Compositions and methods for engineering tregs for treatment of diabetes

Abstract

Binding proteins and high affinity recombinant T cell receptors (TCRs) specific for novel Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitope presented by the class II DRB4 neoantigens are provided herein. Compositions and recombinant host cells encoding and / or expressing the binding proteins and / or high affinity recombinant TCRs are also provided. The compositions and recombinant host cells may be used to treat a subject having Type 1 diabetes, wherein an IGRP neoantigen is a therapeutic target, or an indication wherein IGRP neoantigen is a therapeutic target. Related vaccines, vaccine therapies, and vaccination regimens are also provided.

Description

COMPOSITIONS AND METHODS FOR ENGINEERING TREGSFOR TREATMENT OF DIABETESCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 656358, filed June 5, 2024, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 1351-P31WO-Sequence-Listing.xml. The XML file is 78,392 bytes; was created on May 29, 2025; and is being submitted electronically via Patent Center with the filing of the specification.BACKGROUND

[0003] Type 1 diabetes (T1D), also referred to as juvenile diabetes or insulindependent diabetes, is a chronic condition in which the pancreas produces little or no insulin. The condition is characterized by selective destruction of insulin- secreting beta cells in the pancreas of genetically susceptible individuals. HLA class II genes are the most important genes associated with the risk of inheriting Type I diabetes, accounting for about 40-50% of heritability. Alleles of these genes that affect peptide binding to the MHC class II molecules seem to impact Type I diabetes risk the most. Previously, islet specific T cell receptor (TCR) sequences have been used to generate regulatory T cells by gene editing (EngTreg) as a potential therapy for T1D. However, all the islet specific TCR sequences (TCRs) isolated to date, recognize islet peptides presented by the class II DRB 1*0401 allele, which excludes their use in T1D patients carrying other class II MHC alleles.SUMMARY

[0004] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0005] This disclosure provides compositions and cell-based therapies and related methods for treatment of T1D. The cell-based therapy comprises immune cells, forexample, engineered T regulatory (Treg) cells (EngTregs) that have a stable suppressive phenotype, e.g., against T1D antigen- specific T effector (Tetfs) cells.

[0006] In some embodiments, the present disclosure pertains to islet specific TCR sequences to generate recombinant host cells, for example, regulatory T cells by gene editing, as a potential therapy for T1D. Applicants have successfully isolated TCRs that recognize islet peptides presented by the class II DRB4 molecule which is encoded on the DRB1*O4, DRB1*O7 and DRB1*O9 haplotypes to expand the T1D patient population that can be treated with such cells. TCRs that recognize an islet peptide presented by DRB4 can be used in T1D patients carrying any of these class II DRB1 alleles.

[0007] In some embodiments, the present disclosure relates to a binding protein, a T cell receptor (TCR), or derivatives thereof that recognizes a novel Is let- specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitope presented by the class II DRB4*0103 molecule. In some embodiments, the present disclosure pertains to methods of isolating TCRs that recognize novel epitopes from multiple islet proteins presented by the class II DRB4 molecules for use in engineered T regulatory cells. In some embodiments, the present disclosure also relates to engineered / recombinant host cells, e.g. , T regulatory (EngTreg) cells comprising a binding protein, a TCR, or derivatives thereof recognizing a novel IGRP epitope presented by a class II DRB4*0103 molecule. In some embodiments, the present disclosure pertains to compositions comprising the binding protein, or the host cell, for example, an engineered T regulatory cell comprising a binding protein, a TCR, or derivatives thereof recognizing and binding to the novel IGRP epitope presented by the class II DRB4*0103 molecule. In some embodiments, the present disclosure relates to methods of generating a host cell comprising a binding protein, a TCR, or derivatives thereof recognizing the novel IGRP epitope presented by a class II DRB4*0103 molecule. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a regulatory T cell (Treg).

[0008] In some embodiments, the present disclosure pertains to a method of inhibiting or ameliorating Type 1 Diabetes (T1D). In some embodiments, the method comprises administering to a subject the composition disclosed herein. In some embodiments, the composition comprises the host cell comprising a binding protein, a TCR, or derivatives thereof, for example an engineered Treg, recognizing the novel IGRP epitope presented by the class II DRB4*0103 molecule disclosed herein or a populationhost cells, for example, a population of engineered T regulatory cells made by the methods disclosed herein.

[0009] The TCRs of the present disclosure are isolated by a method comprising stimulating or activating PBMCs obtained from a donor with candidate peptides derived from IGRP followed by assessment of CD 137 expression. In some embodiments the donor suffers from T1D. In some embodiments, the donor is a healthy donor. In some embodiments, the donor is a human being. In an embodiment the method further comprises identifying clonotypes and generating full TCR sequences for the identified islet- specific TCRs. In some embodiments, the method utilizes single cell RNA- sequencing to identify clonotypes and generate full TCR sequences. In some embodiments, the method further comprises generating islet- specific TCR constructs for TCR gene transfer into T cells. In some embodiments, the islet-specific TCR constructs are expressed in CD4 primary T cells. In some embodiments, the method further comprises identifying the MHC class II specific epitopes by T cell proliferation assay. In some embodiments, the specific epitope is identified using T cells transduced with an islet-specific TCR and an APC expressing the DRB4 / DR4 class II MHC alleles.

[0010] In some embodiments, the present disclosure pertains to a binding protein, TCR, or derivatives thereof that recognizes a novel IGRP epitope (amino acid position 25- 44, FLNFMSNVGDPRNIFFIYFP (SEQ ID NO: I) presented by DRB4*0103. In some embodiments, the novel IGRP epitope is identified using APC expressing DRB4 but not DRB 1*0401.

[0011] In some embodiments, there are disclosed herein engineered / recombinant host cells, for example, T regulatory cells expressing a binding protein, a TCR, or derivatives thereof that recognizes a novel IGRP epitope (amino acid position 25-44, FLNFMSNVGDPRNIFFIYFP) (SEQ ID NO: 1) presented by DRB4*0103. In an embodiment, the host cells comprise T regulatory cells (EngTregs).

[0012] The EngTregs of the present disclosure are made using cells isolated from a subject and have a robust suppressive phenotype against T1D antigen-specific T effector cells or Teffs, which robustness is achieved using at least the following three concepts. First, the EngTregs of the present disclosure incorporate a specific method of expressing a TCR specific to a type 1 diabetes (T1D) antigen that includes knocking out the endogenous TCR (or part thereof) and hijacking the nucleic acid in the TRAC locus of the genome that encodes the constant region of the TCRa (herein referred to as TRAC hijacking). Second,the Foxp3 locus of the donor cells is edited to place a heterologous promoter downstream from one or more regulatory elements (e.g., the TSDR) so that F0XP3 is expressed in a manner that is not epigenetically affected. Third, the EngTregs of the present disclosure incorporates a dual-editing approach in which two different loci in the genome, i.e. the Foxp3 locus and the TRAC locus, are edited at the same time incorporating in each of the loci one-half of a chimeric cell-surface receptor that can dimerize in the presence of a ligand (herein referred to as a chemical -inducible signaling complex (CISC) system), such that when the edited cells are expanded in the presence of a dimerizing ligand, only those cells that are dual-edited expand. Moreover, the dimerization of the cell-surface receptor is linked to activation of IL-2 signaling, which again contributes to a suppressive Treg phenotype. The result of dual editing and enrichment for dual edited cells is a population of cells that comprise a subpopulation of dual-edited, CISC containing controllable IL-2 signaling, antigen- specific EngTregs that have a robust antigen- specific suppressive phenotype secondary to T1D antigen- specific TCR and co-expression of high levels of FOXP3 in a manner that is not impacted epigenetically by environmental or other local factors (e.g., an inflammatory environment). The engineered Treg cells of the present disclosure have activity comprising suppression of cytokine production in addition to proliferation.

[0013] In some embodiments, the present disclosure is related to a binding protein comprising: a T cell receptor (TCR) a-chain variable domain (Va); and a TCR P-chain variable domain (V|3). In some embodiments, the binding protein is capable of binding to a FLNFMSNVGDPRNIFFIYFP (SEQ ID NO: Inhuman leukocyte antigen (HLA) complex, and / or an Islet- specific Glucose-6-Phosphatase Catalytic subunit-related peptide (IGRP):HLA complex. In certain embodiments, the IGRP peptide comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO:1. In an embodiment, the HLA comprises DRB4*0103. In some embodiments, the Va comprises a CDR3 amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:4. In some embodiments, the VP comprises a CDR3 amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:7.

[0014] In an embodiment of the present disclosure, there is provided a binding protein comprising: a T cell receptor (TCR) a-chain variable domain (Va) comprising a CDR3 amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:4; and a TCR P-chain variable domain (VP), comprising a CDR3 amino acidsequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:7. In some embodiments, the binding protein is capable of binding to a FLNFMSNVGDPRNIFFIYFP (SEQ ID NO: l):human leukocyte antigen (HLA) complex, and / or an Islet-specific Glucose-6-Phosphatase Catalytic subunit-related peptide (IGRP):HLA complex. In certain embodiments, the IGRP peptide comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO:1. In an embodiment, the HLA comprises DRB4*0103. In some embodiments, the Va comprises a CDR3 amino acid sequence of SEQ ID NO:4, and the V]3 comprises a CDR3 amino acid sequence of SEQ ID NO:7.

[0015] In certain embodiments, the binding protein comprises a CDRla amino acid sequence according to SEQ ID NO:2; a CDR2a amino acid sequence according to SEQ ID NOG; a CDRip amino acid sequence according to SEQ ID NOG; and / or a CDR2[3 amino acid sequence according to SEQ ID NOG. In some embodiments, the binding protein comprises CDRla, XDR2a, CDR3a, CDRip, CDR2[3, and CDR3P amino acid sequences as set forth in: (i) SEQ ID NOs: 2, 3, and 4, respectively; or (ii) SEQ ID NOs: 5, 6, and 7, respectively.

[0016] In some embodiments, the binding protein comprises an amino acid sequence that is at least 85% identical to an amino acid sequence according to TRAV35*02 andTRAJ6*01. In some embodiments, the binding protein comprises an amino acid sequence that is at least 85% identical to an amino acid sequence according to TRBV7- 8*01, TRBJl-3*01, and TRBDl*01.

[0017] In some embodiments, the binding protein further comprises a TCR chain constant domain (CP), a TCR a chain constant domain (Ca), or both. In an embodiment, the TCR P chain constant domain comprises an amino acid sequence according to TRABCl*01 or an amino acid sequence that is at least 85% identical thereto. In an embodiment, the TCRa constant domain comprises an amino acid sequence according to TCRACl*01 or an amino acid sequence that is at least 85% identical thereto. In some embodiments, the Ca has at least 85% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the CP has at least 85% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 11.

[0018] In some embodiments, the binding protein of the present disclosure comprises a TCRa chain that comprises or consists of an amino acid sequence that is atleast 85% identical to an amino acid sequence of SEQ ID NO:8 (CAGPGTSGGSYIPTF). In some embodiments, the binding protein of the present disclosure comprises a TCRP chain that comprises or consists of an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO:9 (CASSLAPLGGSSGNTIYF). In an embodiment, the binding protein comprises: (i) a TCRoc chain that comprises or consists of an amino acid sequence of SEQ ID NO:8; and (ii) a TCRP chain that comprises or consists of an amino acid sequence of SEQ ID NO:9.

[0019] In some embodiments, the binding protein, the TCR, or derivative thereof is capable of binding to a (SEQ ID NO:1):HLA complex, and / or to an Islet-specific Glucose-6-Phosphatase Catalytic subunit-related peptide (IGRP):HLA complex on a cell surface independent of or in the absence of CD4. In some embodiments, the IGRP peptide comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO:1.

[0020] In some embodiments, the binding protein is a TCR, a chimeric antigen receptor, or an antigen-binding fragment of a TCR. In some embodiments, the TCR, the chimeric antigen receptor, or the antigen-binding fragment of a TCR is chimeric, humanized, or human, and / or the antigen-binding fragment of the TCR comprises a single chain TCR (scTCR).

[0021] In some embodiments, the present disclosure provides compositions comprising the binding proteins disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0022] The present disclosure also provides polynucleotides encoding the binding proteins disclosed herein. In some embodiments, the polynucleotide is codon optimized. In certain embodiments, the polynucleotide further comprises a polynucleotide encoding a self-cleaving peptide disposed between the a-chain encoding polynucleotide and the p-chain encoding polynucleotide.

[0023] Provided herein are vectors comprising the polynucleotides disclosed herein. In some embodiments, the vectors of the present disclosure are operably linked to an expression control sequence.

[0024] In some embodiments, the present disclosure provides recombinant host cells comprising the polynucleotides of the present disclosure. Also provided are recombinant host cells comprising the expression vectors of the present disclosure. In all embodiments, the recombinant host cells can express the encoded binding protein on theirsurface. In some embodiments, the polynucleotides are heterologous to the host cells. In some embodiments, the recombinant host cell is a human immune cell. In some embodiments, the recombinant host cell is an immune system cell. In some embodiments, the immune system cell is a CD3+T cell, a CD4+T cell, a CD25+T cell, a FoxP3+T cell, or any combination thereof. In some embodiments, the immune system cell is a T cell, and wherein the T cell is a T regulatory cell (Treg).

