Compositions and methods for treating lupus

Abstract

New or improved treatments for systemic lupus erythematosus are provided. The present invention relates to compositions and methods for the treatment of lupus, particularly systemic lupus erythematosus. The invention involves, inter alia, a binding protein comprising a T-cell receptor (TCR) α chain variable (Vα or V alpha) domain and a TCR β chain variable (Vβ or V beta) domain, which is capable of binding to a complex of a fragment of Smith protein and an HLA-DR15 molecule or an HLA-DR3 molecule.

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for treating lupus, particularly systemic lupus erythematosus.

[0002] Related Applications This application claims priority to Australian Provisional Patent Application No. 2020900864, ​​the contents of which are incorporated herein by reference. [Background technology]

[0003] Systemic lupus erythematosus (SLE) is a chronic, inflammatory autoimmune disease characterized by the production of autoantibodies with specificity against a wide range of autoantigens. SLE autoantibodies mediate organ damage by directly binding to host tissues, depositing in vascular tissues, and forming immune complexes that activate immune cells. Organs targeted in SLE include the skin, kidneys, vasculature, joints, mucous membranes and serous membranes, various blood elements, and the central nervous system (CNS). Disease severity, the spectrum of clinical interventions, and response to treatment vary significantly among patients. This clinical heterogeneity makes lupus difficult to diagnose and manage.

[0004] Due to the great clinical heterogeneity and idiopathic nature of SLE, the management of idiopathic SLE depends on its specific symptoms and severity. Therefore, medications suggested for treating SLE are generally not effective for treating all symptoms of SLE and the complications that arise from them, such as lupus nephritis (LN). LN typically occurs early in the disease course, within five years of diagnosis. The pathogenesis of LN is thought to result from the deposition of immune complexes in the renal glomeruli, which induce an inflammatory response. An estimated 30-50% of patients with SLE develop nephritis, requiring medical evaluation and treatment. LN is a progressive disease that follows a course of clinical exacerbations and clinical remissions.

[0005] Despite significant research into SLE, 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 susceptibility to opportunistic infections, irreversible ovarian failure, baldness, and increased risk of malignancies. Infectious complications occurring concomitantly with active SLE and its treatment with immunosuppressive medications are one of the most common causes of death in patients with SLE. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need for new or improved treatments for SLE. [Means for solving the problem]

[0008] The reference herein to any prior art 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.

[0009] (Summary of the Invention) In one aspect, the invention provides 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 capable of binding to a complex of a fragment of Smith protein and an HLA-DR15 molecule or an HLA-DR3 molecule. Preferably, the HLA-DR15 molecule is an HLA-DR3 molecule. * 01:01 molecule and HLA-DRB1 * Preferably, HLA-DR3 is a 15:01 molecule. * 01:01 molecule and HLA-DRB1 * 03:01 molecule.

[0010] For example, a binding protein of the invention may bind to a peptide consisting of 4, 5, 6, 7, 8, 9, 10 or more consecutive amino acid residues of the sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 258 or 259.

[0011] In one embodiment, a fragment of Smith protein capable of forming a complex with an HLA-DR15 molecule comprises or consists of the amino acid sequence of residues 6-14 or 62-70 of SmB / B' protein or an equivalent amino acid sequence, and preferably, the SmB' protein comprises the sequence of SEQ ID NO: 5. In one embodiment, a fragment of SmB / B' protein comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 4.

[0012] In one embodiment, a fragment of Smith protein capable of forming a complex with an HLA-DR15 molecule comprises or consists of the amino acid sequence of residues 1 to 15 of SmB / B' protein or an amino acid sequence equivalent thereto, and preferably, the SmB' protein comprises the sequence of SEQ ID NO: 5. In one embodiment, a fragment of SmB / B' protein comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0013] In one embodiment, a fragment of Smith protein capable of forming a complex with an HLA-DR15 molecule comprises or consists of the amino acid sequence of residues 58 to 72 of SmB / B' protein or an equivalent amino acid sequence, and preferably, the SmB' protein comprises the sequence of SEQ ID NO: 5. In one embodiment, the SmB / B' protein comprises the amino acid sequence of SEQ ID NO: 2.

[0014] In any embodiment, a fragment of Smith protein capable of forming a complex with an HLA-DR3 molecule comprises or consists of the amino acid sequence of residues 78 to 92 of SmD1 protein or an amino acid sequence equivalent thereto, and preferably the SmD1 protein comprises the sequence of SEQ ID NO: 260. In one embodiment, a fragment of SmD1 protein comprises or consists of the amino acid sequence of SEQ ID NO: 258.

[0015] In one embodiment, a fragment of Smith protein capable of forming a complex with an HLA-DR3 molecule comprises or consists of the amino acid sequence of residues 7 to 21 of SmB / B' protein or an equivalent amino acid sequence, and preferably, the SmB' protein comprises the sequence of SEQ ID NO: 5. In one embodiment, a fragment of SmB / B' protein comprises or consists of the amino acid sequence of SEQ ID NO: 259.

[0016] In any embodiment, the fragment of Smith protein capable of forming a complex with an HLA-DR15 molecule comprises or consists of any one or more of the amino acid sequences of SEQ ID NOs: 1-4.

[0017] In any embodiment, the fragment of Smith protein capable of forming a complex with an HLA-DR3 molecule comprises or consists of the amino acid sequence of SEQ ID NO:258 or 259.

[0018] In another aspect, the present invention also provides 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 Vα domain comprises any "CDR alpha" or any Vα domain amino acid sequence (TRA) as defined in any one of Tables 1 to 4 herein, and / or the Vβ domain comprises any "CDR beta" or any Vβ domain amino acid sequence (TRB) as defined in any one of Tables 1 to 4 herein.

[0019] In another aspect, the present invention also provides 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, the Vα domain comprises a CDR3 comprising an amino acid sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 8, 20, 32, 44, 56, 68, 80, 92, 104, 107, 122, 134, 146, 158, 170, 182, 194, 197, 212, 224, 236, and 248; and / or the Vβ domain comprises a CDR3 comprising an amino acid sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 11, 23, 35, 47, 59, 71, 83, 95, 110, 125, 137, 149, 161, 173, 185, 200, 215, 227, 239, and 251; The binding protein is capable of binding to a complex of a fragment of the Smith protein and an HLA-DR15 molecule. Binding proteins are also provided.

[0020] In another aspect, the present invention also provides 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, the Vα domain comprises a CDR3 comprising an amino acid sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 263, 275, 287, 299, 311, 323, 335, 347, 359, 371, 383, 395, 407, 419, 431, 443, 455, 467, 479, and 491; and / or the Vβ domain comprises a CDR3 comprising an amino acid sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 266, 278, 290, 302, 314, 326, 338, 350, 362, 374, 386, 398, 410, 422, 434, 446, 458, 470, 482, and 494; The binding protein is capable of binding to a complex of a fragment of the Smith protein and an HLA-DR3 molecule. Binding proteins are also provided.

[0021] In another aspect, the present invention also provides 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, the Vα domain comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 20, 32, 44, 56, 68, 80, 92, 104, 107, 122, 134, 146, 158, 170, 182, 194, 197, 212, 224, 236 and 248; and / or the Vβ domain comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 11, 23, 35, 47, 59, 71, 83, 95, 110, 125, 137, 149, 161, 173, 185, 200, 215, 227, 239, and 251; The binding protein is capable of binding to a complex of a fragment of the Smith protein and an HLA-DR15 molecule. Binding proteins are also provided.

[0022] In a particularly preferred embodiment, the Vα domain comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 20 or 32, and the Vβ domain comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 11, 23 or 35.

[0023] In another aspect, the present invention also provides 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, the Vα domain comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 263, 275, 287, 299, 311, 323, 335, 347, 359, 371, 383, 395, 407, 419, 431, 443, 455, 467, 479 and 491; and / or the Vβ domain comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 266, 278, 290, 302, 314, 326, 338, 350, 362, 374, 386, 398, 410, 422, 434, 446, 458, 470, 482, and 494; The binding protein is capable of binding to a complex of a fragment of the Smith protein and an HLA-DR3 molecule. Binding proteins are also provided.

[0024] In a particularly preferred embodiment, the Vα domain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO:263 and the Vβ domain comprises a CDR3 comprising the amino acid sequence of SEQ ID NO:266.

[0025] In another aspect, the present invention also provides 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, T cell receptor (TCR) α chain variable (Vα or V alpha) domain (i) a complementarity determining region (CDR) 1 that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 90, 102, 105, 120, 132, 144, 156, 168, 180, 192, 195, 210, 222, 234, or 246; SEQ ID NO: 7, 19, 31, 43, 55, 67, 79, 91, 103, 106, 121, 133, 145, 157, 169, 181, 193, 196, 211, 223, 235, or 247 and a CDR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 92, 104, 107, 122, 134, 146, 158, 170, 182, 194, 197, 212, 224, 236, or 248; or (ii) CDR1 comprising the sequence set forth in SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 90, 102, 105, 120, 132, 144, 156, 168, 180, 192, 195, 210, 222, 234, or 246; CDR2 comprising the sequence set forth in SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 92, 104, 107, 122, 134, 146, 158, 170, 182, 194, 197, 212, 224, 236, or 248. Including, The TCR β chain variable (Vβ or V beta) domain (i) a CDR1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 9, 21, 33, 45, 57, 69, 81, 93, 108, 123, 135, 147, 159, 171, 183, 198, 213, 225, 237, or 249; a CDR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 11, 23, 35, 47, 59, 71, 83, 95, 110, 125, 137, 149, 161, 173, 185, 200, 215, 227, 239, or 251; or (ii) a CDR1 comprising the sequence set forth in SEQ ID NO: 9, 21, 33, 45, 57, 69, 81, 93, 108, 123, 135, 147, 159, 171, 183, 198, 213, 225, 237, or 249; a CDR2 comprising the sequence set forth in SEQ ID NO: 10, 22, 34, 46, 58, 70, 82, 94, 109, 124, 136, 148, 160, 172, 184, 199, 214, 226, 238, or 250; and a CDR3 comprising the sequence set forth in SEQ ID NO: 11, 23, 35, 47, 59, 71, 83, 95, 110, 125, 137, 149, 161, 173, 185, 200, 215, 227, 239, or 251. Contains Binding proteins are also provided.

[0026] In another aspect, the present invention also provides 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, T cell receptor (TCR) α chain variable (Vα or V alpha) domain (i) a complementarity determining region (CDR) 1 that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 261, 273, 285, 297, 309, 321, 333, 345, 357, 369, 381, 393, 405, 417, 429, 441, 453, 465, 477, or 489; or SEQ ID NO: 262, 274, 286, 298, 310, 322, 334, 346, 358, 370, 382, ​​394, 406, 418, 430, 442, 454, 466, 478, or 490. and a CDR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NOs: 263, 275, 287, 299, 311, 323, 335, 347, 359, 371, 383, 395, 407, 419, 431, 443, 455, 467, 479, and 491; or (ii) CDR1 comprising the sequence set forth in SEQ ID NO: 261, 273, 285, 297, 309, 321, 333, 345, 357, 369, 381, 393, 405, 417, 429, 441, 453, 465, 477, or 489; CDR2 comprising the sequences set forth in SEQ ID NOs: 94, 406, 418, 430, 442, 454, 466, 478 or 490; and CDR3 comprising the sequences set forth in SEQ ID NOs: 263, 275, 287, 299, 311, 323, 335, 347, 359, 371, 383, 395, 407, 419, 431, 443, 455, 467, 479 and 491. Including, The TCR β chain variable (Vβ or V beta) domain (i) a CDR1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 264, 276, 288, 300, 312, 324, 336, 348, 360, 372, 384, 396, 408, 420, 432, 444, 456, 468, 480, or 492; SEQ ID NO: 265, 277, 289, 301, 313, 325, 337, 349, 361, 373, 385, 397, 409, 421, 433, 445, 457, 469, 481, or 493 and a CDR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NOs: 266, 278, 290, 302, 314, 326, 338, 350, 362, 374, 386, 398, 410, 422, 434, 446, 458, 470, 482, and 494; or (ii) CDR1 comprising the sequence set forth in SEQ ID NO: 264, 276, 288, 300, 312, 324, 336, 348, 360, 372, 384, 396, 408, 420, 432, 444, 456, 468, 480, or 492; SEQ ID NO: 265, 277, 289, 301, 313, 325, 337, 349, 361, 373, 385, CDR2 comprising the sequence set forth in SEQ ID NOs: 397, 409, 421, 433, 445, 457, 469, 481, or 493; and CDR3 comprising the sequence set forth in SEQ ID NOs: 266, 278, 290, 302, 314, 326, 338, 350, 362, 374, 386, 398, 410, 422, 434, 446, 458, 470, 482, and 494. Contains Binding proteins are also provided.

[0027] In another aspect, the present invention also provides 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, CDR1 in which the Vα domain comprises any one of the amino acid sequences of SEQ ID NOs: 6, 18, 30, 42, 54, 66, 78, 90, 102, 105, 120, 132, 144, 156, 168, 180, 192, 195, 210, 222, 234, and 246; , 181, 193, 196, 211, 223, 235 and 247; a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 20, 32, 44, 56, 68, 80, 92, 104, 107, 122, 134, 146, 158, 170, 182, 194, 197, 212, 224, 236 and 248; and / or CDR1 in which the Vβ domain comprises any one of the amino acid sequences of SEQ ID NOs: 9, 21, 33, 45, 57, 69, 81, 93, 108, 123, 135, 147, 159, 171, 183, 198, 213, 225, 237, and 249; 172, 184, 199, 214, 226, 238, and 250; and CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 11, 23, 35, 47, 59, 71, 83, 95, 110, 125, 137, 149, 161, 173, 185, 200, 215, 227, 239, and 251, The binding protein is capable of binding to a complex of a fragment of the Smith protein and an HLA-DR15 molecule. Binding proteins are also provided.

