Treatment

Abstract

The present invention relates to methods and compositions for treating autoimmune or inflammatory diseases characterized by an aberrant or inappropriate immune response to one or more of Ro60 protein, MPO protein and Smith protein.

Description

[Technical field]

[0001] The present invention relates to compositions and methods for the treatment of autoimmune or inflammatory diseases, particularly autoimmune diseases characterized by an inappropriate or aberrant immune response to one or more of the Smith, Ro60 and MPO proteins.

[0002] This application claims priority to Australian Provisional Patent Application No. 2021903030, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Autoimmune diseases result from an abnormal immune response against healthy cells, tissues and organs. Over 80 autoimmune diseases have been recognized in humans, collectively affecting over 24 million individuals in the United States alone.

[0004] In some cases, an individual may have more than one autoimmune disease, and autoantibodies to a single "autoantigen" may be associated with more than one condition. Alternatively, an individual may present with multiple autoimmune diseases, where there is no apparent common autoantigen or cause of autoimmunity.

[0005] Despite significant research into autoimmune diseases, effective targeted therapies are lacking. Current treatments, such as corticosteroids, methotrexate, hydroxychloroquine, other immunosuppressants (e.g., cyclosporine, leflunamide, azathioprine, to name a few), and nonsteroidal anti-inflammatory drugs, nonspecifically inhibit immune system activation rather than precisely inhibiting the specific autoimmunity associated with the disorder.

[0006] While many patients do not respond or only partially respond to the standard medical treatments listed above, the long-term use of high-dose corticosteroids and cytotoxic therapies can result in serious side effects such as bone marrow suppression, increased opportunistic infections, irreversible ovarian failure, baldness, and increased risk of malignancies. Infectious complications occurring concomitantly with active autoimmune disease and its treatment with immunosuppressive medications are one of the most common causes of death in patients with autoimmune disease. Summary of the Invention [Problem to be solved by the invention]

[0007] There is a need for new or improved treatments for autoimmune diseases, particularly treatments that target disease-associated autoantigens.

[0008] The reference to any prior art in this specification is not an admission or suggestion that this prior art forms part of the common general knowledge in any legal jurisdiction, or that this prior art would be understood or considered relevant by those skilled in the art and / or could reasonably be expected to be combined with other pieces of prior art. [Means for solving the problem]

[0009] Summary of the Invention The present invention is based on the inventors' surprising discovery of promiscuous self-epitope-specific T cell receptors (TCRs) that are capable of binding multiple self-epitopes and are useful for treating a variety of autoimmune diseases.

[0010] Thus, in a first aspect, the present invention provides a method of treating an autoimmune or inflammatory disease comprising the steps of: administering to a subject in need of treatment for an autoimmune or inflammatory disease a population of regulatory T (Treg) cells that express the binding protein on their cell surface, thereby treating the autoimmune or inflammatory disease; the binding protein comprises a T cell receptor (TCR) alpha chain variable (Valpha) domain and a TCR beta chain variable (Vbeta) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; the Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:16, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:16; The autoimmune disease or inflammatory disease is characterized by an abnormal or inappropriate immune response to Ro60 protein; myeloperoxidase (MPO) protein; Ro60 protein and MPO protein; Smith protein and Ro60 protein; Smith protein and MPO protein; or Smith protein, Ro60 protein and MPO protein, A method is provided.

[0011] In one embodiment of the first aspect of the present invention, there is also provided the use of a population of Treg cells as described above in the manufacture of a medicament for treating an autoimmune disease or an inflammatory disease, preferably wherein the autoimmune disease or inflammatory disease is characterised by an abnormal or inappropriate immune response to Ro60 protein; MPO protein; Ro60 protein and MPO protein; Smith protein and Ro60 protein; Smith protein and MPO protein; or Smith protein, Ro60 protein and MPO protein.

[0012] In a further embodiment of the first aspect of the invention there is provided a population of Treg cells as described above for use in the treatment of an autoimmune or inflammatory disease, preferably wherein the autoimmune or inflammatory disease is characterised by an abnormal or inappropriate immune response to Ro60 protein; MPO protein; Ro60 protein and MPO protein; Smith protein and Ro60 protein; Smith protein and MPO protein; or Smith protein, Ro60 protein and MPO protein.

[0013] In any embodiment, the autoimmune or inflammatory disease may be characterized by an aberrant or inappropriate immune response to a Smith protein.

[0014] In any embodiment, the autoimmune or inflammatory disease may be characterised by an aberrant or inappropriate immune response to a Ro60 protein.

[0015] In any embodiment, the autoimmune or inflammatory disease may be characterized by an aberrant or inappropriate immune response against the Smith protein and against the Ro60 protein.

[0016] In any embodiment, the autoimmune or inflammatory disease may be characterized by an aberrant or inappropriate immune response against the MPO protein.

[0017] In any embodiment, the autoimmune or inflammatory disease may be characterized by an aberrant or inappropriate immune response against the Ro60 protein and against the MPO protein.

[0018] In any embodiment, the autoimmune or inflammatory disease may be characterized by an aberrant or inappropriate immune response against Smith protein, against Ro60 protein and against MPO protein.

[0019] In any embodiment, the abnormal or inappropriate immune response against Smith protein, against Ro60 protein and / or against MPO protein comprises the formation of autoantibodies against Smith protein, against Ro60 protein and / or against MPO protein.

[0020] Herein, autoantibodies against the MPO protein are also referred to as antineutrophil cytoplasmic antibodies (ANCA), in particular anti-MPO ANCA.

[0021] In any embodiment, the autoimmune disease or inflammatory disease is one or more autoimmune diseases or inflammatory diseases selected from the group consisting of systemic lupus erythematosus (SLE), lupus nephritis, Sjogren's syndrome, systemic sclerosis, inflammatory myositis, inflammatory rheumatism, autoimmune vasculitis and microscopic polyangiitis.Preferably, the autoimmune disease or inflammatory disease can be SLE or lupus nephritis.Alternatively, the autoimmune disease or inflammatory disease can be Sjogren's syndrome, preferably primary Sjogren's syndrome.Furthermore, the autoimmune disease or inflammatory disease can be microscopic polyangiitis (MPA).

[0022] In any embodiment, the subject in need of treatment may need treatment for SLE and Sjogren's syndrome, or SLE and MPA, or Sjogren's syndrome and MPA, or all three of SLE, Sjogren's syndrome and MPA.

[0023] In a second aspect, there is provided a method of treating systemic lupus erythematosus (SLE) or lupus nephritis in a subject, comprising: administering to a subject in need of treatment for SLE or lupus nephritis a population of Treg cells expressing the binding protein on their cell surface, thereby treating SLE or lupus nephritis; the binding protein comprises a T cell receptor (TCR) alpha chain variable (Valpha) domain and a TCR beta chain variable (Vbeta) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; the Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:16, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:16; The above SLE or lupus nephritis is Ro60 protein; or characterized by an abnormal or inappropriate immune response to Smith protein and Ro60 protein, A method is provided. Preferably, the method also includes treating Sjogren's syndrome and / or MPA in the subject.

[0024] In one embodiment of the second aspect of the present invention, there is also provided the use of the population of Treg cells as described above in the manufacture of a medicament for treating SLE or lupus nephritis in a subject, preferably wherein the SLE or lupus nephritis is characterized by an abnormal or inappropriate immune response against Ro60 protein; or Smith protein and Ro60 protein. Preferably, the medicament is also a medicament for treating Sjogren's syndrome and / or MPA in a subject.

[0025] In a further embodiment of the second aspect of the invention there is provided a population of Treg cells as described above for use in the treatment of SLE or lupus nephritis, preferably wherein the SLE or lupus nephritis is characterised by an abnormal or inappropriate immune response to Ro60 protein; or Smith protein and Ro60 protein. Preferably the use also comprises treating Sjögren's syndrome and / or MPA in a subject.

[0026] In a further third aspect, there is provided a method of treating Sjogren's syndrome in a subject, comprising: administering to a subject in need of treatment for Sjögren's syndrome a population of Treg cells that express the binding protein on their cell surface, thereby treating Sjögren's syndrome in the subject; the binding protein comprises a T cell receptor (TCR) alpha chain variable (Valpha) domain and a TCR beta chain variable (Vbeta) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; The Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:16, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:16. Methods are provided. Preferably, the methods may also include treating SLE, lupus nephritis and / or autoimmune vasculitis (preferably MPA) in a subject.

[0027] In one embodiment of the third aspect of the present invention, there is also provided the use of the population of Treg cells described above in the manufacture of a medicament for treating Sjögren's syndrome in a subject. Preferably, the medicament is also a medicament for treating SLE, lupus nephritis and / or MPA in a subject.

[0028] In a further embodiment of the third aspect of the invention there is provided a population of Treg cells as described above for use in treating Sjögren's syndrome in a subject. Preferably the use also comprises treating SLE, lupus nephritis and / or MPA in the subject.

[0029] In a fourth embodiment, there is provided a method of treating autoimmune vasculitis in a subject, comprising: administering to a subject in need of treatment for autoimmune vasculitis a population of Treg cells that express the binding protein on their cell surface to treat autoimmune vasculitis in the subject, the binding protein comprises a T cell receptor (TCR) alpha chain variable (Valpha) domain and a TCR beta chain variable (Vbeta) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; The Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:16, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:16. Methods are provided. Preferably, the vasculitis is microscopic polyangiitis (MPA); granulomatosis with polyangiitis (GPA) or eosinophilic granulomatosis with polyangiitis (EGPA), most preferably MPA. The methods may also include treating SLE, lupus nephritis and / or Sjogren's syndrome in the subject.

[0030] In one embodiment of the fourth aspect of the present invention, there is also provided the use of the population of Treg cells described above in the manufacture of a medicament for treating microscopic polyangiitis (MPA) in a subject.Preferably, the medicament is also a medicament for treating SLE, lupus nephritis and / or Sjogren's syndrome in a subject.

[0031] In a further embodiment of the fourth aspect of the invention there is provided a population of Treg cells as described above for use in the treatment of MPA in a subject. Preferably the use also includes treating SLE, lupus nephritis and / or Sjogren's syndrome in the subject.

[0032] In any embodiment of the invention, Sjogren's syndrome or MPA is characterized by an abnormal immune response in a subject against one or more of Smith protein, Ro60 protein and MPO protein.

[0033] In any of the aspects of the invention, the abnormal immune response comprises the formation in the subject of autoantibodies against one or more of the Smith protein, Ro60 protein and MPO protein.

[0034] In any method, use or composition for use of the invention the binding protein preferably comprises: a Vα domain comprising a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 13 or a sequence having no more than one, no more than two, no more than three, no more than four, no more than five amino acid substitutions compared to the sequence of SEQ ID NO: 13; and It comprises a Vβ domain including a CDR3 having an amino acid sequence as set forth in SEQ ID NO:16 or a sequence having not more than one, not more than two, not more than three, not more than four, not more than five amino acid substitutions compared to the sequence of SEQ ID NO:16.

[0035] In any method, use or composition for use of the invention the binding protein preferably comprises: a Vα domain comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO:11, or a sequence which is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:11; and a Vα domain comprising a CDR2 having an amino acid sequence set forth in SEQ ID NO:12, or a sequence which is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:12; and a Vβ domain comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO:14, or a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:14; and The present invention comprises a Vβ domain comprising a CDR2 having an amino acid sequence as set forth in SEQ ID NO:15, or a sequence which is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence as set forth in SEQ ID NO:15.

[0036] In any method, use or composition for use of the invention the binding protein preferably comprises: a Vα domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:11 or a sequence which has no more than one, no more than two, or no more than three amino acid substitutions compared to the sequence of SEQ ID NO:11; a Vα domain comprising a CDR2 having the amino acid sequence set forth in SEQ ID NO: 12 or a sequence which has no more than one, no more than two, or no more than three amino acid substitutions compared to the sequence of SEQ ID NO: 12; a Vβ domain comprising a CDR1 having the amino acid sequence shown in SEQ ID NO: 14 or a sequence which has not more than one, not more than two or not more than three amino acid substitutions compared to the sequence of SEQ ID NO: 14; and / or It comprises a Vβ domain including a CDR2 having the amino acid sequence shown in SEQ ID NO:15 or a sequence having not more than one, not more than two, or not more than three amino acid substitutions compared to the sequence of SEQ ID NO:15.

[0037] In any method, use or composition for use of the invention, the binding protein may comprise a Vα domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set out in SEQ ID NOs: 11, 12 and 13, and a Vβ domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set out in SEQ ID NOs: 14, 15 and 16.

[0038] In any of the methods, uses or compositions for use of the invention described above, the CDRs are determined according to the IMGT method.

[0039] In any method, use or composition for use of the invention, the binding protein may comprise a TCR alpha chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:23, with an insertion, deletion, substitution, addition or combination thereof of at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, and not more than about 10 amino acids, outside the CDR sequences shown.

[0040] In any method, use or composition for use of the invention, the binding protein may comprise a TCR beta chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24, with an insertion, deletion, substitution, addition or combination thereof of at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, and not more than about 10 amino acids, outside the CDR sequences shown.

[0041] In any method, use or composition for use of the invention, the binding protein may comprise a TCR alpha chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:23 and a TCR beta chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24.

[0042] In any method, use, or composition for use of the invention, the Smith protein can be SmB / B', SmD, or any other Smith protein described herein. Thus, in any aspect, the binding protein is capable of binding to SmB / B' and / or SmD. In one embodiment, the binding protein is capable of binding to a fragment of the Smith protein comprising or consisting of the amino acid sequence of residues 78-92 of the SmD1 protein or residues 7-21 of the SmB / B' protein (set forth in SEQ ID NOs: 1 and 3, respectively), or an amino acid sequence equivalent thereto.

[0043] In any method, use or composition for use of the invention, the binding protein is capable of binding to a complex of a fragment of Smith protein, Ro60 protein or MPO protein with an HLA-DR3 molecule and / or an HLA-DR4.5 molecule. Preferably, the binding protein is capable of binding to a fragment of Smith protein comprising or consisting of the amino acid sequence of residues 78-92 of SmD1 protein or residues 7-21 of SmB / B' protein (set forth in SEQ ID NOs: 1 and 3, respectively), or an amino acid sequence equivalent to the above amino acids.

[0044] In any method, use or composition for use of the invention, the binding protein is capable of binding to a complex of a fragment of Ro60 protein and an HLA-DR3 molecule, the fragment of Ro60 protein comprising or consisting of the amino acid sequence of residues 225 to 239 or residues 369 to 383 of Ro60 as set forth in SEQ ID NOs: 5 and 6, respectively, or an amino acid sequence which is equivalent to the above amino acid sequences.

[0045] In any method, use or composition for use of the invention, the binding protein is capable of binding to a complex of a fragment of MPO protein and an HLA-DR3 molecule and / or an HLA-DR4.5 molecule, the fragment of MPO protein comprising or consisting of the amino acid sequence of residues 453 to 467 or residues 724 to 738 of MPO as set forth in SEQ ID NOs: 8 and 9, respectively, or an amino acid sequence which is equivalent to said amino acid sequence.