[0025] In some embodiments, the recombinant host cell of the present disclosure has a biological activity comprising one or more of: (i) inhibition of either or both of activation and proliferation of an effector cell, for example a T effector cell, that recognizes the antigen that is specifically recognized by the binding protein, the TCR, or derivatives thereof of the present disclosure; (ii) inhibition of expression of inflammatory cytokines or inflammatory mediators by an effector cell that recognizes the antigen that is specifically recognized by the binding protein, the TCR, or derivative thereof of the present disclosure; (iii) elaboration of one or more immunosuppressive cytokines, perforin / granzyme, or antiinflammatory products by the recombinant host cell expressing the binding protein, TCR, or derivatives thereof of the present disclosure or induction in the recombinant host cell of at least one of indoleamine 2,3-dioxygenase (IDO), competition for IL2 or adenosine, catabolism of tryptophan, expression of inhibitory receptors; or (iv) inhibition of either or both of activation and proliferation of an effector cell that does not recognize the antigen that is specifically recognized by the binding protein, the TCR, or derivatives thereof disclosed herein.

[0026] Also provided herein is a method of treating a disease or a disorder in a subject in need thereof. In some embodiments, the method comprises administering an effective amount of the compositions disclosed herein. In some embodiments, the method comprises administering a therapeutically effective amount of the recombinant host cells of the present disclosure. In some embodiments, the disease / disorder is associated with the expression of IGRP antigens, such as, for example, Type 1 Diabetes (T1D).DESCRIPTION OF THE DRAWINGS

[0027] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0028] FIGS. 1A-1B depict isolation of islet specific TCR from Tregs using assessment of CD 137 expression following PBMC activation with islet peptide pools (FIG. 1 A). FIG. IB shows the clonotype Tr24 TCR identified using the methods disclosed herein and sequences for TCR alpha and beta chains of Tr24.

[0029] FIGS. 2A-2D depict TCR expression in T cells transduced with Tr24 TCR and screening strategy for identification of antigen specificity. Tr24 TCR expression in CD4+ T cells transduced with lentiviral Tr24 TCR. Flow plot shows mTCR expression gated on CD4+ cells day 7 post-transduction (FIG. 2A) and overall screening strategy and schematic for identification of Tr24, a TCR specific for IGRP25-44 (SEQ ID NO: 14) and restricted to DRB4 (FIG. 2B); Screening strategy to identify antigen specificity of Tr24 by antigen- specific proliferation assay. 101 islet-specific peptides were divided into 11 peptide pools with 10-11 peptides in each pool. After finding a pool where Tr24 Teff proliferate, a second screening was performed with individual peptides within the pool. Priess cells, K562-DRB4 or K562-DR4 cells were used as the APC and each peptide was added at final concentration of 2.5 pg / mL (FIG. 2C). Key steps for antigen- specific proliferation assay. Tr24 Teff were labeled with CTV and then co-cultured with APC and peptide or peptide pool for 3 or 4 days. Cells were harvested and flow cytometry was performed to measure dilution of CTV as proliferation. (FIG. 2D).

[0030] FIGS. 3A-3B depict screening of 11 peptide pools derived from IGRP, ZNT8 or PPI. 101 peptides derived from IGRP, ZNT8 or PPI were split into 11 peptide pools (each pool containing 10-11 peptides) using T cell proliferation assay. Proliferation results from the first screening with DMSO or 11 peptide pools in the presence of Priess as the APC. Tr24 Teff were gated on CD4+ mTCR+. Top panels show histogram of proliferation. Bottom panels show flow plots showing CTV low and CD25+ populations (FIG. 3A). Individual peptides within Pool 8 derived from IGRP were subjected to a second screening (FIG. 3B). Proliferation results from the second screening with DMSO or each peptide within peptide pool 8 in the presence of K562-DRB4, K562-DR4 or Priess cells as the APC. Flow plots showing CTV low and CD25+ populations. Tr24 Teff proliferated specifically in the presence of IGRP p4 when co-cultured with Priess or K562- DRB4, not with K562-DR4. Priess cells (DR4 / DRB4) or K562 (DRB4 or DR4) cells were used as APC.

[0031] FIGS. 4A-4B depict Tr24 TCR specific for IGRP25-44 and restricted toDRB4. Histograms showing the proliferation of Tr24 Teff in the presence of APC andIGRP25-44 with serial dilutions. Irradiated allogeneic PBMC with DRB4 (DRBl*0701 / *0801), K562-DRB4, or K562-DR4 were used as the APC (FIG. 4A). Histograms showing proliferation of Tr24 Teff co-cultured with DRB4 PBMC or K562- DRB4 and IGRP25-44 with or without anti-DR antibody (FIG. 4B).

[0032] FIG. 5 shows that Tr24 TCR is specific for a novel peptide, IGRP25-44, presented by DRB4. Histograms showing proliferation of Tr24 Teff co-cultured with K562-DRB4, allogeneic monocyte derived DC (mDC) with DRB4, or irradiated allogeneic DRB4 PBMC in the presence of DMSO, IGRP17-36, IGRP25-44, IGRP33-52, or IGRP41-60 (left panel).

[0033] Right panel shows amino acid sequences of IGRP peptides used in the proliferation assay in FIG. 5. Amino acids marked in bold (red) show overlapping sequence from IGRP2544.

[0034] FIGS. 6A-6C depict the characterization of Tr24 TCR in comparison with T1D2 and T1D5-1. Comparison of mTCR expression levels in CD4+ T cells transduced with T1D2, T1D5-1, or Tr24 TCR. (FIG. 6A). Comparison of proliferation of CD4+ T cells expressing T1D2, T1D5-1, or Tr24 TCR in the presence of APC and their cognate peptide with serial dilutions. Irradiated allogeneic DRB 1*0401 PBMC were used as APC. (FIG. 6B). FIG. 6C shows graph showing peptide dose response of T cells shown in (FIG. 6B).

[0035] FIG. 7 depicts the timeline to generate polyclonal islet-specific Teff and EngTregs and an in vitro suppression assay to measure suppressive activity of Tr24 EngTregs. CD4+CD25- and CD4 CD25+ cells were isolated from PBMCs of a T1D donor. CD4+CD25- cells were split to generate EngTregs and polyclonal islet- specific T cells. CD4+CD25- T cells were stimulated using irradiated autologous APC (CD4 CD25+) cells loaded with a pool of 10 different islet-specific peptides for 14 days. T cells were expanded with IL-2 from day 7 in 2 to 3 day intervals and harvested at day 14. Polyclonal Islet T cells were used as Teff cells for a suppression assay and cultured for 4 days with no Treg, EngTregs or mock cells in the presence of mDC and a pool of 11 different islet-specific peptides including IGRP2544. Before the co-culture, Teff and EngTregs or Mock cells were labeled with cell trace violet (CTV) and eFluor™ 670, respectively.

[0036] FIGS. 8A-8B depict generation of EngTregs with Tr24 or T1D2 using PBMCs with T1D. Generation of EngTregs expressing T1D2 or Tr24 TCR. CD4+CD25- T cells isolated from T1D PBMCs were activated with CD3 / CD28 expander beads andrecombinant human IL-2 on day 0. Transduction with LV vectors encoding T1D2 or Tr24 TCRs was performed by adding concentrated LV supernatant with protamine sulfate. Beads were removed after a 72-hour incubation, and cells were treated with IL-2 and rested for 16 to 24 hours. For FOXP3 editing, cells were transfected by electroporation with RNP complex combined with Cas9 and guide RNA and then transduced with AAV template. IL-2, TNF-a, and IL-6 were added to the edited cells. Twenty to 24 hours after editing, cells were expanded in media with IL-2, TNF-a, IL-6, and Rapamycin until day 10. Isletspecific LNGFR+ EngTregs were enriched by LNGFR magnetic beads on day 10 and LNGFR- T cells were also collected from the LNGFR+ cell enrichment to be used as mock controls in suppression assays. Cells were aliquoted and frozen down for further experiments (FIG. 8A).

[0037] Flow plots showing mTCR expression and LNGFR / FOXP3 expression gated on CD4+ in edited cells expressing T1D2 or Tr24 TCR on day 7 (FIG. 8B).

[0038] FIGS. 9A-9B show CD3 / CD28 bead-induced suppression by Tr24 EngTregs. Histograms showing proliferation of polyclonal Islet Teff in the presence of CD3 / CD28 beads (top row) and Tr24 Mock, Tr24 EngTregs, or T1D2 EngTregs, (1:1 for Treg:Teff or control :Teff). Histograms were gated on Live, CD3+CD4+ eFluor™ 670- cells (FIG. 9A).

[0039] FIG. 9B shows percent suppression of CD3 / CD28 bead-induced Teff proliferation by Tr24 Mock, Tr24 EngTregs, or T1D2 EngTregs shown in (FIG. 9A).

[0040] FIGS. 10A-10B depicts polyclonal islet-specific Teff suppression by Tr24 EngTregs in comparison with T1D2 EngTregs. Histogram showing proliferation of polyclonal islet Teff in antigen- specific suppression assay. Polyclonal islet Teff were cultured with Tr24 Mock, Tr24 EngTregs, or T1D2 EngTregs (1:1 for Treg:Teff or control: Teff) in the presence of mDC and a pool of 11 islet- specific peptides including IGRP25-44. Histograms were gated on Live, CD3+CD4+ eFluor™ 670“ cells (FIG. 10A). Percent suppression on proliferation of polyclonal islet Teff by Tr24 or T1D2 EngTregs in the presence of mDC and a pool of 1 1 islet-specific peptides described in (A). Data are provided as the mean ± SD of two independent experiments using cells generated from two different T1D donors. **P < 0.01, as determined by paired t-test (FIG. 10B).

[0041] FIGS. 11A-11D show direct suppression of Tr24 Teff by Tr24 EngTregs. Histograms showing proliferation of CTV-labeled Tr24 Teff in the presence of cognate peptide (IGRP25-44), autologous mDC, and the eFluor™ 670-labeled Tr24 Mock or Tr24EngTregs (1:1 for Teff:Treg or Telecontrol ). Histograms were gated on live, CD3+ CD4+, mTCR+ eFluor™ 670“ cells (FIG. 11 A).

[0042] Percent suppression on proliferation of Tr24 Teff by Tr24 Mock or Tr24 EngTregs in the presence of mDC and IGRP25-44 described in (FIG. 11 A). Data are pooled from two independent experiments using cells generated from two different T1D donors (FIG. 11B).

[0043] Direct suppression of Tr24 Teff cytokine production by Tr24 EngTregs. Histograms showing cytokine production (TNF-a, IFN-y, or IL-2) of CTV-labeled Tr24 Teff in the presence of cognate peptide (IGRP25-44), autologous mDC, and the eFluor™ 670-labeled Tr24 Mock or Tr24 EngTregs (1 :1 for Teff:Treg or Telecontrol). Histograms were gated on live, CD3+ CD4+, mTCR+ Efluor™ 670“ cells (FIG. 11C).

[0044] FIG. 1 ID shows percent suppression of Tr24 Teff production of TNF-a, IFN-y, or IL-2 by Tr24 Mock or Tr24 EngTregs in the presence of mDC and IGRP25-44 described in (FIG. 11A).

[0045] FIGS. 12A-12B show bystander suppression of PPI76 Teff by Tr24 EngTregs. Histograms showing proliferation of CTV-labeled PPI76 Teff in the presence of PPI76-90 or a mixture of PPI76-90 and IGRP25-44 plus APC, and the eFluor™ 670-labeled Tr24 Mock or Tr24 EngTregs (1: 1 for TefETreg or Teff: control). Histograms were gated on live, CD3+ CD4+, mTCR+ eFluor™ 670“ cells (FIG. 12A).

[0046] Percent suppression on proliferation of PPI76 Teff by Tr24 Mock or Tr24 EngTregs in the presence of PPI76-90 or a mixture of PPI76-90 and IGRP25-44 plus APC described in (A). Data is pooled from two independent experiments using cells generated from two different T1D donors (FIG. 12B).DETAILED DESCRIPTION

[0047] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

[0048] Previously, Applicant developed a method to identify antigen-specific T helper cells following PBMC activation with a peptide pool followed by assessment of CD 154 expression. The applicant has extended this approach to isolate islet specific T cell receptors (TCRs) from regulatory T cells (Tregs) using assessment of CD 137 expression following PBMC activation with islet peptide pools. This method utilized single cell RNA- sequencing for identifying TCR clonotypes expanded in T1D subjects to generate full TCRsequences (Cerosaletti et al., Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+T Cells in Peripheral Blood of Subjects with Type 1 Diabetes. J. Immunol. 2017 Jul 1 ;199(l):323-335, Linsley et al., Autoreactive T cell receptors with shared germline-like a chains in type 1 diabetes. J CI Insight. 2021 ;6(22):el51349.2021. Based on islet-specific TCR sequences identified by the foregoing method, lentiviral TCR constructs were generated for TCR gene transfer into CD4 primary T cells. These TCR constructs express human TCR variable regions from islet-specific TCRs and mouse TCR constant regions allowing for an improved pairing between the transduced human TCR chains. A specific epitope among candidate peptides was identified by a T cell proliferation assay using T cells transduced with islet-specific TCR and APC expressing the appropriate class II MHC alleles. For example, artificial APC (K562 cells) expressing only the DRB4 or DR4 gene were utilized in proliferation assays. T cells transduced with Tr24 TCR proliferated only in response to IGRP25-44 (SEQ ID NO:34) peptide and APC expressing class II DRB4. Using this approach, a TCR that recognizes a novel IGRP epitope (amino acid position 25-44, FLNFMSNVGDPRNIFFIYFP (SEQ ID NO:1)) presented by DRB4*0103 was isolated using APC that express DRB4 but not DRBl*0401.

