The Therapeutic Effect And Mechanisms Of A Soluble Fusion Protein Of Death Receptor 5 And Immunoglobulin In Models Of Hepatitis

China has the world's largest population of patients with hepatitis,and liver failure caused by hepatitis B virus infection is the most common cause of death of liver diseases in China.In western countries,excessive use of acetaminophen?APAP?is the main cause of acute liver failure.At present,there is still a lack of effective therapeutic drugs for hepatitis,especially those with liver failure.Therefore,it is of great practical value and social significance to develop new drugs for the treatment of hepatitis and liver failure with high efficacy and low toxicity.Since cell death plays a fundamental role in almost all types of liver diseases,targeting apoptosis and necroptosis has become one of the major research directions for the treatment of liver diseases.Tumor necrosis factor-related apoptosis inducing ligand?TRAIL?is one of the most important apoptotic molecules in humans and plays an important role in the initiation and development of various hepatitis.Death receptor 5?DR5?is the TRAIL receptor with the highest affinity,and a soluble form of DR5?sDR5?exists naturally,which can bind TRAIL,but can not transmit signals,thus blocking TRAIL-mediated apoptosis.In this study,a recombinant human sDR5-immunoglobulin Fc?sDR5-Fc?fusion protein was produced,and its pharmacology,safety,pharmacokinetics,efficacy and mechanisms were studied in mice,rats and cynomolgus monkeys.The main research areas include the following:?1?Preparation and characterization of sDR5-Fc protein.By genetic engineering technology,the extracellular domain of DR5 and human IgG1 Fc fragment sequences were fused and transfected into CHO-K1 cells.The stable and high expression sDR5-Fc cell lines were selected.The pharmaceutical-grade sDR5-Fc protein was obtained by large-scale cell culture and protein purification.The purity of sDR5-Fc protein was over 99%and its affinity was E-10 M.It was able to specifically bind TRAIL and effectively block the apoptosis induced by TRAIL.?2?Pharmacodynamic study of sDR5-Fc in concanavalin A?Con A?-induced hepatitis model.sDR5-Fc significantly ameliorated Con A-induced hepatitis or liver failure in mice.Mechanistically,sDR5-Fc inhibited hepatocyte death and alleviated inflammation in vivo.In vitro,sDR5-Fc attenuated the production of inflammatory cytokines by spleen cells activated by Con A or anti-CD3 antibody,through down-regulating the activation of NF-kB.Consistent with these results,the levels of inflammatory cytokines produced by TRAIL^–/–mice injected with Con A or their spleen cells activated by Con A or anti-CD3 antibody were significantly lower than those of TRAIL^+/+mice.?3?Pharmacodynamic study of sDR5-Fc in APAP-induced hepatitis model.sDR5-Fc significantly ameliorated APAP-induced hepatitis or liver failure in mice.The severity and mortality of APAP-induced liver injury in mice were correlated with serum sTRAIL level.APAP induced a large number of CD11b⁺Gr1⁺neutrophils to infiltrate the liver.These cells expressed TRAIL on their surface,and secreted sTRAIL and killed DR5-expressing hepatocytes.In addition,these cells expressed CD80,CD86 and MHC II molecules,which were able to promote T cell proliferation.APAP made hepatocytes more sensitive to TRAIL-induced cell death by up-regulating DR5 expression.Therefore,TRAIL-DR5 signaling pathway plays a key role in APAP-induced hepatitis or liver failure.sDR5-Fc can reduce the apoptosis of hepatocytes,alleviate inflammation and restore liver function.sDR5-Fc can cooperate with N-acetylcysteine in the treatment of APAP-induced hepatitis or liver failure.?4?Study of the safety of sDR5-Fc.The maximum tolerable dose of sDR5-Fc by single intravenous infusion was more than 2198,1500 and 1200 mg/kg in mice,rats and cynomolgus monkeys,respectively.The no observed adverse effect levels of sDR5-Fc in rats and cynomolgus monkeys after repeated intravenous infusion for 13weeks were 200 mg/kg and 100 mg/kg,respectively.The average half-life of sDR5-Fc in cynomolgus monkeys was more than 1.9 days.After 12 weeks of repeated intravenous infusion of sDR5-Fc into cynomolgus monkeys,the exposure of sDR5-Fc increased in a dose-proportional manner,and the mean accumulation ratio ranged from 1.82-to 2.11-fold.After intravenous administration of sDR5-Fc in rats,it mainly distributed in plasma and was excreted through urine.In conclusion,sDR5-Fc,as the first pharmaceutical TRAIL blocker,is safe and effective for the treatment of TRAIL-driven hepatitis or liver failure,and is expected to develop into a new therapeutic drug for hepatitis or liver failure.