[0046] In any method, use or composition for use of the invention, the TCR alpha and beta chains of the binding protein are modified to include cysteine ​​residues that allow for the formation of additional interchain disulfide bonds. Preferably, the residue at Thr48 or an equivalent on the TCR alpha chain and the residue at Ser57 or an equivalent on the TCR beta chain are replaced by cysteine ​​to facilitate the creation of additional disulfide bonds between the constant regions of the TCRs.

[0047] In any method, use or composition for use of the invention, the population of Treg cells may be derived from a subject in need of treatment or from a histocompatible donor. Alternatively, the population of Treg cells may be derived from stem cells, optionally in which case said stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells. In any embodiment, the population of Treg cells may be derived from a mixed population of T cells into which a nucleic acid encoding a binding protein as defined herein has been introduced.

[0048] In any method, use or composition for use of the invention, the population of Tregs comprises: providing a population of T cells exhibiting at least one characteristic of conventional T cells, optionally wherein the population of T cells is a mixed population of T cells; introducing a nucleic acid or vector of the invention into a population of T cells, the nucleic acid or vector encoding a binding protein as described herein; Providing conditions that allow expression of the binding protein on the surface of T cells; Providing conditions that allow the conversion of a population of T cells into regulatory T cells; These methods allow the ex vivo preparation of a population of T cells that exhibit at least one characteristic of regulatory T cells, and that are specific to one or more of the proteins Sm / B', ​​SmD, Ro60, or MPO. It can be obtained by

[0049] In another aspect, the present invention provides a method for preparing a population of regulatory T cells for use in treating an autoimmune disease as described herein, comprising the steps of: Providing a population of regulatory T cells; introducing a nucleic acid or vector encoding a TCR as described herein into a population of regulatory T cells; Providing conditions that allow expression of the TCR on the surface of regulatory T cells Including, This allows for the preparation of populations of regulatory T cells for use in the treatment of autoimmune diseases as described herein. A method is provided.

[0050] Unless the context requires otherwise, as used herein, the term "comprising" and variations of this term, such as "comprising," "including," and "included," are not intended to exclude additional additives, components, integers or steps.

[0051] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0052] [Figure 1]Predominant reactivity with promiscuous self-TCR. T cells from healthy humans were co-cultured with monocyte-derived dendritic cells (DCs) pulsed with six different self-peptides. Each graph shows the number of reactive clones per peptide. Note that only the promiscuous self-TCR sequence was found to be clonally expanded across all peptide stimulations. With regard to the other clonally expanded TCR sequences, none of the TCR sequences cross-react with other peptides, e.g., reacting with SmD1:78-92, other TCR sequences do not react with SmB / B':7-21, Ro60:225-239, Ro60:369-383, MPO:453-467 or MPO:724-738. Black bars = promiscuous self-TCR sequences. White bars = other TCR sequences. [Diagram 2] Experimental protocol supporting the efficacy of Ro60-Tregs in halting disease progression in a new humanized model of Sjogren's syndrome. [Diagram 3] Upregulated Treg genes associated with antigen-specific TCR activation. Upregulated genes associated with TCR activation were FOXP3, CTLA4, IL1R2, CCR10, TIGIT, IKZF2, ITGAM, LGALS1, LGALS3, PI16, IL2RA. Downregulated genes associated with Treg activation were IL13, IFNG, IL7R. These data support that each epitope can induce promiscuous self-TCR engagement and Treg activation. Black dots: upregulated genes; grey dots / shaded dots: downregulated genes. [Figure 4] Expression of promiscuous self-TCR on human Tregs. The transduction protocol allows successful expression of promiscuous self-TCR on the surface of human Tregs. A = mock-transduced human Tregs; B = promiscuous self-TCR-transduced Tregs. Staining for GFP expression and Vβ14TCR expression. [Diagram 5]Promiscuous self-TCR Tregs bind peptide-HLA Dextramers. Promiscuous self-TCR transduced Tregs exhibit binding to SmD1:78-21 / HLA-DRB1*03:01 Dextramer, Ro60:369-383 / HLA-DRB1*03:01 Dextramer and MPO:453-567 / HLA-DRB1*04:05 Dextramer. Filled squares = promiscuous self-TCR transduced Tregs. Open squares = mock transduced Tregs. [Figure 6] Promiscuous self-TCR Tregs are activated following peptide stimulation. Stimulation with SmD1:78-92, Ro60:369-383 and MPO:453-467 induced activation of promiscuous self-TCR transduced Tregs compared to no peptide stimulation. Filled squares = promiscuous self-TCR transduced Tregs. Open squares = mock transduced Tregs. [Figure 7] Promiscuous self-TCR Tregs suppress peptide-specific Tconv cell proliferation. Promiscuous self-TCR-transduced Tregs demonstrated stronger suppression of Tconv cell proliferation across all three self-epitopes. Closed squares = promiscuous self-TCR-transduced Tregs. Open squares = mock-transduced Tregs. [Figure 8] Promiscuous self-TCR engages Sm peptides. Pulsing B-lymphoblastoid cell lines (B-LCL) with Sm epitopes increased the mean pixel intensity of CD3 and phalloidin at the immune synapse. These data indicate that promiscuous self-TCR can engage any HLA-DRB1*03:01-restricted Sm epitope. Arrows indicate immune synapse; BF=bright field; MPI=mean pixel intensity.

[0053] Sequence information

[0054] [Table 1] TIFF2024534506000002.tif235167TIFF2024534506000003.tif232170TIFF2024534506000004.tif78167 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] It is understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0056] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which:

[0057] Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0058] The present inventors have identified a promiscuous self-epitope T cell receptor (self-TCR) capable of binding to multiple self antigens associated with autoimmune diseases, in particular Smith protein, Ro60 protein and myeloperoxidase (MPO) protein.

[0059] Autoantibodies against Smith, Ro60 and MPO proteins are associated with a variety of autoimmune diseases, including systemic lupus erythematosus (SLE), Sjogren's syndrome and autoimmune vasculitis, particularly microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome). Thus, the present invention relates to a single TCR for use in the treatment of a number of autoimmune diseases.

[0060] The particular advantage of the present invention is that it provides a single product (e.g., a population of Treg cells expressing promiscuous TCR) for use in treating a wide range of populations suffering from autoimmune disease, but specific to the specific autoantigen associated with the disease.This is in contrast to existing treatments applied across patient groups, which are non-specific and therefore likely to be less effective, potentially resulting in increased side effects.The approach of the present invention allows for specific targeting of the origin of autoimmune disease within more than one disease population.

[0061] General Throughout this specification, unless specifically stated otherwise or unless the context requires otherwise, reference to a single step, composition, group of steps or group of compositions shall be understood to include one and the plurality (i.e., one or more) of those steps, compositions, group of steps or group of compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. For example, reference to "a" includes the singular as well as two or more; reference to "an" includes the singular as well as two or more; reference to "the" includes the singular as well as two or more, etc.

[0062] Those skilled in the art will understand that the present invention is subject to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes all of the steps, features, compositions and compounds mentioned or indicated herein, individually or collectively, including any and all combinations of said steps or features, or any two or more of them.

[0063] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention, and the present invention is not limited in any way to the methods and materials described.

[0064] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0065] The present invention is not intended to be limited in scope by the detailed examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention.

[0066] Unless specifically stated otherwise, it is to be understood that any example or embodiment of the present invention in this specification applies mutatis mutandis to any other example or embodiment of the present invention.

[0067] Unless otherwise specified, technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., those skilled in the art of cell culture techniques, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0068] Unless otherwise indicated, the recombinant protein, cell culture, and immunological methods utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, vols. 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, vols. 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988; including all current editions); Ed. Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and J. E. Coligan et al. (eds.), Current Protocols in These are described and explained throughout the literature in such sources as "immunology," John Wiley & Sons (including all current editions).

[0069] The description and definition of variable regions and portions thereof, T cell receptors and fragments thereof herein may be further clarified by the IMGT system discussed in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991; Bork et al., J Mol. Biol., 242, 309-320, 1994; Chothia and Lesk, J. Mol Biol., 196:901-917, 1987; Chothia et al., Nature, 342, 877-883, 1989; Al-Lazikani et al., J Mol Biol., 273, 927-948, 1997; or Giudicelli et al., Nucleic Acids Res., 25:206-211, 1997.

[0070] In a preferred embodiment, the CDRs defined herein are defined according to the IMGT method.

[0071] The term "and / or", e.g., "X and / or Y", shall be understood to mean "X and Y" or "X or Y" and shall be understood to give explicit support for both meanings or for either meaning.

[0072] The term "derived from" as used herein shall be understood to indicate that the specified integer may be obtained from a particular source, although not necessarily directly from this source.

[0073] References herein, for example, to ranges of residues, are understood to be inclusive, e.g., a reference to "a region comprising amino acids 1-15" is understood to be inclusive, i.e., the region includes the sequence of amino acids numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 within the specified sequence.

[0074] The term "consisting essentially of" limits the scope of a claim to the specified materials or steps or to materials or steps that do not materially affect the essential characteristics of the claimed invention. For example, a protein domain, region or module (e.g., a binding domain, hinge region, linker module) or protein (which may have one or more domains, regions or modules) "consists essentially of" a particular amino acid sequence if the amino acid sequence of the domain, region, module or protein includes extensions, deletions, mutations, or combinations thereof (e.g., amino acids at the amino or carboxy termini or between domains) that, in combination, contribute 20% or less (e.g., 15% or less, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain, region, module or protein and do not substantially affect the activity of the domain(s), region(s), module(s) or protein (e.g., target binding affinity of a binding protein) (i.e., do not reduce activity by more than 50%, such as a reduction in activity of 40% or less, 30%, 25%, 20%, 15%, 10%, 5% or 1%).

[0075] As used herein, "nucleic acid" or "nucleic acid molecule" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, such as fragments generated by polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any of ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are generated by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-optical isomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modifications or substitutions in sugar moieties or pyrimidine or purine base moieties. The monomers of nucleic acids can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, etc. Nucleic acid molecules can be single-stranded or double-stranded.

[0076] As used herein, the term "recombinant" refers to a cell, microorganism, nucleic acid molecule, or vector that has been genetically engineered by human intervention (i.e., modified by the introduction of an exogenous or heterologous nucleic acid molecule), or to a cell or microorganism that has been altered such that the expression of an endogenous nucleic acid molecule or gene is controlled, deregulated, or constitutive. An artificially created genetic modification can include, for example, a modification that introduces a nucleic acid molecule (which may include expression control elements such as a promoter) that encodes one or more proteins or enzymes, or the addition, deletion, substitution, or other functional disruption or addition of other nucleic acid molecules to the genetic material of a cell. Exemplary modifications include modifications within the coding region of a heterologous or homologous polypeptide, or a functional fragment thereof, derived from a reference or parent molecule.

[0077] Binding Proteins As used herein, "binding protein" refers to a proteinaceous molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein) that possesses the ability to specifically and non-covalently associate with, integrate with, or combine with a target (e.g., Smith protein or fragments thereof, Smith protein fragment:MHC complex). Binding proteins may be purified, substantially purified, synthetic, or recombinant. Exemplary binding proteins include single chain immunoglobulin variable regions (e.g., scTCR, scFv).

[0078] In certain embodiments, any of the binding proteins of the present invention is a T cell receptor (TCR), a chimeric antigen receptor, or an antigen-binding fragment of a TCR, any of which may be a chimeric, humanized, or human TCR. In further embodiments, the antigen-binding fragment of a TCR comprises a single chain TCR (scTCR) or a chimeric antigen receptor (CAR). In certain embodiments, the binding protein is a TCR.

[0079] "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 et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, pp. 4-33, 1997) capable of specifically binding to an antigenic peptide bound to an MHC receptor. TCRs may be found on the surface of a cell or in a soluble form and are generally composed of a heterodimer having an α (alpha) chain and a β (beta) chain (also known as TCRα and TCRβ, respectively) or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively). Like immunoglobulins, the extracellular portions of TCR chains (e.g., α chain, β chain) contain two immunoglobulin domains: a variable domain at the N-terminus (e.g., α chain variable domain or Vα, β chain variable domain or Vβ; typically, amino acids 1-116 according to 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, 5th ed.) and one constant domain adjacent to the cell membrane (e.g., α chain constant domain or Cα, typically, amino acids 117-259 according to Kabat; β chain constant domain or Cβ, typically, amino acids 117-295 according to Kabat). Again, like immunoglobulins, variable domains contain complementarity determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA, 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, the TCR is found on the surface of a T cell (or T lymphocyte) and is associated with the CD3 complex. The source of the TCR used in this disclosure can be from a variety of animal species, such as human, mouse, rat, rabbit, or other mammals.

[0080] In any of the foregoing embodiments, the present disclosure provides for the use of high affinity engineered T cell receptors (TCRs) comprising an alpha chain (α chain) and a beta chain (β chain), the TCR binding to a complex of a fragment of Smith protein, Ro60 protein, or MPO protein and an HLA-DR3 or HLA-DR4 molecule. In certain embodiments, the V beta chain comprises or is derived from an allele of TRBV3, TRBV4, TRBV5, TRBV6, TRBV7, TRBV11, TRBV19, TRBV20, TRBV24, or TRBV28. In a further embodiment, the V alpha chain comprises or is derived from an allele of TRAV1, TRAV2, TRAV3, TRAV4, TRAV8, TRAV9, TRAV12, TRAV14, TRAV17, TRAV21, TRAV23, TRAV25, TRAV26, TRAV27, TRAV29, TRAV38, TRAV39 or TRAV40.In a particular embodiment, the binding protein for use according to the invention comprises: (a) a V beta chain comprising or derived from an allele of TRBV11 (preferably TRBV11-2) and a V alpha chain comprising or derived from an allele of TRAV9 (preferably TRAV9-2); (b) a V beta chain comprising or derived from an allele of TRBV6 (preferably TRBV6-1) and a V alpha chain comprising or derived from an allele of TRAV25; (c) an allele of TRBV7 (preferably TRBV7-9). (d) a V beta chain comprising or derived from an allele of TRBV28 and a V alpha chain comprising or derived from an allele of TRAV23; (e) a V beta chain comprising or derived from an allele of TRBV7 (preferably TRBV7-9) and a V alpha chain comprising or derived from an allele of TRAV26 (preferably TRAV26-1); (f) an allele of TRBV7 (preferably TRBV7-3). (g) a V beta chain comprising or derived from an allele of TRBV20 (preferably TRBV20-1) and a V alpha chain comprising or derived from an allele of TRAV9 (preferably TRAV9-2); (h) a V beta chain comprising or derived from an allele of TRBV3 (preferably TRBV3-1) and a V alpha chain comprising or derived from an allele of TRAV2; (i) a V beta chain comprising or derived from an allele of TRBV4 (preferably TRBV4-2) and a V alpha chain comprising or derived from an allele of TRAV17; (j) a V beta chain comprising or derived from an allele of TRBV4 (preferably TRBV4-2) and a V alpha chain comprising or derived from an allele of TRAV27; (k) a V beta chain comprising or derived from an allele of TRBV6 (preferably TRBV6-5) and a V alpha chain comprising or derived from an allele of TRAV2.

