• This exploratory study describes the effect of eculizumab on multiple physiologic pathways affected by complement dysregulation in aHUS.• The results highlight the importance of sustained terminal complement blockade, even in patients with improved clinical laboratory values.Atypical hemolytic uremic syndrome (aHUS) is a genetic, life-threatening disease characterized by uncontrolled complement activation, systemic thrombotic microangiopathy (TMA), and vital organ damage. We evaluated the effect of terminal complement blockade with the anti-C5 monoclonal antibody eculizumab on biomarkers of cellular processes involved in TMA in patients with aHUS longitudinally, during up to 1 year of treatment, compared with in healthy volunteers. Biomarker levels were elevated at baseline in most patients, regardless of mutational status, plasma exchange/infusion use, platelet count, or lactate dehydrogenase or haptoglobin levels. Eculizumab reduced terminal complement activation (C5a and sC5b-9) and renal injury markers (clusterin, cystatin-C, b2-microglobulin, and liver fatty acid binding protein-1) to healthy volunteer levels and reduced inflammation (soluble tumor necrosis factor receptor-1), coagulation (prothrombin fragment F112 and D-dimer), and endothelial damage (thrombomodulin) markers to near-normal levels. Alternative pathway activation (Ba) and endothelial activation markers (soluble vascular cell adhesion molecule-1) decreased but remained elevated, reflecting ongoing complement activation in aHUS despite complete terminal complement blockade. These results highlight links between terminal complement activation and inflammation, endothelial damage, thrombosis, and renal injury and underscore ongoing risk for systemic TMA and progression to organ damage. Further research regarding underlying complement dysregulation is warranted. This trial was registered at www.clinicaltrials.gov as #NCT01194973. (Blood. 2015;125(21):3253-3262)

Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7–1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.

CD200 is an immunoregulatory protein, highly expressed on activated T and B cells and on various liquid and solid tumors. We have previously shown that treatment with an antagonist anti-CD200 antibody in vivo enhances anti-tumor immunity in a hematologic cancer model. The present studies evaluated whether in vivo anti-CD200 treatment modulates hematopoietic cell subsets and allo- and auto- antibody responses in an AIHD model. Mice were immunized weekly with rat red blood cells (RBC) to prompt robust anti-rat RBC allo- and cross-reactive auto- antibody responses. Immunized mice were randomized into groups receiving either vehicle alone, an anti-CD200 antibody (OX90mG2a), a non-reactive, isotype-matched control antibody (12B4mG2a), cyclosporine (CsA), or CsA plus OX90mG2a or 12B4G2a antibodies. Allo- and auto- antibodies were measured prior to and at the conclusion of treatment. Administration of OX90mG2a with or without CsA significantly impacted CD200+ cell subsets in treated mice. Decreased frequencies of CD200+ T cells, B cells, plasma cells and progenitor bone marrow cells were observed in OX90mG2a-treated mice relative to control mice. All CD200+ populations returned to normal levels after termination of anti-CD200 treatment. OX90mG2a administration also inhibited allo- and auto- antibody production in this model. These data suggest that anti-CD200 antibody therapy is a potent immunomodulator and may provide a therapeutic strategy for antibody-mediated diseases.

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Introduction aHUS is a genetic, life-threatening disease of chronic, uncontrolled complement activation leading to systemic complement-mediated thrombotic microangiopathy (TMA). Ecu (anti-C5 mAb) blocks terminal complement and inhibits TMA, normalizes platelets, and improves renal function in aHUS pts. We describe levels of key biomarkers of complement alternative pathway (CAP) and endothelial activation, as well as inflammation, coagulation, and renal injury that together characterize the disease process in pts with aHUS, prior to and during Ecu treatment. Methods Biomarkers were evaluated using standard methods in healthy subjects (healthy) and adult aHUS pts (N=29-39) at baseline and during 26 weeks (wks) of Ecu treatment in a Phase 2 aHUS clinical trial. Results All biomarkers were elevated at baseline in the majority of pts (Table), including pts with normal platelet counts (Figure), LDH, and/or haptoglobin levels. Following Ecu treatment in all pts, coagulation markers (F1+2 and D-dimer) were significantly reduced by 2.5 wks and decreased by >50% by wk 26. Renal injury markers (U-cystatin C and U-TIMP-1) also reduced significantly by 2.5wks. Interestingly, mean plasma levels of complement Ba, a key marker of CAP activation, were significantly decreased by 4-6 wks following Ecu but remained elevated. Soluble tumor necrosis factor receptor 1 (sTNFR1) and U-clusterin levels decreased by 4-6 wks, while thrombomodulin, VCAM-1, CXCL10, U-FABP-1, U-β2m and IL-6 levels reduced more slowly (12-26 wks). By 26 wks of Ecu, levels of all renal injury markers had normalized (≈78%-90% reduction) but markers of CAP and endothelial activation, inflammation, and coagulation remained elevated relative to levels in healthy subjects. Conclusions At baseline, alternative pathway complement activation and elevated biomarkers of inflammation, coagulation, endothelial activation, and renal injury were evident in the majority of pts with aHUS, including those exhibiting normal markers of TMA. Ecu reduced but did not normalize plasma Ba, suggesting ongoing complement activation (CAP) upstream of C5 and the need for sustained complement inhibition downstream. Ecu treatment rapidly and markedly reduced biomarkers of thrombin generation, fibrinolysis, and inflammation, and normalized markers of renal injury; this reduction was sustained with ongoing Ecu treatment. Endothelial activation biomarkers reduced more slowly, suggesting ongoing effects of CAP C3 activation upstream of C5. These data point to the chronicity of the disease process and support the continuous administration of Ecu in aHUS pts, including those with normal hematological markers of TMA. Disclosures: Cofiell: Alexion Pharmaceuticals: Employment. Kukreja:Alexion Pharmaceuticals: Employment. Bedard:Alexion Pharmaceuticals: Employment. Yan:Alexion Pharmaceuticals: Employment. Mickle:Alexion Pharmaceuticals: Employment. Ogawa:Alexion Pharmaceuticals: Employment. Bedrosian:Alexion Pharmaceuticals: Employment. Faas:Alexion Pharmaceuticals: Employment.