[0028] In another aspect, the present invention also provides 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, CDR1 wherein the Vα domain comprises any one of the amino acid sequences of SEQ ID NOs: 261, 273, 285, 297, 309, 321, 333, 345, 357, 369, 381, 393, 405, 417, 429, 441, 453, 465, 477, or 489; 6, 418, 430, 442, 454, 466, 478 or 490; a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 263, 275, 287, 299, 311, 323, 335, 347, 359, 371, 383, 395, 407, 419, 431, 443, 455, 467, 479 and 491; and / or CDR1 wherein the Vβ domain comprises any one of the amino acid sequences of SEQ ID NOs: 264, 276, 288, 300, 312, 324, 336, 348, 360, 372, 384, 396, 408, 420, 432, 444, 456, 468, 480, or 492; 409, 421, 433, 445, 457, 469, 481 or 493; and CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 266, 278, 290, 302, 314, 326, 338, 350, 362, 374, 386, 398, 410, 422, 434, 446, 458, 470, 482 and 494, The binding protein is capable of binding to a complex of a fragment of the Smith protein and an HLA-DR3 molecule. Binding proteins are also provided.

[0029] In another aspect, the present invention also provides 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, a T cell receptor (TCR) α chain variable (Vα or V alpha) domain comprising CDR1, 2 and 3 according to Table 1 or 2; and / or a T cell receptor (TCR) β chain variable (Vβ or V beta) domain comprising CDR1, 2, and 3 according to Table 1 or 2; Binding proteins are also provided.

[0030] Preferably, the binding protein comprises a TCR1, 2 or 3 sequence according to Table 1 or a TCR1 sequence according to Table 2.

[0031] In any embodiment, the binding protein has a TCR alpha chain comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 501, 503, 505, 507, 509, 511, 513, 515, 517, 518, 520, 522, 524, 526, 528, 530, 532, 533, 535, 537, 539, and 541; and / or a TCR beta chain comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 502, 504, 506, 508, 510, 512, 514, 516, 519, 521, 523, 525, 527, 529, 531, 534, 536, 538, 540, and 542, or any combination thereof.

[0032] In any embodiment, the binding protein has a TCR alpha chain comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621 and 623; and / or a TCR beta chain comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622 and 624, or any combination thereof.

[0033] In any embodiment of the invention, the binding protein comprises a TCR α chain comprising a Vα domain and a TCR β chain comprising a Vβ domain. Preferably, the TCR α chain and the TCR β chain are modified to include cysteine ​​residues that allow for the formation of additional interchain disulfide bonds. The cysteines introduced into each of the TCR α chain and the TCR β chain allow for preferential pairing of the TCR α chain with the TCR β chain when expressed in a cell expressing endogenous TCR α chain and endogenous TCR β chain. Preferably, the residue at Thr48 or an equivalent residue on the TCR α chain and the residue at Ser57 or an equivalent residue on the TCR β chain are replaced with cysteines to facilitate the creation of additional disulfide bonds between the constant regions of the TCRs.

[0034] In another aspect, the present invention provides a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of residues 1-15 or 58-72 of SmB / B' protein, or an amino acid sequence equivalent thereto. In one embodiment, the SmB' protein comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, and 4, preferably the amino acid sequence set forth in SEQ ID NO: 3 or 4.

[0035] In any embodiment, the peptide of the present invention is capable of binding to or forming a complex with an HLA-DR15 molecule, and preferably the HLA-DR15 molecule is HLA-DR15. * 01:01 molecule and HLA-DRB1 * 15:01 molecule.

[0036] In another aspect, the present invention provides a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of residues 7-21 of SmB / B' protein, or an amino acid sequence equivalent thereto. In one embodiment, the SmB' protein comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 259.

[0037] In another aspect, the present invention provides a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of residues 78-92 of SmD1 protein, or an amino acid sequence equivalent thereto. In one embodiment, the SmB / B' protein comprises the amino acid sequence of SEQ ID NO: 260. In one embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 258.

[0038] In any embodiment, the peptide of the present invention is capable of binding to or forming a complex with an HLA-DR3 molecule, and preferably the HLA-DR3 molecule is HLA-DR3. * 01:01 molecule and HLA-DRB1 * 03:01 molecule.

[0039] In another aspect, the invention provides a nucleic acid comprising, consisting essentially of, or consisting of a nucleotide sequence encoding a binding protein or peptide of the invention.

[0040] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding a binding protein or peptide of the present invention. Typically, the vector allows for expression of the nucleotide sequence in a cell, resulting in display of the binding protein on the surface of the cell. The vector may be a retroviral vector, preferably a lentiviral vector. Typically, the vector allows for expression of the nucleotide sequence in T cells, preferably helper T cells, e.g., CD4+ T cells. The CD4+ T cells may be CD4+CD25high T cells.

[0041] In one embodiment, the vector comprises a nucleic acid of the invention operably linked to a promoter.

[0042] In embodiments of the invention directed to binding proteins with a single polypeptide chain, the expression construct may include a promoter linked to the nucleic acid encoding the polypeptide chain.

[0043] In embodiments of the invention directed to multiple polypeptide chains forming a binding protein, the vector comprises, for example, a nucleic acid encoding a polypeptide comprising Vα operably linked to a promoter and a nucleic acid encoding a polypeptide comprising Vβ operably linked to a promoter.

[0044] In another example, the expression construct comprises the following components operably linked, for example, in 5' to 3' order: (i) Promoter (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site; and (iv) a nucleic acid encoding a second polypeptide; A bicistronic expression construct in which the first polypeptide comprises Vα and the second polypeptide comprises Vβ, or vice versa. Preferably, the vector allows for translation of the nucleotide sequence encoding Vβ before translation of the nucleotide sequence encoding Vα.

[0045] In any embodiment, the vector of the invention comprises: (i) EF1α (alpha) promoter; (ii) 2A ribosomal skipping sequence; (iii) woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); (iv) a TCR β chain variable (Vβ or V beta) domain is translated before the (TCR) α chain variable (Vα or V alpha) domain; or (v) An arrangement in which the TCR β chain variable (Vβ or V beta) chain is translated before the (TCR) α chain variable (Vα or V alpha) chain. It may include any one or more or all of the following:

[0046] Preferably, the vector is a lentiviral vector. Even more preferably, the lentiviral vector has any one or more or all of the features shown in Figure 4.

[0047] In another embodiment, the present invention also contemplates separate vectors, one of which encodes a first polypeptide comprising Vα and another of which encodes a second polypeptide comprising Vβ. (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a Vα operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a Vβ operably linked to a promoter; Also provided is a composition comprising:

[0048] In another aspect, the invention provides a cell comprising a vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified, or recombinant. In one example, the cell comprises a vector or nucleic acid of the invention. (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a Vα operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a Vβ operably linked to a promoter; Including, In this case, the first polypeptide and the second polypeptide associate to form the binding protein of the present invention. Preferably, the cell is a T cell, more preferably a helper T cell, e.g., a CD4+ T cell. The CD4+ T cell can be a CD4+ CD25high T cell.

[0049] In another embodiment, the invention provides a cell expressing a binding protein of the invention on its surface. Preferably, the cell is a T cell, more preferably a CD4+ T cell. The CD4+ T cell can be a CD4+ CD25high T cell.

[0050] In another aspect, the present invention provides a method of preparing a population of regulatory T cells for use in treating SLE, comprising: providing a population of regulatory T cells; introducing a nucleic acid or vector of the invention into a population of regulatory T cells; Providing conditions that allow expression of the binding protein on the surface of regulatory T cells. thereby preparing a population of regulatory T cells for use in treating SLE.

[0051] In another aspect, the present invention provides a method of treating SLE in a subject, comprising: administering to the subject an effective amount of regulatory T cells that express on their 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, the binding protein being capable of binding to a complex of a fragment of Smith protein and an HLA-DR15 molecule or an HLA-DR3 molecule, thereby treating SLE in the subject. Preferably, the binding protein is any of the binding proteins of the invention described herein.

[0052] In another aspect, the present invention provides a method for preparing ex vivo a Smith protein-specific T cell population exhibiting at least one characteristic of a regulatory T cell, comprising: providing a population of T cells exhibiting at least one characteristic of regulatory T cells; introducing a nucleic acid or vector of the invention into a population of T cells, wherein the nucleic acid or vector encodes a binding protein of the invention; Providing conditions that allow expression of the binding protein on the surface of the T cells. wherein the Smith protein-specific T cell population exhibits at least one characteristic of a regulatory T cell is prepared ex vivo. 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.

[0053] T cells exhibiting at least one characteristic of regulatory T cells for use in the methods or uses of the invention may be selected from a subject diagnosed with SLE or from a healthy subject. The T cells may be isolated from a histocompatible donor.

[0054] In an alternative embodiment, the invention provides a method for preparing ex vivo a Smith protein-specific T cell population 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 the T cell population being a mixed T cell population; introducing a nucleic acid or vector of the invention into a population of T cells, wherein the nucleic acid or vector encodes a binding protein of the invention; providing conditions that allow expression of the binding protein on the surface of the T cells; Providing conditions that allow for the conversion of a T cell population into regulatory T cells The present invention provides a method for preparing ex vivo a Smith protein-specific T cell population exhibiting at least one characteristic of a regulatory T cell. Preferably, the T cells or mixed T cell population exhibiting at least one characteristic of a conventional T cell are derived from a biological sample derived from a subject with SLE. Alternatively, the T cells can be derived from a histocompatible donor.

[0055] The present invention also relates to compositions of regulatory T cells in which greater than 20% of the cells express a binding protein of the invention. Preferably, the composition comprises greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% of the cells express a binding protein of the invention.

[0056] In another aspect, the present invention provides a method of preparing a population of regulatory T cells for use in treating SLE, comprising: Culturing the population of regulatory T cells in the presence of a peptide of the invention under conditions and for a time sufficient to allow expansion of the subpopulation activated by the peptide. thereby preparing a population of regulatory T cells for use in treating SLE.

[0057] Furthermore, the present invention provides a method for preparing a population of regulatory T cells for use in treating SLE, comprising the steps of: culturing a mixed T cell population or a T cell population exhibiting at least one conventional T cell characteristic in the presence of a peptide of the invention under conditions and for a time sufficient to allow expansion of the subpopulation activated by the peptide; Culturing the T cells under conditions that allow for conversion of the T cells into regulatory T cells. thereby preparing a population of regulatory T cells for use in treating SLE.

[0058] In any embodiment, the conditions for allowing the conversion of conventional T cells or a mixed population of T cells to regulatory T cells can 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 to regulatory T cells. The one or more agents or factors can include TGF-β, Foxp3, or agents for increasing the expression thereof.

[0059] In another aspect, the present invention provides a method of adoptive cell immunotherapy, comprising: extracting a mixed T cell population from a subject diagnosed with a condition associated with an aberrant, unwanted, or otherwise inappropriate immune response to Smith protein; Negative and positive immunoselection and cell sorting were used to isolate CD4 + CD25 + isolating a subpopulation containing T cells (Treg cells); Expanding Treg cells from the subpopulation by contacting the subpopulation with an effective amount of a peptide of the invention; and Introducing the ex vivo expanded Treg cells into a subject The present invention provides immunotherapy comprising:

[0060] In another aspect, the invention provides a composition comprising a binding protein, peptide, cell or vector of the invention and a pharmaceutically acceptable carrier, diluent or excipient.

[0061] 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 Smith protein in a subject, comprising administering to the subject a binding protein, peptide, cell, nucleic acid, or composition of the invention, thereby treating or preventing the condition in the subject.

[0062] 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 Smith 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 of the invention into a population of T cells, wherein the nucleic acid or vector encodes a binding protein of the invention; providing conditions that allow expression of the binding protein on the surface of the T cells; administering T cells expressing the binding protein on their surface and thereby treating or preventing a condition in a subject. Preferably, the T cells exhibiting at least one characteristic of a regulatory T cell are derived from a biological sample from a subject with SLE.

[0063] In another aspect, the invention provides the use of a binding protein, peptide, cell, nucleic acid, or composition of the invention in the manufacture of a medicament for treating or preventing a condition in a subject, wherein the condition is associated with an aberrant, unwanted, or otherwise inappropriate immune response to Smith protein.

[0064] In another aspect, the invention provides a binding protein, peptide, cell, nucleic acid, or composition of the invention for use in treating or preventing a condition associated with an aberrant, unwanted, or otherwise inappropriate immune response to Smith protein in a subject.

[0065] In some embodiments, the condition associated with an abnormal, unwanted, or otherwise inappropriate immune response to Smith protein is systemic lupus erythematosus (SLE). Alternatively, the condition associated with an abnormal, unwanted, or otherwise inappropriate immune response to Smith protein is lupus nephritis (LN). Consequently, a subject in need thereof is a subject diagnosed with SLE or LN.

[0066] Preferably, the subject with SLE has an HLA-DR15 allele or an HLA-DR3 allele, more preferably an HLA-DR1 allele. * 01:01 molecule and HLA-DRB1 * 15:01 molecule or HLA-DRA * 01:01 molecule and HLA-DRB1 * Identified as having the 03:01 molecule.

[0067] Preferably, the peptide for use in the treatment or prevention of SLE is SmB / B':1-15 or peptide SmB / B':58-72 or a fragment thereof as described herein. Preferably, the peptide comprises, consists of, or consists essentially of the sequence set forth in any one of SEQ ID NOs: 1-4.

[0068] 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 further additives, ingredients, integers, or steps.