[0081] In a further embodiment, the binding protein for use according to the invention is selected from the group consisting of: (a) a V beta chain comprising or derived from an allele of TRBV20 (preferably TRBV20-1) and a V alpha chain comprising or derived from an allele of TRAV38 (preferably TRAV38-1); (b) a V beta chain comprising or derived from an allele of TRBV6 (preferably TRBV6-4) and a V alpha chain comprising or derived from an allele of TRAV1 (preferably TRAV1-2); (c) a V beta chain comprising or derived from an allele of TRBV6 (preferably TRBV6-4) and a V alpha chain comprising or derived from an allele of TRAV4; (d) an allele of TRBV4. (e) a V beta chain comprising or derived from an allele of TRBV5 (preferably TRBV5-4) and a V alpha chain comprising or derived from an allele of TRAV21; (f) a V beta chain comprising or derived from an allele of TRBV28 and a V alpha chain comprising or derived from an allele of TRAV27; (g) a V beta chain comprising or derived from an allele of TRBV24 (preferably TRBV24-1) and a V alpha chain comprising or derived from an allele of TRAV1 (preferably TRAV1-1).

[0082] In any aspect or embodiment, the binding protein for use according to the invention comprises: (a) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-7) and a V alpha chain comprising or derived from an allele of TRAJ47; (b) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-3) and a V alpha chain comprising or derived from an allele of TRAJ54; (c) an allele of TRBJ1 (preferably TRBJ1-1). (d) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ44; (e) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-5) and a V alpha chain comprising or derived from an allele of TRAJ38; (f) an allele of TRBJ2 (preferably T (g) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-7) and a V alpha chain comprising or derived from an allele of TRAJ11; (h) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-1) and a V alpha chain comprising or derived from an allele of TRAJ8; (i) an allele of TRBJ2 (j) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-3) and a V alpha chain comprising or derived from an allele of TRAJ49; (k) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-2) and a V alpha chain comprising or derived from an allele of TRAJ7.

[0083] In any aspect or embodiment, the binding protein for use according to the invention comprises: (a) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-4) and a V alpha chain comprising or derived from an allele of TRAJ48; (b) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-5) and a V alpha chain comprising or derived from an allele of TRAJ48; (c) an allele of TRBJ2 (preferably TRBJ2-1). (d) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-7) and a V alpha chain comprising or derived from an allele of TRAJ48; (e) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-1) and a V alpha chain comprising or derived from an allele of TRAJ88; (f) an allele of TRBJ1 (preferably (g) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-2) and a V alpha chain comprising or derived from an allele of TRAJ3; (h) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-3) and a V alpha chain comprising or derived from an allele of TRAJ9; (i) an allele of TRBJ2 (j) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-2) and a V alpha chain comprising or derived from an allele of TRAJ41; (k) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ9.

[0084] In any of the foregoing embodiments, the present disclosure provides a method for the treatment of a TCR comprising administering to a patient a therapeutically effective amount of a TCR comprising administering to said ... * 01:01 molecule and HLA-DRB1 * The present invention presents a high affinity engineered T cell receptor (TCR), which is a 03:01 molecule. In certain embodiments, the V beta chain comprises or is derived from an allele of TRB2, TRBV4, TRBV5, TRB6, TRB7, TRBV9, TRB10, TRBV11, TRB12, TRBV20, TRBV24, TRB27, or TRBV29. In further embodiments, the V alpha chain comprises or is derived from an allele of TRAV1, TRAV2, TRAV8, TRAV9, TRAV10, TRAV12, TRAV20, TRAV26, TRAV30, or TRAV36.

[0085] In a particular embodiment, the binding protein for use according to the invention comprises (a) a V beta chain comprising or derived from an allele of TRBV5 (preferably TRBV5-1) and a V alpha chain comprising or derived from an allele of TRAV20; (b) a V beta chain comprising or derived from an allele of TRBV29 (preferably TRBV29-1) and a V alpha chain comprising or derived from an allele of TRAV12 (preferably TRAV12-1); (c) an allele of TRBV4 (preferably TRBV4- (d) a V beta chain comprising or derived from an allele of TRBV4 (preferably TRBV4-1) and a V alpha chain comprising or derived from an allele of TRAV30; (e) a V beta chain comprising or derived from an allele of TRBV4 (preferably TRBV4-1) and a V alpha chain comprising or derived from an allele of TRAV36 (preferably TRAV36DV7). (f) a V beta chain comprising or derived from an allele of TRBV24 (preferably TRBV24-1) and a V alpha chain comprising or derived from an allele of TRAV12 (preferably TRAV12-1); (g) a V beta chain comprising or derived from an allele of TRBV11 (preferably TRBV11-2) and a V alpha chain comprising or derived from an allele of TRAV12 (preferably TRAV12-3); (h) an allele of TRBV20 (preferably TRBV20-1), or (i) a V beta chain comprising or derived from an allele of TRAV9 (preferably TRAV9-2); (j) a V beta chain comprising or derived from an allele of TRBV20 (preferably TRBV20-1) and a V alpha chain comprising or derived from an allele of TRAV12 (preferably TRAV12-1).

[0086] In a further embodiment, the binding protein for use according to the invention is selected from the group consisting of: (a) a V beta chain comprising or derived from an allele of TRBV27 and a V alpha chain comprising or derived from an allele of TRAV12 (preferably TRAV12-1); (b) a V beta chain comprising or derived from an allele of TRBV6 (preferably TRBV6-1) and a V alpha chain comprising or derived from an allele of TRAV1 (preferably TRAV1-2); (c) a V beta chain comprising or derived from an allele of TRBV7 (preferably TRBV7-9) and a V alpha chain comprising or derived from an allele of TRAV12 (preferably TRAV12-2); (d) a V beta chain comprising or derived from an allele of TRBV2 and a V alpha chain comprising or derived from an allele of TRAV8 (TRAV8-3); (e) an allele of TRBV8 (preferably (f) a V beta chain comprising or derived from an allele of TRBV7 (preferably TRBV7-9) and a V alpha chain comprising or derived from an allele of TRAV10; (g) a V beta chain comprising or derived from an allele of TRBV7 (preferably TRBV7-9) and a V alpha chain comprising or derived from an allele of TRAV19; (h) a V beta chain comprising or derived from an allele of TRBV10 (preferably TRBV10-3) and a V alpha chain comprising or derived from an allele of TRAV2; (i) a V beta chain comprising or derived from an allele of TRBV12 (preferably TRBV12-4) and a V alpha chain comprising or derived from an allele of TRAV20.

[0087] In certain embodiments, the V beta chain comprises or is derived from an allele of TRBJ1 or TRBJ2. In further embodiments, the V alpha chain comprises or is derived from an allele of TRAJ3, TRAJ6, TRAJ9, TRAJ13, TRAJ17, TRAJ23, TRAJ27, TRAJ28, TRAJ31, TRAJ33, TRAJ37, TRAJ42, TRAJ45, TRAJ47, TRAJ48, TRAV49, or TRAV54.

[0088] In a particular embodiment, the binding protein for use according to the invention comprises (a) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-1) and a V alpha chain comprising or derived from an allele of TRAJ6; (b) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-5) and a V alpha chain comprising or derived from an allele of TRAJ45; (c) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-2) and a V alpha chain comprising or derived from an allele of TRAJ54; (d) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ28; (e) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V allele of TRAJ49. or a V alpha chain derived therefrom; (f) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-2) and a V alpha chain comprising or derived from an allele of TRAJ48; (g) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-2) and a V alpha chain comprising or derived from an allele of TRAJ17; (h) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-7) and a V alpha chain comprising or derived from an allele of TRAJ27; (i) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ37; (j) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ3.

[0089] In a particular embodiment, the binding protein for use according to the invention comprises: (a) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-2) and a V alpha chain comprising or derived from an allele of TRAJ9; (b) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-7) and a V alpha chain comprising or derived from an allele of TRAJ33; (c) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ49; (d) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-6) and a V alpha chain comprising or derived from an allele of TRAJ13; (e) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-1) and an allele of TRAJ23, or (f) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ47; (g) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-7) and a V alpha chain comprising or derived from an allele of TRAJ42; (h) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-1) and a V alpha chain comprising or derived from an allele of TRAJ47; (i) a V beta chain comprising or derived from an allele of TRBJ2 (preferably TRBJ2-5) and a V alpha chain comprising or derived from an allele of TRAJ31; (j) a V beta chain comprising or derived from an allele of TRBJ1 (preferably TRBJ1-4) and a V alpha chain comprising or derived from an allele of TRAJ47.

[0090] In any aspect or embodiment, the binding protein of the invention comprises a V beta chain that comprises or is derived from an allele of TRBD1 or TRBD2.

[0091] In any aspect or embodiment, the binding protein of the invention comprises a V beta chain that comprises or is derived from an allele of TRC1 or TRBC2 and a V alpha chain that comprises or is derived from an allele of TRAC.

[0092] In any embodiment of the invention, the binding protein comprises a Vα chain comprising a Vα domain and a Vβ chain comprising a Vβ domain. Preferably, the Vα and Vβ chains are modified to include cysteine ​​residues that allow for the formation of additional interchain disulfide bonds. The cysteines introduced into each of the Vα and Vβ chains allow for preferential pairing of the Vα and Vβ chains when expressed in a cell, resulting in the expression of an endogenous TCR Vα chain and an endogenous TCR Vβ chain. Preferably, the residue at Thr48 or an equivalent on the TCRα chain and the residue at Ser57 or an equivalent on the TCRβ chain are replaced with cysteines to facilitate the creation of additional disulfide bonds between the constant regions of the TCRs. This modification allows for preferential pairing of the introduced TCR and reduces mispairing with the endogenous TCR. This is particularly beneficial for adoptive cell therapy, where regulatory T cells are engineered to express an exogenous TCR.

[0093] By way of example, a method useful for isolating and purifying recombinantly produced soluble TCRs may include obtaining a supernatant from a suitable host cell / vector system that secretes recombinant soluble TCRs into culture medium, and then concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse-phase HPLC steps may be used to further purify the recombinant polypeptide. These purification methods may also be used to separate the immunogen from its native environment. Methods for large-scale production of one or more of the isolated / recombinant soluble TCRs described herein include batch cell culture methods that are monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCRs may be performed according to methods described herein and known in the art.

[0094] The SmB / B' specific binding proteins or SmB / B' specific binding domains described herein (e.g., SEQ ID NOs: 11-24 and variants thereof) can be functionally characterized according to any of a number of art-accepted methods for assaying for T cell activity, including determining T cell binding, activation or induction, and also determining T cell responses that are antigen-specific. Examples include determining T cell proliferation, cytokine release by T cells, stimulation of antigen-specific T cells, MHC-restricted stimulation of T cells, CTL activity (e.g., by detecting release of Cr from preloaded target cells), changes in T cell phenotypic marker expression, and other measures of T cell function. Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: Comprehensive Sourcebook of Techniques, 1998). See also, Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.), Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and references cited therein.

[0095] Sm (Smith) proteins and related nuclear ribonucleoproteins (nRNPs) are targets for autoantibodies in SLE. These antigens reside within intracellular organelles, called spliceosomes, which are composed of peptide containing small RNAs. Anti-Sm antibodies are present in 15-30% of patients with SLE, but are highly specific for SLE. Most Sm proteins occur with high frequency (60%) in young black women with SLE. In healthy individuals or patients with other diseases, Sm proteins are almost never produced. Anti-Sm antibodies should not be confused with anti-smooth muscle antibodies, which have been detected in autoimmune liver disease.

[0096] Sm antigens and nuclear ribonucleoprotein (RNP) antigens are particulate complexes composed of small nuclear RNA (U-RNA) and proteins. These complexes are also called extractable nuclear antigens (ENA) because they are soluble in saline. Autoantibodies against these antigens are produced in systemic lupus erythematosus and mixed connective tissue disease. Among the proteins present in the complexes are the proteins "SmB / B'" and "SmD".

[0097] As used herein, SmB / B' refers to a ribonucleoprotein called "small nuclear ribonucleoprotein-related proteins B and B'," which in humans is a protein encoded by the SNRPB gene. SmB / B' may also be referred to by the alternative names: COD, SNRPB1, snRNP-B, CCMS, and small nuclear ribonucleoprotein polypeptides B and B1.

[0098] The protein encoded by the SNRPB gene is one of several nuclear proteins commonly found among small ribonucleoprotein particles (snRNPs) U1, U2, U4 / U6, and U5. These snRNPs are involved in pre-mRNA splicing, and the encoded protein may also play a role in pre-mRNA splicing or in snRNP structure. Two transcript variants have been found for this gene, encoding different isoforms (B and B').

[0099] All nine core proteins of the Sm complex are targets of anti-Sm autoimmune responses, most frequently the B and D polypeptides.

[0100] As used herein, "Ro60" refers to the 60 kDa SS-A / Ro ribonucleoprotein, an RNA-binding protein. Ro, a 60 kDa autoantigen, is the primary target of the immune response in patients with systemic lupus erythematosus and Sjogren's syndrome, two systemic rheumatic diseases. In lupus patients, anti-Ro antibodies are associated with photosensitive skin lesions and neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with photosensitive skin lesions and third-degree heart block, a cardiac conduction disorder. In vertebrate cells, Ro protein binds to a small RNA of unknown function, known as Y RNA. The function of Ro in cells has long been enigmatic, but recent studies suggest that Ro is involved in two strikingly different processes: quality control of small RNAs and enhancing cell survival after exposure to ultraviolet radiation. Interestingly, mice lacking the Ro protein develop an autoimmune syndrome that shares some features with systemic lupus erythematosus in patients, suggesting that normal function of Ro may be important for the prevention of this autoimmune disease.

[0101] As used herein, myeloperoxidase (MPO) is an enzyme peroxidase that in humans is encoded by the MPO gene on chromosome 17. MPO is most abundantly expressed in neutrophil granulocytes and produces hypohalous acids, including hypochlorous acid. MPO is a lysosomal protein that is stored in azurophilic granules of neutrophils and released into the extracellular space upon degradation. Antibodies to MPO are involved in various types of vasculitis, most notably three clinically and pathologically recognized forms: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA).

[0102] Cells for administration, including adoptive cell therapy The present invention provides methods of treatment that include administering cells, particularly Treg cells expressing a TCR as described herein, either as a population of T cells or as a mixed population of cells.

[0103] In certain embodiments, nucleic acid molecules encoding the binding proteins described herein are used to transfect / transduce host cells (e.g., Treg cells) for use in adoptive transfer therapy. Nucleic acids encoding said binding proteins are described elsewhere in this document.

