Compositions containing HSP60-specific regulatory t cells and methods of treatment using the same

Abstract

Provided here are methods directed to the T cell receptor cloning of heat shock protein 60 (HSP60), the generation of HSP60-specific regulatory T cells (Tregs), and adoptive cell transfer of HSP60-specific Tregs for the treatment of autoimmune and other disorders. Methods of treatment also include administering a therapeutically effective amount of an inhibitor of expression or activity of nucleus accumbens-associated protein- 1 (NAC1) along with the HSP60-specific Tregs.

Description

COMPOSITIONS CONTAINING HSP60-SPECIFIC REGULATORY T CELLS AND METHODS OF TREATMENT USING THE SAMEInventors: Jianxun Jim Song and Paul de FigueiredoCross-reference to related applications

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 653,131, filed on May 29, 2024, which is incorporated by reference herein in its entirety.Government Support

[0002] This invention was made with United States government support under grant no. R01 AI121180 awarded by the National Institutes of Health. The Government has certain rights in the invention.Technical Field

[0003] The present disclosure generally relates to regulatory T cell (Treg)-based adoptive immunotherapy for autoimmune and other diseases. More specifically, the present disclosure relates to the generation of HSP60-specific Tregs and treatment of autoimmune and other disorders by administration of HSP60-specific Tregs.Background

[0004] T cell receptors (TCRs) are present on the surface of T cells and are critical components of antigen recognition. The TCRs specifically recognize peptides of the antigens presented to them by the antigen presenting cells (APCs). Antigen recognition by the TCRs is a key event, which further leads to the activation of downstream immunological signaling cascades like production of cytokines and recruitment of other immune cells. Therefore, TCRs have a crucial role in regulating the immune system. TCRs have been engineered to identify specific target antigens ex-vivo and then, these TCR-bearing T cells are adoptively transferred into patients to treat chronic inflammatory diseases, such as cancer, and autoimmune disorders through adoptive immunotherapy. Immunotherapy has led to remarkable developments in the treatment of human diseases, especially in adoptive immunotherapy of cancer. Despite enormous advances in the field of Treg-based therapy, the clinical efficacy and benefits remain less satisfactory due to a variety of factors that lessen anti-autoimmunity / inflammation, which include ex vivo Treg production, limited in vivo Treg expansion and persistence, and Treg trafficking to inflamed sites.Summary

[0005] Applicant has recognized the need to develop methods to generate highly reactive Tregs for Treg-based adoptive immunotherapy and have addressed these and shortcomings in the art. Embodiments include isolated engineered human regulatory T cells comprising a T cell receptor capable of specifically binding to heat shock protein 60. Embodiments also include methods for producing these isolated engineered regulatory T cells. One such method includes the steps of generating HSP60 T cell receptor (TCR) specific T cells to obtain HSP60 TCR cDNA, amplifying the HSP60 TCR cDNA to obtain a HSP60 specific TCR-a construct and a HSP60 specific TCR-0 construct, and cloning the TCR-a and TCR-0 constructs into an expression vector. The method further includes obtaining regulatory T cells from a human subject and activating them to obtain an isolated regulatory T cell, and introducing the expression vector into the isolated regulatory T cell to obtain the isolated engineered HSP60-specific Tregs. The isolated regulatory T cells can be derived by activation of natural regulatory T cells from the human subject in the presence of transforming growth factor-beta (TGF- 0) and interleukin 2 (IL-2). In certain embodiments, the expression vector is a retroviral vector. In certain embodiments, the retroviral vector contains a self-cleaving peptide sequence between the TCR-a region and TCR-0 region. In certain embodiments, the isolated regulatory T cell is a CD4 FoxP3+regulatory T cell.

[0006] Embodiments include methods for treating an autoimmune disorder in a subject in need thereof by administering to the subject the HSP60-specific Tregs. The autoimmune disorder can be Type 1 diabetes mellitus, Crohn's disease, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis. In certain embodiments, the autoimmune disorder is autoimmune diabetes.

