complexed with MHC Class II. Specifically, 5xl04cells from each cell line or 2xl05PBMCs were individually cultured with the 5e4 TCR-transduced Tregs, lOOOU / mL IL-2, and luM MBP 83-99 peptide overnight for 20 hours in a humidified 37 °C 5% CO2 incubator. TCR-transduced Tregs were also cultured with the cell lines or PBMCs but without MBP 83-99 peptide. As a control, TCR- transduced Tregs were cultured in the absence of cell lines or PBMCs with or without peptide.

[0295] The TCR-transduced Tregs were stained (with CellTrace™ Violet (CTV)) prior to culture. The following day, the activation of transduced TCR-transduced Tregs was measured by flow cytometry by staining with live / dead viability dye, anti-CD3, anti-CD4, anti -TCR Vb2, and anti-CD69 antibody. Data was acquired on a BioRad ZE5 cytometer and analyzed in FlowJo vlO. Cell activation was determined by level of CD69 expression (FIG. 1). Percent CD69 expression was calculated relative to the control. The experiment was performed in duplicate. The %CD69 expression levels with and without peptide are shown in Table 4.

[0296] This data demonstrates that cell lines expressing HLA-DRB1*11 :O4, HLA- DRBl*15:01, HLA-DRBl*04:04, and / or HLA-DRBl*04:02 alleles were capable of activating the population of Tregs expressing TCR-E in the presence of MBP 83-99. Specifically, the data shows that cells homozygous for HLA-DRB1* 15:01 (BLS2b; HH; 525), cells having a single HLA-DRBl*15:01 allele (U266 Bl; DOHH-2; MHH-CALL-2; Cl-1), cells having an HLA-DRBl*15:01 allele and a HLA-DRBl*04:04 allele (L-363); cells having an HLA-DRB1*11:O4 allele (580; 421); cells having an HLA-DRBl*04:04 allele (583; 622); and cells having an HLA-DRB 1*04:02 allele (445; 408) were capable of activating Treg expressing TCR-E (FIG. 1).

[0297] Thus, it has been observed that an MHC Class II comprising an HLA DR2 allele selected from HLA-DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*04: 04, or HLA- DRB 1*04:02 (when complexed with MBP 83-99 peptide) can bind to and activate Tregs expressing TCR-E.Table 4: Percent CD69 expression on representative drug product upon activation by PBMC and cell lines relative to controlExample 2. Binding of regulatory T cells to cells expressing HLA-DRB1 alleles

[0298] This Example involves the generation of EC50 (half maximal effective concentration) curves for TCR-E transduced Treg activation when cocultured with PBMCs expressing MHCs comprising the HLA DRB1 alleles identified in Example 1 (HLA-DRB1*11 :O4, HLA- DRB 1*15:01, HL A-DRB 1*04: 04, or HL A-DRB 1*04: 02) complexed with MBP 83-99 peptideor a Selaginella moellendorffii peptide having the amino acid sequence of SVGKIHFFKMEVVS (SEQ ID NO: 125).

[0299] IxlO5HLA-DR1 -expressing PBMCs (HLA-DRB1*11:O4, HLA-DRBl*15:01, HLA- DRB 1*04:04, or HLA-DRBl*04:02) were cultured in each well of a 96-well round bottom plate 5xl04Tregs transduced with TCR-E and lOOOU / mL of IL-2. PBMCs were stained with CTV prior to culture.

[0300] Either MBP 83-99 or Selaginella moellendorffii peptide (SVGKIHFFKMEVVS (SEQ ID NO: 125)) was diluted to lOuM. An 11-point curve of peptide concentrations was performed starting from lOpM a going down to 9.5pM was added to the 96 well plate.