[0104] The present invention provides a method of preparing a population of regulatory T cells for use in the treatment of an autoimmune disease characterized by an aberrant immune response to one or more of SmB / B', SmD, Ro60 and MPO, comprising: Providing a population of regulatory T cells; introducing a nucleic acid or vector encoding a TCR as described herein into a population of regulatory T cells; Providing conditions that allow expression of the TCR on the surface of regulatory T cells Including, This provides for the preparation of a population of regulatory T cells for use in the treatment of autoimmune diseases characterized by an aberrant immune response to one or more of SmB / B', SmD, Ro60 and MPO. A method is provided.

[0105] The present invention also provides a method for treating an autoimmune disease characterized by an aberrant immune response to one or more of SmB / B', SmD, Ro60, and MPO in a subject, comprising: administering to the subject an effective amount of regulatory T cells that express at the cell surface a binding protein comprising a T cell receptor (TCR) alpha chain variable (Vα or V alpha) domain and a TCR beta chain variable (Vβ or V beta) domain, wherein the binding protein is as described herein. Including, This allows for the treatment of an autoimmune disease characterized by an abnormal immune response to one or more of SmB / B', SmD, Ro60 and MPO in a subject. A method is also provided.

[0106] The present invention further provides a method for preparing ex vivo a population of T cells specific for SmB / B', SmD, Ro60 and MPO and exhibiting at least one characteristic of regulatory T cells, comprising: providing a population of T cells exhibiting at least one characteristic of regulatory T cells; introducing a nucleic acid or vector as described herein into a population of T cells, wherein the nucleic acid or vector encodes a binding protein as described herein; Providing conditions that allow expression of the binding protein on the surface of the T cells. Including, This allows the preparation of a population of T cells ex vivo that are specific to SmB / B', SmD, Ro60 and MPO and exhibit at least one characteristic of regulatory T cells. Methods Preferably, the T cells exhibiting at least one characteristic of a regulatory T cell are derived from a biological sample derived from a subject with SLE.

[0107] The T cells exhibiting at least one characteristic of regulatory T cells used in the method or use of the invention may be selected from a healthy subject. The T cells may be isolated from a histocompatible donor.

[0108] T cells may be reprogrammed from somatic cells or may be differentiated from iPSCs or other stem cells.

[0109] In another aspect, the invention provides a method of treating or preventing a condition associated with an aberrant, unwanted, or otherwise inappropriate immune response to a SmB / B', SmD, Ro60, or MPO protein in a subject, comprising: providing a population of T cells exhibiting at least one characteristic of regulatory T cells; introducing a nucleic acid or vector encoding a binding protein described herein into a population of T cells; Providing conditions that allow expression of the binding protein on the surface of the T cells; administering T cells that express the binding protein on their surface. Including, This allows for the treatment or prevention of a condition in a subject. A method is provided.

[0110] In an alternative embodiment, the present invention provides a method for preparing ex vivo a population of T cells specific for the proteins SmB / B', SmD, Ro60 and MPO and exhibiting at least one characteristic of a regulatory T cell, comprising: providing a population of T cells exhibiting at least one characteristic of conventional T cells, optionally wherein the population of T cells is a mixed population of T cells; introducing a nucleic acid or vector into a population of T cells, the nucleic acid or vector encoding a binding protein as described herein; Providing conditions that allow expression of the binding protein on the surface of the T cells; Providing conditions that allow the conversion of a population of T cells into regulatory T cells Including, This results in the ex vivo preparation of a population of T cells that exhibits at least one characteristic of regulatory T cells and is specific for the proteins SmB / B', SmD, Ro60 and MPO. A method is provided.

[0111] In any embodiment, the conditions to allow for the conversion of conventional T cells or a mixed population of T cells into regulatory T cells may include contacting the conventional T cells or the mixed population of T cells with one or more agents or increasing the expression of one or more factors suitable for the conversion of conventional T cells into regulatory T cells. The one or more agents or factors may include TGF-β, Foxp3, or agents for increasing expression thereof.

[0112] In certain embodiments, one or more of the peptides described herein, derived from SmB / B', SmD, Ro60 and MPO and listed in Table 1, may be used to activate and / or expand a population of T cells to generate T cells (e.g., Treg cells) with specificity for a peptide. For example, the invention provides a method of preparing a population of regulatory T cells for use in treating an autoimmune disease described herein, comprising: i) culturing a population of regulatory T cells in the presence of a first peptide selected from SmD, SmB / B', Ro60 or MPO, which are peptides listed in Table 1 herein, under conditions and for a time sufficient to allow expansion of the subpopulation activated by the first peptide; ii) culturing the subpopulation of regulatory T cells obtained in i) in the presence of a second peptide selected from SmD, SmB / B', Ro60 or MPO, which are peptides listed in Table 1 herein, for a sufficient time under conditions allowing the expansion of the subpopulation activated by the first and second peptides. Including, This allows for the preparation of populations of regulatory T cells for use in the treatment of autoimmune diseases as described herein. Optionally, the method further comprises the step of culturing the subpopulation of regulatory T cells activated by the first and second peptides in the presence of a third or fourth peptide selected from peptides SmD, SmB / B', Ro60 or MPO, listed in Table 1 herein, thereby obtaining a subpopulation of Tregs activated by all four peptides. The first and second peptides are different, e.g., derived from different proteins (and if the method includes a third or fourth peptide, they are derived from a protein different from the protein from which the first and second peptides are derived).

[0113] In one embodiment, the first peptide is derived from a Ro60 protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:5, 6, or 7) and the second peptide is derived from a Smith protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:1, 2, 3, or 4). Optionally, the third peptide is derived from an MPO protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:8, 9, or 10), or, if the second peptide is derived from SmD1 (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:1 or 2), the third peptide may be derived from SmB / B' (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:3 or 4).

[0114] In one embodiment, the first peptide is derived from a Ro60 protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:5, 6, or 7), and the second peptide is derived from an MPO protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:8, 9, or 10). Optionally, the third peptide is a Smith protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO:1, 2, 3, or 4).

[0115] In one embodiment, the first peptide is derived from a Smith protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4) and the second peptide is derived from an MPO protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 8, 9, or 10). Optionally, the third peptide is derived from a Ro60 protein (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 5, 6, or 7), or, if the first peptide is derived from SmD1 (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1 or 2), the third peptide may be derived from SmB / B' (e.g., the peptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 3 or 4).

[0116] Advances in TCR sequencing have been described (e.g., Robins et al., Blood, 114:4099, 2009; Robins et al., Sci. Translat. Med., 2:47-64, 2010; Robins et al. (September 10), J. Imm. Meth., Epub Flash Edition, 2011; Warren et al., Genome Res., 2, 1:790, 2011) and may be utilized in the course of practicing embodiments in accordance with the present disclosure. Similarly, methods for transfecting / transducing a desired nucleic acid into a T cell can be described using adoptive transfer procedures using T cells with a desired antigen specificity (e.g., Schmitt et al., Hum. Gen., 20:1240, 2009; Dossett et al., Mol. Ther., 77:742, 2009; Till et al., Blood, 772:2261, 2008; Wang et al., Hum. Genet. Ther., 75:712, 2007; Kuball et al., Blood, 709:2331, 2007; US2011 / 0243972; US2011 / 0189141; Leen et al., Ann. Rev. Immunol., 25:243, 2007), as well as other methods have been described (e.g., U.S. Patent Application Publication No. 2004 / 0087025), and thus adaptation of these methods to the embodiments disclosed herein is contemplated based on the teachings herein, including the teachings directed to the binding proteins of the invention.

[0117] Cell populations containing regulatory T (Treg) cells can be derived from any source in which Treg cells are present, such as peripheral blood, thymus, lymph nodes, spleen and bone marrow.

[0118] The cell population comprising Treg cells may also be derived from a mixed T cell population or a population of conventional T cells. As described herein, the mixed population or conventional T cells may be contacted with a peptide of the invention to enrich for Sm antigen specificity in the T cells. Alternatively, a nucleic acid encoding a binding protein of the invention may be transduced into the mixed population or conventional T cells. The T cells may then be converted into Treg cells using standard techniques known to those skilled in the art for the production of Treg cells. In certain embodiments, the mixed T cell population or conventional T cells are cultured under conditions that allow for increased expression of TGF-beta, Foxp3. This includes culturing the cells with anti-CD3 / anti-CD28 antibodies, high doses of IL-2, TGF-beta, and inhibition of CDK8 / 19 by rapamycin. In further embodiments, the converted or enriched Treg cell population is stabilized (e.g., by contacting the cells with vitamin C or other agents to stabilize Tregs).

[0119] The Treg cells used for infusion (or indeed the Tconv or mixed T cell population used to generate the Tregs) may be isolated from an allogeneic donor, preferably an HLA-matched donor, or from a subject diagnosed with a condition associated with an abnormal, unwanted or otherwise inappropriate immune response to Smith protein. Preferably, the condition is SLE.

[0120] T cells can also be generated from differentiation of induced pluripotent stem cells (iPSCs) or embryonic stem cells, preferably embryonic stem cell lines. Those skilled in the art are familiar with standard techniques for generating Treg cells from stem cells, including iPSCs. Examples of these techniques are described in Hague et al. (2012), J. Immunol., 189:2338-36; and Hague et al., (2019) JCI Insight, 4:pii 126471.

[0121] Furthermore, in the context of a mixed T cell population, one of skill in the art is familiar with standard techniques for isolating a subpopulation of T cells that are CD4+ CD25+ T cells (Treg cells). For example, CD4+ CD25+ T cells (Treg cells) can be obtained from a biological sample derived from a subject by negative and positive immunoselection and cell sorting.

[0122] In any of the methods of the invention, the Treg cells cultured in the presence of the nucleic acid or vector may be transferred to the same subject from which the cells were obtained. In other words, the cells used in the methods of the invention may be autologous cells, i.e., may be obtained from the subject in which the medical condition is to be treated or prevented. Alternatively, the cells may be allogeneically transferred to another subject. Preferably, the cells are autologous cells to the subject in the method of treating or preventing a medical condition in the subject.

[0123] As used herein, the term "ex vivo" or "ex vivo therapy" refers to therapy in which cells are obtained from the patient or a suitable alternative source, such as a suitable allogeneic donor, and modified such that the modified cells may be used to treat a disease that is ameliorated by the therapeutic benefit provided by the modified cells. The therapy involves the administration or reintroduction of the modified cells into the patient. The benefit of ex vivo therapy is the ability to provide the benefit of the treatment to the patient without exposing the patient to undesirable side effects from the treatment.

[0124] The term "administered" refers to the administration of a therapeutically effective dose of the aforementioned composition comprising the respective cells to an individual. A "therapeutically effective amount" refers to a dose that produces the effect for which it is administered. The exact dose depends on the purpose of the treatment and can be ascertained by the skilled artisan using known techniques. As known in the art and described above, adjustments for systemic delivery versus local delivery, age, weight, general health, sex, diet, number of doses, drug interactions and severity of the condition may be necessary and can be ascertained by the skilled artisan with routine experimentation.

[0125] An "enriched" or "purified" cell population is an increase in the ratio of a particular cell to other cells, e.g., compared to cells found in a subject's body or compared to the ratio before exposure to a peptide, nucleic acid, or vector of the invention. In some embodiments, within an enriched or purified cell population, the particular cells comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% of the total cell population. The cell population may be defined by one or more cell surface markers and / or characteristics.

[0126] Treg cells expressing the binding proteins described herein can be administered to a subject by any method, including, for example, injection, infusion, deposition, implantation, oral ingestion or topical administration, or any combination thereof. The injection can be, for example, intravenous, intramuscular, intradermal, subcutaneous or intraperitoneal, preferably intravenous. A single dose or multiple doses can be administered over a given time period depending on the condition, its severity and the general health of the subject, as can be determined by one of skill in the art without undue experimentation. The injection can be administered at multiple locations.

[0127] Treg cells may be administered alone or in combination with other therapeutic agents. Each dose contains approximately 10 x 10 CD8+ Treg cells. 3 pieces, cells 20×10 3 pieces, cells 50×10 3 pieces, cells 100×10 3 pieces, cells 200×10 3 pieces, cells 500×10 3 pieces, cells 1×10 6 pieces, cells 2×10 6 pieces, cells 20×10 6 pieces, cells 50×10 6 pieces, cells 100×10 6 pieces, cells 200×10 6 pieces, cells 500×10 6 pieces, cells 1×10 9 pieces, cells 2×109 pieces, cells 5×10 9 pieces, cells 10×10 9 The frequency of administration may include, for example, once every week, twice every week, once every two weeks, once every three weeks, once every four weeks, once every month, once every two months, once every three months, once every four months, once every five months, once every six months, etc. The total number of days over which administration is performed may be 1 day, 2 days, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, etc. It is understood that any administration administered may involve two or more infusions on the same day. For administration, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the administered Treg cells exhibit at least one characteristic of Treg cells.

[0128] The Tregs described herein are useful for suppressing an aberrant immune response against self antigens, particularly against SmD, SmB / B', Ro60 or MPO proteins. Treg or mixed Treg populations may be administered in addition to or instead of any accepted protocol for treating autoimmune diseases characterized by an aberrant immune response against one or more of SmD, SmB / B', Ro60 or MPO proteins.

[0129] In certain cases, administration of the immunosuppressant is reduced after administration of the Tregs. The dose of the immunosuppressant may be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% after administration of the Treg or mixed Treg population. In some cases, the dose of the immunosuppressant is reduced by about 50% after treatment with the Treg or mixed Treg population. In another example, administration of the immunosuppressant is stopped after administration of the Treg or mixed Treg population.

[0130] The Tregs described herein may be used in combination with other known drugs and therapies. As used herein, "combined" administration means that two (or more) different therapies are delivered to a subject during the course of the subject's suffering from a disorder (e.g., a disease or condition), e.g., two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or the treatments are stopped for other reasons. In some embodiments, the delivery of one treatment begins while the delivery of a second treatment is still taking place, such that there is an overlap in administration. In the present specification, this is sometimes referred to as "simultaneous" or "contemporaneous" delivery. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In either case, in some embodiments, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., a smaller amount of the second treatment has a comparable effect, or the second treatment relieves symptoms to a greater extent than would be observed if administered in the absence of the first treatment, or a similar situation would be observed with respect to the first treatment. In some embodiments, the delivery is such that the relief of symptoms or other parameters associated with the disorder is greater than would be observed with one treatment delivered in the absence of the other treatment. The effect of the two treatments may be a partial additive effect, a complete additive effect, or a greater than additive effect. The delivery may be such that the effect of the first treatment delivered is still detectable at the time the second treatment is delivered. The Tregs and at least one additional therapeutic agent described herein may be administered simultaneously, by the same composition or separate compositions, or may be administered sequentially. For sequential administration, the Tregs may be administered first and the additional agent may be administered second. Alternatively, the order of administration may be reversed, with the additional agent being administered first and the Tregs being administered second.The Tregs and / or other therapeutic agents, procedures or modalities may be administered when the disorder is active, when the disease is in remission, or when disease activity is declining. The Treg cell therapy may be administered prior to, contemporaneously with, or after another treatment, or when the disorder is in remission.