[0049] In an exemplary embodiment, antigen-specific regulatory T cells (Tregs) were generated by editing Foxp3 expression in CD4+ T cells transduced with islet-TCRs, which resulted in the successful generation of engineered T regulatory cells (EngTregs) expressing islet-specific TCRs. Moreover, antigen- specific and bystander suppressive functions of these EngTregs with islet specific TCRs by in vitro suppression assays was demonstrated. EngTregs expressing Tr24 showed strong suppression on polyclonal isletspecific T effector (Teff) cells derived from T1D PBMCs, comparable to EngTregs expressing T1D2 TCR specific to a peptide presented by the class II DRB 1*0401 allele derived from the islet antigen, IGRP and co-expressed with split CISC elements and a heterologous promoter for stable expression of endogenous FOXP3.

[0050] Collectively, a TCR that recognizes a novel IGRP epitope presented by the class II DRB4*0103 molecule was successfully isolated. Additional TCRs that recognize novel epitopes from multiple islet proteins presented by the DRB4 molecule can be isolated using a similar approach for use in engineered Treg applications in T1D.

[0051] In some aspects the present disclosure provides binding proteins and / or high affinity recombinant TCRs, or derivatives thereof directed to an Islet-specific Glucose-6-Phosphatase Catalytic subunit-related peptide (IGRP) antigen. Compositionsand recombinant host cells including (i.e. , encoding and / or expressing) the binding proteins and / or high affinity recombinant TCRs or derivatives thereof are also provided. Compositions and recombinant host cells according to the present disclosure are useful to treat a subject having Type 1 diabetes, other indications (also referred to herein as a disease or disorder) wherein an IGRP antigen is a therapeutic target. In some embodiments, compositions, and recombinant host cells (e.g., immune cells, such as T cells, that are modified to encode and / or express an IGRP-specific binding protein or high affinity recombinant TCR or derivatives thereof as disclosed herein) with specificity for a IGRP antigen are useful to treat a subject having Type 1 diabetes. In certain embodiments, compositions and recombinant host cells with specificity for an IGRP antigen e.g., immune cells, such as T cells, that are modified to encode and / or express a IGRP-specific binding protein or a high affinity recombinant TCR or derivatives thereof as disclosed herein) may be used to treat a subject having a disease or disorder associated with the expression of IGRP antigen, such as, for example, Type 1 Diabetes (T1D). Immunogenic compositions such as, for example, vaccines, as well as related uses are also provided.

[0052] It will be readily understood that the embodiments, as generally described herein, are exemplary. The following description of various embodiments is not intended to limit the scope of the present disclosure but is merely representative of various embodiments. Moreover, the order of steps or actions of certain methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified.

[0053] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

[0054] Unless specifically defined otherwise, the technical terms, as used herein, have their normal meaning as understood in the art.

[0055] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size, or thickness, is to be understood to include any integer within the recited range, unless otherwise indicated.

[0056] “About,” as used herein, when referring to a measurable value is meant to encompass variations of ±20%, ±10%, ±5%, ±1 %, or ±0.1 % from the specified or indicated value, range, or structure, unless otherwise indicated.

[0057] It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination of the alternatives. As used herein, the terms “include,” “have,” and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0058] “Optional” or “optionally” means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.

[0059] In addition, it should be understood that the individual constructs, or groups of constructs, derived from the various combinations of the structures and subunits described herein, are disclosed by the present application to the same extent as if each construct or group of constructs was set forth individually. Thus, selection of particular structures or particular subunits is within the scope of the present disclosure.

[0060] The term “consisting essentially of” is not equivalent to “comprising” and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, or linker) or a protein (which may have one or more domains, regions, or modules) “consists essentially of’ a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy-terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

[0061] As used herein, “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acidsare those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0062] As used herein, “protein” or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers.

[0063] As used herein, “fusion protein” refers to a protein that, in a single chain, has at least two distinct domains, wherein the domains are not naturally found together in a protein. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized. A fusion protein may further contain other components, such as a tag, a linker, or a transduction marker. In certain embodiments, a protein expressed or produced by a host cell (e.g., a T cell) locates to the cell surface, where the fusion protein is anchored to the cell membrane (e.g. , via a transmembrane domain) and comprises an extracellular portion (e.g. , containing a binding domain) and an intracellular portion (e.g., containing a signaling domain, effector domain, co- stimulatory domain or combinations thereof).

[0064] “Junction amino acids” or “junction amino acid residues” refer to one or more (e.g., about 2-10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein).

[0065] “Nucleic acid molecule” or “polynucleotide” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.

[0066] As used herein, “mutation” refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).

[0067] A “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (lie or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally, or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and lie. Other conservative substitutions groups include:sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W. H. Freeman and Company. In certain embodiments, proline shares certain properties with amino acids that have aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In certain circumstances, substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. Variant proteins, peptides, polypeptides, and amino acid sequences of the present disclosure can, in certain embodiments, comprise one or more conservative substitutions relative to a reference amino acid sequence.

[0068] As understood in the art, “similarity” between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide (e.g., using GENEWORKS™, Align, Clustal™, the BLAST algorithm, or the like).

[0069] Variants of polynucleotides and polypeptides of this disclosure are also contemplated. Variant nucleic acid molecules or polynucleotide are at least 70%, 75%, 80%, 85%, 90%, and are preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to a defined or reference polynucleotide or polypeptide (respectively) as described herein, or that, for a polynucleotide, hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68° C. or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42° C. Nucleic acid molecule variants retain the capacity to encode a fusion protein or a binding domain thereof having a functionality described herein, such as specifically binding a target molecule. For additional details and explanation of stringency of hybridization reactions, see Ausubel, F. M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Moreover, the person skilled in the art may follow the instructions given in the manual Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer,K. H. (1991) International Journal of Systematic Bacteriology 41: 255-260 on how to identify DNA sequences by means of hybridization.

[0070] Variants can also refer to fragments (e.g., a portion resulting from truncation, cleavage, or the like) of a defined or reference sequence, and fragments can be of any length shorter than the length of the defined or reference sequence.

[0071] As used herein, a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A “functional portion” or “active portion” or “functional fragment” or “active fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity), such as an assay for measuring binding affinity or measuring effector function e.g., cytokine release). In certain embodiments, a functional portion refers to a “signaling portion” of an effector molecule, effector domain, costimulatory molecule, or costimulatory domain.

[0072] In certain embodiments, a variant binding protein or a portion or fragment thereof (e.g., binding domain) can comprise one or more amino acid substitutions relative to a parent or reference binding protein or domain, wherein the one or more amino acid substitutions remove, change, or attenuate a potential undesired feature or characteristic, if present, from the parent or reference binding domain or protein; e.g., an amino acid sequence that is potentially immunogenic, or an amino acid sequence that may provide an undesired glycosylation site, an undesired deamidation site, an undesired oxidation site, an undesired isomerization site, or a reduction in thermodynamic stability, or that may result in mis-pairing or mis-folding in a binding protein (e.g., unpaired cysteine residues in close proximity). Amino acid sequences, patterns, and motifs that may provide for an undesired feature or characteristic are known (see, e.g., Seeliger et al., mAbs 7(3): 505-515 (2015)).

[0073] In certain embodiments, an amino acid substitution comprises a substitution to remove a somatic mutation, such as, for example, a reversion to a germ line- encoded amino acid. For example, in certain embodiments, a variant of a reference CDR amino acid sequence, or of a TCR variable domain sequence or TCR constant region sequence, comprises a substitution to remove or attenuate a potential undesired feature or characteristic. It will be understood that such variants are selected so as not to compromise, or substantially compromise, a desired function (e.g., binding specificity and / or affinity for a peptide antigen: HLA complex).

[0074] “Sequence identity,” or “percent sequence identity” as used herein, refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment), if necessary, to achieve, in preferred methods, the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” mean any set of values or parameters which originally load with the software when first initialized. For example, percent sequence identity values can be generated using the NCBI BLAST 2.0 software as defined by Altschul, et al. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402, with the parameters set to default values. Other programs for determining or calculating sequence alignments and percent identity include, for example, BLASTP, BLASTN, and BLASTX.

[0075] A “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide. In otherwords, a functional variant of a polypeptide or encoded polypeptide of this disclosure has “similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant) or avidity; or an assay measuring phosphorylation or activation of, or by, an immune cell protein such as, for example, Lek, ZAP70, Fyn, or the like, including the assays described herein. The ability of a polypeptide or encoded polypeptide of this disclosure (or a functional variant of the same) to initiate, continue, participate in, propagate, or amplify a cell signaling event or events (e.g., T cell signaling in response to antigen-binding) may be determined by examining the activity, structure, chemical state (e.g., phosphorylation), or interactions of or between the variant polypeptide and an immune cell protein that directly acts (e.g., binds to) therewith, or by examining the activity, localization, structure, expression, secretion, chemical state (e.g., phosphorylation), or interactions of or between other biomolecules known or thought to participate in or be affected by the cell signaling event or events. The ability of a polypeptide or encoded polypeptide of this disclosure (or a functional variant of the same) to initiate, continue, participate in, propagate, or amplify a cell signaling event or events may also be determined by using functional assays of host cell activity, including those described herein for measuring the ability of a host cell to release cytokines, proliferate, selectively kill target cells, or treat a subject having a disease or condition expressing or otherwise associated with an antigen bound by a binding protein of this disclosure.

[0076] Variant polypeptides of the present disclosure can, in certain embodiments, include chemical modifications, for example, isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation and the like. Methods to modify polypeptides are known in the art. Modifications are designed so as not to abolish or substantially impair a desired biological activity of the variant.

[0077] An “altered domain” or “altered protein” refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g. , a wild type TCRa chain, TCRP chain, TCRa constant domain, or TCRP constant domain) of at least 85% (e.g., 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%).

[0078] As used herein, the terms “endogenous” or “native” refer to a gene, protein, or activity that is normally present in a host cell.

[0079] As used herein, “heterologous,” “non-endogenous,” and “exogenous” refer to any gene, protein, compound, molecule, or activity that is introduced through manipulation (e.g., genetic manipulation). In certain embodiments, heterologous, non- endogenous, or exogenous molecules (e.g., receptors, ligands, etc.) may not be endogenous to a host cell or subject, but instead nucleic acids encoding such molecules may have been added to a host cell by conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term “homologous” or “homolog” refers to a molecule or activity found in or derived from a host cell, species, or strain. For example, a heterologous, non-endogenous, or exogenous molecule or gene encoding the molecule may be homologous to a native host or host cell molecule or gene that encodes the molecule, respectively, but may have an altered structure, sequence, expression level, or combinations thereof. A non-endogenous molecule may be from the same species, a different species, or a combination thereof.

[0080] The term “expression,” as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post- transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0081] The term “operably-linked” refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably- linked with a coding sequence when it can affect the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). “Unlinked” refers to genetic elements that are not closely associated with one another and the function of one does not affect the other.

[0082] The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g. , a bacterialvector or a viral vector) or may be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector, or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi- synthetic, or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (an episomal vector) or expression of nucleic acid molecules to which they are linked (an expression vector).

[0083] As used herein, “expression vector” refers to a DNA construct containing a nucleic acid molecule that is operably-linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid,” “expression plasmid,” “virus,” and “vector” are often used interchangeably.

[0084] The term “introduced” in the context of inserting a nucleic acid molecule into a cell, means “transfection,” “transformation,” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., a chromosome, a plasmid, a plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). As used herein, the term “engineered” “recombinant” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, a gene, or an operon.

[0085] As described herein, more than one heterologous, non-endogenous, or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. For example, a host cell can be modified to express two or more heterologous, non-endogenous, or exogenous nucleic acid molecules encoding desired TCR specific for a IGRP neoantigen peptide (e.g. , TCRa and TCRP). When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g. , on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.

[0086] As used herein, the terms “host” or “host cell” refer to a cell (e.g., an immune system cell such as, for example, a T cell) or microorganism targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., IGRP-specific binding protein). In certain embodiments, a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to biosynthesis of the heterologous or exogenous protein (e.g. , inclusion of a detectable marker; a deleted, altered, or truncated endogenous TCR; increased co-stimulatory factor expression; etc.). Exemplary host cells and types of cells suitable for use as host cells are described further herein.

[0087] “T cell receptor” (TCR) refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g. , Janeway, el al. , Immunobiology: The Immune System in Health and Disease, 3rdEd., Current Biology Publications, p. 4:33, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and P chains (also known as TCRa and TCR , respectively), or y and 5 chains (also known as TCRy and TCR5, respectively). Like other immunoglobulins, the extracellular portion of TCR chains (e.g., a-chain and -chain) contain two immunoglobulin domains, a variabledomain (e.g., a-chain variable domain or Va, P-chain variable domain or V ; typically amino acids 1 to 1 16 based on Kabat numbering (Kabat, et al., “Sequences of Proteins of Immunological Interest,” US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5thed.)) at the N-terminus, and one constant domain e.g., a-chain constant domain or C«, typically amino acids 117 to 259 based on Kabat, P- chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. Also, like other immunoglobulins, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., lores, et al., Proc. Nat’l Acad. Sci. U.S.A. 57:9138, 1990; Chothia, et al., EMBO J. 7:3745, 1988; see also Lefranc, et al., Dev. Comp. Immunol. 27:55, 2003). In certain embodiments, a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. The source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, cat, dog, goat, horse, or other mammal. In certain embodiments, a TCR complex comprises a TCR or a functional portion thereof; a dimer comprising two CD3 chains, or functional portions or variants thereof; a dimer comprising a CD35 chain and a CDs chain, or functional portions or variants thereof; and a dimer comprising a CD3y chain and a CDs chain, or functional portions or variants thereof, any one or more of which may be endogenous or heterologous to the T cell.