[0069] 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. [Brief explanation of the drawings]

[0070] [Figure 1A] Figure 1 shows the identification of Sm-derived peptides that bind to HLA-DR15. The MHC class II Proimmune REVEAL assay was used to identify Sm-derived peptides (12 amino acid, 15-mer overlaps) that bind to HLA-DR15. The results of the assay are presented as the percentage binding relative to the positive control at 0 h (blue bars) and 24 h (red bars). Based on these scores, a stability index (red bars) for each peptide was derived. The positive control score was 100% at 0 h and 6.4% at 24 h, resulting in a stability index of 6.0. [Figure 1B] Figure 1 shows identification of Sm-derived peptides that bind to HLA-DR15. Binding scores and stability indices of SmB / B'-, SmD1- and SmD3-derived peptides. [Figure 1C-D]Figure 1 shows identification of Sm-derived peptides that bind to HLA-DR15. Binding scores and stability indices of SmB / B'-, SmD1- and SmD3-derived peptides. [Figure 2A] Figure 1 shows human T cell reactivity to the top three HLA-DR15-restricted Sm peptides. To determine whether HLA-DR15-restricted Sm peptides can induce T cell reactivity, three high binders: SmB / B':1-15, SmB / B':58-72, or SmD3:43-57, were cultured with human CD4+ T cells and monocyte-derived dendritic cells derived from HLA-DRB1*15:01 homozygous donors, respectively. T cell reactivity was determined by a cell proliferation assay using Cell Trace Violet (CTV). [Figure 2B] Figure 1 shows human T cell reactivity to the top three HLA-DR15-restricted Sm peptides. Representative FACS plots showing the percentage of CTVlo CD4+ T cells. A robust proliferative response was observed in CD4+ T cells cultured with SmB / B':1-15 and SmB / B':58-72 compared to CD4+ T cells cultured without peptide and with SmD3:43-57. [Figure 3A] Figure 1 shows human T cell reactivity to HLA-DR3-restricted Sm peptides. To determine whether human T cell reactivity to HLA-DR3-restricted Sm peptides can be measured, we investigated the SmD1:78-92 peptide, previously identified by Deshmukh US et al., 2011. The top-ranked peptides in the IEDB (Immune Epitope Database) predicted binding to the SmB / B' and SmB / B':7-21 peptides. These peptides were individually cultured with CD4+ T cells in a co-culture cell proliferation assay, and reactivity was assessed by Cell Trace Violet (CTV) dilution assay. [Figure 3B]Figure 1 shows human T cell reactivity to HLA-DR3-restricted Sm peptides. Representative FACS plots showing the percentage of CTVlo CD4+ T cells. A strong proliferative response was observed only in CD4+ T cells cultured with SmD1:78-92. [Figure 4] FIG. 1 shows a map of the modified lentiviral construct used to transduce TCR into human regulatory T cells, showing the relative positions of the alpha and beta chains, P2A, T2A, as well as the introduced murine mutations and cysteinylations. [Figure 5A] Figure 1 shows TCR transduction into human Tregs. Timeline of the TCR transduction protocol. Human Tregs (CD4+ CD25hi CD127lo) were first sorted by flow cytometry and then stimulated with anti-CD3 and anti-CD28 beads. On day 2, they were transduced with the lentiviral constructs shown in Figure 4. After two rounds of restimulation (days 9 and 26), Tregs were harvested and analyzed for TCR expression and the stability of the Treg phenotype. [Figure 5B] Figure 1 shows TCR transduction into human Tregs. Analysis of TCR expression in human Tregs on day 20 shows that more than 90% of transduced Tregs express the GFP tag. [Figure 5C] Figure 1 shows TCR transduction into human Tregs. Intracellular cytokine staining for the pro-inflammatory cytokine IFN-γ shows that transduced Tregs do not switch to pro-inflammatory cells (staining for IL-17A was also negative). [Figure 5D]Figure 1 shows TCR transduction into human Tregs. The transduced TCR is functional. To determine whether this protocol results in a functional TCR, we transduced a Jurkat T cell line and stimulated the transduced Jurkat T cells with an antigen-presenting cell line (HLA-DR15+ B-LCL) pulsed with the TCR cognate peptide. We demonstrated upregulation of CD69, an early activation marker, after stimulation, confirming that the TCR transduced using this protocol results in a functional TCR on the surface of T cells. [Figure 6A] Figure 1 shows that Sm-TCR-transduced Tregs are more potent suppressors of Sm-specific Tconv cell reactivity than polyclonal Tregs. In vitro T cell proliferation assay. HLA-DR15+ PBMCs isolated from SLE patients were stimulated with the dominant Sm peptide, SmB / B':58-72, and cocultured with polyclonal Tregs (left FACS plot) or Sm-TCR-transduced Tregs (right plot). Proliferation of proinflammatory conventional T cells (Tconv) was assessed by Cell Trace Violet (CTV) dilution assay. Sm-TCR-transduced Tregs more potently inhibited Tconv cell proliferation (12.1% vs. 22.4%). [Figure 6B] Figure 1 shows that Sm-TCR-transduced Tregs are more potent suppressors of Sm-specific Tconv cell reactivity than polyclonal Tregs. Counting of proliferating cells showed that there were more Sm-specific Tconv cells in the polyclonal group compared to the Sm-TCR group (8659 vs. 2053). [Figure 6C]Figure 1 shows that Sm-TCR-transduced Tregs are more potent suppressors of Sm-specific Tconv cell reactivity than polyclonal Tregs. The mean fluorescence intensity (MFI) of Sm-reactive Tconv cells reflects the number of cell divisions. The lower the MFI, the more cell divisions Tconv cells undergo. The MFI of Sm-reactive Tconv cells in the polyclonal Treg-treated group was lower than that in the Sm-TCR Treg group (89.1 vs. 271). Error bars represent SEM. ***P<0.001 by t-test. [Figure 7] Figure 1 shows the expansion of regulatory T cells (Tregs) after stimulation with peptide SmB / B':1-15 or peptide SmB / B':58-72. The proportion of Tregs relative to total CD4+ T cells was determined in the absence of peptide stimulation or after in vitro stimulation with SmB / B':1-15 or SmB / B':58-72. The use of SmB / B':1-15 or SmB / B':58-72 was found to selectively enhance the expansion of Tregs, as evidenced by a significant increase in the proportion of Tregs after peptide stimulation. Data shown are mean values ​​± SD for two independent experiments. ***P<0.001 compared to the no peptide group by one-way ANOVA with Tukey's post-hoc test. [Figure 8] Figure 1 shows that dominant HLA-DR15-restricted Sm-TCRs bind with high affinity to HLA-DR15 dextramers presenting SmB / B':58-72. The TCR binding affinities of the Sm-specific TCRs derived by the inventors were determined using a dextramer-based flow cytometry binding assay. We cloned the top three TCRs (i.e., TCR1, TCR2, and TCR3, identified in Table 1) into a Jurkat T cell line. We measured mean fluorescence intensity (MFI) by flow cytometry and presented the data using Scatchard plots. The relative Bmax and dissociation constant (Kd) are shown in each plot. [Figure 9A]HLA-DR15-restricted Sm-TCR Tregs suppress anti-Sm-specific pro-inflammatory responses and restore tolerance. PBMCs from an HLA-DR15+ / anti-Sm+ SLE patient with lupus nephritis were cocultured with a dominant HLA-DR15-restricted Sm peptide (SmB / B':58-72) and either no Tregs, polyclonal Tregs (pTregs), or HLA-DR15-restricted Sm-specific TCR1-transduced Tregs (Sm-Tregs). In the presence of Sm-Tregs, the number of Sm-specific Tregs was significantly increased relative to the number of Tconv cells. [Figure 9B] HLA-DR15-restricted Sm-TCR Tregs suppress anti-Sm-specific pro-inflammatory responses and restore tolerance. PBMCs from an HLA-DR15+ / anti-Sm+ SLE patient with lupus nephritis were cocultured with a dominant HLA-DR15-restricted Sm peptide (SmB / B':58-72) and either no Tregs, polyclonal Tregs (pTregs), or HLA-DR15-restricted Sm-specific TCR1-transduced Tregs (Sm-Tregs). In the presence of Sm-Tregs, an anti-inflammatory response, i.e., high IL-10 and low IFN-gamma / IL-17A, was dominant (similar to healthy controls), whereas in the absence of Tregs or with pTregs alone, a pro-inflammatory response, i.e., low IL-10 and high IFN-gamma / IL-17A, was dominant (as predicted in patients with autoimmune diseases). These data support the ability of Sm-Tregs to correct abnormal immune responses and restore tolerance to target self-epitopes. Results are expressed as mean ± SEM, *P<0.05, **P<0.01 compared with the no-Treg and pTreg groups using samples from four SLE patients. [Figure 9C]HLA-DR15-restricted Sm-TCR Tregs suppress anti-Sm-specific pro-inflammatory responses and restore tolerance. PBMCs from an HLA-DR15+ / anti-Sm+ SLE patient with lupus nephritis were cocultured with a dominant HLA-DR15-restricted Sm peptide (SmB / B':58-72) and either no Tregs, polyclonal Tregs (pTregs), or HLA-DR15-restricted Sm-specific TCR1-transduced Tregs (Sm-Tregs). In the presence of Sm-Tregs, an anti-inflammatory response, i.e., high IL-10 and low IFN-gamma / IL-17A, was dominant (similar to healthy controls), whereas in the absence of Tregs or with pTregs alone, a pro-inflammatory response, i.e., low IL-10 and high IFN-gamma / IL-17A, was dominant (as predicted in patients with autoimmune diseases). These data support the ability of Sm-Tregs to correct abnormal immune responses and restore tolerance to target self-epitopes. Results are expressed as mean ± SEM, *P<0.05, **P<0.01 compared with the no-Treg and pTreg groups using samples from four SLE patients. [Figure 9D]HLA-DR15-restricted Sm-TCR Tregs suppress anti-Sm-specific pro-inflammatory responses and restore tolerance. PBMCs from an HLA-DR15+ / anti-Sm+ SLE patient with lupus nephritis were cocultured with a dominant HLA-DR15-restricted Sm peptide (SmB / B':58-72) and either no Tregs, polyclonal Tregs (pTregs), or HLA-DR15-restricted Sm-specific TCR1-transduced Tregs (Sm-Tregs). In the presence of Sm-Tregs, an anti-inflammatory response, i.e., high IL-10 and low IFN-gamma / IL-17A, was dominant (similar to healthy controls), whereas in the absence of Tregs or with pTregs alone, a pro-inflammatory response, i.e., low IL-10 and high IFN-gamma / IL-17A, was dominant (as predicted in patients with autoimmune diseases). These data support the ability of Sm-Tregs to correct abnormal immune responses and restore tolerance to target self-epitopes. Results are expressed as mean ± SEM, *P<0.05, **P<0.01 compared with the no-Treg and pTreg groups using samples from four SLE patients. [Figure 10A] Figure 1 shows that HLA-DR15-restricted Sm-Tregs halt the progression of nephritis. NSGMHC-null mice were treated with PBMCs from an SLE patient with lupus nephritis who was also positive for anti-Sm antibodies and HLA-DR15+. At week 3, the onset of functional renal damage (measured by increased proteinuria), mice were administered no Tregs, polyclonal Tregs (pTregs), or HLA-DR15-restricted Sm-Tregs (transduced with HLA-DR15 TCR1). [Figure 10B-C]Figure 1 shows that HLA-DR15-restricted Sm-Tregs prevent the progression of nephritis. Mice that did not receive Tregs or that received pTregs progressed to severe nephritis (i.e., high levels of proteinuria and >50% of glomeruli with necrosis), whereas mice treated with Sm-Tregs did not. Results are expressed as the mean ± SEM of five SLE patient samples. ***P<0.001 compared with the no-Treg and pTreg groups. [Figure 11A-C] Figure 1 shows that HLA-DR3-restricted Sm-Tregs suppress anti-Sm pro-inflammatory cytokine responses and halt the progression of lupus nephritis. PBMCs from HLA-DR3+ / anti-Sm+ SLE patients with lupus nephritis were cocultured with a dominant HLA-DR3-restricted T cell epitope (SmD1:78-92) and no Tregs, polyclonal Tregs (pTregs), or Tregs transduced with an HLA-DR3-restricted TCR (HLA-DR3 TCR1, identified in Table 2) (Sm-Tregs). Cytokine responses were measured on day 8. [Figure 11D-E] Figure 1 shows that HLA-DR3-restricted Sm-Tregs suppress anti-Sm pro-inflammatory cytokine responses and halt the progression of lupus nephritis. NSGMHC-null mice were treated with PBMCs from an SLE patient with lupus nephritis who was also positive for anti-Sm antibodies and HLA-DR3+. At week 3, the onset of functional renal damage (measured by increased proteinuria), mice were treated with no Tregs, polyclonal Tregs (pTregs), or HLA-DR3-restricted Sm-Tregs (transduced with HLA-DR3 TCR1). DETAILED DESCRIPTION OF THE INVENTION

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The present inventors have identified peptides derived from Smith protein that bind to DR15 and DR3, HLA molecules frequently found in individuals with SLE. These peptides, when bound to HLA molecules, result in the proliferation of CD4+ helper T cells, allowing the identification of Smith protein-specific T cell receptors. Thus, the present invention relates to the use of peptide immunotherapy or adoptive cell therapy with regulatory T cells engineered to express Smith protein-specific TCRs to treat SLE.

[0075] An advantage of embodiments of the present invention is that both the identified peptides and TCRs are involved in interactions with HLA-DR subtypes common to lupus patients. Furthermore, antigen-specific regulatory T cell therapy typically exerts a stronger immunosuppressive effect than polyclonal regulatory T cell therapy. Finally, antigen-specific regulatory T cell therapy typically exerts a more limited immunosuppressive effect on protective T cell immunity, e.g., that used to respond to viral infections and / or cancer.

[0076] 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 encompass one and the plurality (i.e., one or more) of that step, composition, group of steps, or group of compositions. Accordingly, 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.

[0077] 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 referred to or indicated herein, individually or collectively, and includes any and all combinations of said steps or features, or any two or more thereof.

[0078] 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. The present invention is not limited in any way to the methods and materials described.

[0079] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0080] 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.

[0081] 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.

[0082] 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).

[0083] 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.G.lover and B.D.Hames (eds.), "DNA Cloning: A Practical Approach," Vols. 1-4, IRL Press (1995 and 1996); and F.M.A.usubel et al. (eds.), "Current Protocols in Molecular Biology," Greene Pub. Associates and Wiley-Interscience (1988; including all current revisions); 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 sources such as "Immunology," John Wiley & Sons (including all current editions).

[0084] The description and definition of variable regions and portions thereof, T-cell receptors and fragments thereof herein may be further clarified by the discussions 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 and / or Al-Lazikani et al., J. Mol. Biol., 273, 927-948, 1997.

[0085] 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.

[0086] As used herein, the term "derived from" shall be understood to indicate that the specified integer may be obtained from a particular source, although it may not necessarily originate directly from this source.