[0301] The plate was transferred to a humidified 5% CO2 37°C incubator. The next day the assay plate was removed from the incubator and centrifuged at 500 x g for 3 minutes. The activation of transduced TCR-transduced Tregs was measured by flow cytometry by staining with live / dead viability dye, anti-CD3, anti-CD4, anti-TCR Vb2, and anti-CD69 antibody. Data was acquired on a BioRad ZE5 cytometer and analyzed in FlowJo vlO. Cell activation was determined by level of CD69 expression. As shown in FIG. 2 and Table 5, each of the tested PBMCs (expressing any one of DRBl*15:01, DRBl*04:04, DRB1*11 :O4, or DRBl*04:02) activated the TCR-transduced Tregs in the presence of either peptide when co-cultured. These data show that PBMCs expressing DRBl*15:01 showed the highest activation of TCR- transduced Treg, followed by DRB 1*04:04, DRB 1*04:02, and then DRB1*11 :O4, and that activation was higher with the MBP83-99 peptide than the Selaginella moellendorffii peptide.Table 5. EC50 values for MBP 83-99 and HLA-DRB1 allele moleculesExample 3. Evaluation of tissue lysates and capability to bind cells from different tissue types expressing HLA-DRB1 alleles

[0302] This Example involves the determination of the ability of Tregs comprising TCR-E to be specifically activated in tissues expressing endogenous MBP peptide.

[0303] Tissue lysates (as shown in FIG. 3) were thawed on ice spun in a centrifuge to pellet any debris. 0.3125mg / mL of lysate was added to HLA-DR1 -expressing PBMCs (HLA-DRB1*11:O4, HLA-DRB1*15:O1, HLA-DRBl*04:04, or HLA-DRBl*04:02) (5xl04cells) per well in a 96 well round bottom plate. The plate was incubated for 1 hour in a humidified 5% CO2 37°C incubator.

[0304] After 1 hour the supernatant was removed and IxlO4Tregs transduced with TCR-E and lOOOU / mL of IL-2 were added. The plate was transferred to a humidified 5% CO2 37°C incubator.

[0305] The next day the assay plate was removed from the incubator and centrifuged at 500 x g for 3 minutes. The activation of transduced TCR-transduced Tregs was measured by flow cytometry by staining with live / dead viability dye, anti-CD3, anti-CD4, anti-TCR Vb2, and anti-CD69 antibody. Data was acquired on a BioRad ZE5 cytometer and analyzed in FlowJo vlO. Cell activation was determined by level of CD69. Percent CD69 levels relative to control are shown in Figure 3 and in Table 6 below:Table 6: CD69 expression of TCR-transduced Treg upon stimulation with tissue lysate loaded PBMCs with HLA haplotypes DRBl*04:04, DRB1*15:O1, DR1*11:O4DRBl*04:02.

[0306] It was found that Tregs were activated in the presence of tissue lysate from the brain and spinal cord, which are known to express MBP 83-99.Example 4. Evaluation capability to bind cells from different tissue types expressing HLA-DRB1 alleles

[0307] Regulatory T cells transduced with a lentiviral vector encoding TCR-A were tested in functional assays for their ability to be activated in response to the MBP83-99 peptide when presented by HLA DRBl*15:01. TCR-A (SEQ ID NO: 17) has an identical amino acid sequence to TCR-E except that two additional cysteine mutations were introduced into the constant region of TCR-E. Both TCRs have the same amino acid sequence in the variable region. Tregs were isolated from PBMCs using the EasySep™ Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit (Stemcell Technologies). Transduced regulatory T cells were differentiated from untransduced regulatory T cells by a GFP integrated in the lentiviral vector.

[0308] To test whether transduced regulatory T cells could be activated, the transduced regulatory T cells were incubated at 37°C with Mitomycin C treated peripheral blood mononuclear cells (PBMCs) isolated from an HLA DRBl*15:01 or HLA DRBl*15:03 donor (or a no PBMC negative control), and 1000 lU / ml IL-2 in T cell culture medium. Cells were unstimulated or stimulated with 1 pM MBP83-99 peptide for TCR-specific stimulation or 30 ng / mL of the OTK3 anti-CD3 antibody as a polyclonal stimulation positive control. The ratio of PBMC to regulatory T cells was 4: 1. Following incubation, regulatory T activation markers (CD69, CD71, and 4-1BB) were measured by FACS and cytokine levels (IL-10) were measured by ELLA after 24 and 72 hours.

