Anti-histone therapy for vascular necrosis in severe glomerulonephritis

Abstract

Severe glomerulonephritis involves cell necrosis as well as NETosis, programmed neutrophil death leading to expulsion of nuclear chromatin and neutrophil extracellular traps (NETs). Histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells. This was prevented by histone-neutralizing agents anti-histone IgG, activated protein C and heparin. Histone toxicity on glomeruli was TLR2/4-dependent. Anti-GBM glomerulonephritis involved NET formation and vascular necrosis. Pre-emptive anti-histone IgG administration significantly reduced all aspects of glomerulonephritis, including vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment and activation of glomerular leukocytes and glomerular crescent formation. Subjects with established glomerulonephritis treated with anti-histone IgG, recombinant activated protein C, or heparin all abrogated severe glomerulonephritis suggesting that histone-mediated glomerular pathology is a subsequent, not initial event in necrotizing glomerulonephritis. Neutralizing extracellular histones is therapeutic in severe experimental glomerulonephritis.

What is claimed is: 
     
         1 . A method of treating glomerulonephritis comprising administering to a subject with glomerulonephritis (GN) at least one anti-histone agent selected from the group consisting of an anti-histone antibody or antigen-binding fragment thereof, activated protein C (APC), and heparin. 
     
     
         2 . The method of  claim 1 , wherein the anti-histone antibody binds to a human histone selected from the group consisting of histone H1/H5, histone H2A, histone H2B, histone H3 and histone H4. 
     
     
         3 . The method of  claim 1 , wherein the anti-histone antibody is an anti-histone H4 antibody. 
     
     
         4 . The method of  claim 1 , wherein the anti-histone antibody is selected from the group consisting of BWA-3, LG2-1 and LG2-2. 
     
     
         5 . The method of  claim 1 , wherein the glomerulonephritis is rapidly-progressive glomerulonephritis (RPGN). 
     
     
         6 . The method of  claim 1 , wherein the glomerulonephritis is anti-neutrophil cytoplasmic antibody (ANCA) associated glomerulonephritis. 
     
     
         7 . The method of  claim 1 , wherein the anti-histone agent inhibits activity of toll-like receptor 2 (TLR-2) and TLR-4. 
     
     
         8 . The method of  claim 1 , wherein administration of anti-histone antibody reduces vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment and activation of glomerular leukocytes and glomerular crescent formation. 
     
     
         9 . The method of  claim 1 , wherein the anti-histone antibody is a chimeric, humanized or human antibody. 
     
     
         10 . The method of  claim 1 , wherein the antibody fragment is selected from the group consisting of F(ab′) 2 , Fab′, F(ab) 2 , Fab, Fv, sFv, scFv and single domain antibody (nanobody). 
     
     
         11 . The method of  claim 1 , further comprising administering two or more anti-histone agents to the subject. 
     
     
         12 . The method of  claim 11 , wherein the two or more agents are selected from the group consisting of an anti-histone antibody, activated protein C and heparin. 
     
     
         13 . The method of  claim 1 , wherein the subject is a human subject. 
     
     
         14 . The method of  claim 1 , wherein administration of the anti-histone agent is effective to treat vascular necrosis in severe glomerulonephritis. 
     
     
         15 . The method of  claim 1 , further comprising administering to the subject an anti-TNF-α antibody. 
     
     
         16 . The method of  claim 1 , wherein the subject is a human subject. 
     
     
         17 . The method of  claim 1 , further comprising administering to the subject an antibody against toll-like receptor 2 (TLR-2) or TLR-4. 
     
     
         18 . The method of  claim 1 , wherein the anti-histone antibody or antigen-binding fragment thereof is not conjugated to a therapeutic agent. 
     
     
         19 . The method of  claim 1 , wherein the anti-histone antibody or antigen-binding fragment thereof is conjugated to at least one therapeutic agent. 
     
     
         20 . The method of  claim 19 , wherein the therapeutic agent is selected from the group consisting of a second antibody, an antigen-binding fragment of a second antibody, a radionuclide, an immunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, a cytokine, a chemokine, a drug, a toxin, a hormone, an siRNA and an enzyme. 
     
     
         21 . The method of  claim 20 , further comprising administering to the subject an immunomodulator selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interleukin (IL), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α, interferon-β, interferon-γ, interferon-λ, TGF-α, TGF-β, interleukin-1 (IL-1), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin and lymphotoxin. 
     
     
         22 . The method of  claim 21 , wherein the cytokine is selected from the group consisting of human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors, interferon-α, interferon-β, interferon-γ, macrophage-CSF (M-CSF), IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and lymphotoxin. 
     
     
         23 . The method of  claim 1 , further comprising administering to the subject a second antibody or antigen-binding fragment thereof, where the second antibody binds to an antigen selected from the group consisting of histone H2B, histone H3, histone H4, a proinflammatory effector of the innate immune system, a proinflammatory effector cytokine, a proinflammatory effector chemokine, TNF-α, MIF, CD74, HLA-DR, IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, CD40L, CD44, CD46, CD55, CD59, CCL19, CCL21, mCRP, MCP-19, MIP-1A, MIP-1B, RANTES, ENA-78, IP-10, GRO-β, lipopolysaccharide, lymphotoxin, HMGB-1, tissue factor, a complement regulatory protein, a coagulation factor, thrombin, a complement factor, C3, C3a, C3b, C4a, C4b, C5, C5a, C5b, Flt-1 and VEGF. 
     
     
         24 . The method of  claim 1 , wherein the antibody is a bispecific antibody comprising a first binding site for a histone and a second binding site for a non-histone antigen. 
     
     
         25 . The method of  claim 1 , wherein the anti-histone antibody or antigen-binding fragment thereof is a fusion protein.