[0087] References herein to ranges of residues, for example, 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.

[0088] 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%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and do not substantially affect (i.e., do not reduce activity by more than 50%, such as by no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s) or protein (e.g., target binding affinity of a binding protein).

[0089] As used herein, "nucleic acid" or "nucleic acid molecule" refers to 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-enantiomer forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modified or replaced 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.

[0090] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if the material is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a nucleic acid may be part of a vector and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), but such a vector or composition may still be isolated, in the sense that it is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" refers to the segment of DNA involved in producing a polypeptide chain, and includes the regions preceding and following the coding region, the "leader and trailer," as well as intervening sequences (introns) between individual coding segments (exons).

[0091] 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 so that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated, or constitutive. Human-made genetic alterations can include, for example, alterations that introduce nucleic acid molecules (which may include expression control elements such as promoters) that encode 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 alterations include alterations within the coding region of a heterologous or homologous polypeptide, or functional fragment thereof, derived from a reference or parent molecule.

[0092] In the art, "conservative substitution" is recognized as a substitution of one amino acid with another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO97 / 09433, p. 10; Lehninger, "Biochemistry", 2nd ed., Worth Publishers, Inc., NY, NY, pp. 71-77, 1975; Lewin, "Genes IV", Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

[0093] 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 fragment thereof, Smith protein fragment:MHC complex). Binding proteins can be purified, substantially purified, synthetic, or recombinant. Exemplary binding proteins include single chain immunoglobulin variable regions (e.g., scTCR, scFv).

[0094] 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 TCR, a humanized TCR, or a 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.

[0095] "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) that is capable of specifically binding to an antigenic peptide bound to an MHC receptor. TCRs may be found on the surface of cells 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," U.S. 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.

[0096] In any of the foregoing embodiments, the present disclosure provides a method for treating a TCR comprising an alpha chain (α chain) and a beta chain (β chain), wherein the TCR binds to a complex of a fragment of Smith protein and an HLA-DR15 molecule, preferably the HLA-DR15 molecule is HLA-DR15. * 01:01 molecule and HLA-DRB1 *The present invention presents a high-affinity engineered T cell receptor (TCR) that is a 15:01 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 further embodiments, 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 certain embodiments, the binding proteins of the invention comprise: (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), or (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) (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 TRAV17; (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.

[0097] In a further embodiment, the binding protein of 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; and (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).

[0098] In any aspect or embodiment, the binding protein of 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 TRBJ2 (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.

[0099] In any aspect or embodiment, the binding protein of 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 TRBJ (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.

[0100] In any aspect or embodiment, the binding proteins of the invention comprise a V beta chain that comprises or is derived from an allele of TRBD1 or TRBD2.

[0101] In any aspect or embodiment, the binding protein of the invention comprises a V beta chain comprising or derived from an allele of TRC1 or TRBC2 and a V alpha chain comprising or derived from an allele of TRAC.

[0102] In any of the foregoing embodiments, the present disclosure provides a method for treating a TCR comprising an alpha chain (α chain) and a beta chain (β chain), wherein the TCR binds to a complex of a fragment of Smith protein and an HLA-DR3 molecule, preferably the HLA-DR3 molecule is HLA-DR3. * 01:01 molecule and HLA-DRB1 * The present invention presents a high-affinity engineered T cell receptor (TCR) that 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.

[0103] In certain embodiments, the binding proteins of the invention comprise: (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 TRBV4-1), (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) (i) a V beta chain derived from or comprising an allele of TRBV9 and a V alpha 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).

[0104] In a further embodiment, the binding protein of 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 TRBV (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.

[0105] In certain embodiments, the V beta chain comprises or is derived from an allele of TRBJ1 or TRBJ2, hi 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.

[0106] In certain embodiments, the binding proteins of the invention are selected from the group consisting of: (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 TRAJ49 allele. (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.

[0107] In certain embodiments, the binding proteins of the invention are selected from the group consisting of: (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 a TRAJ23 allele. (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.

[0108] In any aspect or embodiment, the binding proteins of the invention comprise a V beta chain that comprises or is derived from an allele of TRBD1 or TRBD2.

[0109] In any aspect or embodiment, the binding protein of the invention comprises a V beta chain comprising or derived from an allele of TRC1 or TRBC2 and a V alpha chain comprising or derived from an allele of TRAC.

[0110] 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 chain and Vβ chain are modified to include cysteine ​​residues that allow for the formation of additional interchain disulfide bonds. The cysteines introduced into each of the Vα chain and Vβ chain allow for preferential pairing of the Vα chain and Vβ chain, resulting in the expression of an endogenous TCR Vα chain and an endogenous TCR Vβ chain, when expressed in a cell. Preferably, the residue at Thr48 or its equivalent on the TCR α chain and the residue at Ser57 or its equivalent on the TCR β 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, in which regulatory T cells are engineered to express an exogenous TCR.

[0111] By way of example, a useful method for isolating and purifying recombinantly produced soluble TCRs may involve obtaining a supernatant from a suitable host cell / vector system that secretes recombinant soluble TCRs into the culture medium, followed by 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 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 immunogens from their 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 soluble TCRs may be performed according to methods described herein and known in the art.

[0112] The SmB / B'-specific binding proteins or SmB / B'-specific binding domains described herein (e.g., SEQ ID NOS: 6-257 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, as well as determining antigen-specific T cell responses. 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 Cr release 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.

[0113] As used herein, SmB / B' refers to the ribonucleoprotein called "Smith protein" or "small nuclear ribonucleoprotein-associated 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.

[0114] The protein encoded by the SNRPB gene is one of several nuclear proteins commonly found among the 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 snRNP structure. Two transcript variants encoding different isoforms (B and B') have been found for this gene.

[0115] 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.

[0116] 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. Sm proteins are frequently expressed in young black women with SLE (60%). Sm proteins are almost never expressed in healthy individuals or patients with other diseases. Anti-Sm antibodies should not be confused with anti-smooth muscle antibodies, which have been detected in autoimmune liver diseases.

[0117] Systemic lupus erythematosus (SLE) is characterized by the presence of diverse autoantibodies directed against multiple intracellular antigens. Among the different candidate autoantigens recognized by autoantibodies in SLE, the Sm antigen of the small nuclear ribonucleoprotein U-1 complex is considered to be a pathological hallmark of SLE. Antibodies against these autoantigens are sufficiently distinctive to be part of the American College of Rheumatology (ACR) classification criteria for SLE.

[0118] Adoptive Cell Therapy The present invention provides methods for preparing cells for adoptive cell therapy, methods for treating subjects with these cells, and the cells themselves.

[0119] In certain embodiments, nucleic acid molecules encoding the binding proteins of the invention are used to transfect / transduce host cells (e.g., Treg cells) for use in adoptive transfer therapy.

[0120] In an alternative embodiment, one or more peptides of the invention are used to activate and / or expand T cell populations to generate T cells with specificity for the peptides (e.g., Treg cells).

[0121] 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. (Sep 10), J. Imm. Meth., Epub Preprint, 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 T cells with a desired nucleic acid include 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. Gene 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. US2004 / 0087025), and therefore, adaptation of these methods to the embodiments disclosed herein is contemplated based on the teachings herein, including teachings directed to the binding proteins of the invention.

[0122] Cell populations containing regulatory T (Treg) cells can be derived from any source where Treg cells are present, such as peripheral blood, thymus, lymph nodes, spleen, and bone marrow.

[0123] A cell population containing Treg cells can 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 can be contacted with a peptide of the present invention to enrich for Sm antigen specificity in the T cells. Alternatively, a nucleic acid encoding a binding protein of the present invention can be transduced into the mixed population or conventional T cells. The T cells can then be converted into Treg cells using standard techniques known to those skilled in the art for generating 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 and Foxp3. This includes culturing the cells with anti-CD3 / anti-CD28 antibodies, high doses of IL-2, TGF-beta, and inhibition of CDK8 / 19 with 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 for stabilizing Tregs).

[0124] 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 aberrant, unwanted, or otherwise inappropriate immune response to Smith protein. Preferably, the condition is SLE.

[0125] 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.

[0126] 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.

[0127] In any of the methods of the present invention, Treg cells cultured in the presence of a nucleic acid or vector can be transferred into the same subject from which the cells were obtained. In other words, the cells used in the methods of the present invention can be autologous, i.e., obtained from the subject in whom a medical condition is to be treated or prevented. Alternatively, the cells can be allogeneically transferred into another subject. Preferably, the cells are autologous to the subject in the method of treating or preventing a medical condition in the subject.

[0128] As used herein, the terms "ex vivo" or "ex vivo therapy" refer to therapy in which cells are obtained from the patient or a suitable alternative source, such as a suitable allogeneic donor, and modified so that the modified cells can be used to treat a disease that is ameliorated by the therapeutic benefit provided by the modified cells. Treatment 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.

[0129] 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 will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques. As known in the art and described above, adjustments for systemic 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 one of ordinary skill in the art with routine experimentation.

[0130] An "enriched" or "purified" cell population is an increase in the ratio of particular cells 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.

[0131] Treg cells expressing the binding proteins of the present invention 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 period of time 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. Injections can be given at multiple locations.

[0132] Treg cells can be administered alone or in combination with other therapeutic agents. Each dose contains approximately 10 x 10 CD8+ T 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 2×10 9 pieces, cells 5×109 pieces, cells 10×10 9 The frequency of administration may include, for example, once weekly, twice weekly, 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 occurs 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 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%, or at least 99% of the administered Treg cells exhibit at least one characteristic of Treg cells.

[0133] peptide The present invention relates to a method for identifying HLA-DR15, specifically HLA-DR15, derived from Smith proteins. * 01:01 molecule and HLA-DRB1 * Peptides are provided that bind to the 15:01 molecule and can induce proliferation of CD4+ T cells. These peptides have particular application in immunotherapy for treating conditions associated with an aberrant, unwanted, or otherwise inappropriate immune response to Smith proteins. Preferably, the condition is SLE.

[0134] In another aspect, the present invention provides a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of residues 1-15 or 58-72 of SmB / B' protein, or an amino acid sequence equivalent thereto. In one embodiment, the SmB' protein comprises the amino acid sequence of SEQ ID NO: 5. In a further embodiment, the peptide comprises, consists of, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, or 4.

[0135] In any embodiment, the peptide of the present invention is capable of binding to or forming a complex with an HLA-DR15 molecule, and preferably the HLA-DR15 molecule is HLA-DR15. * 01:01 molecule and HLA-DRB1 * 15:01 molecule.

[0136] Furthermore, the present invention relates to a method for the treatment of HLA-DR3 and HLA-DR4, which are derived from Smith proteins. * 01:01 molecule and HLA-DRB1 * Peptides capable of binding to the 03:01 molecule and inducing proliferation of CD4+ T cells are provided. These peptides have particular application in immunotherapy for treating conditions associated with an aberrant, unwanted, or otherwise inappropriate immune response to Smith proteins. Preferably, the condition is SLE.

[0137] In another aspect, the present invention provides a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of residues 7-21 of SmB / B' protein, or an equivalent amino acid sequence thereof, or a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of residues 78-92 of SmD1 protein, or an equivalent amino acid sequence thereof. In one embodiment, the SmB' protein comprises the amino acid sequence of SEQ ID NO:5, in which case preferably the peptide comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:259. In one embodiment, the SmD1 protein comprises the amino acid sequence of SEQ ID NO:260, in which case preferably the peptide comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:258.

[0138] In any embodiment, the peptide of the present invention is capable of binding to or forming a complex with an HLA-DR3 molecule, and preferably the HLA-DR3 molecule is HLA-DR3. * 01:01 molecule and HLA-DRB1 * 03:01 molecule.

[0139] Reference to a "peptide" includes reference to a peptide, polypeptide, or protein, or portions thereof. Peptides may be glycosylated or non-glycosylated and / or may contain a range of other molecules fused, linked, conjugated, or otherwise associated with proteins such as amino acids, lipids, carbohydrates, or other peptides, polypeptides, or proteins. References hereinafter to a "peptide" include peptides comprising a sequence of amino acids as well as peptides in association with other molecules, such as amino acids, lipids, carbohydrates, or other peptides, polypeptides, or proteins.

[0140] "Derivatives" include fragments, parts, portions, and variants derived from natural, synthetic, or recombinant sources, including fusion proteins. Parts or fragments include, for example, active regions of the subject peptide. Derivatives can be derived from amino acid insertions, deletions, or substitutions. Amino acid insertion derivatives include amino- and / or carboxyl-terminal fusions of one or more amino acids as well as intrasequence insertions. Insertional variants of an amino acid sequence are those in which one or more amino acid residues are introduced into a predetermined site within the protein, although random insertions are also possible, with appropriate screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence.

[0141] Amino acid substitution variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place. An example of an amino acid substitution variant is a conservative amino acid substitution. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides, or proteins. In one embodiment, a cysteine ​​residue is substituted with serine, as exemplified herein.

[0142] Chemical and functional equivalents of the subject peptides shall be understood as molecules that exhibit any one or more of the functional activities of these molecules and may be derived from any source, including those that are chemically synthesized or those that are identified through screening processes such as natural product screening.

[0143] Analogs contemplated herein include, but are not limited to, side chain modifications, incorporation of unnatural amino acids and / or their derivatives during peptide, polypeptide or protein synthesis, and the use of cross-linking agents and other methods to impart conformational constraints to proteinaceous molecules or their analogs.

[0144] Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methyl acetimidate; acylation with acetic anhydride; carbamylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and periodoxylation of lysine with periodoxal-5'-phosphate followed by reduction with NaBH4.

[0145] The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal. Carboxyl groups may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatization, for example, to the corresponding amide. Sulfhydryl groups may be modified by carboxymethylation with iodoacetic acid or iodoacetamide; performation to cysteic acid; mixed disulfide formation with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; the formation of mercuric derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercuric chloride, 2-chloromercuric-4-nitrophenol, and other mercurials; and carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides, while tyrosine residues may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

[0146] Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbonethoxylation with diethylpyrocarbonate.

[0147] Examples of incorporation of unnatural amino acids and derivatives during protein synthesis include, but are not limited to, the use of norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids.

