Currently used anticoagulants prevent thrombosis but increase bleeding. We show an anticoagulation therapy without bleeding risk based on a plasma protease factor XII function-neutralizing antibody. We screened for antibodies against activated factor XII (FXIIa) using phage display and demonstrated that recombinant fully human antibody 3F7 binds into the FXIIa enzymatic pocket. 3F7 interfered with FXIIa-mediated coagulation, abolished thrombus formation under flow, and blocked experimental thrombosis in mice and rabbits. We adapted an extracorporeal membrane oxygenation (ECMO) cardiopulmonary bypass system used for infant therapy to analyze clinical applicability of 3F7 in rabbits. 3F7 provided thromboprotection as efficiently as heparin, and both drugs prevented fibrin deposition and thrombosis within the extracorporeal circuit. Unlike heparin, 3F7 treatment did not impair the hemostatic capacity and did not increase bleeding from wounds. These data establish that targeting of FXIIa is a safe mode of thromboprotection in bypass systems, and provide a clinically relevant anticoagulation strategy that is not complicated by excess bleeding.

G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF- and G-CSF receptor-deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti-G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSF receptor-treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti-G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1β, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti-G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

We report the generation of a chimeric monoclonal antibody (ch806) with specificity for an epitope on the epidermal growth factor receptor (EGFR) that is different from that targeted by all other anti-EGFR therapies. Ch806 antibody is reactive to both de2-7 and overexpressed wild-type (wt) EGFR but not native EGFR expressed in normal tissues at physiological levels. Ch806 was stably expressed in CHO (DHFR À/À) cells and purified for subsequent characterisation and validated for use in preliminary immunotherapy investigations. Ch806 retained the antigen binding specificity and affinity of the murine parental antibody. Furthermore, ch806 displayed enhanced antibody-dependent cellular cytotoxicity against target cells expressing the 806 antigen in the presence of human effector cells. Ch806 was successfully radiolabelled with both iodine-125 and indium-111 without loss of antigen binding affinity or specificity. The radioimmunoconjugates were stable in the presence of human serum at 371C for up to 9 days and displayed a terminal half-life (T 1/2b ) of approximately 78 h in nude mice. Biodistribution studies undertaken in BALB/c nude mice bearing de2-7 EGFR-expressing or amplified EGFR-expressing xenografts revealed that 125 I-labelled ch806 failed to display any significant tumour retention. However, specific and prolonged tumour localisation of' 111In-labelled ch806 was demonstrated with uptake of 31%ID g À1 and a tumour to blood ratio of 5 : 1 observed at 7 days postinjection. In vivo therapy studies with ch806 demonstrated significant antitumour effects on established de2-7 EGFR xenografts in BALB/c nude mice compared to control, and both murine 806 and the anti-EGFR 528 antibodies. These results support a potential therapeutic role of ch806 in the treatment of suitable EGFR-expressing tumours, and warrants further investigation of the potential of ch806 as a therapeutic agent.

, and ERDF. Z.D. was supported by a fellowship from Fondation Française pour la Recherche contre le My elome et les Gammapathies monoclonales (F.F.R.M.G.

The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (βc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human βc receptor. The binding epitope of CSL311 on the βc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human βc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human βc receptor is central to pathogenesis. The coordinates for the βc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).