[0088] “CD3” is a multi-protein complex of six chains (see, Borst J, et al. , J. Biol. Chem. 258(8):5135-41 , 1983 and laneway, et al., p. 172 and 178, 1999 supra). In mammals, the complex includes a CD3y chain, a CD33 chain, two CD3s chains, and a homodimer of CD3^ chains. The CD3y, CD36, and CD3e chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD38, and CD3s chains are negatively charged, which is thought to allow these chains to associate with positively charged regions of TCR chains. The intracellular tails of the CD3y, CD36, and CD3e chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or IT AM, whereas each CD3 chain has three. Without being bound by theory, it is believed the ITAMs are important for the signaling capacity of a TCR complex. CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.

[0089] As used herein, “TCR complex” refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3ychain, a CD35 chain, two CD3e chains, a homodimer of CD3^ chains, a TCRa chain, and a TCR0 chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3(^ chains, a TCRy chain, and a TCR5 chain. A “component of a TCR complex,” as used herein, refers to a TCR chain ( / .<?., TCRa, TCR0, TCRy, or TCR8), a CD3^ chain (z.e., CD3y, CD35, CD3a, or CD3Q or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCR , CD3y, CD35, and two CD3s chains).

[0090] “Major histocompatibility complex” (MHC) refers to glycoproteins that deliver peptide antigens to a cell surface. MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non-covalently associated P2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and p, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8+T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4+T cells. Human MHC is referred to as human leukocyte antigen (HLA).

[0091] “CD4” refers to an immunoglobulin co-receptor glycoprotein that assists the TCR in communicating with antigen-presenting cells (see, Campbell and Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKB P01730). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and includes four immunoglobulin domains (DI to D4) that are expressed at the cell surface. During antigen presentation, CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII P2, while the TCR complex binds MHCII al / pi).

[0092] As used herein, the term “CD8 co-receptor” or “CD8” means the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer. The CD8 co-receptor assists in the function of cytotoxic T cells (CD8+) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21 :630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1:81-88, 2004). In humans, there are five (5) different CD8 beta chains (see UniProtKB identifier P10966) and a single CD8 alpha chain (see UniProtKB identifier P01732).

[0093] “Chimeric antigen receptor” (CAR) refers to a fusion protein engineered to contain two or more naturally occurring amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. CARs of the present disclosure include an extracellular portion comprising an antigen binding domain (i.e., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a scFv or scTCR derived from an antibody or TCR specific for a cancer antigen, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain etal., Cancer Discov. 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci. 37(3):220 (2016); Stone et al., Cancer Immunol. Immunother. 63(11): 1163 (2014)). In certain embodiments, a binding protein comprises a CAR comprising an antigen specific TCR binding domain (see, e.g., Walseng et al., Scientific Reports 7:10713, (2017); the TCR CAR constructs and methods of which are hereby incorporated by reference in their entirety).

[0094] The term “variable region” or “variable domain” refers to the domain of a TCR a-chain or fl-chain (or y-chain and 5-chain for y5 TCRs), or of an antibody heavy or light chain, that is involved in binding to antigen. The variable domains of the a-chain and P-chain (Va and V , respectively) of a native TCR generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs. Variable domains of antibody heavy (VH) and light (VL) chains each also generally comprise four generally conserved framework regions (FRs) and three CDRs. In some instances, variable domains of both a TCR a-chain or P-chain (or y-chain and 5-chain for y5 TCRs), or of an antibody heavy or light chain, are involved in binding. In some instances, a variable domain of one of a TCR a-chain or P-chain (or y-chain and 5-chain for y5 TCRs), or of an antibody heavy or light chain, is involved in binding.

[0095] The terms “complementarity determining region,” and “CDR,” are synonymous with “hypervariable region” or “HVR,” and are known in the art to refer to sequences of amino acids within TCR or antibody variable regions, which confer antigen specificity and / or binding affinity and are separated in primary sequence from one another by framework amino acids. In general, there are three CDRs in each variable region (i.e., three CDRs in each of the TCRa-chain and P-chain variable regions; 3 CDRs in each of the antibody heavy chain and light chain variable regions). In the case of TCRs, CDR3 isthought to be the main CDR responsible for recognizing processed antigen. In general, CDR1 and CDR2 mainly, or in some cases, exclusively, interact with the MHC. Variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, EU, International Immunogenetics Information System (IM GT), Contact, and Aho), which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (Bioinformatics 15:298-300 (2016)). In certain embodiments of the present disclosure, CDRs are determined using IM GT numbering. IM GT determination of CDRs from a TCR sequence can be achieved using, for example, IMGT V-Quest (imgt.org / IMGTindex / V- QUEST.php). It will be understood that a CDR from a, for example, TCR Va or VP region or domain may have a particular sequence according to a particular numbering scheme, and may have a shorter, longer, or shifted (e.g., partially overlapping) sequence by a different numbering scheme.

[0096] “Antigen” or “Ag” as used herein refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells (e.g., T cells), or both. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen.

[0097] The term “epitope” or “antigenic epitope” includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes can be comprised of consecutive amino acids (e.g., a linear epitope), or amino acids from different parts of a protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding and / or processing by the cellular immune system.

[0098] A “binding domain” (also referred to as a “binding region” or “binding moiety”), as used herein, refers to a molecule, such as a peptide, oligopeptide, polypeptide, or protein that possesses the ability to specifically and non-covalently associate, unite, or combine with a target molecule (e.g., IGRP peptide (SEQ ID NO: 1, or an immunogenic fragment thereof comprising or consisting of about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, or 20, contiguous amino acids of SEQ ID NO: 1); IGRP peptide:MHC complex, wherein the MHC allele can be DRB4*0103. A binding domain includes any naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partner for a biological molecule or other target of interest. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or TCR or functional binding domain or antigen-binding fragment thereof. Exemplary binding domains include single chain antibody variable regions (e.g., single domain antibodies, sFv, scFv, and Fab), receptor ectodomains (e.g., TNF-a), ligands (e.g., cytokines and chemokines), antigen-binding regions of TCRs, such as single chain TCRs (scTCRs), synthetic polypeptides selected for the specific ability to bind to a biological molecule, aptamers, or single domain antibodies (e.g., camelid or fish-derived single domain antibodies; see, e.g., Arbabi-Ghahroudi M. Front. Immunol. 8:1589 (2017)).

[0099] A “linker” refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex). In certain embodiments, a linker is comprised of about two to about 35 amino acids, about four to about 20 amino acids, about eight to about 15 amino acids, about 15 to about 25 amino acids, or another suitable number of amino acids. Exemplary linkers include glycine-serine linkers, wherein one or more consecutive glycines are followed by a serine, which sequence may be repeated two, three, four, or more times.

[0100] Any binding domain of the present disclosure may be engineered in a single chain format so that the C-terminal end of a first domain is linked by a short peptide sequence to the N-terminal end of a second domain, or vice versa (e.g., in the case of a scTCR, (N)VP(C)-linker-(N)Va(C) or (N)Va(C)-linker-(N)VP(C). In certain embodiments, the binding domain is chimeric, human, or humanized.

[0101] As used herein, the term “IGRP- specific binding protein” refers to a protein or polypeptide that specifically binds to and / or that is specific for an IGRP antigen.

[0102] In some embodiments, a binding protein or polypeptide binds to IGRP, such as an IGRP peptide complexed with an MHC or HLA molecule, e.g. , on a cell surface, with a, or at least about a particular affinity. An IGRP-specific binding protein may bind to an IGRP antigen, a variant thereof, or a fragment thereof. For example, the IGRP- specific binding protein may bind to an amino acid sequence according to SEQ ID NO: 1, or to an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:1. In certain embodiments, a IGRP- specific binding protein binds a IGRP-derived peptide:HLA complex (or IGRP-derived peptide:MHC complex) with an affinity that is about the same as, at least about the same as, or is greater than at or about the affinity exhibited by an exemplary IGRP-specific binding protein provided herein, such as any of the IGRP-specific TCRs provided herein, for example, as measured by the same assay. Kd can be measured to assess the affinity of an IGRP-specific binding protein.

[0103] Assays for assessing affinity or apparent affinity or relative affinity are known. For example, apparent affinity of a TCR for antigemHLA can be measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers. In some examples, apparent Kd of a TCR is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent Kd being determined as the concentration of ligand that yielded half-maximal binding.

[0104] As used herein, “specifically binds” refers to an association or union of a binding protein (e.g., a T cell receptor or a chimeric antigen receptor) or a binding domain (or a fusion protein thereof), to a target molecule with an affinity or Ka(i.e. , an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M-1, while not significantly associating or uniting with any other molecules or components in a sample. Binding domains (or fusion proteins thereof) may be classified as “high affinity” binding domains (or fusion proteins thereof) or “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Kaof at least 107M-1, at least 10sM-1, at least 109M-1, at least 1010M-1, at least 10nat least 1012M-1, or at least 1013M-1. “Low affinity” binding domains refer to those binding domains with a Kaof up to 107M-1, up to 106M-1, or up to105M. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5M to 10-13M). In certain embodiments, a binding domain may have “enhanced affinity,” which refers to a selected or engineered binding domain with stronger binding to a target antigen than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka(equilibrium association constant) for the target antigen that is higher than the wild type binding domain, or due to a Kd for the target antigen that is less than that of the wild type binding domain, or due to an off-rate (KOff) for the target antigen that is less than that of the wild type binding domain. A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, el al., Ann. N. Y. Acad. Sci. 57:660 (1949); and U.S. Pat. Nos. 5,283,173, 5,468,614, or the equivalent).

[0105] The IGRP antigen- specific binding proteins, TCRs, or derivatives thereof as described herein, may be functionally characterized according to any of a large number of art accepted methodologies for assaying host cell activity, including determination of host cell binding, activation or induction and also including determination of host cell responses that are antigen- specific. Examples include determination of host cell proliferation, host cell cytokine release, antigen-specific host cell stimulation, MHC / HLA restricted host cell stimulation, cytotoxic T lymphocyte (CTL) activity (e.g., by detecting51Cr release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T-cell functions. Procedures for performing these and similar assays may be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998; see also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, Mass. (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, Calif. (1979); and Green and Reed, Science 281:1309 (1998) and references cited therein).

[0106] By way of further illustration, in the case of a host cell that expresses a binding protein of the present disclosure, avidity of the host cell for antigen can be determined by, for example, exposing the host cell to the peptide, or to a peptide:HLA complex (e.g., organized as a tetramer or other multimer), or to an antigen-presenting cell (APC) that presents the peptide to the host cell, optionally in a peptide:HLA complex, andthen measuring an activity of the host cell, such as, for example, production or secretion of cytokines e.g., IFNy; TNFa); increased expression of host cell signaling or activation components (e.g., CD 137 (4- IBB)); proliferation of the host cell; or killing of the APC (e.g., using a labeled-chromium release assay).

[0107] A target molecule, which is specifically bound by a binding protein, a TCR, or a derivative thereof, of the present disclosure, may be found on or in association with a cell of interest (“target cell”). Exemplary target cells include any cell in a subject or sample from a subject, or a cell for research purposes, that expresses an antigen (IGRP) of the present disclosure, such as, for example, an islet cell, a cell associated with an autoimmune disease or disorder.

[0108] In certain embodiments, any host cell of the present disclosure e.g., a recombinant host cell expressing and / or encoding a heterologous binding protein as provided herein) can be an immune system cell. As used herein, the terms “immune system cell” and “immune cell” refer to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which gives rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and a lymphoid progenitor cell (which gives rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a stem cell memory T cell, a y8 T cell, a regulatory T cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

[0109] A “T cell” is an immune system cell that matures in the thymus and produces TCRs. T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). Tvi can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD 127 as compared to naive T cells or TCM).Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzyme and perforin as compared to TCM. Other exemplary T cells include regulatory T cells, such as CD4+ CD25+ (Foxp3+) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8+CD28-, and Qa-1 restricted T cells.

[0110] “Hematopoietic stem cells” or “HSCs” refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to other cell types including cells of the T cell lineage. HSCs may be isolated, for example, but not limited to, from fetal liver, bone marrow, and cord blood.

[0111] “Embryonic stem cells,” “ES cells,” or “ESCs” refer to undifferentiated embryonic stem cells that have the ability to integrate into and become part of the germ line of a developing embryo. Embryonic stem cells are capable of differentiating into hematopoietic progenitor cells and any tissue or organ. Embryonic stem cells that are suitable for use herein include cells from the J 1 ES cell line, 129J ES cell line, murine stem cell line D3 (American Type Culture Collection), the R1 or E14K cell lines derived from 129 / Sv mice, cell lines derived from Balb / c and C57B 1 / 6 mice, and human embryonic stem cells (e.g., from WICELL® Research Institute, WI; or ES cell International, Melbourne, Australia).

[0112] “Cells of T cell lineage” refer to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g., CD3+, CD4+, and CD8+), or a physiological, morphological, functional, or immunological feature specific for a T cell. For example, cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25+immature and inactivated T cells; cells that have undergone CD4 or CD8 linage commitment; thymocyte progenitor cells that are CD4+CD8+double positive; single positive CD4+or CD8+; TCRaP or TCRyb; or mature and functional or activated T cells.