[0007] Embodiments include methods for treating an autoimmune disorder in a subject in need thereof by administering to the subject the HSP60-specific Tregs along with a therapeutically effective amount of an inhibitor of expression or activity of nucleus accumbens-associated protein- 1 (NAC1). Inhibitors of NAC1 can include a chemical agent, such as a composition containing NIC3, or a biological agent that inhibit the function of NAC1 protein. The inhibitor of NAC1 can be a compound corresponding to Formula I (referred to as NIC3):

[0008] The inhibitor ofNACl can be a NAC1 -targeted siRNA. The NAC1 -targeted siRNA can be administered as a nanoliposome formulation. The inhibitor of NACl can be a CRISPR / Cas- based genome editing composition comprising one or more vectors encoding: (a) one or more guide RNAs (gRNAs) that are complementary to one or more target sequences in a NAC1 gene and (b) a nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the one or more gRNAs hybridize to the NAC1 gene and the CRISPR-associated endonuclease cleaves the NAC1 gene, and wherein the NAC1 gene is non-functional in the subject relative to a subject to whom the CRISPR / Cas-based genome editing composition is not administered. The inhibitor of NAC1 can be an isolated antibody or its binding fragment thereof that binds to a NAC1 protein. Contents of the PCT application No. PCT / US2022 / 080367 are incorporated herein by reference in their entirety.

[0009] Aspects and advantages of these exemplary embodiments and other embodiments, are discussed in detail herein. Moreover, it is to be understood that both the foregoing information and the following detailed description provide merely illustrative examples of various aspects and embodiments, and are intended to provide an overview or framework for understanding the nature and character of the claimed aspects and embodiments. Accordingly, these and other objects, along with advantages and features of the present disclosure, will become apparent through reference to the following description and the accompanying drawings. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and permutations.Brief Description of the Drawings

[0010] The accompanying drawings, which are included to provide a further understanding of the embodiments of the present disclosure, are incorporated in and constitute a part of this specification, illustrate embodiments of the present disclosure, and together with the detailed description, serve to explain principles of the embodiments discussed herein. No attempt is made to show structural details of this disclosure in more detail than may be necessary for a fundamental understanding of the embodiments discussed herein and the various ways in which they may be practiced.

[0011] FIG. 1A is an illustration of the protocol adopted to generate HSP60 TCR. HSP60 peptide was intraperitoneally injected into C57BL / 6 mice on Day 0 and Day 15 post-start of the protocol. The animals were the housed under specific pathogen free conditions with ad libitumaccess to sterile food and water. The mice were then sacrificed on Day 45 and the spleen and LNs were pooled under sterile conditions. The single-cells were then sorted onto each well of a 96-well plate using APC conjugated MHC-type 2 HSP60 tetramer. FIG. IB is an illustration of the nucleic acid containing the HSP60-TCR-a-P2A-TCR-p. The single cells isolated using APC conjugated MHC-type 2 HSP60 tetramer were then amplified using a two-step PCR method with primers designed to the TCR-constant and variable region. The primers were designed to amplify a previously unknown sequence. The sequence information retrieved from the paired HSP60 TCR- a and TCR-P regions were used to synthesize the full-length HSP60-TCR-a-P2A-TCR-P in the multiple cloning site of MSC-V IRES retroviral vector.

[0012] FIG. 2A demonstrates the identification and amplification of HSP60 TCR-a and TCR- p. APC-conjugated MHC II (I-Ab)-restricted HSP60 tetramer that was used to retrieve HSP60 specific TCR-a and TCR-p. Flowcytometric dot-plot assay shows the expression of HSP60 TCR- a and TCR-P regions on single-cells isolated from the pooled spleen and lymph-nodes of HSP60- peptide injected C57BL / 6 mice. FIG. 2B is a photograph of representative PCR amplicons (550 base-pairs) of HSP60 TCR-a and TCR-P regions conducted from single-cells sorted from MHC type 2 HSP60 tetramer. The PCR amplicons of TCR-a and TCR-P were sequenced and the inserts were synthesized on an MSV-IRES- retroviral vector as described previously. The retroviral vector was then transduced to CD4+FoxP3+Tregs, which were induced from nTregs by exogenous addition of TGF- p and IL-2. FIG. 2C presents the flowcytometric dot-plot analysis of LAb- restricted HSP60 tetramer positive cells which were transduced.

