Novel VISTA-Ig constructs and the use of VISTA-Ig for Treatment of Autoimmune, Allergic and Inflammatory Disorders

Abstract

The present invention relates to a fusion proteins comprising regulatory T cell protein, VISTA (V-domain Immunoglobulin Suppressor of T cell Activation (PD-L3) and an immunoglobulin protein (Ig), preferably also containing a flexible linker intervening the VISTA and Ig Fc polypeptide. The invention also provides the use of VISTA polypeptides, multimeric VISTA polypeptides, VISTA-conjugates (e.g., VISTA-Ig), and VISTA antagonists for the treatment of autoimmune disease, allergy, and inflammatory conditions, especially lupus, multiple sclerosis, psoriasis, psoriatic arthritis, multiple sclerosis, Crohn's disease, inflammatory bowel disease and type 1 or type 2 diabetes.

1 - 226 . (canceled) 
     
     
         227 . An isolated VISTA-Ig fusion protein comprising (i) at least one polypeptide with at least about 80% sequence identity to the extracellular domain of the VISTA polypeptide sequence in SEQ ID NO: 2, 4, 5, 16-25, 36, 37, 68, 69, 72 or 73 or 75 or a fragment thereof which comprises at least 50 amino acids; (ii) at least one linker which comprises at least 5 amino acids; and (iii) at least one Ig Fc protein or fragment thereof, wherein said linker intervenes a VISTA polypeptide and a Ig Fc protein and the resultant VISTA-Ig fusion protein elicits more potent immunosuppressive activity in vivo than an otherwise identical fusion protein lacking the linker. 
     
     
         228 . The VISTA-Ig fusion protein of  claim 227 , wherein said polypeptide (i) comprises at least one polypeptide having at least 90% or at least 95% sequence identity to the extracellular domain of human or murine VISTA or a fragment thereof which is at least 50 amino acids. 
     
     
         229 . The VISTA-Ig fusion protein of  claims 227 , comprising a human IgG1, IgG2, IgG3 or IgG4 Fc region or fragment or variant thereof. 
     
     
         230 . The VISTA-Ig fusion protein of  claim 227 , containing at least one Fc region that comprises one or more modifications that modulate complement binding, FcR binding, glycosylation and/or effector function. 
     
     
         231 . The VISTA-Ig fusion protein of  claim 227 , containing at least one linker that comprises about 4 glycine residues to about 15 glycine residues. 
     
     
         232 . The VISTA-Ig fusion protein of  claim 227 , containing at least one linker that comprises at about 8 to about 50 amino acid residues. 
     
     
         233 . The VISTA-Ig fusion protein of  claim 227 , comprising at least 2, 3 or 4 polypeptides which each possess at least about 90% or about 95% sequence identity to the extracellular domain of the polypeptide sequence of SEQ ID NO: 2, 4, 5, 16-25, 36, or 37 or a fragment thereof which comprises at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 amino acids. 
     
     
         234 . The VISTA-Ig fusion protein of  claim 227 , comprising at least one linker wherein about 30% to about 90% of the linker is comprised of glycine and/or serine residues. 
     
     
         235 . The VISTA-Ig fusion protein of  claim 227 , which comprises at least one human or murine Fc region. 
     
     
         236 . The VISTA-Ig fusion protein of  claim 235 , which comprises at least one human IgG1, IgG2, IgG3 or IgG4 Fc region or a murine IgG2a or IgG2b constant region. 
     
     
         237 . The VISTA-Ig fusion protein of  claim 235 , which comprises at least one human IgG1 or IgG3 Fc region. 
     
     
         238 . The VISTA-Ig fusion protein of  claim 227 , which exhibits about 30% to about 100% more immunosuppressive activity in an assay that detects T cell proliferation than an otherwise identical VISTA-Ig fusion protein lacking said at least one linker. 
     
     
         239 . The VISTA-Ig fusion protein of  claim 227 , which exhibits about 1.0 fold to about 10 fold more immunosuppressive activity in an assay that detects T cell proliferation than an otherwise identical VISTA-Ig fusion protein lacking said at least one linker. 
     
     
         240 . The VISTA-Ig protein of  claim 227 , comprising the extracellular domain of VISTA comprising amino acid residues 32-190 or amino acid residues 16-194. 
     
     
         241 . The VISTA-Ig protein of  claim 227 , comprising at least two copies of a VISTA protein or fragment thereof comprising at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 amino acids and at least two copies of a VISTA protein and IgG1 Fc or non-FcR-binding IgG1. 
     
     
         242 . The VISTA-Ig protein of  claim 227 , comprising at least two, three, four, five, six, seven, or eight copies of a VISTA protein or fragment thereof comprising at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 amino acids of SEQ ID NO: 2, 4, 5, 16-25, 36, 37, 68, 69, 72 or 73 or 75 or a fragment thereof and at least one IgG1 or IgG2a. 
     
     
         243 . The VISTA-Ig protein of  claim 227 , comprising at least two copies of a VISTA protein or fragment thereof which each at least possess about 90, 95, 96, 97, 98 or 99% sequence identity to the extracellular domain comprising the polypeptide sequence of SEQ ID NO: 2, 4, or 25 or fragment thereof comprising at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 amino acids. 
     
     
         244 . The VISTA-Ig protein of  claim 227 , comprising at least one extracellular domain or fragment of VISTA attached to the N-terminus of an oligomerization domain. 
     
     
         245 . The VISTA-Ig protein of  claim 244 , wherein the oligomerization domain is GCN4, COMP, SNARE, CMP, MAT, LLR containing 1 NLRC, NOD2 nucleotide-binding NLRC2, LRR containing 1 NLRC, NOD2 nucleotide-binding NLRC2, or PSORAS1. 
     