[0131] When administered in combination, the Tregs and additional agents (e.g., second or third agents) described herein, or all of these, may be administered in higher, lower, or the same amounts or dosages as the amounts or dosages of each agent used individually, e.g., as monotherapy. In certain embodiments, the amounts or dosages of the Tregs, additional agents (e.g., second or third agents), or all of these administered are lower or less (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amounts or dosages of each agent used individually. In other embodiments, the amount or dosage of the Tregs, additional agent (e.g., second agent or third agent), or all of these that results in a desired effect (e.g., treatment of cancer) is lower or at a lower dose (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent individually required to achieve the same therapeutic effect.

[0132] For example, the additional therapeutic agent(s) may include one or more immunosuppressants commonly administered for the treatment of autoimmune diseases (SLE, Sjogren's syndrome, APA, and other diseases described herein). The immunosuppressant(s) may be agents administered immediately after transplantation to prevent acute rejection (e.g., methylprednisolone, atgam, thymoglobulin, basiliximab, or alemtuzumab) or immunosuppressant(s) used for maintenance (e.g., prednisone, calcineurin inhibitors (e.g., cyclosporine or tacrolimus), mycophenolate mofetil, azathioprine, sirolimus, or everolimus). Other immunosuppressants administered following organ transplantation include CTLA-4 fusion proteins (e.g., belatacept or abatacept), corticosteroids (e.g., methylprednisolone, dexamethasone, or prednisolone), cytotoxic immunosuppressants (e.g., azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, or methotrexate), immunosuppressive antibodies (e.g., antithymocyte globulin, basiliximab, or infliximab), sirolimus derivatives (e.g., everolimus or sirolimus), and antiproliferative agents (e.g., mycophenolate mofetil, mycophenolate sodium, or azathioprine). Additional immunosuppressants suitable for use in the present invention as described herein will be known to those of skill in the art, and the invention is not limited in this respect.

[0133] An effective amount of a therapeutic agent (e.g., Tregs or mixed Treg populations specific for donor alloantigens or autoantigens) for the treatment or prevention of an autoimmune disorder described herein can be administered to a subject by standard methods. For example, the agent can be administered by any of a number of different routes, including, for example, intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, transdermal injection, oral, transdermal (topical), intraarterial, intratumoral, intralymph node, intramedullary, or transmucosal.

[0134] In some embodiments, the agent (e.g., Tregs or a mixed population of Tregs including Tregs specific for an autoantigen) may be administered directly (e.g., by injection or infusion) into the affected tissue. In one embodiment, the compositions described herein are administered into a body cavity or fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid). For example, the therapeutic agent (e.g., Tregs or a mixed population of Tregs specific for a donor alloantigen or autoantigen) may be administered by injection or infusion, e.g., intramuscular, subcutaneous, intraperitoneal, or intravenous injection. The most suitable route of administration in any given case will depend on the particular agent being administered, the patient, the particular disease or condition being treated, the pharmaceutical formulation, the method of administration (e.g., number of doses and route of administration), the age, weight, sex of the patient, the severity of the disease being treated, the diet of the patient, and the excretion rate of the patient. The agent (e.g., Tregs or mixed Treg populations specific for donor allo- or auto-antigens) may be encapsulated or injected, e.g., in a viscous form, for delivery to the selected site. The agent may be administered by a matrix capable of delivering the agent to the selected site. The matrix provides sustained release of the agent and provides proper presentation and a suitable environment for cell infiltration. The matrix may be formed from materials currently used for other implant medical applications. The selection of the matrix material is based on any one or more of biocompatibility, biodegradability, mechanical and cosmetic properties, and interface properties. One example is a collagen matrix.

[0135] The therapeutic agent (e.g., Tregs specific for donor alloantigens or autoantigens or a mixed Treg population) can be incorporated into a pharmaceutical composition suitable for administration to a subject, e.g., a human. Such compositions typically include an agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and all such carriers compatible with pharmaceutical administration.

[0136] The use of such media and agents for pharmaceutical active substances is well known. Except insofar as any conventional media or agent is incompatible with the active compound, such media may be used in the applications described herein. Supplementary active compounds may also be incorporated into the compositions.

[0137] As used herein, the term "unit dosage form" refers to a dosage amount suitable for one administration.For example, a unit dosage form can be the amount of therapeutic agent contained in a delivery device, for example, in a syringe or in an intravenous bag.For example, a unit dosage form is administered in a single administration.In another example, more than one unit dosage form can be administered at the same time.

[0138] In some embodiments, the Treg or mixed Treg population is administered as a monotherapy, i.e., another treatment for the condition is not administered contemporaneously to the subject. The Treg or mixed Treg population composition may be administered once to the patient. If necessary, the Treg cell composition may also be administered multiple times. The Treg or mixed Treg population may be administered by using injection methods commonly known in immunotherapy (see, e.g., Rosenberg et al., New England Journal of Medicine., 319:1676 (1988)).

[0139] Dosages of the above treatments administered to a patient will vary depending on the condition being treated and the exact characteristics of the recipient of the treatment. Scaling of dosages for human administration can be performed according to art-accepted practices.

[0140] In some embodiments, a single treatment regimen is required. In other embodiments, one or more subsequent doses or treatment regimen administrations can be performed. For example, after 3 months of treatment every other week, treatment is repeated once a month for 6 months or 1 year or more. In some embodiments, after the initial treatment, no further treatment is performed.

[0141] The dosage of the compositions described herein can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect. A skilled physician will typically monitor the subject to determine when the treatment is providing therapeutic benefit with respect to duration and frequency of treatment and to determine whether to administer additional cells, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. In general, the dosage will vary with the age, condition, and sex of the patient, and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the unlikely event of a complication.

[0142] Nucleic Acids and Vectors In another aspect, the invention provides the use of a nucleic acid molecule, construct or composition comprising one or more nucleic acid molecules encoding or complementary to a sequence encoding the binding proteins described herein or homologs or analogs thereof. The nucleic acid molecules may be used to make the binding proteins described herein or may be used for cell therapy to treat the diseases or conditions described herein.

[0143] The term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule. Constructs may be present in a vector (e.g., bacterial vectors, viral vectors) or may be integrated into a genome. A "vector" is a nucleic acid molecule capable of carrying another nucleic acid. A vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule that may contain chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acid. Exemplary vectors are vectors capable of autonomous replication (episomal vectors) or vectors capable of expressing nucleic acid molecules to which they are linked (expression vectors).

[0144] Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-stranded RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and herpes simplex virus type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, fowlpox virus, and canarypox virus). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma virus, mammalian type C retroviruses, mammalian type B retroviruses, mammalian type D retroviruses, HTLV-BLV group retroviruses, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, in Fundamental Virology, 3rd ed., BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0145] As used herein, "lentiviral vector" refers to an HIV-based lentiviral vector for gene delivery, which may be integrative or nonintegrative, may have a relatively large packaging capacity, and may transduce a range of different cell types. Lentiviral vectors are usually generated after transient transfection of three or more plasmids (packaging plasmid, envelope plasmid, and transfer plasmid) into producer cells. Similar to HIV, lentiviral vectors also enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0146] In any embodiment, the vector for use in accordance with the methods of the present invention comprises: · EF1α (alpha) promoter; · 2A ribosomal skipping sequence; · Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); Arrangement that places the TCR beta chain variable (Vβ or Vbeta) domain before the (TCR) alpha chain variable (Vα or Vα) domain; or Arrangement to translate the variable TCR beta chain (Vβ or Vbeta) before the variable (TCR) alpha chain (Vα or Vα) may include any one or more or all of the following:

[0147] Preferably, the vector is a lentiviral vector.

[0148] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of this coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely related to each other so that the function of one does not affect the other.

[0149] As used herein, "expression vector" refers to a nucleic acid construct containing a nucleic acid molecule operably linked to a suitable control sequence capable of effecting 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 a suitable mRNA ribosome binding site, and sequences to control the termination of transcription and translation. A vector can be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can be integrated into the genome itself. In the present specification, "plasmid", "expression plasmid", "virus" and "vector" are often used interchangeably.

[0150] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on a coding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof.

[0151] The term "introduced" in the context of inserting a nucleic acid molecule into a cell means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be integrated into the genome of the cell (e.g., chromosomal DNA, plasmid DNA, plastid DNA, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0152] As used herein, a "heterologous" nucleic acid molecule, a "heterologous" construct or a "heterologous" sequence or an "exogenous" nucleic acid molecule, an "exogenous" construct or a "exogenous" sequence refers to a nucleic acid molecule or a portion of a nucleic acid molecule that is not native to the host cell, but may be homologous to a nucleic acid molecule or a portion of a nucleic acid molecule derived from the host cell. The source of the heterologous nucleic acid molecule, the heterologous construct or the heterologous sequence or the exogenous nucleic acid molecule, the exogenous construct or the exogenous sequence may be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule is added to a host cell or host genome (i.e., is not endogenous or native), for example, by conjugation, transformation, transfection, electroporation, etc., where the added molecule may be integrated into the host genome, may be present as extrachromosomal genetic material (e.g., as a plasmid or other form of autonomously replicating vector), or may be present in multiple copies. In addition, "heterologous" refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity.

[0153] As described herein, more than one heterologous or exogenous nucleic acid molecule may be introduced into a host cell as separate nucleic acid molecules, as multiple genes that are individually regulated, as polycistronic nucleic acid molecules, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. For example, as disclosed herein, a host cell may be modified to express two or more heterologous or exogenous nucleic acid molecules that encode a desired TCR (e.g., TCRα and TCRβ) specific to a peptide described herein. When two or more exogenous nucleic acid molecules are introduced into a host cell, the two or more exogenous nucleic acid molecules may be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into a host chromosome at a single site or multiple sites, or any combination thereof. The number of heterologous nucleic acid molecules or heterologous protein activities referred to refers to the number of encoding nucleic acid molecules or the number of protein activities, and not the number of separate nucleic acid molecules introduced into the host cell.

[0154] As used herein, the term "endogenous" or "natural" refers to a gene, protein, or activity that is normally present in a host cell. Furthermore, a gene, protein, or activity that is mutated, overexpressed, shuffled, duplicated, or otherwise altered compared to a parent gene, protein, or activity is also considered to be endogenous or natural to this particular host cell. For example, an endogenous control sequence (e.g., promoter, translational repression sequence) from a first gene may be used to alter or regulate the expression of a second natural gene or nucleic acid molecule, where the expression or regulation of the second natural gene or nucleic acid molecule differs from the normal expression or regulation in the parent cell.

[0155] The term "homologous" or "homolog" refers to a molecule or activity found in or derived from a host cell, host species or host strain. For example, a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene, and optionally may have altered expression levels, may differ in sequence, may have an altered activity, or any combination thereof.

[0156] Host cells can be transformed to express the nucleic acids of the invention using conventional techniques, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambruck et al. (1989) and other laboratory textbooks. The nucleic acid sequences of the invention may also be chemically synthesized using standard techniques.

[0157] As used herein, the term "host" refers to a cell (e.g., a Treg cell) or microorganism that has been targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., a high or enhanced affinity TCR). In certain embodiments, the host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties (e.g., incorporation of a detectable marker; deletion, alteration, or truncation of an endogenous TCR; increased expression of a costimulatory factor) that are associated or unrelated to the biosynthesis of the heterologous or exogenous protein. In some embodiments, the host cell is genetically modified to express a binding protein described herein.

[0158] Treatment conditions The present invention relates to methods for treating a variety of autoimmune diseases, in particular autoimmune diseases characterized by an aberrant immune response (such as the formation of autoantibodies) against one or more of the proteins SmB / B', SmD, Ro60 and MPO. The methods typically involve administering in an individual in need of treatment a population of Treg cells, where the Treg cells express a TCR binding protein as described herein.

[0159] The phrase "therapeutically effective amount" generally refers to an amount of cells expressing a binding protein or peptide of the invention that (i) treats a particular disease, condition or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition or disorder described herein.

[0160] As used herein, "preventing" or "prevention" is intended to refer to at least a reduction in the likelihood or risk of (or susceptibility to) a disease or disorder (i.e., the failure to develop at least one clinical symptom of the disease in an individual who may have been exposed to or may be predisposed to the disease, but has not yet undergone or exhibited symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are well known to physicians.

[0161] In particularly preferred embodiments, the methods of the invention may prevent or reduce the severity of, or inhibit or minimize the progression, recurrence or symptoms of, a disease or condition described herein. Thus, the methods of the invention have therapeutic as well as prophylactic utility.

[0162] The term "treatment" of a subject or "treating" includes the purpose of delaying, slowing, stabilizing, curing, healing, palliating, alleviating, altering, repairing, ameliorating, improving, or influencing a disease or condition, symptoms of a disease or condition, or the risk of (or susceptibility to) a disease or condition. The term "treating" refers to any indicia of successful treatment or amelioration of an autoimmune disease described herein (e.g., SLE, Sjogren's syndrome, vasculitis, microscopic polyangiitis, or sclerosis), including any objective or subjective parameter, such as palliative; remission; slowing the rate of progression; reducing the severity of the condition; stabilizing, reducing, or making the condition tolerable by an individual; slowing the rate of degeneration or decline; reducing the debilitating nature of the degenerative end point; or improving the physical or mental well-being of the subject.

[0163] It is understood that the methods described herein can also be used in combination with existing standard treatments / standard therapies for the targeted autoimmune disease requiring treatment.Those skilled in the art are familiar with existing standard therapies for the treatment of autoimmune disease requiring treatment, including but not limited to the use of steroids, antimalarials (hydroxychloroquine, chloroquine), immunosuppressants (azathioprine, methotrexate, mycophenolate mofetil, mycophenolic acid, tacrolimus, voclosporin, cyclosporin), kinase inhibitors (baricitinib, tofacitinib, upadacitinib) and biologics (belimumab, rituximab, anifrolumab, ustekinumab, obinutuzumab).The present invention includes the combination of existing standard therapies with the specific methods of the present invention.

[0164] A "subject" herein is preferably a human subject. Although the present invention has application in humans, the present invention is also useful for veterinary purposes. The present invention is useful for domestic or farm animals such as cattle, sheep, horses and poultry; companion animals such as cats and dogs; and zoo animals. The terms "subject" and "individual" are understood to be individuals in need of treatment according to the present invention.

[0165] Systemic lupus erythematosus The present invention includes methods for treating systemic lupus erythematosus, particularly in cases associated with an abnormal immune response against one or more of the proteins SmB / B', SmD or Ro60, or the MPO protein.