[0013] FIG. 3 presents a pairwise Sequence Alignment of TCR-a and TCR-P regions. The pairwise sequence alignment results of the amplicons of HSP60 TCR-a and TCR-P with the NCBI database indicates a 100% match with the constant regions of TCR-a and TCR-P regions.

[0014] FIG. 4 presents the pairwise sequence alignment of HSP60-TCR-a-P2A-TCR-p.

[0015] FIG. 5 is an illustration of a representative method of making highly reactive Tregs forTreg-based adoptive immunotherapy.Detailed Description

[0016] Regulatory T (Treg) cells suppress a variety of immune responses to self-antigens and are essential for maintaining peripheral tolerance, preventing autoimmunity, and limiting chronic inflammatory diseases. Treg cells, like all CD4+T cells, possess a somatically rearranged TCR. The TCRs of Treg cells herein have been engineered to identify specific target antigens ex-vivo.Provided here are methods of generating TCR modified Tregcells for treatment of chronic inflammatory diseases like cancer and autoimmunity through adoptive immunotherapy. These methods are also used for treatment of other debilitating diseases such as rheumatoid arthritis and diabetes. In the following description, numerous details are set forth in order to provide a thorough understanding of the various embodiments. In other instances, well-known processes, devices, and systems may not been described in particular detail in order not to unnecessarily obscure the various embodiments. Additionally, illustrations of the various embodiments may omit certain features or details in order to not obscure the various embodiments.

[0017] Stress induced heat shock proteins (HSPs) are known to be excellent target antigens for anti-cancer immunotherapy, and are rapidly emerging as promising targets for the treatment of autoimmune diseases. HSP60 is a self-antigen which is highly upregulated in a host of pathological autoimmune conditions, such as autoimmune diabetes, multiple sclerosis, and RA. HSP60 was selected by the inventors as a target antigen for modification of the TCRs of Treg cells to combat RA and other autoimmune diseases. Interestingly, immunologically presented HSP60 was used to enhance the differentiation of cord blood cells into CD4+CD25+FoxP3+Tregs, an immune cell type that suppresses inflammatory and autoimmune responses. Similarly, therapeutic administration of HSP60 has been shown to increase the presence of regulatory T (Treg) cells with a concomitant decrease in atherosclerotic plaques accompanied by an increase in the production of TGF-p.

[0018] Embodiments include isolated engineered human regulatory T cells comprising a T cell receptor capable of specifically binding to heat shock protein 60. Embodiments also include methods for producing these isolated engineered regulatory T cells. One such method includes the steps of generating HSP60 T cell receptor (TCR) specific T cells to obtain HSP60 TCR cDNA, amplifying the HSP60 TCR cDNA to obtain a HSP60 specific TCR-a construct and a HSP60 specific TCR-P construct, and cloning the TCR-a and TCR-P constructs into an expression vector. The method further includes obtaining regulatory T cells from a human subject and activating them to obtain an isolated regulatory T cell, and introducing the expression vector into the isolated regulatory T cell to obtain the isolated engineered HSP60-specific Tregs. The isolated regulatory T cells can be derived by activation of natural regulatory T cells from the human subject in the presence of transforming growth factor-beta (TGF- P) and interleukin 2 (IL-2). In certain embodiments, the expression vector is a retroviral vector. In certain embodiments, the retroviral vector contains a self-cleaving peptide sequence between the TCR-a region and TCR-P region. Incertain embodiments, the isolated regulatory T cell is a CD4+FoxP3+regulatory T cell.

[0019] Embodiments include methods for treating an autoimmune disorder in a subject in need thereof by administering to the subject the HSP60-specific Tregs. The autoimmune disorder can be Type 1 diabetes mellitus, Crohn's disease, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis. In certain embodiments, the autoimmune disorder is autoimmune diabetes.