[0148] The crosslinking agent is, for example, n=1 to n=6 (CH2) nHomobifunctional cross-linkers such as bifunctional imidoesters with spacer groups, glutaraldehyde, N-hydroxysuccinimide esters, and heterobifunctional reagents that typically contain an amino-reactive moiety and another group-specific reactive moiety, such as N-hydroxysuccinimide, can be used to stabilize the three-dimensional conformation.

[0149] The structure of the peptides according to the invention can be modified for a variety of purposes, such as to increase solubility, enhance therapeutic or prophylactic efficacy, increase stability, or increase resistance to proteolysis. Modified peptides can be made in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition, to modify immunogenicity. Similarly, components can be added to the peptides of the invention to achieve the same result.

[0150] For example, a peptide can be modified so that it exhibits the ability to induce T cell anergy. In this case, the most important binding residues for a T cell receptor can be determined using known techniques (e.g., substituting each residue and determining the presence or absence of T cell reactivity). In one example, a residue shown to be essential for interacting with a T cell receptor can be modified by replacing the essential amino acid with another, preferably similar, amino acid residue (conservative substitution), the presence of which has been shown to alter T cell reactivity or T cell function. In addition, an amino acid residue that is not essential for T cell receptor interaction can be modified by replacing it with another amino acid, the incorporation of which may alter T cell reactivity or T cell function but does not, for example, abolish binding to an MHC protein of interest.

[0151] Exemplary conservative substitutions are detailed below and include:

[0152] [Table 1]

[0153] Such modifications result in the creation of molecules that fall within the scope of "mutants" of the subject peptides as defined herein. "Mutant" shall be understood as a reference to a peptide that exhibits one or more structural features or functional activities that differ significantly from those exhibited by the non-mutated peptide counterpart.

[0154] The peptides of the present invention may also be modified to incorporate one or more polymorphisms arising from natural allelic variation, and peptides may be substituted with D-amino acids, unnatural amino acids, or amino acid analogs to result in modified peptides within the scope of the present invention. Peptides may also be modified via conjugation with polyethylene glycol (PEG) using known techniques. Reporter groups may also be added to facilitate purification of peptides of the present invention and potentially increase their solubility. Other well-known types of modifications, including the insertion of specific endoprotease cleavage sites, the addition of functional groups, or the replacement of hydrophobic residues with less hydrophobic residues, as well as site-directed mutagenesis of DNA encoding the peptides of the present invention, may also be used to introduce alterations that may be useful for a wide range of purposes. The various modifications to the peptides of the present invention mentioned above are mentioned by way of example only and are intended solely to indicate the breadth of modifications that may be made.

[0155] The peptides of the present invention may be prepared by recombinant means or by chemical synthesis. According to a preferred embodiment of the present invention, a recombinant peptide or mutant thereof is provided that is preferentially immunoreactive with T cells derived from an individual with autoreactivity against Smith protein and is expressed by a host cell transformed with a vector encoding the peptide sequence of the present invention. The peptide may be fused to another peptide, polypeptide, or protein. Alternatively, the peptide may be prepared by chemical synthesis, such as the Merrifield solid-phase synthesis procedure. Furthermore, while synthetic peptides of the sequences presented above represent a preferred embodiment, the present invention also extends to biologically pure preparations of naturally occurring peptides or fragments thereof. By "biologically pure" is meant a preparation that contains at least about 60%, preferably at least about 70%, or preferably at least about 80%, and even more preferably at least about 90% or more, as determined by weight, activity, or other appropriate means.

[0156] Nucleic acids and vectors In another aspect, the invention provides nucleic acid molecule compositions comprising one or more nucleic acid molecules encoding or complementary to sequences encoding the binding proteins and peptides of the invention, or derivatives, homologs, or analogs thereof. The nucleic acid molecules of the invention may be used to make the binding proteins or peptides of the invention, or may be used for cell therapy to treat the diseases or conditions described herein.

[0157] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule. Constructs may be present in a vector (e.g., bacterial vector, viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule capable of carrying another nucleic acid. Vectors can be, for example, plasmids, cosmids, viruses, RNA vectors, or linear or circular DNA or RNA molecules that may contain chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. Exemplary vectors are vectors capable of autonomous replication (episomal vectors) or vectors capable of expression of nucleic acid molecules to which they are linked (expression vectors).

[0158] Viral vectors include negative-strand 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-strand RNA viruses such as picornaviruses and alphaviruses, as well as adenoviruses, double-stranded DNA viruses including 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 viruses, 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).

[0159] As used herein, "lentiviral vector" refers to an HIV-based lentiviral vector for gene delivery, which may be integrative or non-integrative, may have a relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are typically generated after transient transfection of three or more plasmids (packaging plasmid, envelope plasmid, and transfer plasmid) into producer cells. Like HIV, lentiviral vectors also enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, the 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.

[0160] In any embodiment, the vector of the invention comprises: (i) EF1α (alpha) promoter; (ii) 2A ribosomal skipping sequence; (iii) woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); (iv) a TCR β chain variable (Vβ or V beta) domain is translated before the (TCR) α chain variable (Vα or V alpha) domain; or (v) An arrangement in which the TCR β chain variable (Vβ or V beta) chain is translated before the (TCR) α chain variable (Vα or V alpha) chain. It may include any one or more or all of the following:

[0161] Preferably, the vector is a lentiviral vector. Even more preferably, the lentiviral vector has any one or more or all of the features shown in Figure 4.

[0162] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of that 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.

[0163] As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to a suitable control sequence(s) 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 that control the termination of transcription and translation. A vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Upon transformation of a suitable host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Herein, "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.

[0164] 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.

[0165] 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 cell's genome (e.g., chromosomal DNA, plasmid DNA, plastid DNA, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0166] As used herein, a "heterologous" nucleic acid molecule, "heterologous" construct, or "heterologous" sequence, or an "exogenous" nucleic acid molecule, "exogenous" construct, or "exogenous" sequence, refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to the host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule derived from the host cell. The source of a heterologous nucleic acid molecule, heterologous construct, or heterologous sequence, or an exogenous nucleic acid molecule, exogenous construct, or exogenous sequence, may be from a different genus or species. In certain embodiments, a heterologous nucleic acid molecule or exogenous nucleic acid molecule is added to a host cell or host genome (i.e., is not endogenous or native) by, for example, conjugation, transformation, transfection, electroporation, etc., where the added molecule may be integrated into the host genome, may exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), or may exist in multiple copies. Additionally, "heterologous" refers to a non-native enzyme, protein, or other activity that is encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity.

[0167] 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 individually regulated multiple genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. For example, as disclosed herein, a host cell can be engineered to express two or more heterologous or exogenous nucleic acid molecules encoding desired TCRs (e.g., TCRα and TCRβ) specific for a WT-1 antigenic peptide. 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 protein activities, not the number of separate nucleic acid molecules introduced into the host cell.

[0168] As used herein, the term "endogenous" or "native" 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 endogenous or native to that 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, native gene or nucleic acid molecule, where the expression or regulation of the second, native gene or nucleic acid molecule differs from its normal expression or regulation in the parent cell.

[0169] The term "homologous" or "homologue" 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.

[0170] As used herein, "sequence identity" refers to the percentage of amino acid residues in one sequence that are identical to those in another reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps, if necessary, and excluding conservative substitutions from the sequence identity. Percent sequence identity values ​​may be generated using the NCBI BLAST 2.0 software, with parameters set to default values, as defined by Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res., 25:3389-3402.

[0171] 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-affinity or enhanced anti-WT-1 TCR). In certain embodiments, the host cell may 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), optionally associated or unrelated to the biosynthesis of the heterologous or exogenous protein. In some embodiments, the host cell is genetically modified to express a protein or fusion protein that modulates immune signaling in the host cell, e.g., promoting a survival and / or expansion advantage of the modified cell (see, e.g., immunomodulatory fusion proteins in WO2016 / 141357, incorporated herein by reference in their entirety).

[0172] The nucleic acid molecule can be ligated into an expression vector capable of expression in prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeast, fungal, insect, mammalian, or plant cells). The nucleic acid molecule can be ligated, fused, or otherwise associated with a nucleic acid molecule encoding another entity, such as, for example, a signal peptide. The nucleic acid molecule can also include additional nucleotide sequence information fused, linked, or otherwise associated therewith at either the 3'-terminal portion or the 5'-terminal portion, or at both the 3'-terminal portion and the 5'-terminal portion. The nucleic acid molecule can also be part of a vector, such as an expression vector. The latter embodiment facilitates the production of recombinant forms of the binding proteins or peptides of the invention.

[0173] Such nucleic acids may be useful for the recombinant production of the binding proteins or peptides of the invention, or proteins comprising them, by insertion into a suitable vector and transfection into a suitable cell line. Such expression vectors and host cell systems also form aspects of the present invention.

[0174] In recombinant peptide production, host cells transformed with a nucleic acid having a sequence encoding a binding protein or peptide according to the invention, or a functional equivalent of the nucleic acid sequence, are cultured in a medium appropriate for the particular cell of interest. The binding protein or peptide can then be purified from the cell culture medium, the host cells, or both, using techniques well known in the art, such as ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, or immunopurification with antibodies specific for the binding protein or peptide.

[0175] Nucleic acids encoding the binding proteins or peptides of the invention can be expressed in bacterial cells such as E. coli, insect cells, yeast cells, or mammalian cells such as Chinese hamster ovary cells (CHO). Suitable expression vectors, promoters, enhancers, and other expression control elements are discussed in Sambruck et al. (1989). Other suitable expression vectors, promoters, enhancers, and other expression elements are well known to those skilled in the art. Examples of suitable expression vectors in yeast include YepSec1 (Balderi et al., 1987, Embo J., 6:229-234); pMFa (Kurjan and Herskowitz, 1982, Cell., 30:933-943); JRY88 (Schultz et al., 1987, Gene., 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). These vectors, as well as baculovirus and mammalian expression systems, can be used without restriction. For example, baculovirus systems are commercially available for expression in insect cells (ParMingen, San Diego, CA), while pMsg vectors are commercially available for expression in mammalian cells (Pharmacia, Piscataway, NJ).

[0176] Expression vectors suitable for expression in E. coli include, inter alia, pTrc (Amann et al., 1998, Gene., 69:301-315); pGex (Amrad Corporation, Melbourne, Australia); pMal (NE Biolabs, Beverley, MA); pRit5 (Pharmacia, Piscataway, NJ); pEt-11d (Novagen, Maddison, WI) (Jameel et al., 1990, J. Virol., 64:3963-3966), and pSem (Knapp et al., 1990, BioTechniques., 8:280-281). Use of pTRC and pEt-11d, for example, results in expression of non-fusion proteins. The use of pMal, pRit5, pSem, and pGex results in the expression of proteins or peptides fused to maltose E-binding protein (pMal), protein A (pRit5), truncated galactosidase (PSEM), or glutathione S-transferase (pGex). When the binding protein or peptide is expressed as a fusion protein, it is particularly advantageous to introduce an enzyme cleavage site at the fusion junction between the carrier protein and the peptide of interest. The binding protein or peptide of the invention can then be recovered from the fusion protein via enzymatic cleavage at the enzyme site and biochemical purification using conventional techniques for purifying proteins and peptides. Different vectors also have different promoter regions that allow constitutive or inducible expression or temperature induction. Additionally, it may be appropriate to express recombinant peptides in different E. coli hosts with altered abilities to degrade recombinantly expressed proteins. Alternatively, it may be advantageous to modify the nucleic acid sequence to use codons preferentially utilized by E. coli, such nucleic acid modifications having no effect on the amino acid sequence of the expressed protein.

[0177] 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. Nucleic acid sequences of the invention can also be chemically synthesized using standard techniques.

[0178] In addition to recombinant production of peptides according to the invention, the nucleic acids may also be utilized as probes for experimental or purification purposes.

[0179] Condition being treated The identification and synthesis of the binding proteins, peptides, cells, nucleic acids, vectors, and compositions of the invention disclosed herein facilitates the development of a range of prophylactic and therapeutic treatment protocols for use with immune conditions associated with Smith proteins. It also facilitates the development of reagents for use in these protocols. It should be understood, therefore, that the invention extends to the use of peptides, or functional derivatives, homologs, or analogs thereof, in the therapeutic and / or prophylactic treatment of patients. Such treatment methods include, but are not limited to, the following:

[0180] Administration of cells expressing a subject peptide or a binding protein of the invention to a patient as a means of inducing desensitization or immunological tolerance. This can be achieved, for example, by inducing Th2 anergy or apoptosis directed toward Smith protein. Treatment protocols based on the administration of specific concentrations of given cells or peptides expressing the binding protein according to specific regimens to induce tolerance can be utilized. Such methods can eliminate hypersensitivity to Smith protein or reduce the severity of hypersensitivity or sensitivity to Smith protein.

[0181] Preferably, such treatment regimens are capable of modifying the T cell response or both B cell and T cell responses of a target individual. As used herein, modification of a subject's autoimmune response can be defined as the induction of immune non-responsiveness or diminished immunity to Smith protein or other autoantigens, as determined by standard clinical procedures. In particular, since immunosuppressive cells (e.g., Tregs and myeloid-derived suppressor cells) mobilized as a result of Sm-specific Treg therapy exhibit non-antigen-specific immunosuppressive capabilities, creating a tolerant environment to multiple autoantigens, it is expected that the use of Sm-specific Tregs can induce immune tolerance to autoantigens beyond Smith protein.

[0182] Exposure of an individual to the binding proteins, peptides, cells, nucleic acids, vectors, and compositions of the invention may tolerize or anergize appropriate T cell subpopulations so that they become unresponsive to Smith protein and other self-antigens and do not participate in stimulating the immune response upon such exposure.

[0183] In one embodiment, the method desensitizes or induces immunological tolerance to Smith protein.

[0184] In another embodiment, the desensitization or tolerance is achieved by inducing anergy or apoptosis of T cells.

[0185] In yet another embodiment, the desensitization or tolerance is achieved by inducing Smith-specific Treg cells.

[0186] 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.

[0187] 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 who has not yet experienced or exhibited symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are well known to physicians.