[0166] Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. At least 5 million people worldwide have SLE, 90% of those diagnosed are women, and most develop SLE between the ages of 15 and 44. In Australia, SLE is diagnosed in approximately 1 in 1000 people, with more frequent and severe disease in Aboriginal Australians and Asian Australians. SLE patients suffer from chronic immune-mediated inflammatory damage in the brain, kidneys, heart, lungs, joints, skin, and other organs, resulting in a marked shortening of life expectancy, exemplified by a standardized mortality rate of over 3. In a UK cohort, the mean age at death of 14% of patients who died during follow-up was only 52 years. The clinical course is often characterized by episodic flares associated with increasing irreversible organ damage and subsequent death.

[0167] Other forms of lupus include discoid lupus, drug-induced lupus and neonatal lupus Of these, systemic lupus erythematosus (also known as SLE) is the most common and severe form. A more complete classification of lupus includes the following types: acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, discoid lupus erythematosus (chronic cutaneous), childhood discoid lupus erythematosus, generalized discoid lupus erythematosus, localized discoid lupus erythematosus, chilblain-like lupus erythematosus (Hutchinson's disease), lupus erythematosus-lichen planus overlap syndrome, lupus erythematosus panniculitis (lupus erythematosus profundus), tumescent lupus erythematosus, verrucous lupus erythematosus (hypertrophic lupus erythematosus), cutaneous lupus mucinosis, complement deficiency syndrome, drug-induced lupus erythematosus, neonatal lupus erythematosus, and systemic lupus erythematosus.

[0168] Cutaneous lupus erythematosus (CLE) is seen in the majority of SLE cases and is often observed on skin exposed to sunlight, manifesting as variably severe and sometimes disfiguring skin erythema. Lupus can also manifest as a purely cutaneous form, also known as incomplete lupus erythematosus. Although not all factors that lead to the development of SLE and its pattern of periodic exacerbations are known, it is clear that exposure to sunlight is important in the exacerbation of the cutaneous disease as well as the systemic disease.

[0169] Among the symptoms common to patients diagnosed with lupus, nearly all patients have joint pain and / or swelling (i.e., arthritis). The joints most frequently affected are those of the fingers, palms, wrists, and knees. Other common symptoms include pleuritic chest pain, oral and nasal ulcers, fatigue, fever without other causes, general discomfort, anxiety or feeling sick (malaise), hair loss, sensitivity to sunlight, skin erythema ("butterfly" erythema) in about half of SLE sufferers, as well as cicatricial "disciform" lesions and swollen lymph nodes. Those skilled in the art are familiar with the various other important symptoms of lupus, including, but not limited to, nephritis, CNS lesions, blood lesions, gastrointestinal lesions, and vasculitis.

[0170] As used herein, photosensitivity or abnormal photosensitivity in individuals with CLE or SLE includes skin erythema resulting from an aberrant response to sunlight. Beyond the onset of skin erythema, exposure to sunlight may cause individuals living with lupus to experience increased disease activity, with symptoms such as joint pain, weakness, fatigue, and fever. Two-thirds of lupus sufferers have increased sensitivity to ultraviolet light from sunlight or from artificial indoor light, such as fluorescent light, or both.

[0171] Sjögren's syndrome The present invention includes methods for treating Sjogren's syndrome, particularly in cases associated with an abnormal immune response against one or more of the proteins SmB / B', SmD or Ro60, or the MPO protein.

[0172] Sjögren's syndrome is an autoimmune disorder estimated to affect between 0.5% and 1% of the general population, with primary Sjögren's syndrome (pSS) being such an autoimmune disorder, with 9 out of 10 patients with Sjögren's syndrome being women. The majority of women with pSS are characterized by mild to moderate disease with symptoms of fatigue, joint pain, and dry eyes and / or mouth. The disease is characterized by lymphocytic infiltration of the salivary and lacrimal glands with subsequent inflammation, damage, and loss of function of the salivary and lacrimal glands causing dry eyes and dry mouth. Involvement of major organ systems, including lungs, kidneys, and liver, is a common systemic manifestation of pSS. At the biochemical level, pSS is associated with elevated immunoglobulin levels and production of antinuclear antibodies against ribonucleoprotein complexes such as SSA / Ro and SSB / La.

[0173] Fatigue is one of the most common extraglandular symptoms of Sjogren's syndrome and is defined by persistent generalized fatigue. It has been reported that an estimated 70% of pSS patients suffer from profound fatigue, which negatively impacts their quality of life. Serologically, about 80% of these patients have anti-Ro / SSA autoantibodies that bind to autoantigens containing small non-coding RNA molecules. Fatigue can be characterized in terms of intensity, duration, and impact on daily function. Especially in the first-line treatment setting, fatigue is strongly associated with depression. Thus, there is a need for a means to improve fatigue in patients with autoimmune diseases such as Sjogren's syndrome.

[0174] As used herein, the terms "primary Sjogren's syndrome (pSS)", "Sjogren's syndrome", "Sjogren's disease" and "Sjogren's" may be used interchangeably.

[0175] In any embodiment, the treatment of Sjogren's syndrome may include alleviating or reducing fatigue in a subject in need of treatment. A variety of PRO (patient reported outcome) scales have been used and validated in measuring fatigue in subjects with chronic diseases. Such PROs are known in the art and may be used to evaluate the efficacy of treatment with a population of Treg cells as described herein. The European League Against Rheumatism (EULAR) Sjogren's Syndrome (SS) Patient Reported Index (ESSPRI) was developed to evaluate symptoms in patients with primary Sjogren's syndrome (Seror et al., Ann. Rheum. Dis., 2011, 70:968-972). The ESSPRI was developed as a global score to measure all important and disabling symptoms of primary Sjogren's syndrome: dryness, pain in extremities, and fatigue. The ESSPRI has been shown to be sufficient to measure each of the symptoms without losing content validity, and the score is easy to calculate. The ESSPRI is a patient-administered questionnaire that assesses symptoms in patients with primary Sjogren's syndrome. The questionnaire includes three scales, one for each of the following symptoms: (1) dryness, (2) pain in extremities, and (3) fatigue. Each component of the ESSPRI is measured by a single numeric scale ranging from 0 to 10, and the global ESSPRI score is the average of the three scales: (dryness + pain in extremities + fatigue) / 3. A reduction of at least 1 point in the ESSPRI score is clinically meaningful.

[0176] The Functional Assessment by Chronic Illness Therapy Fatigue scale (FACIT-Fatigue) is used to assess an individual's level of fatigue during their usual daily activities over the past week. The FACIT-Fatigue questionnaire and Scoring & Interpretation Materials are available at FACIT.org (Elmhurst, Ill., USA). The FACIT-Fatigue questionnaire provides a series of overall and target scales. The FACIT-Fatigue scale has many benefits, including high internal validity, high test-retest reliability, reliability and sensitivity to change in patients with various chronic health conditions, ease of use, and use in a variety of settings (KF Tennant, Try This: best Practices, Nursing Care to Older Adults, No. 30, 2012; Chandran et al., Ann. Rheum. Dis., 2007, 66:936-939). The FACIT-Fatigue is a 13-item questionnaire originally developed to measure fatigue in patients with cancer and is currently used to measure fatigue in patients with Sjögren's disease. Patients are asked to answer 13 questions rated from 0 to 4 (0=not at all, 1=slightly, 2=somewhat, 3=very much, 4=extremely). The fatigue scale has 13 items with a maximum possible score of 52. A high score on the fatigue scale corresponds to a low level of fatigue and points to an improved quality of life. To calculate the FACIT-Fatigue score, response scores to the negatively worded questions are reversed and then the responses for the 13 items are added together. Eleven items with responses have their scores reversed (if no response is missing, item score = 4-response), and two items (items 7-8) have their responses unchanged. All items are added together with a high score corresponding to a lower level of fatigue. If an individual question is omitted, the score is prorated using the average of the other responses in the scale: FACIT-Fatigue = 13 × [total (reversed items) + total (items 7–8)] / number of items answered.

[0177] The Profile of Fatigue (ProF) was developed to establish a valid assessment tool in characterizing fatigue associated with primary Sjögren's syndrome. The ProF has been shown to be a reliable and valid scale for measuring the severity of fatigue and overall discomfort in patients with primary Sjögren's syndrome. The ProF is a 16-item self-administered questionnaire divided into two domains, one for physical fatigue and one for mental fatigue. The physical fatigue domain includes 12 items divided into four dimensions: (a) need for rest (4 items), (b) poor starting (3 items), (c) reduced stamina (3 items), and (d) muscle weakness (2 items). The mental fatigue domain includes 4 items divided into two dimensions: (a) reduced concentration (2 items), and (b) reduced memory (2 items). Patients rate each item on a scale of 0 to 7 (0 = "no problem at all" to 7 = "the worst possible condition") based on how they felt at their worst during the past two weeks. A score for each dimension may be obtained by adding the scores of the items within each dimension and dividing the sum by the number of items within each dimension. A score for each domain (e.g., physical domain, mental domain) may be obtained by adding the scores of the aspects within each domain and dividing the sum by the number of aspects within each domain. Higher scores indicate increased fatigue (Bowman et al., Rheumatology, 2004, 43:758-764; Strombeck et al., Scand. J. Rheumatol. 2005, 34:455-459; Segal et al., Arthritis Rheum. 2008 Dec. 15, 59(12):1780-1787).

[0178] In some embodiments, patient status is assessed by measuring fatigue in the patient via one or more patient-reported indices (e.g., ESSPRI, PROF, FACIT), compared to the level of fatigue in the patient prior to treatment, or compared to the level of fatigue in similarly affected untreated or control patients.

[0179] For example, a human subject in need of treatment is selected or identified (e.g., a patient who meets the American College of Rheumatology criteria for SLE or a patient who meets the American-European Consensus Sjogren's Classification Criteria). The subject may need relief from the causes or symptoms of SLE or Sjogren's syndrome, such as fatigue. The subject may be identified in a clinical setting or elsewhere, such as in the subject's home, via the subject's own use of a self-test kit. The patient's condition is assessed at baseline (day 1) and after a period following the first administration, such as days 8, 15, 29, 43, 57, 71, 85, 99, or at the end of the study, for example, by the ESSPRI index, PROF, and / or FACIT-Fatigue scale. Other appropriate criteria may also be measured. The number and intensity of administrations are adjusted according to the needs of the subject. After treatment, one or more of the following outcomes are observed: (1) improvement in ESSPRI index compared to ESSPRI index before treatment or compared to similarly affected but untreated subjects / control subjects; (2) improvement in PROF compared to PROF before treatment or compared to similarly affected but untreated subjects / control subjects; (3) improvement in FACIT-Fatigue scale compared to FACIT-Fatigue scale before treatment or compared to similarly affected but untreated subjects / control subjects. In some embodiments, the improvement in ESSPRI index is a clinically meaningful improvement. A clinically meaningful improvement in ESSPRI index is a reduction in ESSPRI score of at least 1 point.

[0180] A variety of neuropsychological assays known in the art can also be used to assess the effectiveness of the treatment methods disclosed herein, including assessing the improvement of Sjogren's syndrome-related cognitive function.For example, the digit symbol substitution test (DSST) provides a valid and sensitive test for measuring cognitive dysfunction that affects many domains.The DSST is sensitive to both the presence of cognitive dysfunction and changes in cognitive function across a range of clinical populations, including patients with Sjogren's syndrome.This neuropsychological test is a widely used, highly validated, and highly sensitive test readout for executive function-related inputs.

[0181] The DSST is a timed, written cognitive test administered on a single sheet of paper. The test requires the patient to match symbols with numbers according to a legend at the top of the form. The patient transfers the symbols into the space below the number sequence, and the number of correct symbols within an allowed time (e.g., 90 or 120 seconds) is calculated. The test provides data on the accuracy and speed of performing the task. Patients' DSST performance correlates with real-life functional outcomes, such as the ability to perform everyday tasks, and recovery from functional incapacity in a range of psychiatric conditions. The DSST test can be used to assess attention and / or concentration in patients.

[0182] The DSST is a multifactorial test that provides a practical and valid way to measure cognitive performance and monitor cognitive function over time. To perform well on the DSST, patients must have intact motor speed, scanning, or writing or drawing abilities (i.e., basic dexterity), attention, and visual perception functions. The DSST provides high sensitivity for detecting cognitive impairment and has many benefits, including brevity, reliability, sensitivity to change, and minimal influence of language, culture, and education on test performance (Jaeger, J., Journal of Clinical Psycopharmacology, 38(5), 513-518, October 2018).

[0183] systemic sclerosis The present invention includes methods for treating systemic sclerosis, particularly in cases associated with an abnormal immune response against one or more of the proteins SmB / B', SmD or Ro60, or the MPO protein.

[0184] Systemic sclerosis (Ssc; also known as scleroderma) is a non-genetic, non-infectious, multi-organ system autoimmune disease that can result in progressive fibrosis of the skin and / or internal organs. The condition is characterized by excess collagen deposition. One form of the condition, known as CREST syndrome (also called localized scleroderma), can result in the following features: calcinosis, Raynaud's phenomenon, esophageal dysmotility, digital sclerosis, and telangiectasia.

[0185] SSc has a wide variety of symptoms, caused by excessive deposition of extracellular matrix in the dermis, resulting in skin fibrosis. In later stages, SSc is characterized by progressive tissue fibrosis, affecting other internal organs, such as the stomach, lungs, or kidneys. Thus, scleroderma is a disease characteristic that also includes, for example, pulmonary fibrosis, renal fibrosis, cardiac, gastric, or vascular fibrosis. It is suggested that inflammation, autoimmune disorders, or vascular damage activate fibroblasts. Fibroproliferation, accompanied by excessive extracellular matrix production dominated by type I collagen, results in progressive tissue fibrosis, which can cause end-organ failure and lead to high morbidity and mortality in patients with end-stage SSc.

[0186] The present invention contemplates the treatment of systemic sclerosis (SSc), diffuse systemic sclerosis (dSSc), localized systemic sclerosis (lSSc), overlapping systemic sclerosis, undifferentiated systemic sclerosis, systemic sclerosis sine scleroderma (ssSSc), dermal fibrosis, scleroderma, nephrogenic fibrosing derma (NFD) and / or keloid formation.

[0187] In some embodiments, an individual in need of treatment for scleroderma is evaluated for inflammation, fibrosis, vasculopathy, and / or autoimmunity. In some embodiments, an individual is evaluated for at least one of the following: skin thickening, skin thickening proximal to the metacarpophalangeal (MCP) joint, skin thickening of the fingers, finger swelling, finger sclerosis, fingertip lesions, fingertip ulcers, pitting scars, telangiectasias, nail fold capillary abnormalities, calcinosis, esophageal dilation, scleroderma renal crisis, interstitial lung disease, pulmonary arterial hypertension and / or interstitial lung disease, Raynaud's phenomenon, associated antibodies (e.g., anti-centromere antibodies, anti-Scl-70 antibodies / anti-topoisomerase antibodies, anti-fibrillary autoantibodies, anti-type III RNA polymerase autoantibodies, anti-Th / To, PM-Scl, anti-Ro, anti-Ul-ribonucleoprotein, etc.), and collagen mRNA levels in the skin. In some embodiments, scleroderma is localized to the skin. In some embodiments, scleroderma involves at least one organ other than the skin. In some embodiments, scleroderma is referred to as systemic sclerosis. In some embodiments, scleroderma is CREST syndrome.