[0020] Embodiments include methods for treating an autoimmune disorder in a subject in need thereof by administering to the subject the HSP60-specific Tregs along with a therapeutically effective amount of an inhibitor of expression or activity of nucleus accumbens-associated protein- 1 (NAC1). Inhibitors of NAC1 can include a chemical agent, such as a composition containing NIC3, or a biological agent that inhibit the function of NAC1 protein. The inhibitor of NAC1 can be a NACl-targeted siRNA. The NACl-targeted siRNA can be administered as a nanoliposome formulation. The inhibitor of NAC1 can be a CRISPR / Cas-based genome editing composition comprising one or more vectors encoding: (a) one or more guide RNAs (gRNAs) that are complementary to one or more target sequences in a NAC1 gene and (b) a nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPRj-associated endonuclease, whereby the one or more gRNAs hybridize to the NAC1 gene and the CRISPR- associated endonuclease cleaves the NAC1 gene, and wherein the NAC1 gene is non-functional in the subject relative to a subject to whom the CRISPR / Cas-based genome editing composition is not administered. The inhibitor of NAC1 can be an isolated antibody or its binding fragment thereof that binds to a NAC1 protein. Contents of the PCT application No. PCT / US2022 / 080367 are incorporated herein by reference in their entirety.

[0021] Methods herein include retroviral transduction of HSP60 TCR-a and TCR- TCRs into in-vitro activated (by anti CD3 and anti-CD28 antibodies) Tregcells. These TCR modified Tregcells are administered for adoptive immunotherapy of various autoimmune diseases. Methods disclosed here include steps to generate antigen-specific T cells for adoptive immunotherapy. These methods can be used both for generating Treg cells for research purposes and for clinical use in patients.

[0022] The description may use the phrases “in certain embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous. The term “about” or “approximately” is defined as being close to as understood byone of ordinary skill in the art. In one non-limiting embodiment, the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. The terms “removing,” “removed,” “reducing,” “reduced,” or any variation thereof, when used in the claims and / or the specification includes any measurable decrease of one or more components in a mixture to achieve a desired result. The use of the words “a” or “an” when used in conjunction with any of the terms “comprising,” “including,” “containing,” or “having,” in the claims or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The term “plurality” as used herein refers to two or more items or components. The terms “wt.%”, “vol.%”, or “mol.%” refers to a weight, volume, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, which includes the component. In a non-limiting example, 10 grams of component in 100 grams of the material is 10 wt.% of component.

[0023] A “subject” refers an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey) and a non-primate (such as a mouse). In some aspects of the invention, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, an infant, or a child. In other aspects, the subject is an adult subject.

[0024] As used herein, the terms “treating”, “treatment” and the like, shall include the management and care of a subject or patient for the purpose of combating a disease, condition, or disorder and includes the administration of a composition to prevent the onset of the symptoms or complications, alleviate the symptoms or complications, reduce at least one associated sign, symptom, or condition, or eliminate the disease, condition, or disorder. Treatment also refers to a prophylactic treatment, such as prevention of a disease (e.g., autoimmune disorders) or prevention of at least one sign, symptom, or condition associated with the disease (e.g., autoimmune disorders). Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0025] As used herein, a “vector” is any agent capable of delivering or maintaining nucleic acids to a host cell, and includes viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors), plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules.

[0026] Provided here are methods of introducing the HSP60 peptide for expression in Tregcells. These antigen-specific Treg cells may be used for the treatment of autoimmune diseases, such asType 1 diabetes mellitus (T1D), Crohn's disease, multiple sclerosis, rheumatoid arthritis, and ulcerative colitis, where the expression of the HSP60 protein may enhance desired Treg activities. Provided here are methods for treatment of an autoimmune disorder in a subject by administering to the subject HSP60-specific Tregcells. Provided here are methods for producing HSP60-specific Treg cells for the treatment of an autoimmune disorder. One such method can include generating HSP60 TCR specific T cells in a subject to obtain HSP60 TCR cDNA. The method also includes amplifying the HSP60 TCR cDNA to obtain a HSP60 specific TCR-a construct and a HSP60 specific TCR-P construct; cloning the TCR-a and TCR-P constructs into a retroviral vector; isolating regulatory T cells from the subject; and introducing the retroviral vector into the isolated regulatory T cells from the subject to obtain the HSP60-specific regulatory T cells. The autoimmune disorder may be rheumatoid arthritis, T1D, Crohn's disease, multiple sclerosis, and ulcerative colitis. In some embodiments, the retroviral vector contains a self-cleaving peptide sequence between the TCR-a region and TCR-P region. One such embodiment includes generating a construct containing an immunodominant HSP60 specific TCR- a and TCR-P separated by a P2A linker and cloning this construct into a vector for expression in Treg cells. The constructs may be integrated and packaged into non-replicating, defective viral genomes like adenovirus, adeno- associated virus, or herpes simplex virus or others, including retroviral vectors or lentiviral vectors, for infection or transduction into cells. The constructs may include viral sequences for transfection, if desired. In certain embodiments, the HSP60 specific TCR- a and TCR-P are delivered via a retroviral vector for expression in Treg cells