[0188] In particularly preferred embodiments, the methods of the present invention may prevent, or reduce the severity of, or inhibit or minimize the progression, recurrence, or symptoms of, the diseases or conditions described herein. Thus, the methods of the present invention have therapeutic as well as prophylactic utility.

[0189] The term "treatment" or "treating" a subject 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 risk of (or susceptibility to) a disease or condition. The term "treating" refers to any indicator of successful treatment or amelioration of SLE and related conditions described herein, including any objective or subjective parameter, such as sedation; remission; reduction in the rate of exacerbation; reduction in the severity of the condition; stabilization, reduction in symptoms, or increasing the tolerability of the condition to an individual; slowing the rate of degeneration or decline; prevention of debilitating end-point degeneration; or improvement in the physical or mental well-being of the subject.

[0190] It is also understood that the methods described herein can be used in combination with existing standard treatments / therapies for SLE. Those skilled in the art are familiar with existing standard therapies for treating SLE, including, but not limited to, the use of steroids, antimalarials (hydroxychloroquine, chloroquine), immunosuppressants (azathioprine, methotrexate, mycophenolate mofetil, mycophenolic acid, tacrolimus, voclosporin, cyclosporine), kinase inhibitors (baricitinib, tofacitinib, upadacitinib), and biologics (belimumab, rituximab, anifrolumab, ustekinumab, obinotuzumab). The present invention encompasses the combination of existing standard therapies with the specific methods of the present invention.

[0191] 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 who require treatment according to the present invention.

[0192] Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. At least 5 million people worldwide have SLE, with 90% of those diagnosed being women, and most developing SLE between the ages of 15 and 44. In Australia, SLE is diagnosed in approximately 1 in 1,000 people, with higher prevalence and severity in Aboriginal Australians and Asian Australians. SLE patients suffer from chronic immune-mediated inflammatory damage to the brain, kidneys, heart, lungs, joints, skin, and other organs, resulting in a significant shortening of life expectancy, exemplified by a standardized mortality rate of greater than 3. In a UK cohort, the mean age at death for 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.

[0193] 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 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.

[0194] Cutaneous lupus erythematosus (CLE), seen in the majority of SLE cases, is often observed on sun-exposed skin and manifests 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 leading to the onset of SLE and its pattern of intermittent exacerbations are known, it is clear that exposure to sunlight is important in exacerbating the cutaneous disease as well as the systemic disease.

[0195] Among the symptoms common to patients diagnosed with lupus, nearly all patients experience joint pain and / or swelling (i.e., arthritis). Commonly affected joints 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 a feeling of illness (malaise), hair loss, sensitivity to sunlight, and, in approximately half of SLE patients, skin erythema ("butterfly" rash), 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 involvement, hematological involvement, gastrointestinal involvement, and vasculitis.

[0196] 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 can 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, ultraviolet light from artificial indoor light, such as fluorescent lighting, or both.

[0197] composition Administration of the compositions of the present invention (referred to herein as "medicines") in the form of pharmaceutical compositions can be carried out by any convenient means. In certain embodiments, the medicament is a peptide described herein, preferably a peptide comprising, consisting of, or consisting essentially of a sequence set forth in any one of SEQ ID NOS: 1-4. It is expected that the medicament of the pharmaceutical composition will exhibit therapeutic activity when administered in an amount that depends on the particular case. Variations will depend, for example, on the human or animal and the selected medicament. A wide range of dosages may be applicable. For example, about 0.01 μg to about 1 mg of medicament may be administered per administration, depending on the patient. Dosage regimens may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, weekly, monthly, or at other appropriate time intervals, with the dose being proportionally reduced as indicated by the requirements of the situation. In another example, the composition is first administered to induce tolerance, followed by additional administrations of the composition, if necessary, to maintain tolerance. These booster doses may be administered, for example, monthly, and may be administered for any period of time, including for the life of the patient.

[0198] The agent may be administered in any convenient manner, such as orally, intravenously (if water soluble), intraperitoneally, intramuscularly, subcutaneously, intradermally (with or without the use of a conventional needle or other transdermal delivery device), transdermally, intranasally, sublingually, or via suppository routes, or by implantation (e.g., using sustained-release molecules). Preferably, the composition is administered intradermally. The agent may be administered in the form of a pharmaceutically acceptable non-toxic salt, such as an acid addition salt or a metal complex, e.g., zinc, iron, etc. (considered to be a salt for purposes of this application). Examples of such acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, etc. When the active ingredient is administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch, or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. In the context of peptides for administration, compositions comprising said peptides may be in the form of liposomes or conjugated to nanoparticles. Those skilled in the art are familiar with standard methods for formulating peptides for administration to a subject in need thereof.

[0199] Pharmaceutical forms suitable for use in injections include sterile aqueous solutions (if soluble in water) or sterile aqueous dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, or may be in the form of creams or other forms suitable for topical application. Pharmaceutical forms must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Osmotic agents are useful for keeping the preparation isotonic with human plasma, thereby avoiding tissue damage. Commonly used isotonic agents include dextrose, trehalose, glycerin, and mannitol. Glycerol and sodium chloride are other options, but are less commonly used. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Delayed absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0200] Sterile injectable solution can be prepared by incorporating active compound in a suitable solvent with various other ingredients as listed above as required in the required amount, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile medium that contains the basic dispersion medium and other ingredients listed above as required.For the preparation of sterile injectable powder, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of active ingredient and any other desired ingredients from the solution that has already been sterile-filtered.

[0201] The active ingredient, when protected in a suitable form, may be orally administered, for example, with an inert diluent or an assimilable edible carrier, enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% of the active compound by weight. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 and about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit contains between about 0.1 μg and 1000 μg of active compound.

[0202] Tablets, troches, pills, capsules, and the like may also contain the ingredients listed below: binders such as gum, acacia, corn starch, or gelatin; excipients such as calcium diphosphate; disintegrating agents such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint, oil of wintergreen, or cherry flavoring. In the case of capsules, the unit dosage form may also contain a liquid carrier in addition to the above types of materials. Various other materials may be present as coatings or to otherwise modify the physical unit dosage form. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup or elixir may contain the active compound, sucrose as a sweetener, methylparaben and propylparaben as preservatives, a dye, and a flavoring such as cherry or orange flavoring. Of course, any material used in preparing any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts employed. Additionally, the active compound(s) may be incorporated into sustained-release preparations and formulations.

[0203] The pharmaceutical composition may also include a genetic molecule, such as a vector capable of transfecting a target cell, the vector carrying a nucleic acid molecule encoding a modulatory agent. The vector may be, for example, a viral vector.

[0204] Routes of administration include, but are not limited to, respiratory (e.g., via aerosol, intranasal, or oral), intratracheal, nasopharyngeal, intravenous, intraperitoneal, subcutaneous, intracranial, intradermal, transdermal, intramuscular, intraocular, intrathecal, intracerebral, intranasal, infusion, oral, rectal, via IV drip patch, implant, and sublingual. Preferably, the route of administration is intravenous, subcutaneous, intradermal, transdermal, or intranasal, and more preferably, intravenous.

[0205] Yet another aspect of the present invention relates to a composition as hereinbefore defined when used in any method of the present invention.

[0206] [Table 2] TIFF2026041767000003.tif229165TIFF2026041767000004.tif230165TIFF2026041767000005.tif227168TIFF202 6041767000006.tif228165TIFF2026041767000007.tif229167TIFF2026041767000008.tif229166TIFF20260417670 00009.tif229167TIFF2026041767000010.tif227165TIFF2026041767000011.tif227165TIFF2026041767000012.t if226167TIFF2026041767000013.tif226166TIFF2026041767000014.tif228169TIFF2026041767000015.tif170166

[0207] [Table 3] TIFF2026041767000017.tif228166TIFF2026041767000018.tif220166TIFF2026041767000019.tif226166TIFF2026041767000020.tif227169TIFF2026041767000021.tif228168TIFF2026041767000022.tif226168TIFF2026041767000023.tif228168TIFF2026041767000024.tif226166TIFF2026041767000025.tif225168TIFF2026041767000026.tif228166TIFF2026041767000027.tif227167TIFF2026041767000028.tif226168TIFF2026041767000029.tif98165

[0208]

Table 4

[0209]

Table 5

[0210] [Example 1] Epitope mapping To identify Sm-derived peptides that bind to HLA-DR15, we utilized the REVEAL MHC-peptide Binding Assay from ProImmune. Briefly, 149 peptides (15-mer overlaps of 12 amino acids) were synthesized spanning three known immunogenic Sm proteins: SmB / B', SmD1, and SmD3 (Migliorini, 2005, Autoimmunity, 38:47-54). The HLA-DR15 complex (HLA-DR1) was also involved in the synthesis of peptides derived from Sm. * 01:01+HLA-DRB1 * The relative affinity of each peptide to the antibody (15:01) was measured against a known positive control. Peptides that bind with high affinity give a high signal by forming a tertiary complex that can be detected by antibody.

[0211] HLA typing Healthy human whole-donor blood was HLA typed at high resolution by the Victorian Transplant and Immunogenetics Service, Red Cross, Melbourne. Typing of common and well-documented alleles (CWD) was performed using sequence-based typing (SBT) using the IMGT / HLA reference database and SSO / SSP methods or next-generation sequencing (NGS) (Mack et al., 2013, Tissue Antigens, 81:194-203).

[0212] Isolation of primary human cells Freshly drawn, fully HLA-typed human whole blood was collected, and PBMCs were isolated using Lymphoprep density gradient medium in Sepmate-50 tubes according to the manufacturer's instructions (Stemcell). Monocytes were purified from PBMCs using an EasySep magnet and a Human Monocyte Isolation Kit according to the manufacturer's instructions (Stemcell). Monocytes were differentiated into mature dendritic cells (DCs) for 7 days using an ImmunoCult Dendritic Cell Culture Kit (Stemcell).

[0213] CD4+ cells were purified directly from whole blood using RosetteSep Human CD4+ T Cell Enrichment Cocktail according to the manufacturer's instructions (Stemcell).

[0214] Naive regulatory T cells were purified by first enriching for CD4+ cells using RosetteSep Human CD4+ T Cell Enrichment Cocktail, and then sorting CD4+, CD25high, CD127low, CD45RA+, and PI- cells on a FACS Aria Fusion flow cytometer (BD) using the following antibodies: anti-human CD4 Pacific Blue (Biolegend), anti-human CD25 APC (Biolegend), anti-human CD127 PE (Biolegend), and anti-human CD45RA PE Cy7 (BD).

[0215] In vitro co-culture 200,000 freshly isolated CD4+ enriched cells were stained with 5 μM Cell Trace Violet (CTV) cell proliferation dye (Invitrogen) and plated in one well of a 96-well flat-bottom tissue culture plate (Corning) with 100 μg / mL of the peptide, SmD1, in RPMI 1640 medium (Gibco) supplemented with 10% human AB serum, 2 mM L-glutamine (Gibco), and 1% penicillin / streptomycin (Gibco). 78-92 (HLA-DRB1:0301), SmB / B' 7-21 (HLA-DRB1:0301), SmB / B' 1-15 (HLA-DRB1:1501) or SmB / B' 58-72 To determine the efficacy of Sm-TCR transduced Tregs, 100,000 HLA-matched mature dendritic cells were co-cultured with 100,000 Sm-TCR transduced Tregs in the presence of HLA-DRB1:1501 (Mimotopes). 5 10 PBMCs 3 10 Sm-TCR-transduced Tregs or 10 4 The cells were cultured with control polyclonal Tregs. Duplicate wells were seeded and co-cultured for 5 days in a 5% CO2, 37°C incubator.

[0216] Flow cytometry After 5 days of coculture, cells were harvested and stained with custom dCODE Dextramer-PE according to the manufacturer's instructions (Immudex). Cells were further stained with anti-human CD4 APC (eBioscience), anti-human CD8 Alexa Fluor 488 (Biolegend), and propidium iodide (PI) (Sigma). CD8-, PI-, CD4+, and CTV-low cells were sorted using a FACS Aria Fusion flow cytometer (BD) and counted by trypan blue staining on a hemocytometer. Immediately afterward, cells were sent for 10x sequencing.

[0217] 10x Sequencing To generate cDNA libraries for single-cell whole-transcriptome sequencing for V(D)J / Dextramer enrichment, FACS-sorted cells were resuspended at a concentration of 700–1200 cells per 1 μL and loaded into a Chromium Controller (10× Genomics) according to the manufacturer's protocol for the Chromium Single Cell Reagent Kit, along with the Chromium V(D)J human T cell enrichment kit and the Chromium Single Cell Feature Barcode Library Kit (all from 10× Genomics). The cell recovery target was set at 10,000 cells. Single-cell cDNA libraries were sequenced on an Illumina NextSeq Sequencer using 150-bp paired-end (V(D)J library) reads or single-end (transcriptome) reads. Whole-transcriptome data, along with V(D)J paired reads, were processed using Cell Ranger Version 3.1 (10× Genomics) and the Seurat R Package. After data processing, the data were visualized in Loupe Cell Browser and Loupe VDJ Browser (10x Genomics), and clonally expanded TCR sequences were selected for further analysis.

[0218] Plasmid design A lentiviral plasmid backbone (Creative Biolabs) was used containing the EF1 alpha promoter 5' to an EcoRI restriction site and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) 3' to an XbaI restriction site. These elements were flanked by 5' long terminal repeat (LTR) and 3' long terminal repeat (LTR) sequences, respectively. The TCR transgene sequence was designed in SnapGene (GSL Biotech LLC) to contain a 5' EcoRI restriction site, followed by the TCR beta chain, spaced apart by a P2A ribosomal skipping sequence, followed by the TCR alpha chain, spaced apart by a T2A ribosomal skipping sequence, followed by an enhanced green fluorescent protein (eGFP) sequence, and finally, a 3' XbaI restriction site. Using the GeneOptimiser tool (Invitrogen), the TCR alpha and beta chains were subjected to minimal murine (Sommermeyer, J Immunol, 2010) and cysteinylation (Cohen, Cancer Res, 2007) codon optimization and gene optimization for humans (Homo sapiens). The TCR transgene cassette was synthesized by GeneArt (Invitrogen) and ligated into the lentiviral backbone at the EcoRI and XbaI restriction sites.