[0113] The term “isolated” refers to material that is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all the co-existingmaterials in the natural system is isolated. Such nucleic acid could be part of a vector and / or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term “gene” refers to the segment of DNA involved in producing a polypeptide chain. It includes regions preceding and following the coding region “leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).

[0114] As used herein to describe a cell, microorganism, nucleic acid molecule, or vector, the term “recombinant” or “modified” or “engineered” refers to a cell, microorganism, nucleic acid molecule, or vector that has been modified by introduction of an exogenous nucleic acid molecule (e.g., DNA, RNA) or protein, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated, or constitutive, where such alterations or modifications may be introduced by genetic engineering. Genetic alterations may include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell’s genetic material. Exemplary modifications include those in coding regions or functional fragments thereof of heterologous or homologous polypeptides from a reference or parent molecule.

[0115] Additional definitions are provided throughout the present disclosure. Binding Proteins Specific for IGRP-related peptide

[0116] In one aspect, the present disclosure provides binding proteins (e.g., an immunoglobulin superfamily binding protein or a portion thereof) that include a TCR V domain and a V|3 domain, wherein the binding protein is configured to bind to, is capable of binding to, and / or is specific for an IGRP-related peptide.

[0117] In certain embodiments, an IGRP-specific binding protein is configured to bind to, capable of binding to, or is specific for FLNFMSNVGDPRNIFFIYFP (SEQ ID NO:1):HLA complex, or a peptide:HLA complex wherein the peptide comprises or consists of about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, or 20 contiguous amino acids of SEQ ID NO:1. In some embodiments, the HLA comprises DRB4*0103.In some embodiments, the TCR Va domain comprises a CDR3 amino acid sequence that is at least about 85% (i.e., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identical to the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the TCR Va domain CDR3 amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the TCR V0 domain comprises a CDR3 amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, the TCR Va domain CDR3 amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.

[0118] In any of the presently disclosed embodiments, an IGRP-specific binding protein comprises a CDRla amino acid sequence that is at least about 85% identical to the amino acid sequence set forth in SEQ ID NO:2, a CDR2a amino acid sequence that is at least about 85% identical to the amino acid sequence set forth in to SEQ ID NO:3, a CDRip amino acid sequence that is at least about 85% identical to the amino acid sequence set forth in to SEQ ID NO:5, and / or a CDR2P amino acid sequence that is at least about 85% identical to the amino acid sequence set forth in to SEQ ID NO:6.

[0119] In further embodiments, an IGRP-specific binding protein comprises: CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences as set forth in SEQ ID NOs:2, 3, 4, 5, 6, and 7, respectively.

[0120] In certain embodiments, an IGRP-specific binding protein comprises a TCR Va domain that comprises or consists of an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, an IGRP-specific binding protein comprises a TCR VP domain that comprises or consists of an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13.

[0121] In further embodiments, any one or more of the p or a CDR amino acid sequences as provided herein can be present in the VP domain and / or the Va domain, respectively.

[0122] In certain embodiments, at least three or four of the complementary determining regions (CDRs) may have no change in sequence, and the CDRs that do have sequence changes may have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof.

[0123] In certain embodiments, an IGRP-specific binding protein comprises a TCR Va domain and a TCR VP domain according to SEQ ID NO: 12, or according to SEQ ID NO: 13, respectively.

[0124] In any of the embodiments described herein, a binding protein (e.g., IGRP-specific binding protein as discussed herein) can comprise a “signal peptide” (also known as a leader sequence, leader peptide, or transit peptide). Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell. A signal peptide may be removed from the polypeptide during, or once localization or secretion is completed. Polypeptides that have a signal peptide are referred to herein as a “pre-protein” and polypeptides having their signal peptide removed are referred to herein as “mature” proteins or polypeptides. In certain embodiments, a binding protein of this disclosure comprises a mature VP domain, a mature Va domain, or both. In some embodiments, a binding protein of this disclosure comprises a mature TCR -chain, a mature TCR a chain, or a mature TCR P-chain and a mature TCR a chain.

[0125] Exemplary binding proteins and fusion proteins of this disclosure expressed by a cell may include a signal peptide (e.g., as binding pre-proteins), and the cell may remove the signal peptide to generate a mature binding protein. In certain embodiments, a binding protein comprises two components, such as an a chain and a P chain, which can associate on the cell surface to form a functional binding protein. The two associated components may comprise mature proteins.

[0126] In certain embodiments, a binding protein comprises a TCR variable domain comprising an amino acid sequence encoded by a human TCR V, D, and / or J allele. By way of background, during lymphocyte development, Va exons are assembled from different variable and joining gene segments (V-J), and VP exons are assembled from different variable, diversity, and joining gene segments (V-D-J). The TCRa chromosomal locus has 70-80 variable gene segments and 61 joining gene segments. The TCRP chromosomal locus has 52 variable gene segments, and two separate clusters of each containing a single diversity gene segment, together with six or seven joining gene segments. Functional Va and VP gene exons are generated by the recombination of a variable gene segment with a joining gene segment for Va, and a variable gene segment with a diversity gene segment and a joining gene segment for Vp. Nucleotide and amino acid sequences according to TCR gene segments of various alleles are known in the art andcan be found on the ImMunoGeneTics website; for example, at imgt.org / IMGTrepertoire / LocusGenes / listIG_TR / TR / human / Hu_TRgroup.html.

[0127] It will be understood that while a polynucleotide encoding a binding protein can comprise a same nucleotide sequence according to a TCR gene segment as disclosed herein, any nucleotide sequence that encodes the amino acid sequence of the referenced gene segment may be used.

[0128] In any of the herein disclosed embodiments, the TCR VOL domain of an IGRP-specific binding protein comprises an amino acid sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 consecutive amino acids, or more) according to TRAV35*02 or TRAJ6*01, or an amino acid sequence that is at least 85% identical thereto. In certain embodiments, the TCR Vp domain of an IGRP-specific comprises an amino acid sequence of TRBV7-8*01, TRBJ1-3*O1, and TRBD*01. Nucleotide and amino acid sequences of human T cell receptor variable region alleles (e.g., TRAV, TRBV, TRAJ, TRBJ, TRBD), including of the alleles provided herein, are known and are available, for example, through the IM GT (ImMunoGeneTics) Information System®; e.g., at imgt.org / IM GTrepertoire / Proteins / alleles / list_alleles.php?species=Homo %20sa piens&group=TRAV ; at imgt.org / IM GTrepertoire / Proteins / alleles / list_alleles.php?species=Homo %20sa piens&group=TRB V ; at imgt.org / IMGTrepertoire / Proteins / alleles / list_alleles.php?species=Homo %20sa piens&group=TRAJ; at imgt.org / IM GTrepertoire / Proteins / alleles / list_alleles.php?species=Homo %20sa piens&group=TRBJ ; and at imgq.org / IMGTrepertoire / Proteiris / alleles / list_alleles.php7speciesM lomo %20sa piens&group=TRBD.

[0129] In some embodiments, an IGRP-specific binding protein comprises an amino acid sequence encoded by a TCR a-chain joining (J a) domain gene segment and an amino acid sequence encoded by TCR P-chain joining (JP) gene segment. A TCR Ja domain can comprise an amino acid sequence according to TRAJ6*01, or an amino acid sequence that is at least 85% identical thereto. A TCR JP domain can comprise an aminoacid sequence according to TRBJ1-3*O1, or an amino acid sequence that is at least 85% identical thereto.

[0130] These human T cell receptor variable domain allele polynucleotide and amino acid sequences are incorporated by reference herein.

[0131] In any of the presently disclosed embodiments (i.e., IGRP-specific binding protein), a binding protein can further comprise a TCR P chain constant domain (C ), a TCR a chain constant domain (Ca), or both. In an embodiment, the TCR P chain constant domain comprises an amino acid sequence according to TRABCl*01 or an amino acid sequence that is at least 85% identical thereto. In an embodiment, the TCRa constant domain comprises an amino acid sequence according to TCRAC 01 or an amino acid sequence that is at least 85% identical thereto. Exemplary amino acid sequences of human TCR Ca and CP can be found at, for example, UniProtKb P01848 (Ca) and UniProtKb P01850 and A0A5B9 (CP). Exemplary amino acid sequences of murine TCR constant regions can be found at UniProtKb A0A0A6YWV4, A0A075B5J4, and A0A075B5J3. These amino acid sequences are incorporated by reference herein.

[0132] In any of the presently disclosed embodiments (i.e., IGRP-specific binding protein), the binding protein further comprises a CP and a Ca, wherein the VP and the CP together comprise a TCR P chain, and wherein the Va and the Ca together comprise a TCR a chain, and wherein the TCR P chain and the TCR a chain are capable of associating to form a dimer.

[0133] In certain embodiments, a TCR Ca has at least about 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, a TCR CP has at least about 85% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 11.

[0134] Also contemplated are binding proteins that comprise a TCRa chain and / or a TCRP chain having at least 85% identity to an amino acid sequence comprised in SEQ ID NO:8 or 9, respectively.

[0135] In certain embodiments, an IGRP-specific binding protein may be a TCR, a chimeric antigen receptor, or an antigen-binding fragment of a TCR. In certain embodiments, the TCR, the chimeric antigen receptor, or the antigen-binding fragment of the TCR may be chimeric, humanized, or human. In further embodiments, the antigenbinding fragment of the TCR comprises or consists of a single-chain TCR (scTCR).

[0136] Also provided herein are high affinity recombinant TCRs that are configured to bind to, are capable of binding to, and / or are specific for an IGRP antigen. A high affinity recombinant TCR can comprise a Va domain that is at least about 85% (i.e. , at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identical to an amino acid sequence of SEQ ID NO: 12. In some embodiments, a high affinity recombinant TCR can comprise a VP domain that is at least about 85% i.e., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identical to an amino acid sequence of SEQ ID NO: 13. In some embodiments, a high affinity recombinant TCR comprises a TCR Va domain having at least about 85% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 12, and / or comprises a TCR VP domain having at least about 85% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 13.

[0137] In some embodiments, the TCR Va domain comprises a CDR3 amino acid sequence that is at least about 85% (i.e., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identical to the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the TCR Va domain CDR3 amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the TCR VP domain comprises a CDR3 amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, the TCR VP domain CDR3 amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.

[0138] In certain embodiments, at least three or four of the complementary determining regions (CDRs) may have no change in sequence, and the CDRs that do have sequence changes may have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof.

[0139] In any of the presently disclosed embodiments, an IGRP-specific high affinity recombinant TCR comprises a CDRla amino acid sequence according to SEQ ID NO:2, a CDR2a amino acid sequence according to SEQ ID NO:3, a CDRip amino acid sequence according to SEQ ID NO:5, and / or a CDR2P amino acid sequence according to SEQ ID NO:6.

[0140] In further embodiments, An IGRP-specific high affinity recombinant TCR comprises: CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences as set forth in SEQ ID NOs:2, 3, 4, 5, 6 and 7, respectively.

[0141] In any of the presently disclosed embodiments, an IGRP-specific binding protein or high affinity recombinant TCR is capable of binding to a FLNFMSNVGDPRNIFFIYFP(SEQ ID NO: 1):DRB4*0103 complex or a peptide:DRB4*0103 complex, wherein the peptide comprises or consists of about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, or 20 contiguous amino acids of SEQ ID NO:1.

[0142] In some embodiments, an IGRP-specific binding protein or high affinity recombinant TCR is capable of binding to a FLNFMSNVGDPRNIFFIYFP (SEQ ID NQ:l):DRB4*0103 complex or a peptide:DRB4*0103 complex, wherein the peptide comprises or consists of about7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, or 20 contiguous amino acids of SEQ ID NO: 1 , on a cell surface independent or in the absence of CD8 and / or CD4.

[0143] In some embodiments, an IGRP-specific binding protein or high affinity recombinant TCR can be provided in soluble form (see, e.g., Walseng et al., PLoS One doi:10.1371 / journal.pone.0119559 (2015)), optionally conjugated to a detectable agent. Methods useful for isolating and purifying recombinantly produced soluble TCR, by way of example, may include obtaining supernatants from suitable host cell / vector systems that secrete the recombinant soluble TCR into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix and / or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated / recombinant soluble TCR described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.Compositions

[0144] Another aspect of the disclosure is directed to a composition including a binding protein or high affinity recombinant TCR, or derivatives thereof, as describedabove. The composition may further include a pharmaceutically acceptable carrier, diluent, and / or excipient, as described further herein.

[0145] Also provided herein are immunogenic compositions (e.g., for use in a vaccine).

[0146] In some embodiments, an immunogenic composition comprises immune cells, for e.g., regulatory T cells, expressing the binding protein, the TCR, or derivatives thereof, that specifically bind to an isolated peptide comprising an IGRP antigen or a fragment thereof. In certain embodiments, the peptide has an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to FLNFMSNVGDPRNIFFIYFP (SEQ ID NO: 1 ), or an immunogenic fragment thereof. The isolated peptide can comprise or be contained in a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acids. Furthermore, the polypeptide can include a sequence of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, contiguous amino acids from the IGRP amino acid sequence set forth in SEQ ID NO:1.