[0309] CD69, CD71, 4- IBB, and IL- 10 levels in each population after 24 and 72 hours are shown in Figure 4. As is shown, in the absence of antigen, little activation of TCR-A expressing Tregs is shown, either in the presence or absence of PBMC, regardless of HLA haplotype. Incontrast, in the presence of MBP83-99, TCR-A expressing Tregs are activated when MBP is presented by either HL A DRB 1*15:01 or HL A DRB 1*15:03 -expressing PBMC .Example 5. Antigen-specific Activation of TCR-E expressing Treg by HLA- DRB1*15:O1, HLA-DRB1* 15:02 and HLA-DRB1* 15:03

[0310] HLA-DRBl*15:01 was selected due to the increased risk of multiple sclerosis (MS) in Caucasians that carry this high-risk allele. HLA-DRB 1 * 15 :02 and 15 :03, two additional HLA- DRB1*15 alleles that each differ from *15:01 by a single amino acid, were selected because both have been shown to present the immunodominant epitope of myelin basic protein, MBPss- 99, similar to that of 15:01. Further, similar to HLA-DRB 1*15:01, HLA-DRB 1*15:03 is linked to an increased MS risk while HLA-DRB 1*15:02 is a common allele found in populations of Southeast Asian and Oceania ancestry.

[0311] The activity of TCR-E Treg was assessed when MBP83-99 was presented in the context HLA-DRB 1*15:01, HLA-DRB 1 * 15 : 02 and HLA-DRB 1 * 15 : 03 in vitro. TCR-E Treg were cocultured with HLA-DRB l*15:03+, HLA-DRB l*15:02+or HLA-DRBl*15:01+PBMCs and titrated concentrations of MBP83-99 (0-10pM) in the presence of recombinant human IL-2 (1000 lU / mL) Cells were stained with a panel of fluorescently labeled antibodies specific for T cell identity (CD3 and CD4) and Treg activation markers (CD69) for analysis by multicolor flow cytometry to assess Treg activation.

[0312] As is shown in FIG. 5A-FIG. 5B, TCR-E Tregs demonstrated an on-target response to MBP83-99 when presented by both DRB 1 * 15 :02 (FIG. 5 A) and DRB 1 * 15 :03 (FIG. 5B) relative to DRB1* 15:01, which has previously been shown to activate TCR-E expressing Tregs when complexed with MBP83-99. TCR-E Tregs showed average EC50 values of 11-14 nM for HLA- DRBl*15:01, 78.5nM for HLA-DRB 1*15:02 and 40.4 nM for HLA-DRB1* 15:03.

[0313] These results demonstrate that TCR-E expressing Tregs are capable of being potently activated by MBP83-99 presented in the context of HLA-DRB 1* 15:02 (FIG. 5 A) and HLA- DRB 1*15:03 (FIG. 5B), in addition to HLA-DRB 1*15:01, thereby broadening the patient segment that can receive TCR-E expressing Tregs.Example 6: Evaluation of tissue lysates and capability to bind cells from different tissue types expressing HLA-DRB1*15:O1, HLA-DRB1* 15:02 and HLA-DRB1* 15:03 alleles

[0314] TCR-E expressing Tregs were evaluated for activation by tissue lysates spanning all major human organs. Cells were stained with a panel of fluorescently labeled antibodies specific for T cell identity (CD3 and CD4) and Treg activation markers (CD69) for analysis bymulticolor flow cytometry to assess activation of TCR-E expressing Tregs. DRB1*15:O1, DRB 1*15:02 and DRB 1*15:03 PBMC cells were pulsed with tissue lysate and cultured with TCR-E expressing Tregs. Activation of TCR-E expressing Tregs was detected by CD69 expression after overnight culture. Negative control was with medium alone and positive control was with 1 pM of MBP83-99.

[0315] As is shown in FIG. 6A-FIG. 6B, TCR-E expressing Tregs displayed no off-target activation from tissue extracts presented by these haplotypes. Only lysates from the spinal cord and brain where MBP83-99 is specifically expressed activated TCR-E expressing Tregs.

[0316] 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 can encompass the entirety of the document.

[0317] 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.”

[0318] 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.

[0319] In the claims, as well as in the specification above, 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, z.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.

[0320] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0321] 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.

Claims

CLAIMS1. A method comprising: administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with a Major Histocompatibility Complex (MHC) Class II comprising HLA-DRB 1*11 :04, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1 * 11 :04 allele.