[0219] Virus production TCR-encoding lentiviral particles were prepared by transient transfection of HEK 293T cells using Lipofectamine 3000 reagent according to the manufacturer's instructions (Life Technologies). pLenti-TCR, a lentiviral vector containing the alpha-beta TCR insert and eGFP, and the LentiArt viral packaging plasmids pHelp1, pHelp2, and pHelp3 (Creative Biolabs), were mixed at a 3:1:1:1 ratio (pLenti-TCR:pHelp1:pHelp2:pHelp3) and transfected at 25.9 μg per 55 cm Petri dish. At 24 and 52 hours posttransfection, supernatants were collected, passed through 0.45 μm PVDF Millex-HV filters, and concentrated with Lenti-X Concentrator reagent according to the manufacturer's instructions (Clontech). Viral particles were resuspended in PBS and frozen in aliquots at −80° C. until use. HIV-1 Gag p24 antigen concentration was measured by HIV-1 p24Antigen ELISA kit (Abcam).

[0220] Viral transduction To transduce primary human naive regulatory T cells (Tregs), sorted Tregs were placed in RPMI-1640 (Gibco) supplemented with 10% human AB serum, 2 mM L-glutamine (Gibco), and 50 μM 2-mercaptoethanol and incubated with T Cell Activator aCD2, aCD3, aCD28 Microbeads (Miltenyi Biotech) at a 1:2 bead-to-cell ratio and 300 IU IL-2 per mL (Stemcell) for 48 hours. Lentiviral particles (400 ng HIV-1 p24 Gag per cell) were added at 5 μg / cm for 2 hours at 32°C and 1,500 × g. 2Activated Tregs (0.25 × 10 cells per well) were spinoculated onto a 24-well plate coated with RetroNectin (Takara Bio Inc.). 6 ) was added, spun at 1,500 x g at 32°C for 2 hours, and then placed in an incubator at 37°C with 5% CO2 for 48 hours.

[0221] Expansion and phenotypic analysis of transduced Tregs Forty-eight hours after transduction and every 48 hours thereafter, 50% of the cell culture medium was aspirated and replaced with fresh RPMI-1640 supplemented with 10% human AB serum, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 1% penicillin / streptomycin, and 300 IU IL-2 per mL. Cell cultures were expanded at ≥80% confluency. To assess phenotypic stability after 2 weeks of expansion, Treg cells were analyzed on an LSR Fortessa ×20 flow cytometer (BD) after staining with Live / Dead Fixable Near-IR (Invitrogen), CD4-BUV496 (BD), CD25-BUV395 (BD), CD127-PE CF594 (BD), TCRVbx-PE (where x denotes an antibody specific for a particular TCR clone), FoxP3-BV421 (Biolegend), Latency Associated Peptide (LAP)-APC (eBioscience), GARP-BV786 (BD), Helios-PECy7 (Biolegend), IL10-BV650 (BD), IFN-gamma-BB700 (BD), IL17A-APCR700 (BD), and IL2-BV711 (BD).

[0222] [Example 2] Identification of Sm-derived peptides that bind to HLA-DR15 The identification of Sm-derived peptides that bind to HLA-DR15 is shown in Figure 1. Specifically, in Figure 1A, the MHC class II Proimmune REVEAL assay was used to identify Sm-derived peptides (15-mer overlaps of 12 amino acids) that bind to HLA-DR15. The results of the assay are presented as the percentage binding relative to the positive control at 0 h (blue bars) and 24 h (red bars). Based on these scores, a stability index (red bars) for each peptide was derived. The positive control score was 100% at 0 h and 6.4% at 24 h, resulting in a stability index of 6.0. Figures 1B-D show the binding scores and stability indexes of the SmB / B'-, SmD1-, and SmD3-derived peptides.

[0223] [Example 3] Human T cell reactivity to the top three HLA-DR15-restricted Sm peptides Human T cell reactivity to the top three HLA-DR15-restricted Sm peptides is shown in Figure 2. Specifically, as shown in Figure 2A, to determine whether HLA-DR15-restricted Sm peptides could induce T cell reactivity, the top three high binders, SmB / B':1-15, SmB / B':58-72, or SmD3:43-57, were each cultured with human CD4+ T cells. T cell reactivity was determined by a cell proliferation assay using Cell Trace Violet (CTV).

[0224] Figure 2B shows a representative FACS plot showing the percentage of CTVlo CD4+ T cells. A strong proliferative response was observed in CD4+ T cells cultured with SmB / B':1-15 and SmB / B':58-72 compared to CD4+ T cells cultured without peptide and with SmD3:43-57.

[0225] [Example 4] Human T cell reactivity to HLA-DR3-restricted Sm peptides Human T cell reactivity to HLA-DR3-restricted Sm peptides is shown in Figure 3. Specifically, as shown in Figure 3A, to determine whether human T cell reactivity to HLA-DR3-restricted Sm peptides could be measured, we investigated the SmD1:78-92 peptide, previously identified by Deshmukh US et al., 2011, and found that the top peptides in the IEDB (Immune Epitope Database) predicted binding to the SmB / B' and SmB / B':7-21 peptides. These peptides were individually cultured with CD4+ T cells in a co-culture cell proliferation assay, and reactivity was assessed by Cell Trace Violet (CTV) dilution assay.

[0226] Figure 3B shows a representative FACS plot showing the percentage of CTVlo CD4+ T cells. A strong proliferative response was observed only in CD4+ T cells cultured with SmD1:78-92.

[0227] Example 5: Modified lentiviral constructs A map of the modified lentiviral construct used to transduce the TCR into human regulatory T cells is shown in Figure 4. The relative positions of the alpha and beta chains, P2A, T2A, as well as the introduced murine mutations and cysteinylations are shown.

[0228] [Example 6] TCR transduction into human Treg TCR transduction of human Tregs is shown in Figure 5. In detail, Figure 5A shows the timeline of the TCR transduction protocol. Human Tregs (CD4+ CD25hi CD127lo) were first sorted by flow cytometry and then stimulated with anti-CD3 / anti-CD28 beads, followed by lentiviral TCR transduction on day 2. After two rounds of restimulation (days 9 and 26), Tregs were harvested and analyzed for TCR expression and the stability of the Treg phenotype.

[0229] FIG. 5B shows that analysis of TCR expression in human Tregs at day 20 indicates that more than 90% of transduced Tregs express the GFP tag.

[0230] Intracellular cytokine staining for the pro-inflammatory cytokine IFN-γ is shown in Figure 5C, which reveals that transduced Tregs do not switch into pro-inflammatory cells (staining for IL-17A was also negative).

[0231] The results in Figure 5D show that the transduced TCR is functional. To determine whether this protocol results in a functional TCR, we transduced a Jurkat T cell line and stimulated the transduced Jurkat T cells with an antigen-presenting cell line (HLA-DR15+ B-LCL) pulsed with the TCR cognate peptide. We observed upregulation of CD69, an early activation marker, after stimulation, confirming that the TCR transduced using this protocol results in a functional TCR on the surface of the T cells.

[0232] [Example 7] Transduction of Sm-TCR is a more potent suppressor of Sm-specific Tconv cell reactivity HLA-DR15+ PBMCs were stimulated with the dominant Sm peptide, SmB / B':58-72, and cocultured with polyclonal Tregs or Sm-TCR-transduced Tregs (Tregs were transduced with the lentiviral vector shown in Figure 4, and the TCR corresponds to HLA-DR15 TCR#1 with the CDR3α sequence of CALSSYGNKLVF (SEQ ID NO: 8) and the CDR3β sequence of CASSSLSGSSYEQYF (SEQ ID NO: 11)). In vitro T cell proliferation assays were performed. Proliferation of proinflammatory conventional T cells (Tconv) was assessed by Cell Trace Violet (CTV) dilution assay. Sm-TCR-transduced Tregs more potently inhibited Tconv cell proliferation (12.1% vs. 22.4%).

[0233] Proliferating cell counts showed that there were more Sm-specific Tconv cells in the polyclonal group compared to the Sm-TCR group (8659 compared to 2053).

[0234] The mean fluorescence intensity (MFI) of Sm-reactive Tconv cells reflects the number of cell divisions. The lower the MFI, the more cell divisions the Tconv cells undergo. The MFI of Sm-reactive Tconv cells in the polyclonal Treg-treated group was lower than that in the Sm-TCR Treg group (89.1 vs. 271). The data are shown in Figure 6. Error bars are SEM. *** P<0.001 by t-test.

[0235] These data support that Tregs transduced with Sm-specific TCRs more potently suppress autoreactive proinflammatory responses to Sm antigens. The inventors believe that these data provide proof-of-concept that Tregs transduced with Sm-specific TCRs are better at suppressing autoreactive T cell responses to Sm autoantigens and that fewer Tregs are required to achieve antigen-specific suppression. In addition, the ability to use fewer Tregs reduces the risk of side effects in patients receiving Tregs.

[0236] Example 8 Stimulation with peptide SmB / B':1-15 or peptide SmB / B':58-72 induces expansion of regulatory T cells CD4+ T cells from DR15 homozygous donors were cocultured with autologous monocyte-derived dendritic cells pulsed with peptide SmB / B':1-15 or peptide SmB / B':58-72, or no peptide (control). After 8 days, CD4+ cells were subjected to single-cell sequencing using the 10x Genomics Human Immune Repertoire Single Cell Profiling kit. Cell clusters with high expression of Foxp3 and TIGIT, as well as clusters with high expression of CD52 and LTB, were designated Tregs.

[0237] The results shown in Figure 7 confirm that stimulation with peptide SmB / B':1-15 or peptide SmB / B':58-72 results in the selective expansion of Tregs, as evidenced by the significant increase in the proportion of Tregs after peptide stimulation. Data shown are means ± SD for two independent experiments. *** P<0.001 compared with the no peptide group by one-way ANOVA with Tukey's post-hoc test.

[0238] [Example 9] Dominant HLA-DR15-restricted Sm-TCR binds with high affinity to HLA-DR15 dextramers presenting SmB / B':58-72 To determine the binding affinity of dominant Sm-specific TCRs, we performed a Dextramer-based flow cytometry binding assay. Dextramers contain 10 peptide-MHC complexes linked together on a dextran backbone. These fluorescent dye-labeled Dextramers allow for the detection of Sm-specific T cells and can be used to determine the relative affinities of Sm-specific TCRs.

[0239] First, using custom lentiviral vectors, we cloned the top three TCRs obtained in Example 8 into a Jurkat T cell line. These TCRs are identified as TCR1 to TCR3 in Table 1.

[0240] We then measured mean fluorescence intensity (MFI) by flow cytometry and presented the data using a Scatchard plot. The results are shown in Figure 8. The lower the concentration at which the MFI plateaus, the lower the dissociation constant (Kd), indicating higher affinity. We found that the top-ranked Sm TCR bound with the highest affinity compared to the second- and third-ranked Sm-specific TCRs. This result is important for validating this novel method of identifying high-affinity TCRs for disease treatment using high-throughput single-cell TCR sequencing.

[0241] Example 10: Sm-TCR Tregs suppress anti-Sm-specific pro-inflammatory responses and restore tolerance To determine the effectiveness of Sm-Tregs in suppressing anti-Sm-specific proinflammatory responses, we used Tregs from SLE patients to generate Sm-Tregs and examined them in in vitro cocultures. We compared patient anti-Sm responses with or without Tregs or with polyclonal Tregs (pTregs).

[0242] First, we used a proliferation assay to measure the effect of Sm-Tregs on the expansion of pro-inflammatory Sm-specific conventional T cells (Tconv). The results (Fig. 9A) show that in the presence of Sm-Tregs, the number of Tregs is significantly increased relative to the number of Sm-specific Tconvs. This result indicates that Sm-Tregs potently suppress the expansion of Sm-specific Tconv cells. Furthermore, the ratio of autoantigen-specific Tregs to Tconvs of >10 is similar to our other data showing that healthy individuals have 10-fold more autoantigen-specific Tregs than Tconvs.

[0243] Next, we measured cytokine production and found that in the presence of Sm-Tregs, an anti-inflammatory response, i.e., high IL-10 and low IFN-γ / IL-17A, was dominant (as in healthy individuals), whereas without Tregs or with only pTregs, a pro-inflammatory response, i.e., low IL-10 and high IFN-γ / IL-17A, was dominant (as predicted in patients with autoimmune diseases). These data (shown in Figures 9B-9D) support the ability of Sm-Tregs to correct abnormalities in immune responses and restore tolerance of targeted self-epitopes. Results are shown as mean ± SEM, using samples from four SLE patients, compared with the no-Treg and pTreg groups. * P<0.05, ** Expressed as P<0.01.

[0244] [Example 11] HLA-DR15-restricted Sm-Treg prevents progression of nephritis To support the efficacy of Sm-Tregs in halting disease progression, we devised a new humanized model for lupus nephritis.

[0245] In this model, adoptive transfer of PBMCs from patients with lupus nephritis into immune-compromised NSGMHC null mice results in the development of functional renal damage (measured by increased urinary protein) and intrasegmental glomerular necrosis (measured by histological staining of kidney sections). At week 3, the time of development of functional renal damage, mice received no Tregs, polyclonal Tregs (pTregs), or Sm-Tregs. Figure 10A provides an outline of the experimental protocol.

[0246] Mice that did not receive Tregs or that received pTregs progressed to severe nephritis (i.e., high levels of proteinuria and >50% of glomeruli with necrosis), whereas nephritic mice treated with Sm-Tregs did not exhibit further disease progression (see Figures 10B-10C). Results are expressed as the mean ± SEM of five SLE patient samples. ***P<0.001 compared with the no Treg group and the pTreg group.

[0247] [Example 12] HLA-DR3-restricted Sm-Treg prevents progression of nephritis Similar to the approach outlined in Example 11, the inventors determined whether HLA-DR3-restricted Sm-Tregs also have therapeutic efficacy.