[0147] In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier, discussed further herein. The pharmaceutically acceptable carrier may be a non-naturally occurring pharmaceutically acceptable carrier. In certain embodiments, the non-naturally occurring pharmaceutically acceptable carrier may include a cream, emulsion, gel, liposome, nanoparticle, or ointment. In some other embodiments, the vaccine may include an immuno-effective amount of an adjuvant such as, for example, poly-ICLC, CpG, GM-CSF, or alum.Polynucleotides, Vectors, and Host Cells

[0148] Also provided are polynucleotides that encode a binding protein, high- affinity recombinant TCR, immunogenic composition, or a functional fragment or portion thereof, as disclosed herein. It will be appreciated by those of ordinary skill in the art that, due to the degeneracy of the genetic code, there are numerous nucleotide sequences that encode a binding protein, TCR, or immunogenic composition as described herein. Some such polynucleotides can bear limited or minimal sequence identity to the nucleotide sequence of a native, original, or identified polynucleotide sequence. Nonetheless, polynucleotides that vary due to differences in codon usage are expressly contemplated by the present disclosure. In certain embodiments, sequences that have been codon-optimized for expression in a mammalian host cell are specifically contemplated. Codon optimizationcan be performed using known techniques and tools, e.g., using the GenScript® OptimiumGene™ tool. Codon-optimized sequences include sequences that are partially codon-optimized (i.e., at least one codon is optimized for expression in the host cell) and those that are fully codon-optimized. Codon optimization for expression in certain immune host cells is disclosed in, for example, Scholten et al., Clin. Immunol. 119: 135, (2006).

[0149] In some embodiments, a single polynucleotide encodes a binding protein as described herein, or, alternatively, the binding protein may be encoded by more than one polynucleotide. In other words, components or portions of a binding protein may be encoded by two or more polynucleotides, which may be contained on a single nucleic acid molecule or may be contained on two or more nucleic acid molecules.

[0150] In certain embodiments, a polynucleotide encoding two or more components or portions of a binding protein or TCR of the present disclosure comprises the two or more coding sequences operatively associated in a single open reading frame. Such an arrangement can advantageously allow coordinated expression of desired gene products, such as, for example, contemporaneous expression of alpha and beta chains of a TCR, such that they are produced in about a 1 : 1 ratio. In certain embodiments, two or more substituent gene products of a binding protein of this disclosure, such as a TCR (e.g., alpha and beta chains), are expressed as separate molecules and associate post-translationally. In further embodiments, two or more substituent gene products of a binding protein of this disclosure are expressed as a single peptide with the parts separated by a cleavable or removable segment. For instance, self-cleaving peptides useful for expression of separable polypeptides encoded by a single polynucleotide or vector are known in the art.

[0151] Accordingly, in certain embodiments, a heterologous polynucleotide encoding a TCR a-chain and a heterologous polynucleotide encoding a TCR P-chain are contained in a single open reading frame, wherein the single open reading frame further comprises a polynucleotide encoding a self-cleaving peptide disposed between the a-chain- encoding polynucleotide and the -chain-encoding polynucleotide. It will be understood that either orientation (e.g., P-chain-encoding polynucleotide-self-cleaving peptide-a- chain-encoding polynucleotide; a-chain-encoding polynucleotide-self-cleaving peptide-P- chain-encoding polynucleotide) is contemplated.

[0152] In certain embodiments, a polynucleotide of the present disclosure comprises or consists of a polynucleotide having at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 14 and 15.

[0153] Isolated or recombinant nucleic acid molecules encoding a binding protein or high affinity recombinant TCR or a derivative thereof specific for IGRP as described herein may be produced and prepared according to various methods and techniques of the molecular biology or polypeptide purification arts.

[0154] In further embodiments, a binding protein or TCR is expressed as part of a transgene construct that encodes, and / or a host immune cell can further encode: one or more additional accessory protein, such as a safety switch protein; a tag, a selection marker; a CD8 co receptor p chain; a CD8 co-receptor a chain or both; or any combination thereof. Polynucleotide and transgene construct useful for encoding and expressing binding proteins and accessory components (e.g. , one or more of a safety switch protein, a selection marker, CD8 co-receptor -chain, or a CD8 co-receptor a-chain) are described in PCT application PCT / US2017 / 053112, the polynucleotides, transgene constructs, and accessory components, including the nucleotide and amino acid sequences, all of which are hereby incorporated by reference. It will be understood that any or all of a binding protein of the present disclosure, a safety switch protein, a tag, a selection marker, a CD8 co-receptor P chain, or a CD8 co-receptor a-chain may be encoded by a single nucleic acid molecule or may be encoded by polynucleotide sequences that are, or are present on, separate nucleic acid molecules.

[0155] Other accessory components useful for modified immune cells of the present disclosure comprise a tag or selection marker that allows the cells to be identified, sorted, isolated, enriched, or tracked. For example, marked immune cells having desired characteristics (e.g., an antigen-specific TCR and a safety switch protein) can be sorted away from unmarked cells in a sample and more efficiently activated and expanded for inclusion in a product of desired purity.

[0156] As used herein, the term “selection marker” comprises a nucleic acid construct (and the encoded gene product) that confers an identifiable change to a cell permitting detection and positive selection of immune cells transduced with a polynucleotide comprising a selection marker. RQR is a selection marker that comprises a major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, an RQR-encoding polynucleotide comprises a polynucleotide that encodes the 16-amino-acid CD34 minimal epitope. In some embodiments, the CD34 minimalepitope is incorporated at the amino terminal position of a CD8 co-receptor stalk domain (Q8). In further embodiments, the CD34 minimal binding site sequence can be combined with a target epitope for CD20 to form a compact marker / suicide gene for T cells (RQR8) (Philip et al., 2014, incorporated by reference herein). This construct allows for the selection of immune cells expressing the construct, with for example, CD34 specific antibody bound to magnetic beads (Miltenyi) and that utilizes clinically accepted pharmaceutical antibody, rituximab, that allows for the selective deletion of a transgene expressing engineered T cell (Philip et al., 2014).

[0157] Also provided are expression vectors comprising a polynucleotide according to the present disclosure. Any suitable expression vector, including an exemplary expression vector as disclosed herein, may be used. Furthermore, the expression vector may be configured to or capable of delivering the polynucleotide to a host cell.

[0158] A typical vector may include a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked, or which is capable of replication in a host organism. As discussed herein, some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell upon introduction into the host cell and thereby replicate along with the host genome. Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as “expression vectors”). According to related embodiments, it is further understood that, if one or more agents (e.g., polynucleotides encoding immunoglobulin superfamily binding proteins or high affinity recombinant TCRs specific for IGRP, or variants thereof, as described herein) is co-administered to a subject, that each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.

[0159] Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno- associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, but are not limited to, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott- Raven Publishers, Philadelphia, 1996).

[0160] Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing chimeric antigen receptor transgenes are known in the art and have been previous described, for example, in U.S. Pat. No. 8,119,772; Walchli, et al., PLoS One 6:327930, (2011); Zhao, et al., J. Immunol. 174:4415, (2005); Engels, et al., Hum. Gene Ther. 14: 1155, (2003); Frecha, et al., Mol. Ther. 75: 1748, (2010); and Verhoeyen, et al. , Methods Mol. Biol. 506:91, (2009). Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0161] Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing TCR or CAR transgenes are known in the art and have been previously described, for example, in: U.S. Pat. No. 8,119,772; Walchli et al., PLoS One 6:327930 (2011); Zhao et al., J. Immunol. 174:4415 (2005); Engels et al., Hum. Gene Ther. 14: 1155 (2003); Frecha et al., Mol. Ther. 18: 1748 (2010; and Verhoeyen et al., Methods Mol. Biol. 506:97 (2009). Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5:1517 (1998)).

[0162] Other vectors developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors. Pp 209-40 inFriedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0163] When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicis tronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.

[0164] In certain embodiments, the nucleic acid encoding a binding protein or high affinity recombinant TCR specifically for IGRP antigen may be operatively linked to one or more certain elements of a vector. For example, polynucleotide sequences that are needed to affect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e.. Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. In some embodiments, a viral or plasmid vector further includes a transduction marker (e.g., green fluorescent protein, tEGFR, tCD19, tNGFR, etc.).

[0165] In certain embodiments, a vector is capable of delivering a polynucleotide construct to a host cell (e.g., a hematopoietic progenitor cell or a human immune system cell). In specific embodiments, a vector is capable of delivering a construct to human immune system cell, such as, for example, a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y5 T cell, a natural killer cell, a dendritic cell, or any combination thereof. In further embodiments, a vector is capable of delivering a construct to a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. In some embodiments, a vector that encodes a construct of the present disclosure may further comprise a polynucleotide that encodes a nuclease that can be used to perform a chromosomal knockout in a host cell (e.g., a CRISPR-Cas endonuclease or another endonuclease as disclosed herein) or that can be used to deliver a therapeutic transgene orportion thereof to a host cell in a gene therapy replacement or gene repair therapy. Alternatively, a nuclease used for a chromosomal knockout or a gene replacement or gene repair therapy can be delivered to a host cell independent of a vector that encodes a construct of this disclosure.

[0166] Also provided are host cells that encode (e.g., comprise a heterologous polynucleotide encoding) and / or express a binding protein or high-affinity recombinant TCR, or derivatives thereof, as disclosed herein. In some embodiments, the host cell may be a hematopoietic progenitor cell, or an immune system cell, such as a human immune system cell. In any of the presently disclosed embodiments, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8-double negative T cell, a y5 T cell, a natural killer cell, a dendritic cell, or any combination thereof. Additionally, the T cell may be a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, a regulatory T cell, or any combination thereof. In certain embodiments, the host cell is modified to comprise or contain the heterologous polynucleotide using a vector as disclosed herein.

[0167] In any of the presently disclosed embodiments, a host cell, such as a host immune cell, can comprise a chromosomal gene knockout of an endogenous immune cell protein, such as, for example, PD-1, TIM3, LAG3, CTLA4, TIGIT, an HLA component, or a TCR component, or any combination thereof. As used herein, the term “chromosomal gene knockout” refers to a genetic alteration or introduced inhibitory agent in a host cell that prevents (e.g., reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0168] Also provided herein are unit doses that comprise an effective amount of a modified immune cell or of a composition comprising the modified immune cell. In certain embodiments, a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified / engineered Treg cells, wherein the unit dose contains a reduced amount or substantially no naive T cells (i.e. , has less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, less than about 10%, less than about 5%, or less then about 1 % the population of naive T cells present in a unit dose as compared to a patient sample having a comparable number of PBMCs).

[0169] It will be appreciated that a unit dose of the present disclosure may comprise a binding protein, TCR, or derivatives thereof, or recombinant host cell as described herein (i.e., expressing a binding protein, TCR, or derivatives thereof, specific for an IGRP antigen or a fragment thereof).Uses

[0170] In another aspect, the present disclosure provides methods of treating a subject in need thereof (i.e., having or suspected of having a disease or disorder associated with an IGRP antigen or a portion thereof. In some embodiments, the method comprises administering to the subject an effective amount of a composition (e.g., binding protein or a derivative thereof, TCR or a derivative thereof, recombinant host cell expressing the binding protein or a derivative thereof or host cells expressing TCR or a derivative thereof, immunogenic compositions, polynucleotides, vectors, or related compositions) as described herein. Such diseases include Type 1 Diabetes (T1D).

[0171] “Treat,” “treatment,” or “ameliorate” refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, dog, mouse, or rat). In general, an appropriate dose or treatment regimen including a host cell of this disclosure, and optionally an adjuvant, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof.

[0172] A “therapeutically effective amount” or “effective amount” of a composition (e.g., binding protein or host cell expressing or encoding the same) of this disclosure refers to that amount of compound or cells sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner. When referring to an individual active ingredient or a cell expressing a single active ingredient, administered alone, a therapeutically effective dose refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, atherapeutically effective dose refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously. A combination may also be a cell expressing more than one active ingredient, such as two different binding proteins that specifically bind to the same or different antigens.

[0173] As used herein, “statistically significant” refers to a p-value of 0.050 or less when calculated using the Student’s t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.

[0174] In certain embodiments, a method comprises administering an effective amount of a composition comprising binding proteins, high affinity recombinant TCRs, host cells, immunogenic compositions, polynucleotides, or vectors as described herein to the subject, in certain embodiments, the subject may have or be suspected of having TID or an (other) indication wherein an IGRP antigen is a therapeutic target.

[0175] In general, an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide a benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to nontreated subjects.

[0176] For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.

[0177] Also contemplated are pharmaceutical compositions (compositions) that comprise a binding protein, high-affinity recombinant TCRs, host (i.e., modified) immune cells, immunogenic compositions, polynucleotides, or vectors as disclosed herein and a pharmaceutically acceptable carrier, diluents, or excipient. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. In embodiments, compositions comprising fusion proteins or host cells as disclosed herein further comprise a suitable infusion media. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol®-R (Abbott) or Plasma-Lyte® A (Baxter),5% dextrose in water, Ringer’s lactate can be utilized. An infusion medium can be supplemented with human serum albumin or other human serum components.

[0178] Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art.

[0179] An effective amount of a pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein. An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term “therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.

[0180] In the case of an adoptive cell therapy, a therapeutically effective dose is an amount of host cells encoding and / or expressing a binding protein or high affinity recombinant TCR specific for an IGRP antigen, or derivatives thereof) used in adoptive transfer that is capable of producing a clinically desirable result ( / .<?., a sufficient amount to induce or enhance a specific T cell immune response against cells expressing IGRP antigen, e.g., a cytotoxic T cell response, in a statistically significant manner) in a treated human or non-human mammal. In various embodiments, the therapeutically effective dose is an amount of engineered Treg cells only. In particular embodiments, a T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0181] The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until infusion into the patient.