[0188] In some embodiments, the condition is assessed using a plasma sample. In some embodiments, the condition is assessed using a blood sample. In some embodiments, the condition is assessed using a skin biopsy sample. In some embodiments, the condition is assessed using a fibrotic biomarker. In some embodiments, the condition is assessed using detection of condition-specific autoantibodies.

[0189] In some embodiments, the condition is evaluated using the modified Rodnan skin score (MRSS). In some embodiments, the MRSS score for each body site is as follows: 0=no skin lesions; 1=mild thickening; 2=moderate thickening and 3=severe thickening. In some embodiments, the MRSS at a body site is at least 2. In some embodiments, the individual has moderate skin thickening. In some embodiments, skin thickness is measured at 17 different body sites. In some embodiments, the individual has a whole body MRSS score of at least 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 or 51.

[0190] In some embodiments, the condition is assessed using palpation. In some embodiments, the condition is assessed using palpation in 17 different body regions. In some embodiments, the body regions include at least one of the following: fingers, palms, forearms, arms, feet, legs and thighs (bilaterally), and face, chest and abdomen (singlely).

[0191] In some embodiments, the condition is assessed using the localized Scleroderma cutaneous assessment tool (LoSCAT). In some embodiments, LoSCAT assesses 18 anatomical skin sites.

[0192] In some embodiments, LoSCAT captures disease activity (mLoSSI) and damage (LoSDI) parameters. In some embodiments, LoSCAT considers skin lesions resulting from the disease localized scleroderma during the inactive phase of the disease. In some embodiments, the score for each site is based on the most severe score for each parameter. In some embodiments, skin changes are compared to contralateral or ipsilateral skin areas to minimize inter-subject variability.

[0193] In some embodiments, the Leroy / Medsger criteria are used. In some embodiments, the 1988 Leroy / Medsger criteria are used. In some embodiments, the 2001 Leroy / Medsger criteria are used. In some embodiments, the condition is evaluated using the American College of Rheumatology (ACR) criteria. In some embodiments, the condition is evaluated using the European League Against Rheumatism (EULAR) criteria. In some embodiments, the 2013 ACR / EULAR diagnostic criteria are used. In some embodiments, an individual is diagnosed with systemic sclerosis using the 2013 ACR / EULAR diagnostic criteria if they have a total score of at least 9.

[0194] In some embodiments, treatment is evaluated using a change in a score as described herein. In some embodiments, treatment is evaluated using a percentage change in a measurement as described herein. In some embodiments, treatment is evaluated using a change in a remote thermography profile of the skin lesion after treatment. In some embodiments, treatment is evaluated using a change in an ultrasound profile of the target skin lesion after treatment.

[0195] In some embodiments, the condition is assessed using a computed tomography (CT) scan of the lungs. In some embodiments, the condition is assessed by measuring renal function. In some embodiments, the condition is assessed by measuring blood creatinine levels. In some embodiments, the condition is assessed using a pulmonary function test. In some embodiments, the condition is assessed using forced vital capacity (FVC), a measure of lung capacity. In some embodiments, the condition is assessed using diffusion capacity (DLCO), a measure of oxygen exchange in the alveoli.

[0196] In some embodiments, the condition is assessed using the Dermatology Life Quality Index (DLQI). In some embodiments, the condition is assessed using the Health Assessment Questionnaire-Disability Index (HAQ-DI). In some embodiments, the condition is assessed using a physician global assessment (PGA).

[0197] inflammatory myositis The present invention includes methods for treating inflammatory myositis, particularly in cases associated with an abnormal immune response against one or more of the proteins SmB / B', SmD or Ro60, or the MPO protein.

[0198] Inflammatory myositis is a systemic autoimmune disease characterized by muscle inflammation. Examples of inflammatory myositis conditions that can be treated as described herein include, but are not limited to, polymyositis, dermatomyositis, inclusion body myositis, and juvenile myositis.

[0199] Any suitable method can be used to determine whether a subject has an inflammatory myositis condition. For example, a subject (e.g., a human) can be identified as having an inflammatory myositis condition using standard diagnostic methods. In some cases, a tissue biopsy can be collected and analyzed to determine whether a subject has an inflammatory myositis condition.

[0200] Any suitable method can be used to determine whether the severity of the inflammatory myositis condition is reduced following treatment according to the methods of the invention, for example, the severity of the inflammatory myositis condition can be assessed visually, by diagnostic methods, or by patient interview.

[0201] Autoimmune Vasculitis, Including Microscopic Polyangiitis The present invention includes methods for treating autoimmune vasculitis, including microscopic polyangiitis, particularly in cases associated with an aberrant immune response against the MPO protein, or one or more of the proteins SmB / B', SmD or Ro60.

[0202] Autoimmune vasculitis is an autoimmune disease that causes inflammation and narrowing of blood vessels (arteries, veins, and capillaries). In severe cases, the condition can cause organ damage or death. Types of vasculitis are grouped according to the size of the affected vessels. Most types of vasculitis are rare and involve large vessels (polymyalgia rheumatica, Takayasu's arteritis, temporal arteritis (giant cell arteritis)), medium vessels (Buerger's disease, cutaneous vasculitis, Kawasaki disease, polyarteritis nodosa), and small vessels (Behcet's syndrome, Churg-Strauss syndrome, cutaneous vasculitis, Henoch-Schönlein purpura, microscopic polyangiitis, granulomatosis with polyangiitis, Golf's vasculitis, cryoglobulinemia). Vasculitis symptoms may occur once or several times over a period of several years. The disease affects people of all ages, races, and genders.

[0203] The signs and symptoms of vasculitis vary and can range from mild to fatal. The signs and symptoms of vasculitis depend on the type of vasculitis, the organs affected, and how severe the condition is. Some people have few signs and symptoms, while others can be extremely severe. In some cases, symptoms develop slowly over a period of months, while in other cases, signs and symptoms begin rapidly, over a period of days or weeks.

[0204] Common symptoms include fever, loss of appetite, weight loss, fatigue, and generalized aches and pains. Vasculitis can affect various organ systems and body tissues, causing a range of signs and symptoms, including the skin (purpura or erythema or bumps; clusters of small spots, spots, bruises, or hives; itching), joints (pain or arthritis in one or more joints), lungs (shortness of breath; hemoptysis), digestive tract (mouth pain; stomach pain; blockage of blood flow to the intestines, which in severe cases can cause intestinal weakness or rupture), sinuses, nose, throat, and ears (sinus infection or chronic middle ear infection; sinus pain; occasionally hearing loss), eyes (red, itchy, sore eyes; light sensitivity; blurred vision; rarely, blindness), brain (headaches; confused thinking; altered mental function; stroke-like symptoms such as muscle weakness and paralysis), nerves (numbness, tingling and weakness in various parts of the body; loss of feeling or strength in the palms and feet; lancinating pains in the arms and legs). In severe cases, vasculitis can cause blockages in blood vessels, possibly resulting in aneurysms.

[0205] Microscopic polyangiitis is a necrotizing vasculitis involving small caliber vessels that may contribute to glomerular and pulmonary capillary damage contributing to the pulmonary-renal syndrome in addition to the systemic symptoms associated with vasculitis.

[0206] Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies formed against antigens in the cytoplasm of neutrophil granulocytes and monocytes. They are associated with many autoimmune disorders, particularly systemic vasculitis and ANCA-associated vasculitis (AAV). ANCA are divided into four patterns when visualized by immunofluorescence (IF): cytoplasmic ANCA (c-ANCA), C-ANCA (atypical), perinuclear ANCA (p-ANCA) and atypical ANCA (a-ANCA), also known as x-ANCA. c-ANCA shows granular fluorescence in the cytoplasm with central interlobular highlighting. C-ANCA (atypical) shows cytoplasmic staining that is usually uniform and without interlobular highlighting. p-ANCA has three subtypes: classical p-ANCA, p-ANCA without nuclear extension and granulocyte-specific antinuclear antibodies (GS-ANA). Classical p-ANCA shows perinuclear staining with intranuclear extension, p-ANCA without intranuclear extension has perinuclear staining without intranuclear extension, GS-ANA shows intranuclear staining only on granulocytes, and a-ANCA often shows a combination of both cytoplasmic and perinuclear staining.

[0207] p-ANCA antigens include myeloperoxidase (MPO). Currently, the diagnosis of ANCA-positive vasculitis is further based on biopsy, whether it is a skin biopsy, a kidney biopsy, a neuromuscular biopsy, etc. ANCA constitute an important diagnostic aid for systemic vasculitis. Thus, anti-myeloperoxidase (MPO) ANCA is present in 60-75% of patients with microscopic polyangiitis and in 38% of patients with Churg-Strauss syndrome.

[0208] In a preferred embodiment of the invention, the treatment of autoimmune vasculitis includes the treatment of patients with anti-MPO ANCA. Examples of anti-MPO ANCA positive vasculitis include microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), and in rare cases, granulomatosis with polyangiitis (Wegener's granulomatosis). EXAMPLES

[0209] Example 1: Identification of promiscuous TCRs We co-cultured T cells from healthy individuals with six different self-peptides derived from three known different self-antigens (two Smith; two Ro60 and two MPO autoantigens). In all six cultures, the most reactive T cell clone (i.e., TCR) was found to be the same. In more detail,

[0210] Methods Approximately 50 ml of whole blood was obtained via venipuncture into EDTA-coated Vacutainer tubes (BD Biosciences). Mononuclear cells (MNC) were isolated using density centrifugation with Lymphoprep in 50 ml SepMate tubes (StemCell Technologies) according to the manufacturer's protocol. Monocyte isolation was then performed for MNC using the EasySep Human Monocyte Isolation Kit (StemCell Technologies).

[0211] Isolated monocytes were stained with the proliferation dye CellTrace Far Red (Life Technologies), counted, and cultured at 10 cells / ml in ImmunoCult DC Differentiation Medium (StemCell Technologies). 6 Cells were resuspended at 10 cells / well in 96-well polystyrene flat-bottom plates (In-Vitro Technologies). 5 The medium was replaced on day 3 of culture, and dendritic cell maturation supplement (Stemcell Technologies) was added on day 5 of culture according to the manufacturer's instructions.

[0212] On day 7 of monocyte culture, another 50 ml of whole blood was obtained via venipuncture from the same healthy individual expressing both HLA-DR3 and HLA-DR4.5. CD4+ T cells were isolated using the RosetteSep Human CD4+ T Cell Enrichment Kit (Stemcell Technologies). The resulting CD4+ T cells were labeled with CellTrace Violet (ThermoFisher Scientific), resuspended in warmed supplemented RPMI medium (10% human male AB serum, 2% penicillin / streptomycin, 4 mM L-glutamine and 50 μM 2-mercaptoethanol) and transferred to wells containing dendritic cells at a ratio of 1 CD4+ T cells to 1 dendritic cells.

[0213] Co-cultures were supplemented with 100 μg / ml peptide and 80 units / ml IL-2 (StemCell Technologies).

[0214] The peptides used were SmD1:78-92, SmB / B':7-21(HLA-DR3(DRA1 * 01:01+DRB1 * 03:01), Ro60:225-239, Ro60:369-383 (HLA-DR3 (DRA1 * 01:01+DRB1 * 03:01); MPO:453-467 and MPO:724-738 (HLA-DR4.5 (DRA1 * 01:01+DRB1 * He was arrested at 04:05. The sequences of the tested peptides are presented in Table 1 herein.

[0215] On day 6 of culture, cells were harvested and surface stained with BUV496-labeled anti-human CD4 (BD Biosciences), APC-H7-labeled anti-human CD8 (BD Biosciences), BV711-labeled anti-human CD69 (BD Biosciences), AF488-labeled anti-human HLA-DR (Biolegend), and propidium iodide (Sigma).

[0216] After staining, cells were filtered through a 20 μm filter into ice-cold sterile MACS buffer in a sterile polypropylene tube, and then CD4+ CTVlo cells were sorted using a FACS Aria cell sorter from BD Biosciences. Sorted cells were then sent for single-cell TCR10× sequencing.

[0217] All HLA types in healthy individuals: HLA-A * 11:01 / 33:03;HLA-B * 58:01;HLA-C * 03:02;HLA-DRB1 * 03:01 / 04:05;HLA-DRB3 * 02:02;HLA-DRB4 * 01:03;HLA-DPB1 * 04:01 / 05:01;HLA-DPA1 * 01:03 / 02:02;DQB1 * 02:01 / 04:01;DQA1 * The time is 03:03 / 05:01.

[0218] Cultures were performed on different days for different peptides (ie, Sm peptide, Ro60 peptide and MPO peptide).

[0219] result Single cell TCR sequencing revealed that the same Treg-derived TCR (same TCR as defined in Table 1 herein) was the dominant clonotype in all subjects (see FIG. 1). The results indicate that a single TCR recognizes and predominantly expands six different self-peptide-MHC conformations.

[0220] Example 2 To confirm the promiscuous nature of the TCR, the TCR was transduced into a Jurkat T cell line and stimulated individually with Sm, Ro60 and MPO peptides in the presence of DR3 / DR4.5 expressing B-LCL as antigen presenting cells. Positive responses were determined by measuring upregulation of the T cell activation marker CD69, IL-2 production in the supernatant and / or proliferation on transduced Jurkat T cells.

[0221] To determine the effectiveness of the TCRs identified in Example 1 in suppressing anti-Ro60-specific pro-inflammatory responses, a proliferation assay is used to measure the effect of TCR-expressing Tregs on the expansion of pro-inflammatory Ro60-specific conventional T cells (Tconv). The results are expected to show that in the presence of Ro60-Tregs, the number of Tregs against Ro60-specific Tconv is significantly increased. This result means that Ro60-Tregs strongly suppress the expansion of Ro60-specific Tconv cells.

[0222] Cytokine production is then measured and the results are expected to show that in the presence of Ro60-Tregs, an anti-inflammatory response will predominate, i.e., high IL-10, low IFN-g and IL-17A (as in healthy individuals), whereas without Tregs or with only pTregs, a pro-inflammatory response will predominate, i.e., low IL-10, high IFN-gamma and IL-17A (as would be expected in an autoimmune disease patient). These data support that Ro60-Tregs reset the aberrant immune response and restore tolerance to the targeted self-epitopes.

[0223] Example 3: HLA-DR3-restricted Ro60-Tregs halt the progression of Sjögren's syndrome To support the efficacy of Ro60-Tregs in halting disease progression, we devise a new humanized model for Sjogren's syndrome based on a similar published model (Young NA et al., Clin Immunol. 2015 Jan 156(1):1-8).