[0248] As shown in Figure 11, HLA-DR3-restricted Sm-Tregs were shown to suppress anti-Sm pro-inflammatory cytokine responses and halt the progression of lupus nephritis. Briefly, PBMCs from HLA-DR3+ / anti-Sm+ SLE patients with lupus nephritis were cocultured with a dominant HLA-DR3-restricted T cell epitope (SmD1:78-92) and no Tregs, polyclonal Tregs (pTregs), or Tregs transduced with an HLA-DR3-restricted TCR (HLA-DR3 TCR1, identified in Table 2) (Sm-Tregs). Cytokine responses were measured on day 8.

[0249] NSGMHC Null mice were also treated with PBMCs from an SLE patient with lupus nephritis who was positive for anti-Sm antibodies and HLA-DR3+. At week 3, the onset of functional renal damage (measured by increased proteinuria), mice were treated with no Tregs, polyclonal Tregs (pTregs), or HLA-DR3-restricted Sm-Tregs (transduced with HLA-DR3 TCR1).

[0250] conclusion Taken together, the results in this example confirm that we have identified highly reactive T cell receptors specific for the Smith (Sm) antigen, a key target autoantigen in lupus, and show that these T cell receptors can be transduced into human Tregs that can be used to specifically suppress autoimmunity against the Sm antigen.

[0251] The present inventors have demonstrated that both HLA-DR15-restricted SmTCRs and HLA-DR3-restricted SmTCRs are therapeutically effective and can be used to halt the progression of autoimmune diseases.

[0252] Clinically, the present invention enables a novel antigen-specific regulatory cell-based treatment in which autologous regulatory T cells specific for Sm antigens are adoptively transferred into lupus patients to suppress their underlying disease causes and halt disease progression.

[0253] Current treatments for lupus are nonspecific and have toxic side effects. The current standard of care for lupus involves the use of corticosteroids, which themselves cause significant side effects, including diabetes and osteoporosis, and nonspecific immunosuppressants, which have adverse side effects and limited efficacy. In the last 50 years, the only newly approved add-on treatment for lupus is the anti-BAFF antibody, belimumab. However, to date, belimumab has limited clinical efficacy, and its contraindications include active nephritis and central nervous system symptoms. Therefore, with existing treatments, increasing irreversible organ damage over time is a common outcome, resulting in a standardized mortality rate approximately two to three times higher than that of the healthy community. Therefore, the need for better treatments for lupus remains clear and unmet.

[0254] The treatments described herein, i.e., the use of Sm-specific Tregs and peptides disclosed herein, are predicted to enhance Treg potency and induce enhanced immunosuppression while minimizing the suppressive effect on protective immunity. The use of Sm-specific Tregs (and peptides that activate / expand such Tregs) is predicted to induce immune tolerance to self-antigens beyond Smith proteins, as immunosuppressive cells (e.g., Tregs and myeloid-derived suppressor cells) recruited as a result of Sm-specific Treg therapy will exhibit additional non-antigen-specific immunosuppressive capabilities, creating a tolerant environment to multiple self-antigens.

[0255] 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 binding protein comprising a T cell receptor (TCR) α chain variable (Vα or V-alpha) domain and a TCR β chain variable (Vβ or V-beta) domain, wherein the binding protein is capable of binding to a complex of a Smith protein fragment and an HLA-DR3 molecule. (1) The Vα domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 261, CDR2 containing the amino acid sequence shown in SEQ ID NO: 262, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 263; the Vβ domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 264, CDR2 containing the amino acid sequence shown in SEQ ID NO: 265, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 266; (2) The Vα domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 273, CDR2 containing the amino acid sequence shown in SEQ ID NO: 274, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 275; the Vβ domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 276, CDR2 containing the amino acid sequence shown in SEQ ID NO: 277, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 278; (3) The Vα domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 285, CDR2 containing the amino acid sequence shown in SEQ ID NO: 286, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 287; the Vβ domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 288, CDR2 containing the amino acid sequence shown in SEQ ID NO: 289, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 290; (4) The Vα domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 297, CDR2 containing the amino acid sequence shown in SEQ ID NO: 298, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 299; the Vβ domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 300, CDR2 containing the amino acid sequence shown in SEQ ID NO: 301, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 302; (5) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 309, CDR2 containing the amino acid sequence shown in SEQ ID NO: 310, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 311; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 312, CDR2 containing the amino acid sequence shown in SEQ ID NO: 313, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 314; (6) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 321, CDR2 containing the amino acid sequence shown in SEQ ID NO: 322, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 323; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 324, CDR2 containing the amino acid sequence shown in SEQ ID NO: 325, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 326; (7) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 333, CDR2 containing the amino acid sequence shown in SEQ ID NO: 334, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 335; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 336, CDR2 containing the amino acid sequence shown in SEQ ID NO: 337, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 338; (8) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 345, CDR2 containing the amino acid sequence shown in SEQ ID NO: 346, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 347; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 348, CDR2 containing the amino acid sequence shown in SEQ ID NO: 349, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 350; (9) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 357, CDR2 containing the amino acid sequence shown in SEQ ID NO: 358, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 359; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 360, CDR2 containing the amino acid sequence shown in SEQ ID NO: 361, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 362; (10) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 369, CDR2 containing the amino acid sequence shown in SEQ ID NO: 370, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 371; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 372, CDR2 containing the amino acid sequence shown in SEQ ID NO: 373, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 374; (11) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 381, CDR2 containing the amino acid sequence shown in SEQ ID NO: 382, ​​and CDR3 containing the amino acid sequence shown in SEQ ID NO: 383; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 384, CDR2 containing the amino acid sequence shown in SEQ ID NO: 385, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 386; (12) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 393, CDR2 containing the amino acid sequence shown in SEQ ID NO: 394, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 395; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 396, CDR2 containing the amino acid sequence shown in SEQ ID NO: 397, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 398; (13) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 405, CDR2 containing the amino acid sequence shown in SEQ ID NO: 406, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 407; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 408, CDR2 containing the amino acid sequence shown in SEQ ID NO: 409, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 410; (14) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 417, CDR2 containing the amino acid sequence shown in SEQ ID NO: 418, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 419; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 420, CDR2 containing the amino acid sequence shown in SEQ ID NO: 421, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 422; (15) The Vα domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 429, CDR2 containing the amino acid sequence shown in SEQ ID NO: 430, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

431. The Vβ domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 432, CDR2 containing the amino acid sequence shown in SEQ ID NO: 433, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

434. (16) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 441, CDR2 containing the amino acid sequence shown in SEQ ID NO: 442, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 443; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 444, CDR2 containing the amino acid sequence shown in SEQ ID NO: 445, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 446; (17) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 453, CDR2 containing the amino acid sequence shown in SEQ ID NO: 454, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 455; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 456, CDR2 containing the amino acid sequence shown in SEQ ID NO: 457, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 458; (18) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 465, CDR2 containing the amino acid sequence shown in SEQ ID NO: 466, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 467; the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 468, CDR2 containing the amino acid sequence shown in SEQ ID NO: 469, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 470; (19) The Vα domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 477, CDR2 containing the amino acid sequence shown in SEQ ID NO: 478, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 479, and the Vβ domain comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 480, CDR2 containing the amino acid sequence shown in SEQ ID NO: 481, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 482; or (20) The Vα domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 489, CDR2 containing the amino acid sequence shown in SEQ ID NO: 490, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 491; the Vβ domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 492, CDR2 containing the amino acid sequence shown in SEQ ID NO: 493, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

494. Binding proteins.

2. The HLA-DR3 molecule, HLA-DRA ＊ 01:01 molecule and HLA-DRB1 ＊ A binding protein according to claim 1, which is a 03:01 molecule.

3. A binding protein according to claim 1 or 2, which binds to a peptide consisting of 4, 5, 7, 8, 9, 10 or more consecutive amino acid residues of the sequence specified in either one of Sequence ID No. 258 or 259.

4. The binding protein according to claim 1 or 3, wherein a fragment of Smith protein capable of forming a complex with an HLA-DR3 molecule comprises or is derived from the amino acid sequence of residues 7-21 of the SmB / B' protein or residues 78-92 of the SmD1 protein.

5. The binding protein according to claim 4, wherein the SmD protein comprises the amino acid sequence of SEQ ID NO:

260.

6. (1) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 585, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 586; (2) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 587, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO: 588; (3) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 589, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO: 590; (4) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 591, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO: 592; (5) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 593, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO: 594; (6) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 595, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO: 596; (7) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 597, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO: 598; (8) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 599, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO: 600; (9) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 601, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 602; (10) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 603, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 604; (11) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 605, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 606; (12) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 607, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 608; (13) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 609, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 610; (14) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 611, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 612; (15) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 613, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 614; (16) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 615, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 616; (17) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 617, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 618; (18) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 619, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 620; (19) The TCRα chain comprises the amino acid sequence shown in SEQ ID NO: 621, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO: 622; or (20) The TCRα chain contains the amino acid sequence shown in SEQ ID NO: 623, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO:

624. A binding protein according to any one of claims 1 to 5.

7. The binding protein according to any one of claims 1 to 6, wherein the TCRα chain and the TCRβ chain are modified to include cysteine ​​residues that enable the formation of further interchain disulfide bonds.

8. The binding protein according to claim 7, wherein a residue at position 180 of the TCRα chain containing the sequence shown in SEQ ID NO: 605, or an equivalent residue, and a residue at position 189 of the TCRβ chain containing the sequence shown in SEQ ID NO: 606, or an equivalent residue, are replaced with cysteine ​​to facilitate the creation of further disulfide bonds between the TCR constant regions.

9. A peptide consisting of the amino acid sequence shown in Sequence ID No.

259.

10. A peptide consisting of the amino acid sequence shown in Sequence ID No.

258.

11. A nucleic acid comprising, or consisting of, a nucleotide sequence encoding a binding protein according to any one of claims 1 to 8, or a nucleotide sequence encoding a peptide according to claim 9 or 10.

12. A vector comprising the nucleic acid described in claim 11.

13. (a) enabling the expression of nucleotide sequences within cells, resulting in the presentation of binding proteins on the cell surface, (b) It is a retroviral vector, or (c) Below: (i) EF1α (alpha) promoter; (ii) 2A ribosome skipping sequence; (iii) Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); (iv) A configuration in which the TCRβ chain variable (Vβ or Vbeta) domain is translated before the (TCR)α chain variable (Vα or Valpha) domain; or (v) Arrangement that translates the TCRβ chain variable (Vβ or Vbeta) chain before the (TCR)α chain variable (Vα or Valpha) chain. Including one or more or all of the following: The vector according to claim 12.

14. A cell comprising the vector according to claim 12 or 13 or the nucleic acid according to claim 11.

15. A method for preparing a population of regulatory T cells for use in the treatment of systemic lupus erythematosus (SLE), wherein the regulatory T cells are obtained from a subject, and the method is Steps to prepare a population of regulatory T cells; A step of introducing the nucleic acid according to claim 11 or the vector according to claim 12 or 13 into a population of regulatory T cells; A step that provides conditions that enable the expression of binding proteins on the surface of regulatory T cells. including, or A step of preparing a mixed T cell population or T cell population that exhibits at least one characteristic of a normal T cell; A step of introducing the nucleic acid according to claim 11 or the vector according to claim 12 or 13 into a population of T cells; A step of providing conditions that enable the expression of binding proteins on the surface of T cells; The steps of isolating regulatory T cells from a mixed T cell population, or culturing cells within a population under conditions that promote the conversion of cells into regulatory T cells; A step to selectively stabilize converted regulatory T cells. including, or A step of culturing a T cell population in the presence of the peptide described in claim 10, under conditions that allow for the proliferation of a subpopulation of cells activated by the peptide, and for a sufficient amount of time; The step of optionally separating regulatory T cells from a mixed cell population or converting T cells to regulatory T cells if the T cell population includes a mixed cell population or includes normal T cells. Includes, This provides a method for preparing a population of regulatory T cells for use in the treatment of SLE.

16. The method according to claim 15, wherein the T cells are derived from a biological sample derived from a subject having SLE or from an allogeneic donor not having SLE.

17. The method according to claim 15, wherein the T cells are derived from a subject requiring treatment for SLE.

18. The method according to claim 15, wherein the T cells are derived from stem cells, and optionally the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells.

19. A composition comprising a binding protein according to any one of claims 1 to 8, a peptide according to claim 9 or 10, a cell according to claim 14, a nucleic acid according to claim 11, or a vector according to claim 12 or 13, and a pharmaceutically acceptable carrier, diluent, or excipient.

20. A composition for use in a method for treating or preventing a lupus condition associated with an abnormal, undesirable, or otherwise inappropriate immune response to Smith protein in a subject, wherein the composition is A step of preparing a population of T cells that exhibit at least one characteristic of regulatory T cells; The step of introducing the nucleic acid described in claim 11 or the vector described in claim 12 or 13 into a population of T cells; and A step that provides conditions that enable the expression of binding proteins on the surface of T cells. The method includes T cells that express a binding protein on the surface of T cells obtained by a method including the following: The method includes the step of administering T cells that express a binding protein on the surface of the T cells, thereby treating or preventing lupus in a subject. composition.

21. Use of a binding protein according to any one of claims 1 to 8, a peptide according to claim 9 or 10, a cell according to claim 14, a nucleic acid according to claim 11, or a vector according to claim 12 or 13 in the manufacture of a pharmaceutical for treating or preventing a lupus condition in a subject, wherein the lupus condition is associated with an abnormal, undesirable, or otherwise inappropriate immune response to Smith protein.

22. A composition comprising a binding protein according to any one of claims 1 to 8, a peptide according to claim 9 or 10, a cell according to claim 14, or a nucleic acid according to claim 11, or a vector according to claim 12 or 13, for use in treating or preventing a lupus condition associated with an abnormal, undesirable, or otherwise inappropriate immune response to Smith protein in a subject.

23. The composition according to claim 20 or 22, wherein the lupus condition associated with an abnormal, undesirable, or otherwise inappropriate immune response to Smith protein is systemic lupus erythematosus (SLE) or lupus nephritis (LN).

24. The use according to claim 21, wherein the lupus condition associated with an abnormal, undesirable, or otherwise inappropriate immune response to Smith protein is systemic lupus erythematosus (SLE) or lupus nephritis (LN).

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