[0182] If the subject composition is administered parenterally, the composition may also include sterile aqueous or oleaginous solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer’ s solution, isotonic salt solution, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycols in mixtures with water. Aqueous solutions or suspensions may further include one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition,the active compounds may be incorporated into sustained-release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired therapeutic effect in association with an appropriate pharmaceutical carrier.Engineered T regulatory' Cells

[0183] In some embodiments the compositions and methods disclosed herein include engineered T regulatory cells comprising a constitutively expressed FoxP3 gene product expressed at a level equal to or greater than the level of FoxP3 expression in natural T regulatory (Treg or suppressor T) cells, and a transduced (e.g., artificially engineered by gene editing, viral vector transduction, transfection or other genetic engineering methodologies) the binding protein, the T cell receptor (TCR) or derivatives thereof disclosed herein. Methods of producing engineered T regulatory cells are disclosed in U.S. Patent Publication No. 20230279351 Al , the disclosure of which is hereby expressly incorporated by reference in its entirety. In some embodiments, the method of producing engineered regulatory T cells comprises gene editing a locus in the genome of a T cell to obtain the engineered T regulatory cell of the present disclosure. In some embodiments, the methods disclosed herein contemplate editing a first and a second locus in the genome of a T cell to obtain the engineered T regulatory cell disclosed herein. In some embodiments, the locus comprises TRACI loci. In some embodiments, the locus comprises FOXP3 loci. In some embodiments, where a first and a second loci are edited, the first locus comprises a TRACI and a second locus comprises a FOXP3. In some embodiments, the T cell is a CD4 T cell.

[0184] In some embodiments, the present disclosure provides an engineered T regulatory cell comprising a heterologous polynucleotide or a fragment thereof. In some embodiments, the heterologous polynucleotide or a fragment thereof is incorporated into the genome of the engineered T regulatory cell. In some embodiments, the heterologous polynucleotide or a fragment thereof is incorporated into the TRACI locus in the genome of the engineered regulatory T cell. In some embodiments, the heterologous polynucleotide or a fragment thereof is incorporated into the FOXP3 locus in the genome of the engineered regulatory T cell. In some embodiments, the heterologous polynucleotide encodes a chimeric molecule comprising TCR specific to a type 1 diabetes (T1D) antigen presented by the class II DRB4 molecule (human V-(D)-J) and murine constant regions.

[0185] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding TCRP or a functional fragment thereof; and a nucleic acid encoding TRAV and / or TRAJ or functional fragment thereof, wherein the TCRP and TRAV and / or TRAJ form parts of a TCR specific to a type 1 diabetes (T1D) antigen presented by the class II DRB4 molecule. In some embodiments, the vector encodes a chimeric molecule comprising TCR specific to a type 1 diabetes (T1D) antigen presented by the class II DRB4 molecule (human V-(D)-J) and murine constant regions. In some embodiments, the vector is a lentiviral vector. In some embodiments, the present disclosure provides a T cell comprising the vectors disclosed herein.

[0186] In some embodiments, the present disclosure provides an engineered T regulatory cell comprising a first polynucleotide and a second polynucleotide. In some embodiments, the first polynucleotide or fragment thereof is incorporated into a first locus of the genome of the engineered T regulatory cell. In some embodiments, the first locus comprises the TRAC locus. In some embodiments, the second polynucleotide or fragment thereof is inserted into a second locus of the genome of the engineered T regulatory cell. In some embodiments the second locus comprises the FOXP3 locus. In some embodiments the first polynucleotide comprises: (i) a first promoter, wherein the first promoter is MND; (ii) a first nucleic acid encoding a first CISC component comprising rapamycin binding domain of FK-binding protein 12 (FKBP) or a functional fragment thereof, and an intracellular signaling domain or functional fragment of IL2Ry; (iii) a nucleic acid encoding TCRP or a functional fragment thereof; and (iv) a nucleic acid encoding TRAV and / or TRAJ or functional fragment thereof, wherein the TCRP and TRAV and / or TRAJ form parts of a TCR specific to a type 1 diabetes (T1D) antigen presented by the class II DRB4 molecule. In some embodiments, the second polynucleotide comprises: (i) a second promoter, wherein the second promoter is MND; (ii) a second nucleic acid encoding a second CISC component comprising a second extracellular binding domain that comprises rapamycin binding domain of FKBP12-Rapamycin Binding domain of mTOR (FRB) or functional fragment thereof, and a second intracellular signaling domain comprising an intracellular signaling domain or functional fragment of IL2RP; and (iii) a third nucleic acid encoding a cytosolic FRB or function fragment thereof.

[0187] Provided herein is a method of engineering a T regulatory cell (Treg), or a population of Treg cells for the treatment, inhibition, or amelioration of T1D. In some embodiments, such a method comprises editing a population of T cells isolated from a firstsubject by contacting the population of T cells with any of the polynucleotides provided herein. In some embodiments, the method comprises dual-editing a population of T cells isolated from a first subject by contacting the population of T cells with any of the first polynucleotides as provided herein, and any of the second polynucleotides as provided here. In some embodiments, method comprises further contacting the population of T cells with an endonuclease or a vector comprising a nucleic acid encoding an endonuclease that can cleave within a locus of the genome of the T cell (e.g., a TRACI or a Foxp3 locus) such that a polynucleotide or fragment thereof is incorporated into the locus.

[0188] In some embodiments, method comprises further contacting the population of T cells with a first endonuclease, or nucleic acid encoding the first endonuclease, that can cleave within a first locus e.g., TRAC locus); and a second endonuclease, or nucleic acid encoding the second endonuclease, that can cleave within a second locus e.g., Foxp3 locus), such that the first polynucleotide or fragment thereof is incorporated into the first locus, and the second polynucleotide or fragment thereof is inserted into the second locus. In some embodiments, if the endonuclease is an RNA- guided endonuclease, then dual-editing comprises further contacting the population of T cells with a first gRNA or a nucleic acid encoding the first gRNA that guides cleavage within the first locus, and a second gRNA or a nucleic acid encoding the second gRNA that guides cleavage within the second locus.

[0189] In some embodiments, a method of engineering a population of Treg cells for the treatment of T1D further comprises enriching the cells population for edited or dualedited cells. In some embodiments, the method comprises allowing the cells to expand, by contacting the population of cells with a dimerizing ligand that will dimerize the first CISC component and the second CISC component on dual-edited engineered Treg cells comprising editing in both the first and second locus and thus express both the first CISC component and the second CISC component. Because CISC dimerization causes IL2 signaling, those cells with dual editing will multiply faster than those cells without dual editing (e.g. , cells with no editing or only editing in a single locus). In some embodiments, a population of T cells is contacted with 0.01 nM - 100 nM rapamycin (e.g., 0.01-100, 0.05-100, 0.05-0.5, 0.1-10, 0.1-100, 0.05-0.1, 0.5-20, 0.5-10, 1-100, 5-50, 5-15, 10-20 nM). In some embodiments, a population of T cells is contacted with 10 nM rapamycin.

[0190] In some embodiments, a method of engineering a population of Treg cells for the treatment of T1D further comprises isolating cells from a first subject so that theycan be edited or dual-edited. In some embodiments, cells are isolated from the blood of a first subject. In some embodiments, cells isolated from the blood of a first subject are further purified to isolate CD4+ T cells. In some embodiments, a first subject is human. In some embodiments, a first subject is a human suffering from diabetes (e.g., T1D).

[0191] In some embodiments, a method of engineering a population of Treg cells for the treatment of T1D further comprises stimulation using CD3 / CD28 (e.g., on a solid substrate such as a bead). In some embodiments, a population of cells is again stimulated using CD3 / CD28 (e.g., on a solid substrate such as a bead) after being contacted with a dimerization ligand.

[0192] FIG. 7 provides an example protocol for engineering a population of Treg cells for the treatment of T1D.

[0193] Provided herein are methods of inserting only a TCR-encoding nucleic acid into the TRAC locus, i.e., without inserting one or more of other components, e.g., a CISC component and / or a promoter. In some embodiments, the disclosure provides a vector comprising the TCR-encoding nucleic acid. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector comprises a nucleic acid encoding a chimeric TCR. In some embodiments, the chimeric TCR comprises human V-(D)-J and murine constant regions.

[0194] In some embodiments, any of the methods described herein to engineer Tregs is performed, at least in part (e.g., contacting cells with any of the polynucleotides as described herein, or enrichment / expansion of edited or dual-edited cells), in vivo.

[0195] Provided herein is a composition comprising an engineered Treg cell (EngTregs) produced by any one of the methods described herein. In some embodiments, a composition of EngTregs as provided herein comprises a population of cells comprising a subpopulation comprising edited or dual-edited EngTregs having a suppressive phenotype. In some embodiments, the subpopulation comprises at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45%,) of the population of T cells within 8 hours of being edited or dual-edited. In some embodiments, the subpopulation comprises at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45%,) of the population of T cells within 2 days of being dual-edited. In some embodiments, the subpopulation comprises at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%,) of the population of T cells within 18 days of being edited or dual-edited.

[0196] In some embodiments, a composition of EngTregs as provided herein is formulated for therapeutic use. In some embodiments, a composition comprising EngTregs further comprises pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient can include DMSO (e.g., 2-15% DMSO) and / or albumin (e.g., up to 5% human albumin).

[0197] In some embodiments, a composition for EngTregs comprises at least 103EngTreg cells. In some embodiments a composition administered to a subject comprises at least 10 mL of fluid, or 10-200 mL of fluid.

[0198] Provided herein is a method of treating, inhibiting, or ameliorating T1D in a subject. Both allogeneic and autologous approaches are contemplated herein. In some embodiments, cells are isolated from a first subject, engineered according to any one of the methods of engineering Treg cells as provided herein, and then administered into the first subject. In some embodiments, cells are isolated from a first subject, engineered according to any one of the methods of engineering Treg cells as provided herein, and then administered into a second subject. In some embodiments, the first subject suffers from or is at risk of suffering from T1D. In some embodiments, the first subject does not suffer from T1D, or does not suffer from any form of diabetes. In some embodiments, the second subject suffers from or is at risk of suffering from T ID. In some embodiments a subject to which a composition of EngTregs is administered is a human subject.

[0199] In some embodiments, a method of treating, inhibiting, or ameliorating T1D in a subject comprises administering to the subject any one of the compositions described herein that comprises EngTregs that can be made by any one of the methods of engineering Tregs cells described herein.

[0200] Any particular route of administration is contemplated herein. For example, a composition can be administered by infusion. In some embodiments, a method comprises direct injection into the pancreas. In some embodiments, at least 103EngTregs per kg of body weight in approximately 10-200 mL suspension is administered to a subject. In some embodiments, a method comprises administering EngTreg cells to the subject more than once. In some embodiments, EngTreg cells are administered to the subject 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, or more times.EXAMPLES

[0201] The following examples are illustrative of disclosed methods, uses, and compositions. In light of this disclosure, those of skill in the art will recognize that variations of these examples and other examples of the disclosed methods and compositions are possible without undue experimentation.EXAMPLESExample 1 : Isolation and characterization of human Tr24 TCRIsolation of islet specific TCR

[0202] PBMCs obtained from healthy control donor (wb242765749) were activated using an islet specific peptide pool followed by assessment of CD 137, CD25 and CD 127 expression to identify islet reactive CD4 Treg cells (FIG. 1A). Single cell RNA sequencing of islet specific Treg cells was performed to generate full TCR sequences. FIG. IB shows the identified Treg-derived TCR clonotype expanded in a healthy control subject and the variable and joining gene segments and CDR3 TCR sequence for the Tr24 TCR obtained using the single cell RNA- sequencing approach. Lentiviral TCR constructs for Tr24 TCR gene transfer into CD4+ primary T cells were generated. These TCR constructs express human the Tr24 TCR variable regions and mouse TCR constant regions allowing for an improved pairing between the transduced human TCR chains. To determine islet specificity, Tr24 TCR was expressed in human CD4+ T cells and surface expression was confirmed by flow cytometry (FIG. 2A). Tr24+ CD4+ T cells were then stimulated using 101 peptides from IGRP, ZNT8, or PPI islet proteins, divided into 11 pools of 10-11 peptides each (FIG. 2B, C and 3A), followed by an individual peptide screen for the pool identified in the first screen (FIG. 2C and 3B). Priess cells (DR4 / DRB4) or K562 cells expressing DR4 or DRB4 were used as antigen presenting cells (APC). Stimulation was performed using 2.5 pg / mL of each peptide and antigen specific cell proliferation was assessed after 3-4 days. The screening strategy for determining the specificity of Tr24 TCR is detailed in FIGS. 2A-2D. Using this approach, Tr24 TCR that recognizes a novel IGRP epitope (amino acids 24-44; SEQ ID NO:34) was successfully isolated.Tr24 TCR is specific for IGRP2544 and restricted to DRB4

[0203] A specific epitope among candidate peptides was identified using an antigen specific proliferation assay with the CD4+ primary T cells transduced with isletspecific TCR labeled with CTV, APC expressing the appropriate class II MHC alleles, and islet-specific peptide. T cells transduced with Tr24 TCR (Tr24 Teff) showed higher proliferation in the presence of APC and one of 11 peptide pools, pool 8, compared toDMSO or other 10 peptide pools, from the first screening (FIG. 3A). Tr24 Teff proliferated only in response to IGRP25_44 peptide and Preiss cells (DR4 / DRB4) or K562 cells expressing DRB4, APC expressing class II DRB4, not K562-DR4 cells (FIGS. 3B). FIGS. A-B further show that Tr24 TCR is specific for IGRP25 44peptide and restricted to DRB4. Proliferation of Tr24 Teff increased with higher concentration of IGRP25-44, only in the presence of allogeneic PBMC with DRB4, not DR4 or K562-DRB4 cells (FIG. 4A). Antigen-specific proliferation of Tr24 Teff in the presence of DRB4 PBMC or K562-DRB4 was completely inhibited by incubation with anti-DR antibody (FIG. 4B). Tr24 Teff did not respond to adjacent peptides containing overlapping sequence of IGRP25-44, including IGRP17-36, which was previously reported as DR4 epitope in T1D (FIG. 5). Tr24 TCR is specific for a novel epitope, IGRP25-44. presented by DRB4 (FIG. 5).Characterization of Tr24 TCR in comparison with T1D2 and TID5-1 TCRs