[0027] In certain embodiments, the methods integrate several steps such as isolation of Treg cells and preparation of these Tregcells for transduction. In an embodiment, the Platinum-E (Plat- E) packaging cell lines are utilized to stably produce a high-titer of retroviral particles. In certain embodiments, the methods involve an RT-PCR step to capture the full-spectrum diversity of TCRs followed by normal PCR steps for amplification and cloning of HSP60 specific TCR-a and TCR- P sequences.

[0028] A schematic diagram depicting the workflow for an embodiment is shown in FIG. 5. Briefly, intraperitoneal injection of HSP60 peptide was done on Day 0 and boosted again on Day 15. The HSP60 peptide injection primes the CD4+T cells to express HSP60 antigen specific TCR- a and TCR-p. These antigen specific CD4+T cells are then single cell sorted using MHC-type 2 HSP60 tetramer. The TCR-a and TCR-P regions are amplified using reverse transcriptase PCRwith a set of primers described herein. The amplicons are sequenced to identify the unique sequence of HSP60 specific TCR-a and TCR-P regions. The TCR-a and TCR-P sequences are then synthesized onto a retroviral vector with a P2A sequence between TCR-a and TCR-P regions. The P2A sequence causes ribosomal skipping during translation resulting in the effective separation of TCR-a and TCR-P proteins. Finally, the new retroviral plasmid is exogenously transduced on CD4 FoxP3+Tregs, which are sorted by using the same MHC-type 2 HSP60 tetramer.

[0029] In certain embodiments, antigen-specific Tregs from induced pluripotent stem cells are combined with a therapeutically effective amount of an inhibitor of expression or activity of nucleus accumbens-associated protein-1 (NAC1). NAC1 acts as a trigger of the immune response through destabilization of Tregs and suppression of tolerance induction. Therapeutic targeting of NAC1 provides a tolerogenic strategy for treatment of autoimmune disorders. Provided herein are methods of treating a disease by administering HSP60-specific Treg cells along with a therapeutically effective amount of an inhibitor of NAC1. Embodiments of an inhibitor of NAC1 include both chemical and biological agents that inhibit the function of NAC 1. Inhibitors of NAC 1 can include a chemical agent, such as a composition containing NIC3, or a biological agent that inhibit the function of NAC 1 protein, such as an isolated antibody or its binding fragment thereof that binds to NAC 1. Inhibitors of NAC 1 can include a biological agent that reduces the expression of NAC1 gene, such as a NAC 1 -targeted siRNA or a CRISPR / Cas-based genome editing composition targeting the NAC1 Gene. Contents of the PCT application No. PCT / US2022 / 080367 are incorporated herein by reference in their entirety.Examples

[0030] Various examples are described to illustrate selected aspects of the various embodiments for production of antigen-specific regulatory T cells for the treatment of an autoimmune disorder.

[0031] Generation of HSP60 TCR specific T cells. FIG. 1A is an illustration of the protocol adopted to generate HSP60 TCR. The C57BL / 6 mice are injected intraperitoneally ( . / ?.) with HSP60 peptide at the start of the protocol (Day 0). The i.p. injection of the HSP60 peptide was repeated on Day 15 and Day 30 post-initiation of the experiment. On Day 45, the mice were humanely euthanized with CO2 following the animal facility guidelines. The inguinal, branchial,mesenteric, cervical lymph-nodes and spleen were then pooled from the mice and homogenized using 70 pM cell strainer. The homogenized cell-suspension was then re-suspended in lx red blood cell (lx RBC) lysis buffer. The suspension was kept in room temperature for 5 minutes and then the lysis buffer was removed by centrifugation at 1200 rpm for 5 minutes. The lysate was then dissolved in Phosphate-buffered Saline (lx PBS). The cell-suspension was stained with anti-MHC HSP60 tetramer, and the cells were single- cell sorted into 96-well plates for recovering single- CD4+T cells using BD FACSAria high sensitivity Flowcytometer.

