The modified light chain of inter-alpha inhibitor/antibody fusion protein, MR1007, improves survival in the rabbit sepsis models
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KeywordsLight Chain Serine Protease Human Umbilical Vein Endothelial Cell Antithrombin Thrombin Generation
Inter-alpha inhibitor is an endogenous serine protease inhibitor and is markedly reduced in severe sepsis. Therapeutic inter-alpha inhibitor replacement showed a survival advantage in several animal models. The light chain is responsible for the serine protease inhibitory activity of inter-alpha inhibitor. Since pro-coagulant and proinflammatory proteases as well as innate immune cells are activated in sepsis, we genetically engineered a novel fusion protein, MR1007, which consists of the modified light chain of inter-alpha inhibitor and the anti-CD14 antibody, and evaluated the potential of MR1007 as an anti-sepsis agent.
Inhibitory activity against serine proteases was assayed using purified enzymes and chromogenic substrates. Anticoagulant activity was measured using human or rabbit plasma. The inhibitory effect on endothelial cell injury was assessed using human umbilical vein endothelial cells. Binding to CD14 and leukocytes was analysed using Biacore or radiolabeling. The survival benefit was evaluated in the endotoxin shock model and the cecal ligation and puncture (CLP) model.
MR1007 inhibited the thromboplastin-induced thrombin generation by inhibiting activities of coagulation factors Xa and XIa at 10–100 μg/ml. It also prevented the contact pathway generation of bradykinin at 10–30 μg/ml. Additionally, it inhibited the leukocyte elastase-induced endothelial cell injury at 10–100 μg/ml. MR1007 had a high affinity for CD14 and bound to leukocytes, but did not block lipopolysaccharide binding to CD14. In the rabbit endotoxin shock model, MR1007 (3 mg/kg, i.v.) even when given 8 hours after the injection of endotoxin improved the survival (n = 12, P < 0.05), whereas both antithrombin and prednisolone exhibited less efficacy. Moreover, MR1007 (10 mg/kg, i.v.) given at 2 hours post-CLP improved the survival (n = 9, P < 0.05) in the CLP model.
These results suggest that the modified light chain of inter-alpha inhibitor fusion protein, MR1007, can effectively suppress not only the serine protease-mediated coagulation, but also leukocyte-induced inflammation, so that MR1007 may become a promising anti-sepsis agent.