[0204] Tr24 TCR expression level, functional avidity, and T cell proliferation using cognate peptide in comparison to two other IGRP specific TCRs, T1D2 and T1D5- 1, are shown in FIG. 6). Tr24 expression was similar to T1D5-1 and lower than T1D2 (FIG. 6A). Functional avidity, measured by serial dilution of their cognate peptide in antigen- specific proliferation, was higher than T1D2 (low) but still lower than T1D5-1 (FIGS. 6B-C).Example 2: Generation of EngTregs expressing Tr24 TCR and Generation of Polyclonal Islet-specific Teff cells

[0205] FIG. 7 shows a schematic for the timeline and key steps to generate polyclonal islet- specific T effector cells and EngTregs and an in vitro suppression assay to measure suppressive activity of EngTregs. Engineered Treg cells (EngTregs) products were generated for use in human subjects for prevention and / or treatment of Type 1 Diabetes (T1D) by combination of lentiviral TCR delivery and HDR-based FOXP3 editing.CD4+CD25‘ and CD4 CD25+ cells were isolated from PBMCs of T1D donor and CD4+CD25- cells were split to generate EngTregs and polyclonal islet- specific T cells. For generation of polyclonal Islet-specific T cells, CD4+CD25- T cells were stimulated using irradiated autologous APC (CD4 CD25+) cells loaded with a pool of 10 different isletspecific peptides for 14 days. T cells were expanded with IL-2 from day 7 in 2 to 3-day intervals and harvested at day 14. Polyclonal Islet T cells were used as Teff for suppression assay and cultured for 4 days with no Treg, EngTregs or mock cells in the presence of mDC and a pool of 11 different islet-specific peptides including IGRP25-44. Before the co-culture,Teff and EngTregs or Mock cells were labeled with cell trace violet (CTV) and eFluor™ 670, respectively.Generation of EngTregs expressing Tr24 or T1D2 TCR using PBMCs with T1D

[0206] FIG. 8A shows a schematic for the generation of EngTregs with Tr24 or T1D2 TCR using PBMCs obtained from TID donor. CD4+CD25- T cells isolated from T1D PBMCs were activated with CD3 / CD28 expander beads and recombinant human IL- 2 on day 0. Transduction with LV vectors encoding T1D2 or Tr24 TCRs was performed by adding concentrated LV supernatant with protamine sulfate. Beads were removed after a 72-hour incubation, and cells were treated with IL-2 and rested for 16 to 24 hours. For FOXP3 editing, cells were transfected by electroporation with RNP complex combined with Cas9 and guide RNA and then transduced with AAV template (AAV 3080). IL-2, TNF-a, and IL-6 were added to the cells on day 4. Twenty to 24 hours after editing, cells were expanded in media with IL-2, TNF-a, IL-6, and Rapamycin until day 10. Islet-specific LNGFR+ EngTregs were enriched by LNGFR magnetic beads on day 10 and LNGFR- T cells were also collected from the LNGFR+ cell enrichment to be used as mock controls in suppression assays. Cells were aliquoted and frozen down for further experiments. Edited cells with T1D2 or Tr24 TCR on day 7 showed 88.6% or 70.1% of TCR expression and 26.3% or 26.7% of LNGFR+ FOXP3+, respectively (FIG. 8B).Suppressive activity of Tr24 EngTregs in comparison to Tr24 Mock or T1D2 EngTregs

[0207] Polyclonal islet Teff cells were cultured with Tr24 Mock, Tr24 EngTregs, or T1D2 EngTregs in the presence of CD3 / CD28 beads to measure suppressive activity of Tr24 EngTregs. Both Tr24 and T1D2 EngTregs, but not Mock cells showed robust suppression of bead-induced Teff proliferation and the suppression by Tr24 and T1D2 EngTregs was comparable (FIG. 9A-B).Polyclonal islet-Teff suppression by Tr24 EngTregs in comparison to T1D2 EngTregs

[0208] In the setting of antigen stimulation, polyclonal islet Teff proliferated in the presence of mDC and a mixture of 11 islet peptides that include IGRP25-44, a cognate peptide for Tr24. Proliferation of islet peptide-specific Teff was specifically suppressed by both Tr24 and T1D2 EngTregs, not by Tr24 Mock cells (FIG. 10A). Both Tr24 and T1D2 EngTregs exhibited strong suppression, but slightly lower suppression was observed by Tr24 EngTregs than T1D2 EngTregs (FIG. 10B).Direct suppression of Tr24 Teff by Tr24 EngTregs

[0209] Autologous Tr24 Teff were prepared by transducing primary human CD4+ T cells with LV expressing Tr24 TCR. IGRP25-44 specific proliferation of Tr24 Teff was inhibited by Tr24 EngTregs, but not by Tr24 Mock cells (FIGS. 11A-B). Tr24 EngTregs also suppressed production of TNF-a, IFN-y, or IL-2 in Tr24 Teff cells and Tr24 Mock cells did not mediate suppression (FIGS. 11C-D). The findings demonstrate a potent direct, antigen- specific, Teff suppression by EngTregs.Bystander suppression ofPPI76 Teff by Tr24 EngTregs

[0210] Further, a bystander suppression phenotype was observed. To determine whether Tr24 EngTregs exert bystander suppression, autologous PPI76 Teff that express PPI76 TCR and specific for PPI76-90 in the context of DR4. Tr24 EngTregs or Mock cells were co-cultured with PPI76 Teff in the presence of APC pulsed with either PPI76-90 or with a mixture of PPI76 -90 plus IGRP25 44. PPI76 Teff cell proliferation was only suppressed by Tr24 EngTregs when both PPI76-90 and IGRP25-44 peptides were present (FIGS. 12A-B). Tr24 Mock cells did not show bystander suppression of PPI76 Teff proliferation.

[0211] These findings show that dual-HDR edited Ag-specific EngTregs have suppressive function that enable cell-based therapy for T1D.

[0212] The EngTreg cells generated by the methods disclosed herein have distinct advantages. Specifically, the methods disclosed herein overcome the scaling limitations of alternative methods of preparing Treg cells e.g., sorting human cells to isolate Tregs) by starting with more abundant T cell sources (e.g., bulk CD4+ T cells), and specifically enriching for edited cells with an engineered receptor that provides IL-2 proliferative signaling in the presence of rapamycin or a rapalog. Moreover, in vivo engraftment of such engineered cells may be supported by administration of rapamycin or a rapalog. Such engineered cells also display Treg-associated markers, cytokine production phenotypes, and suppressive functions in vitro.

[0213] Certain nucleotide sequences and amino acid sequences useful with certain embodiments provided herein are listed in TABLE 1, TABLE 2, TABLE 3, and TABLE 4, respectively.TABLE 1TABLE 2TABLE 3TABLE 4More embodiments

[0214] Certain aspects disclosed in the APPENDIX are useful with certain methods and compositions disclosed herein. All the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0215] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit andscope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0216] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0217] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0218] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0219] 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.”

[0220] The phrase “and / or” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0221] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0222] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yetanother embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0223] 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.

[0224] 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, i.e. , to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A binding protein comprising: a T cell receptor (TCR) a-chain variable domain (Va); and a TCR b-chain variable domain (VP), wherein the binding protein is capable of binding to a FLNFMSNVGDPRNIFFIYFP (SEQ ID NO: 1):human leukocyte antigen (HLA) complex, and / or an Islet- specific Glucose-6-Phosphatase Catalytic subunit-related peptide (IGRP):HLA complex, wherein the IGRP peptide comprises or consists of 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO:1, and wherein the HLA comprises DRB4*0103.

2. The binding protein of claim 1, wherein the Va comprises a CDR3 amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:4, and / or the VP comprises a CDR3 amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:7.

3. A binding protein comprising: a T cell receptor (TCR) a-chain variable domain (Va) comprising a CDR3 amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:4; and a TCR P-chain variable domain (VP) comprising a CDR3 amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:7, wherein the binding protein is capable of binding to a FLNFMSNVGDPRNIFFIYFP (SEQ ID NO: 1):human leukocyte antigen (HLA) complex, and / or an Islet-specific Glucose-6-Phosphatase Catalytic subunit-related peptide (IGRP):HLA complex, wherein the IGRP peptide comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO:1, and wherein the HLA comprises DRB4*0103.

4. The binding protein of any one of claims 1-3, wherein the Va comprises a CDR3 amino acid sequence of SEQ ID NO:4, and the VP comprises a CDR3 amino acid sequence of SEQ ID NO:7.

5. The binding protein of any one of claims 1-3 further comprising:(i) a CDRla amino acid sequence according to SEQ ID NO:2;(ii) a CDR2a amino acid sequence according to SEQ ID NO: 3;(iii) a CDRi amino acid sequence according to SEQ ID NO:5; and / or(iv) a CDR2 amino acid sequence according to SEQ ID NO:6.

6. The binding protein of claim 5, comprising CDRla, CDR2a, CDR3a, CDRi , CDR2 , and CDR3 amino acid sequences as set forth in:(i) SEQ ID NOs:2, 3, and 4, respectively; or(ii) SEQ ID NOs:5, 6, and 7, respectively.

7. The binding protein of claim 1, wherein:(i) the Va comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO: 12; and / or(ii) the V comprises or consists of an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 13.

8. The binding protein of claim 7, wherein at least three or four of the complementary determining regions (CDRs) of the Va and / or the VP have no change in sequence, and wherein the CDRs that do have sequence changes have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof.

9. The binding protein of any one of claims 1-3, wherein:(i) the Va comprises an amino acid sequence that is at least 85% identical to an amino acid sequence according to TRAV35*02 and TRAJ6*01 ; and / or(ii) the VP comprises an amino acid sequence that is at least 85% identical to an amino acid sequence according to TRBV7-8*01, TRBJ1-3*O1, and TRBD1*O1 ; and / or(iii) the binding protein further comprises an amino acid sequence that is at least 85% identical to an amino acid sequence according to TRBJ1-3*O1.

10. The binding protein of any one of claims 1-3, further comprising a TCR P chain constant domain (C ), a TCR a chain constant domain (Ca), or both.

11. The binding protein of claim 10, comprising:(i) a TCRa chain that comprises or consists of an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO:8 and a TCRP chain that comprises or consists of an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO:9; or(ii) a TCRa chain that comprises or consists of an amino acid sequence of SEQ ID NO: 8 and a TCRP chain that comprises or consists of an amino acid sequence of SEQ ID NO:9.

12. The binding protein of claim 10, wherein:(i) the Ca has at least 85% identity to, comprises, or consists of the amino acid sequence of TRAC*01 or as set forth in SEQ ID NO: 10; and / or(ii) the CP has at least 85% identity to, comprises, or consists of the amino acid sequence of TRBC1 *01 or as set forth in SEQ ID NO: 11.

13. The binding protein of any one of claims 1-3, wherein the binding protein is capable of binding to a (SEQ ID NO:1):HLA complex, and / or to an Islet-specific Glucose-6-Phosphatase Catalytic subunit-related peptide (IGRP):HLA complex, wherein the IGRP peptide comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO:1, on a cell surface independent of or in the absence of CD4.

14. The binding protein of any one of claims 1-3, wherein the binding protein is a TCR, a chimeric antigen receptor, or an antigen-binding fragment of a TCR, wherein optionally, the TCR, the chimeric antigen receptor, or the antigen-binding fragment of a TCR is chimeric, humanized, or human, and / or the antigen-binding fragment of the TCR comprises a single chain TCR (scTCR).

15. A composition comprising the binding protein of claim 1 or claim 3 and a pharmaceutically acceptable carrier, diluent, or excipient.

16. A polynucleotide encoding the binding protein of any one of claims 1-3.

17. The polynucleotide of claim 16, wherein the polynucleotide is codon optimized.

18. The polynucleotide of claim 17, wherein the polynucleotide further comprises a polynucleotide encoding a self-cleaving peptide disposed between the a-chain encoding polynucleotide and the b-chain encoding polynucleotide.

19. An expression vector, comprising the polynucleotide of any one of claims 16-18 operably linked to an expression control sequence.

20. A recombinant host cell, comprising the polynucleotide of any one of claims 16-18, or the expression vector of claim 19, wherein the recombinant host cell is capable of expressing on its cell surface the encoded binding protein, wherein the polynucleotide is heterologous to the host cell.

21. The recombinant host cell of claim 20, wherein the recombinant host cell:(i) is a human immune cell;(ii) is an immune system cell, wherein the immune cell is a CD3+T cell, a CD4+T cell, a CD25+T cell, a FoxP3+T cell, or any combination thereof; or(iii) is an immune system cell, wherein the immune system cell is a T cell, and wherein the T cell is a T regulatory cell (Treg).

22. A method of treating a subject in need thereof, the method comprising: administering an effective amount of a composition comprising the recombinant host cell of claim 20 to the subject, wherein the subject has T1D.

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