2. The method of claim 1, wherein the HLA-DRB1 *11 :04 allele is identified by HLA haplotyping.

3. A method comprising: detecting an HLA-DRB1*11 :O4 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

4. The method of claim 3, wherein the HLA-DRB 1 * 11 :04 allele is detected by HLA haplotyping.

5. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subj ect for treatment based on the presence of an HLA-DRB 1 * 11 : 04 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

6. The method of any one of the preceding claims, wherein the subject has been identified as also having an HLADRB1 allele selected from HLA-DRB1*11 :O4, HLA- DRB1*15:O1, HLA-DRB1*15:O2, DRB 1* 15:03, HLA-DRB1 *04:04, and HLA- DRB 1*04:02.

7. The method of claim 5 or 6, wherein one or more HLADRB1 allele is identified byHLA haplotyping.

8. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB1*11 :O4 allele in the subject and (ii) based on the presence of an HLADRB1 allele selected from HLA-DRB1*11:O4, HLA-DRB1*15:O1, HLA-DRB 1*15:02, HLA-DRB 1*15:03, HLA- DRB 1*04:04, and HLA-DRB 1*04:02 in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1 * 11 :04.

9. The method of any one of claims 2 or 4-7, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

10. The method of any one of the preceding claims, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRB 1*15:01, HLA-DRB 1 * 15 : 02, HLA-DRB 1*15:03, HL A- DRB 1*04:04, and HLA-DRB 1*04:02.

11. A method comprising: administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04: 02, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1*04: 02 allele.

12. The method of claim 11, wherein the HLA-DRB 1 *04: 02 allele is identified by HLA haplotyping.

13. A method comprising: detecting an HLA-DRBl*04:02 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04:02.

14. The method of claim 13, wherein the HLA-DRB 1 *04:02 allele is detected by HLA haplotyping.

15. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on the presence of an HLA-DRB 1 *04:02 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04:02.

16. The method of any one of claims 11-15, wherein the subject has been identified as having an HLADRB1 allele selected from HLA-DRB 1*04: 02, HLA-DRBl*15:01, HLA- DRB 1 * 15 : 02, HLA-DRB 1*15:03, HLA-DRB 1 * 04 : 04, and HLA-DRB 1 * 11 : 04.

17. The method of claim 15 or 16, wherein the HLA-DRB 1*04: 02 allele is identified by HLA haplotyping.

18. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB 1*04: 02 allele in the subject and (ii) the presence of an HLADRB1 allele selected from HLA- DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*15:02, HLA-DRB 1*15:03, HLA- DRB 1*04:04, and HLA-DRB 1*04:02 in the subject; andadministering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04:02.

19. The method of any one of claims 12 or 14-18, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods .

20. The method of any one of claims 11-19, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HL A selected from HLA-DRB 1*15:01, HLA-DRB 1 * 15 : 02, HLA-DRB 1*15:03, HL A-DRB 1*04:04, and HLA-DRB1*11 :O4.

21. A method comprising: administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04: 04, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1*04: 04 allele.

22. The method of claim 21, wherein the HLA-DRB 1 *04:04 allele is identified by HLA haplotyping.

23. A method comprising: detecting an HLA-DRB 1*04: 04 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04:04.

24. The method of claim 23, wherein the HLA-DRB 1 *04:04 allele is detected by HLA haplotyping.

25. A method comprising:human leukocyte antigen (HL A) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on the presence of an HLA-DRB 1 *04:04 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04:04.

26. The method of any one of claims 21-25, wherein the subject has been identified as having an HLADRB1 allele selected from HLA-DRB 1*04: 02, HLA-DRBl*15:01, HLA- DRB 1 * 15 : 02, HLA-DRB 1*15:03, HLA-DRB 1 * 04 : 04, and HLA-DRB 1 * 11 : 04.

27. The method of claim 25 or 26, wherein the HLA-DRB 1*04: 04 allele is identified by HLA haplotyping.

28. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB 1*04: 04 allele in the subject and (ii) the presence of an HLADRB1 allele selected from HLA- DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*15:02, HLA-DRB 1*15:03, HLA- DRB 1*04:04, and HLA-DRB 1*04:02 in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*04:04.

29. The method of any one of claims 22 or 24-28, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

30. The method of any one of claims 21-29, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRB 1*15:01, HLA-DRB 1 * 15 : 02, HLA-DRB 1*15:03, HL A-DRB 1*04:02, and HLA-DRB1*11 :O4.