[0032] Amplification of HSP60 TCR cDNA. The HSP60 TCR cDNA was amplified by using multiplex one-step RT-PCR containing 80 different primers binding to the leader sequence of TCR initiation codon. In order to validate the primer-set, a second cycle of PCR was performed subsequent to the first PCR for amplification of HSP60 TCR-a and TCR-P at 550 base-pairs size. The single cell RT-PCR reaction mix and conditions were performed under conditions presented in Table 1.

[0033] Table 1: Single cell RT-PCR reaction mix and conditions

[0034] The second PCR was conducted with TCR-a and TCR-P specific primers by following the conditions as outlined here in Table 2.

[0035] Table 2: TCR- a and TCR- ft PCR reaction mix and conditions

[0036] The PCR product was sequenced to delineate the exact sequences of HSP60 TCR-a and TCR-P and the dominant sequence (-60%) was identified as the positive sequence for HSP60 TCR-a and TCR-0. The HSP60 TCR-a and TCR-P gene products were synthesized and cloned into the retroviral mammalian vector MSCV-IRES-GFP plasmid with the P2A self-cleaving peptide inserted between the HSP60 TCR-a and TCR-p. The P2A self- cleaving peptide functions by cleaving the fusion protein of HSP60 TCR-a and TCR- into distinct and functional TCR-a and TCR-P fragments. The plasmid map generated from the resultant construct is shown in FIG. IB

[0037] Transduction of HSP60 TCR-a and TCR- gene products into regulatory T cells. A MSCV-IRES-GFP- TCR-a-P2A-TCR-P plasmid construct was expanded using high efficiency NEB-Stable competent A. coli (cat no. C3040H) following the manufacturer’s protocol. On Day 1, the Platinum-E (PLAT-E) retroviral packaging cells were dislodged using trypsin-EDTA and neutralized using DMEM medium. The cells were centrifuged at 1,200g for 5 min and resuspended in DMEM media after removing the supernatant. The cell number was determined, and the cells were resuspended in DMEM media at a concentration of 1 x 106cells / ml. The cells were then plated on a 25mm cell-culture grade dish. The PLAT-E cells are to be transfected the next day, after reaching 80% confluency. The media was removed from the plate gently and the cells werewashed once with lx PBS. DMEM medium with antibiotics was supplemented into the plate and the transfection complex was prepared as shown in Table 3.

[0038] Table 3: Transduction of PLA T-E cells.

[0039] Mixture A and Mixture B are incubated at room temperature separately for 5 minutes, and then mixed and incubated for 20 min at room temperature. The mixture is then transferred to the PLAT-E cells and evenly distributed. The plate is incubated 37 °C / 5% CO2 in a cell incubator for 8 to 12 hours and then assessed for GFP expression under the microscope. The packaging cell line PLAT-E creates viral particles carrying HSP60-TCR-U-P2A-TCR-P which are ready to be transduced into Treg cells isolated from the spleen and lymph-nodes of C57BL / 6 mice.

[0040] Preparation of Tregs for transduction. The C57BL / 6 mouse spleen and lymph-nodes (LNs) are isolated and pooled together. The spleen and LNs are then placed on a 70pM cell strained and mashed to obtain single cell suspension. The cells are rinsed off the cell strainer by using RPMI medium and the single cell suspension is resuspended in RPMI medium. The single cell suspension is then centrifuged at 1200 x g for 5 min. The supernatant is discarded, and the cells are resuspended in lx RBC lysis buffer for 5 mins at room temperature. The cells are centrifuged again at 1200 x g for 5 min and the lysis buffer (supernatant) is removed. The cells are then resuspended in Mojo-Sort™ buffer (BioLegend; cat no 480137) and the Treg cells are isolated by negative selection using the BioLegend Treg cell isolation kit. The cells are then plated on anti-CD3 Ab coated 48 well plate and anti-CD28 Ab along with IL-2, which is exogenously supplemented into the RPMI medium.