[0224] In this model, immunocompromised NSG-MHC expression of PBMCs from patients with anti-Ro60 antibodies null Adoptive transfer into mice results in tissue damage measured histologically by infiltration of human T cells and loss of structural integrity of normal tissue in lacrimal and salivary glands. At the onset of disease, mice are given no Treg, polyclonal Treg (pTreg) or Ro60-Treg. Figure 2 provides an outline of the experimental protocol.

[0225] Mice that did not receive Tregs or that received pTregs developed dacryoadenitis and sialadenitis that progressed to destruction of the lacrimal and salivary glands, whereas mice treated with Ro60-Tregs displayed only minimal disease.

[0226] To confirm that the disease observed in this model was mediated by an anti-Ro60 immune response, monocyte-derived DCs isolated from Sjögren's syndrome patients were pulsed with Ro60 peptide and injected into mice 7 days after PBMC transfer. In this way, anti-Ro60-specific T cells were preferentially expanded, accelerating anti-Ro60 T cell-mediated disease.

[0227] Example 4: Upregulation of Treg genes associated with antigen-specific TCR activation Method: HLA-DRB1 * 03:01 / DRB1 *04:05 CD4+ T cells from healthy donors were co-cultured with monocyte-derived dendritic cells (DCs) in the presence of the following individual self-epitopes: SmD1:78-92, SmB / B':7-21, Ro60:369-383, MPO:453-467, MPO:724-738. Expanded T cells were subjected to single-cell sequencing analysis (10x Genomics). To determine whether the clonal expansion of promiscuous self-TCRs is linked to TCR activation, we analyzed single-cell transcriptome data to compare gene expression profiles with other T cells and identify genes associated with TCR activation. In addition, because the identified promiscuous self-TCRs are derived from Tregs, we also identified genes associated with Treg activation.

[0228] Results: As shown in Figure 3, the upregulated genes associated with TCR activation were FOXP3, CTLA4, IL1R2, CCR10, TIGIT, IKZF2, ITGAM, LGALS1, LGALS3, PI16, and IL2RA. The downregulated genes associated with Treg activation were IL13, IFNG, and IL7R.

[0229] Conclusions: These data confirm that each epitope can induce promiscuous self-TCR engagement and activation of Tregs.

[0230] Example 5: Expression of promiscuous self-TCR on human Treg Methods: To study the beneficial immunosuppressive effect of adding promiscuous self-TCR to human Tregs, we developed a lentiviral transduction process that allows the expression of promiscuous self-TCR on the surface of human Tregs. First, the TCR sequence of the promiscuous self-TCR was cloned into a lentiviral vector to obtain a lentiviral stock. Human Tregs were then isolated from PBMCs by magnetic separation and flow sorting, expanded in the presence of anti-CD2, anti-CD3, anti-CD28 and IL-2, and then transduced with lentivirus at an MOI of 30. Transduction efficiency was measured by flow cytometry. GFP expression reflects the transduction efficiency, and Vβ14 expression reflects the percentage of Tregs expressing promiscuous self-TCR on the surface of the cells.

[0231] Results: As shown in Figure 4, the transduction efficiency of the Treg transduction protocol was 30.8%. Among the transduced GFP+ Tregs, 32.3% were positive for Vβ14. In non-transduced GFP- cells, endogenous Vβ14 expression was 3.84%.

[0232] Conclusion: The present inventors demonstrated that it is possible to express the promiscuous self-TCR of the present invention on the surface of human Treg.

[0233] Example 6: Promiscuous self-TCR Tregs bind peptide-HLA dextramers Methods: To confirm that the promiscuous self-TCR on Tregs can bind to Sm, Ro and MPO autoantigens, we used the following peptide-HLA dextramer-SmD1:78-21 / HLA-DRB1 * 03:01, Ro60:369-383 / HLA-DRB1 * 03:01 and MPO:453-567 / HLA-DRB1 * 04:05 was generated and binding was then measured by flow cytometry.

[0234] Results: As shown in Figure 5, Tregs transduced with promiscuous self-TCR expressed SmD1:78-21 / HLA-DRB1 * 03:01 Dextramer, Ro60:369-383 / HLA-DRB1 * 03:01 Dextramer and MPO: 453-567 / HLA-DRB1 * 04:05 Exhibits binding to dextramer.

[0235] Conclusion: Tregs transduced with promiscuous self-TCRs are able to promiscuously bind to Sm, Ro60 and MPO autoantigens.

[0236] Example 7: Promiscuous self-TCR Tregs are activated following peptide stimulation Methods: To confirm that promiscuous self-TCR Tregs are activated by peptide stimulation, we transduced promiscuous self-TCR-transduced Tregs with HLA-DRB1 expressing B-LCL. * 03:01 / HLA-DRB1 * Co-culture with 04:05 and SmD1:78-92, Ro60:369-383 or MPO:453-467, and then the early T cell activation marker CD69 and the Treg-specific activation marker GARP were measured by flow cytometry. Upregulation of CD69 or GARP was compared to mock-transduced Tregs.

[0237] Results: As shown in FIG. 6, stimulation with SmD1:78-92, Ro60:369-383 and MPO:453-467 induced activation of promiscuous self-TCR transduced Tregs compared to no peptide stimulation.

[0238] Conclusion: Promiscuous self-TCR-transduced Tregs are activated when stimulated with Sm, Ro60 and MPO autoantigens.

[0239] Example 8: Promiscuous self-TCR engages Sm peptide Methods: To confirm that promiscuous self-TCR can engage Sm peptide-HLA complexes, we used imaging flow cytometry to identify promiscuous self-TCR expressing J76 T cells and HLA-DRB1 expressing B-LCL as antigen-presenting cells. * The immune synapse between the 0.01 and 0.03 peptides was visualized. BLCLs were pulsed with SmD1:78-92 or SmB / B':7-21 without peptide for 2 hours in serum-free RPMI at 37°C. Pulsed B-LCLs were then mixed with promiscuous self-TCR-transduced J76 cells at a ratio of 1:1 in serum-free RPMI and incubated for 2 hours at 37°C. Cells were then fixed and stained with an antibody / phalloidin cocktail containing anti-HLA-DR BV421 (clone G46-6; BD), anti-CD3e PE (clone OKT3; Invitrogen) and phalloidin AF647 (Invitrogen) in permeabilization buffer. Cells were resuspended in PBS / propidium iodide, which was added immediately prior to analysis to identify and quantify the fluorescence of immune synapses of B-LCL-J76 doublets using Ideas Software ver.6.2 (Luminex) following acquisition on an Amnis Imagestream X Mark II imaging flow cytometer (Luminex). Increases in mean pixel intensity of CD3 or phalloidin at the immune synapse represent TCR binding and activation.

[0240] Results: As shown in FIG. 8, pulsing B-LCLs with the Sm epitope increased the mean pixel intensity of CD3 and phalloidin at the immune synapse.

[0241] Conclusion: Promiscuous self-TCR is a type of TCR that is highly specific to any HLA-DRB1 antigen. * 03:01 It can also engage the restricted Sm epitope.

[0242] It is understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

Claims

1. A pharmaceutical composition for use in treating an autoimmune or inflammatory disease, comprising a population of regulatory T (Treg) cells expressing a binding protein on the cell surface, the binding protein comprises a T cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; the Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 16, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 16; The autoimmune disease or inflammatory disease is - Ro60 protein; MPO protein; - Ro60 and MPO proteins; Smith protein and Ro60 protein; Smith protein and MPO protein, or Smith protein, Ro60 protein and MPO protein 10. A pharmaceutical composition characterized by an abnormal or inappropriate immune response to

2. an autoimmune or inflammatory disease, (i) Smith protein; (ii) Ro60 protein, or Smith protein and Ro60 protein; (iii) MPO protein, or Ro60 protein and MPO protein, or (vi) Smith protein, Ro60 protein, and MPO protein The pharmaceutical composition of claim 1, characterized by an abnormal or inappropriate immune response to

3. 2. The pharmaceutical composition of claim 1, wherein the abnormal or inappropriate immune response against Smith protein, Ro60 protein, and / or MPO protein comprises the formation of autoantibodies against Smith protein, Ro60 protein, and / or MPO protein.

4. 2. The pharmaceutical composition according to claim 1, wherein the autoimmune disease or inflammatory disease is one or more autoimmune diseases or inflammatory diseases selected from the group consisting of systemic lupus erythematosus (SLE), lupus nephritis, Sjogren's syndrome, systemic sclerosis, inflammatory myositis, inflammatory rheumatoid arthritis, autoimmune vasculitis, and microscopic polyangiitis.

5. an autoimmune or inflammatory disease, (i) SLE or lupus nephritis, (ii) Sjögren's syndrome, or (iii) Vasculitis The pharmaceutical composition according to claim 4, wherein

6. The pharmaceutical composition described in claim 5, wherein the autoimmune disease or inflammatory disease is primary Sjogren's syndrome or microscopic polyangiitis (MPA).

7. 5. The pharmaceutical composition of claim 4, wherein the subject is in need of treatment for SLE and Sjogren's syndrome, or SLE and MPA, or Sjogren's syndrome and MPA, or all three of SLE, Sjogren's syndrome and MPA.

8. A pharmaceutical composition for use in treating systemic lupus erythematosus (SLE) or lupus nephritis in a subject, comprising a population of regulatory T (Treg) cells expressing a binding protein on their cell surface, comprising: the binding protein comprises a T cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; the Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 16, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 16; The SLE or lupus nephritis is Ro60 protein or Smith protein and Ro60 protein 10. A pharmaceutical composition characterized by an abnormal or inappropriate immune response to 9. A pharmaceutical composition for use in treating Sjogren's syndrome in a subject, comprising a population of regulatory T (Treg) cells that express a binding protein on their cell surface, comprising: the binding protein comprises a T cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; the Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 16, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 16; Pharmaceutical compositions.

10. A pharmaceutical composition for use in treating autoimmune vasculitis in a subject, comprising a population of regulatory T (Treg) cells expressing a binding protein on their cell surface, comprising: the binding protein comprises a T cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain; the Vα domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO:13, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:13; the Vβ domain comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 16, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 16; Pharmaceutical compositions.

11. The pharmaceutical composition described in claim 10, wherein the autoimmune vasculitis is microscopic polyangiitis (MPA).

12. 9. The pharmaceutical composition of claim 8, which is also for use in treating Sjogren's syndrome and / or MPA in a subject.

13. 10. The pharmaceutical composition of claim 9, which is also for use in treating SLE, lupus nephritis and / or MPA in a subject.

14. 11. The pharmaceutical composition of claim 10, which is also for use in treating SLE, lupus nephritis and / or Sjogren's syndrome in a subject.

15. The pharmaceutical composition according to any one of claims 9 and 11 to 14, wherein Sjogren's syndrome or MPA is characterized by an abnormal immune response in the subject against one or more of Smith protein, Ro60 protein, and MPO protein.

16. 16. The pharmaceutical composition of claim 15, wherein the abnormal immune response comprises the formation of autoantibodies in the subject against one or more of Smith protein, Ro60 protein, and MPO protein.

17. The binding protein is a Vα domain comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO:11, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:11; and a Vα domain comprising a CDR2 having an amino acid sequence set forth in SEQ ID NO:12, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:12; and a Vβ domain comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO:14, or a sequence which is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:14; and a Vβ domain comprising a CDR2 having an amino acid sequence set forth in SEQ ID NO:15, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:15; The pharmaceutical composition according to any one of claims 1 to 14, comprising:

18. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the binding protein comprises a Vα domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13, and a Vβ domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15 and 16.

19. the binding protein comprises a Vα domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13, and a Vβ domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15 and 16; 4. The pharmaceutical composition of claim 3, wherein the abnormal or inappropriate immune response against Smith protein, Ro60 protein, and MPO protein comprises the formation of autoantibodies against Smith protein, Ro60 protein, and MPO protein.

20. the binding protein comprises a Vα domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13, and a Vβ domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15 and 16; 10. The pharmaceutical composition of claim 9, wherein Sjogren's syndrome is characterized by an abnormal immune response in the subject against one or more of Smith protein, Ro60 protein, and MPO protein.

21. the binding protein comprises a Vα domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13, and a Vβ domain having CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15 and 16; 11. The pharmaceutical composition of claim 10, wherein MPA is characterized by an abnormal immune response in the subject against one or more of Smith protein, Ro60 protein, and MPO protein.

22. the binding protein comprises a TCR alpha chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23, with 1 to 10 amino acid insertions, deletions, substitutions, additions or combinations thereof outside the indicated CDR sequences; the binding protein comprises a TCR β chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24, with 1-10 amino acid insertions, deletions, substitutions, additions, or combinations thereof, outside the indicated CDR sequences; or the binding protein comprises a TCR alpha chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23 and a TCR beta chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 24, The pharmaceutical composition according to any one of claims 1 to 14.

23. The binding protein is (i) A complex of a fragment of Smith protein, Ro60 protein, or MPO protein with an HLA-DR3 molecule or an HLA-DR4 molecule. (ii) a complex of a fragment of Smith protein and an HLA-DR3 molecule, wherein the fragment of Smith protein comprises or consists of the amino acid sequence of residues 78-92 of SmD1 protein or residues 7-21 of SmB / B' protein (set forth in SEQ ID NOS: 1 and 3, respectively), or an amino acid sequence equivalent to said amino acids; (iii) a complex of a fragment of Ro60 protein and an HLA-DR3 molecule, wherein the fragment of Ro60 protein comprises or consists of the amino acid sequence of residues 225 to 239 or residues 369 to 383 of Ro60 as set forth in SEQ ID NOs: 5 and 6, respectively, or an amino acid sequence equivalent to said amino acid sequence; or (iv) a complex of a fragment of the MPO protein and an HLA-DR4 molecule, wherein the fragment of the MPO protein comprises or consists of the amino acid sequence of residues 453-467 or residues 724-738 of MPO set forth in SEQ ID NOs: 8 and 9, respectively, or an amino acid sequence equivalent to said amino acid sequence; The pharmaceutical composition according to any one of claims 1 to 14, which is capable of binding to

24. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the TCR alpha and TCR beta chains are modified to include cysteine ​​residues that allow for the formation of additional interchain disulfide bonds, preferably wherein the residue at Thr48 on the TCR alpha chain, or a residue equivalent thereto, and the residue at Ser57 on the TCR beta chain, or a residue equivalent thereto, are substituted by cysteine ​​to facilitate the creation of additional disulfide bonds between the TCR constant regions.

25. The population of Treg cells is derived from a subject in need of treatment. the population of Treg cells is derived from stem cells, preferably said stem cells being induced pluripotent stem cells (iPSCs) or embryonic stem cells, or The population of Treg cells is derived from a mixed population of T cells into which a nucleic acid encoding a binding protein as defined in any one of claims 1 to 14 has been introduced. The pharmaceutical composition according to any one of claims 1 to 14.

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