31. A method comprising: administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB1*15:O1, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB1* 15:01 allele.

32. The method of claim 31, wherein the HLA-DRBl *15:01 allele is identified by HLA haplotyping.

33. A method comprising: detecting an HLA-DRBl*15:01 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRBl*15:01.

34. The method of claim 33, wherein the HLA-DRBl *15:01 allele is detected by HLA haplotyping.

35. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subj ect for treatment based on the presence of an HL A-DRB 1 *15:01 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRBl*15:01.

36. The method of any one of claims 31-35, wherein the subject has been identified as having an HLADRB1 allele selected from HLA-DRBl*04:02, HLA-DRBl*15:01, HLA- DRB 1 * 15 : 02, HL A-DRB 1*15:03, HL A-DRB 1 * 04 : 04, and HL A-DRB 1 * 11 : 04.

37. The method of claim 35 or 36, wherein the HLA-DRB1*15:O1 allele is identified by HLA haplotyping.

38. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB1* 15:01 allele in the subject and (ii) the presence of an HLADRB1 allele selected from HLA- DRB1*11:O4, HLA-DRBl*15:01, HLA-DRB 1*15:02, HLA-DRB 1*15:03, HLA- DRB 1*04:04, and HLA-DRB 1*04:02 in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRBl*15:01.

39. The method of any one of claims 32 or 34-38, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

40. The method of any one of claims 31-39, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRB 1*04: 02, HLA-DRB 1*04: 04, HLA-DRB 1* 15:02, HLA-DRB 1*15:03 and HLA-DRB1*11 :O4.

41. A method comprising: administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1* 15:03, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1* 15:03 allele.

42. The method of claim 41, wherein the HLA-DRB1 * 15:03 allele is identified by HLA haplotyping.

43. A method comprising:detecting an HLA-DRB1* 15:03 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*15:03.

44. The method of claim 43, wherein the HLA-DRB 1 * 15 :03 allele is detected by HLA haplotyping.

45. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on the presence of an HLA-DRB 1 * 15:03 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*15:03.

46. The method of any one of claims 41-45, wherein the subject has been identified as having an HLADRB1 allele selected from HLA-DRB 1*04: 02, HLA-DRBl*15:01, HLA- DRB 1*15:02, DRB 1* 15:03, HLA-DRB 1 *04:04, and HLA-DRB1*11 :O4.

47. The method of claim 45 or 46, wherein the HLA-DRB 1* 15:03 allele is identified by HLA haplotyping.

48. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB 1* 15:03 allele in the subject and (ii) the presence of an HLA DRB 1 allele selected from HLA- DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*15:02, DRBl*15:03, HLA-DRB 1*04: 04, and HLA-DRB 1*04: 02 in the subject; andadministering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*15:03.

49. The method of any one of claims 42 or 44-48, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

50. The method of any one of claims 41-49, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HL A selected from HLA-DRB 1*04: 02, HLA-DRB 1*04: 04, HLA-DRB1* 15:02, DRBl*15:01 and HLA-DRB1*11 :O4.

51. A method comprising: administering to a subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1* 15:02, wherein the subject has been diagnosed with multiple sclerosis and has been identified as having an HLA-DRB 1* 15:02 allele.

52. The method of claim 51, wherein the HLA-DRB1 * 15:02 allele is identified by HLA haplotyping.

53. A method comprising: detecting an HLA-DRB1* 15:02 allele in a subject diagnosed with multiple sclerosis; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*15:02.

54. The method of claim 53, wherein the HLA-DRB 1 * 15 :02 allele is detected by HLA haplotyping.

55. A method comprising:human leukocyte antigen (HL A) haplotyping a subject diagnosed with multiple sclerosis; selecting the subj ect for treatment based on the presence of an HL A-DRB 1 * 15 : 02 allele in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*15:02.

56. The method of any one of claims 51-55, wherein the subject has been identified as having an HLADRB1 allele selected from HLA-DRB 1*04: 02, HLA-DRBl*15:01, HLA- DRB 1*15:02, DRB 1* 15:03, HLA-DRB 1 *04:04, and HLA-DRB1*11 :O4.