     
         246 . A recombinant cell which expresses a VISTA-Ig fusion protein according to  claim 227 . 
     
     
         247 . The cell of  claim 246 , which is a yeast, bacterial, fungal, insect, avian,  Xenopus , or mammalian cell. 
     
     
         248 . A pharmaceutically acceptable composition containing a therapeutically effective amount of a VISTA-Ig fusion protein according to  claim 227 . 
     
     
         249 . A method of inhibiting T cell, neutrophil, monocyte and/or leukocyte proliferation in a subject in need thereof, comprising the administration of a VISTA-Ig fusion protein according to  claim 227 . 
     
     
         250 . The method of  claim 249 , wherein the T cells include one or more of rested or activated CD4+ T cells, CD8+ T cells, memory cells, and/or effector cells. 
     
     
         251 . A method of inhibiting T cell activation in a subject in need thereof, comprising the administration of a VISTA-Ig fusion protein according to  claim 227 . 
     
     
         252 . A method of inducing the expression of Foxp3 on T cells in a subject in need thereof and/or inducing tolerance to an autoantigen or foreign antigen or allergen, comprising the administration of a VISTA-Ig fusion protein according to  claim 227 . 
     
     
         253 . A method of inhibiting T cell infiltration and/or Th17 cell proliferation in a subject in need thereof, comprising the administration of a VISTA-Ig fusion protein according to  claim 227 . 
     
     
         254 . An isolated siRNA molecule that targets VISTA comprising the nucleic acid sequence of any one of SEQ ID NOs: 38-67, wherein (i) the isolated siRNA molecule comprising the amino acid sequence of any one of SEQ ID NO: 38-47 targets either the ORF or UTR region of VISTA comprising the amino acid sequence of any one of SEQ ID NO: 38-47; (ii) the isolated siRNA molecule comprising the amino acid sequence of any one of SEQ ID NO: 48-57 targets the UTR region only of VISTA; and (iii) the isolated siRNA molecule comprising the amino acid sequence of any one of SEQ ID NO: 58-67 targets the ORF region only of VISTA. 
     
     
         255 . A composition comprising at least one siRNA molecule according to  claim 254 . 
     
     
         256 . A method for treating an allergic disorder, an inflammatory disorder, an autoimmune disorder, graft-versus-host disease, multiple sclerosis, follicular hyperplasia, myelopoiesis and/or an allergic respiratory disorder comprising administering at least one siRNA molecule of  claim 254  that targets VISTA. 
     
     
         257 . A method of treating or preventing lupus and/or one or more of the symptoms associated with lupus comprising administering a therapeutically or prophylactically effective amount of the VISTA-Ig protein of  claim 227 , wherein the symptom associated with lupus includes splenomegaly, proteinuria, loss of kidney function, macrophage infiltration into the kidneys, Ig deposition in glomeruli, proteinuria, increased pro-inflammatory cytokine production, weight loss or impaired renal function. 
     
     
         258 . The method of  claim 257 , which further includes:
 (i) the administration of another drug for treating lupus, wherein the lupus drug is a Nonsteroidal anti-inflammatory drug such as naproxen, ibuprofen, an Antimalarial drugs such as hydroxychloroquine (Plaquenil), a Corticosteroid such as Prednisone, an Immune suppressant such as cyclophosphamide (Cytoxan), azathioprine (Imuran, Azasan), mycophenolate (Cellcept), leflunomide (Arava), methotrexate (Trexall); and/or   (ii) the administration of another immunosuppressant, or agent that improves kidney function, wherein said other immunosuppressive agent is PD-1, PD-L1, PD-L2, CTLA4, or ICOS protein or at least one PD-1, PD-L1, PD-L2, CTLA4, or ICOS fusion protein comprising the entire extracellular region or fragment of said extracellular region that is at least 50, 100, 150, 200, 250 or 300 amino acids or a variant that possesses at least 80-90 or 95% sequence identity to any of the extracellular regions of one PD-1, PD-L1, PD-L2, CTLA4, or ICOS or to a fragment thereof that is at least 50, 100, 150, 200, 250 or 300 amino acids or an antibody s or antibody fragment specific to any of PD-1, PD-L1, PD-L2, CTLA4, or ICOS.   
     
     
         259 . A VISTA-Ig fusion that possesses at least 90%, at least 95%, at least 98% or 100% sequence identity to the VISTA polypeptide in SEQ ID NO:68, 69, 72 or 73. 
     
     
         260 . A method of specifically targeting T cells, NK cells and/or myeloid cells in a patient in need thereof by administration of a VISTA-Ig polypeptide according to  claim 227 . 
     
     
         261 . The VISTA-Ig polypeptide of  claim 227 , comprising a human IgG1 Fc region that is mutated to introduce at least one mutation selected from E269R, E233P, D265A, K322A, P331G and P331/K322A. 
     
     
         262 . A method of administering a VISTA-Ig fusion protein according to  claim 227  to treat a subject in need thereof, said subject having an autoimmune, inflammatory, or allergic disorder, wherein the administered VISTA-Ig fusion protein agonizes the effects of VISTA on immunity and thereby suppresses autoimmunity, allergy or inflammation in said subject. 
     
     
         263 . The method of  claim 262 , wherein the subject has an autoimmune disorder. 
     
     
         264 . The method of  claim 262 , wherein the subject has an inflammatory disorder. 
     
     
         265 . The method of  claim 262 , wherein the subject has an allergic disorder. 
     
     
         266 . The method of  claim 262 , wherein the subject has a disorder selected from rheumatoid arthritis, lupus, psoriasis, graft-versus-host disease, multiple sclerosis, follicular hyperplasia, myelopoiesis and an allergic respiratory disorder.