[0041] Transduction of activated Tregs. The supernatant collected from PLAT-E cells after transfection contains packaged viral particles carrying the HSP60-TCR-a-P2A-TCR-P construct. The supernatant is added to activated Treg on Day 2 post plating of these cells. The viral particles are added along with 10 pg / mL of polybrene to facilitate transduction. The plate is centrifuged at 2,000 rpm for 2h to allow the viruses to infect the Tregs.

[0042] The single cell sorting report identifying the HSP60 activated CD4+T cells is shown in FIG. 2A. The cells were sorted by using APC tagged anti-MHC HSP60 tetramer. FIG. 2A demonstrates the identification and amplification of HSP60 TCR-a and TCR-0. APC -conjugated MHC II (I-Ab)-restricted HSP60 tetramer was used to retrieve HSP60 specific TCR-a and TCR- p. Flowcytometric dot-plot assay shows the expression of HSP60 TCR-a and TCR-P regions on single-cells isolated from the pooled spleen and lymph-nodes of HSP60-peptide injected C57BL / 6 mice. The efficacy of the two-step nested PCR reaction as indicated in step 1.1.1 to 1.1.6 was checked by running 10 pL of the PCR mix in 1% agarose gel (FIG. 2B). FIG. 2B shows representative PCR amplicons (550 base-pairs) of HSP60 TCR-a and TCR-P regions conducted from single-cells sorted from MHC type 2 HSP60 tetramer.

[0043] FIG. 2C shows the PCR amplicons of TCR-a and TCR-P were sequenced and the inserts were synthesized on an MSV-IRES- retroviral vector as described previously. The retroviral vector was then transduced to CD4+FoxP3+Tregs, which were induced from nTregs by exogenous addition of TGF- P and IL-2. The figure depicts the flowcytometric dot-plot analysis of I- Ab-restricted HSP60 tetramer positive cells which were transduced. FIG. 3 depicts a pairwise Sequence Alignment of TCR-a and TCR-P regions. The pairwise sequence alignment results of the amplicons of HSP60 TCR-a and TCR-P with the NCBI database indicates a 100% match with the constant regions of TCR-a and TCR-P regions. All the PCR products were sequenced to obtain the TCR sequence and repertoire information. The pairwise sequence alignment of HSP60-TCR- a-P2A-TCR-P is shown in FIG. 4. The sequences of the primers used in this study are shown in the Tables below.

[0044] Table 4 - Leader-primers for mouse TCR-a

[0045] Table 5 - Leader-primers for mouse TCR-0

[0046] Table 6: Primers for mouse TCR constant and adapter

[0047] The isolated engineered human regulatory T cells described herein contain the expression components for a T cell receptor capable of specifically binding to heat shock protein 60. A method for producing these isolated engineered HSP60-specific Tregs includes the steps of generating HSP60 T cell receptor (TCR) specific T cells to obtain HSP60 TCR cDNA, amplifyingthe HSP60 TCR cDNA to obtain a HSP60 specific TCR-a construct and a HSP60 specific TCR-P construct, and cloning the TCR-a and TCR-P constructs into an expression vector. The method further includes obtaining regulatory T cells from a human subject and activating them to obtain an isolated regulatory T cell, and introducing the expression vector into the isolated regulatory T cell to obtain the isolated engineered HSP60-specific Tregs. The isolated regulatory T cells can be derived by activation of natural regulatory T cells from the human subject in the presence of TGF- P and IL-2. In certain embodiments, the expression vector is a retroviral vector and can include a sequence coding for a self-cleaving peptide between the TCR-a region and TCR-P region. In certain embodiments, the isolated regulatory T cell is a CD4+FoxP3+regulatory T cell.