57. The method of claim 55 or 56, wherein the HLA-DRB 1* 15:02 allele is identified by HLA haplotyping.

58. A method comprising: human leukocyte antigen (HLA) haplotyping a subject diagnosed with multiple sclerosis; selecting the subject for treatment based on (i) the presence of an HLA-DRB 1* 15:02 allele in the subject and (ii) the presence of an HLA DRB 1 allele selected from HLA- DRB1*11 :O4, HLA-DRBl*15:01, HLA-DRB 1*15:02, DRBl*15:03, HLA-DRB 1*04: 04, and HLA-DRB 1*04: 02 in the subject; and administering to the subject a regulatory T cell comprising an engineered T cell receptor (TCR) that binds to and is activated by a myelin basic protein (MBP) peptide complexed with an MHC Class II comprising HLA-DRB 1*15:02.

59. The method of any one of claims 52 or 54-58, wherein the haplotyping is performed using next-generation sequencing (NGS), Sanger-based sequencing, and / or polymerase chain reaction (PCR) methods.

60. The method of any one of claims 51-59, wherein the engineered TCR is also activated when bound to the MBP peptide complexed with an MHC Class II comprising an HLA selected from HLA-DRB 1*04: 02, HLA-DRB 1*04: 04, HLA-DRBl*15:01, HLA-DRB 1*15:03 and HLA-DRB1*11 :O4.

61. The method of any one of the preceding claims, wherein the MBP peptide is anMBP 83-99 peptide comprising the amino acid sequence of ENP VVHFFKNIVTPRTP (SEQ ID NO: 61).

62. The method of any one of the preceding claims, wherein the engineered TCR is encoded as a single polypeptide, optionally comprising a self-cleaving peptide sequence.

63. The method of any one of the preceding claims, wherein the administering comprises administering to the subject a cellular composition comprising a therapeutically effective amount of regulatory T cells comprising the engineered TCR.

64. The method of claim 63, wherein the administering comprises one or more infusions of the cellular composition.

65. The method of claim 63 or 64, wherein the regulatory T cells are autologous relative to the subject.

66. The method of claim 63 or 64, wherein the regulatory T cells are allogeneic relative to the subject.

67. The method of any one of claims 63-66, wherein the regulatory T cells are derived from stem cells.

68. The method of claim 67, wherein the stem cells are pluripotent stem cells69. The method of claim 68, wherein the pluripotent stem cells are embryonic stem cells or hematopoietic stem cells.

70. The method of claim 68, wherein the pluripotent stem cells are induced pluripotent stem cells.

71. The method of any one of claims 63-66, wherein the regulatory T cells are derived from polarized T cells.

72. The method of any one of claims 63-66, wherein the regulatory T cells are derived from thymocytes.

73. The method of claim 72, wherein the regulatory T cells are isolated from peripheral blood or cord blood.

74. The method of any one of claims 63-66, wherein the regulatory T cells are induced regulatory T cells.

75. The method of any one of the preceding claims, wherein the subject is diagnosed with multiple sclerosis.

76. The method of any one of the preceding claims, wherein the subject has Progression Independent of Relapse Activity (PIRA).

77. The method of claims 75 or 76, wherein the subject is diagnosed with progressive multiple sclerosis, relapsing remitting multiple sclerosis, or multiple sclerosis.

78. The method of claim 77, wherein the progressive multiple sclerosis is primary progressive multiple sclerosis (PPMS) or non-relapsing progressive multiple sclerosis.

79. The method of any one of the preceding claims, wherein the engineered TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and / or the beta chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

80. The method of claim 79, wherein the alpha chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and the beta chain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

81. The method of claim 80, wherein:(i) the alpha chain comprises 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(ii) the beta chain comprises 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.

82. The method of claim 81, wherein: the alpha chain comprises an amino acid sequence having at least 90%identity to SEQ ID NO: 4 or SEQ ID NO: 5; and the beta chain comprises an amino acid sequence having at least 90%identity to SEQ ID NO: 10 or SEQ ID NO: 11.

83. The method of any one of the preceding claims, wherein an endogenous FOXP3 locus of the regulatory T cell is not engineered.

84. The method of any one of the preceding claims, wherein the regulatory T cell comprising the engineered TCR has a hypomethylated Treg-specific demethylated region (TSDR) at an endogenous FOXP3 locus.

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