[0048] Embodiments include methods for treating an autoimmune disorder in a subject in need thereof by administering to the subject the HSP60-specific Tregs. The autoimmune disorder can be Type 1 diabetes mellitus, Crohn's disease, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis. In certain embodiments, the autoimmune disorder is autoimmune diabetes. Embodiments include methods for treating an autoimmune disorder in a subject in need thereof by administering to the subject the HSP60-specific Tregs along with a therapeutically effective amount of an inhibitor of expression or activity of nucleus accumbens-associated protein- 1 (NAC1). Inhibitors ofNACl can include a chemical agent, such as a composition containing NIC3, or a biological agent that inhibit the function of NACl protein. The NAC1 inhibitor may be administered concurrently or subsequently to the administration of the HSP60-specific Tregs. The inhibitor of NACl can be a NAC1 -targeted siRNA. The NAC1 -targeted siRNA can be administered as a nanoliposome formulation. The inhibitor ofNACl can be a CRISPR / Cas-based genome editing composition that renders the NAC1 gene non-functional in the subject relative to a subject to whom the CRISPR / Cas-based genome editing composition is not administered. The inhibitor ofNACl can be an isolated antibody or its binding fragment thereof that binds to a NAC1 protein.

[0049] Other objects, features and advantages of the disclosure will become apparent from the foregoing figures, detailed description, and examples. It should be understood, however, that the figures, detailed description, and examples, while indicating specific embodiments of the disclosure, are given by way of illustration only and are not meant to be limiting. Additionally, it is contemplated that changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the detailed description. In further embodiments, features from specific embodiments may be combined with features from other embodiments. Forexample, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be added to the specific embodiments described herein.

Claims

ClaimsWhat is claimed is:

1. An isolated engineered human regulatory T cell comprising: a T cell receptor capable of specifically binding to heat shock protein 60.

2. A method for producing the isolated engineered regulatory T cell of Claim 1, the method comprising: generating HSP60 T cell receptor (TCR) specific T cells to obtain HSP60 TCR cDNA; amplifying the HSP60 TCR cDNA to obtain a HSP60 specific TCR-a construct and a HSP60 specific TCR-P construct; cloning the TCR-a and TCR-P constructs into an expression vector; obtaining regulatory T cells from a human subject and activating them to obtain an isolated regulatory T cell; and introducing the expression vector into the isolated regulatory T cells from the subject to obtain the isolated engineered regulatory T cell of Claim 1.

3. The method of Claim 2, wherein the expression vector is a retroviral vector.

4. The method of Claim 3, wherein the retroviral vector contains a self-cleaving peptide sequence between the TCR-a region and TCR-P region.

5. The method of Claim 2, wherein the isolated regulatory T cell is a CD4+FoxP3+regulatory T cell.

6. The method of Claim 5, wherein the isolated regulatory T cell is derived by activation of natural regulatory T cells from a human subject in the presence of transforming growth factor-beta (TGF- P) and interleukin 2 (IL-2).

7. A method for treating an autoimmune disorder in a subject in need thereof, the method comprising: administering to the subject the isolated engineered human regulatory T cell of Claim 1.

8. The method of Claim 7, wherein the autoimmune disorder is Type 1 diabetes mellitus, Crohn's disease, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis.

9. The method of Claim 7, wherein the autoimmune disorder is autoimmune diabetes.

10. The method of Claim 7, further comprising:administering a therapeutically effective amount of an inhibitor of expression or activity of nucleus accumbens-associated protein-1 (NAC1).

11. The method of Claim 10, wherein the inhibitor of NAC1 is a compound corresponding to Formula I:(Formula I)12. The method of Claim 10, wherein the inhibitor of NAC1 is a NACl-targeted siRNA.

13. The method of Claim 12, wherein the NACl-targeted siRNA is administered as a nanoliposome.

14. The method of Claim 10, wherein the inhibitor of NAC1 is a CRISPR / Cas-based genome editing composition comprising one or more vectors encoding: (a) one or more guide RNAs (gRNAs) that are complementary to one or more target sequences in a NAC 1 gene and (b) a nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the one or more gRNAs hybridize to the NAC1 gene and the CRISPR-associated endonuclease cleaves the NAC1 gene, and wherein the NAC1 gene is non-functional in the subject relative to a subject to whom the CRISPR / Cas-based genome editing composition is not administered.

15. The method of Claim 10, wherein the inhibitor of NAC1 is an isolated antibody or its binding fragment thereof that binds to a NAC1 protein.

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