The Development Of Recombinant Flagellin CBLB502 And Its Application Studies In Preventing Immune-mediated Liver Injury

Liver is an innate immune organ which shares a unique dual blood supplying system,therefore,it was anatomically exposed to various kinds of gut bacterial components including TLRs ligands(e.g.bacterial lipopeptide/lipoprotein,dsRNA,LPS,and Flagellin)and it is reported that TLRs-mediated signaling was extensively implicated in the onset and progression of inflammatory liver diseases.As the only known TLR that sense a protein ligand,TLR5 activation exerted fabulous protective efficacy against ion-radiation,chemotherapeutic drugs,oxygen stress,and grat-versus-host response induced tissue and/or organ injury.However,no reports have been involved in the effect of TLR5 signaling on the pathogenesis of inflammatory liver diseases by now.Here we developed an artificially modified novel TLR5 agonist CBLB502 and evaluated the effect of CBLB502-mediated immune mechanism on concanavalin A-induced hepatic injury.The major results are shown as following:Based on the previous work,we successfully established a pilot-scale purification procedural for recombinant CBLB502 by using DEAE negative-ion exchanging chromatography.About 200-230 g wet bacteria per batch was obtained and purified to >95% purity recombinant CBLB502 protein with a yield of nearly 2 g in the established conditions.The quality control studies of recombinant CBLB502 bulk drug substance(BDS)including physicochemical properties(Peptide mass fingerprinting,N-terminal sequence,correct mass spectra),content,purity,specific activity,residues check(antibiotics residues,exogenous DNA residues,host cell protein residues)and validation of analytical procedures(accuracy,precision,characteristics,linearity,range)were carried out according to the Chinese Pharmacopoeia(version 2010),and ensured all the above items met the quality requirements of recombinant protein drugs in Chinese Pharmacopoeia.Subsequently,we made the production and quality verification procedurals of recombinant CBLB502 BDS based on the above assays and verified the CBLB502 efficacy in a nonhuman primate severe marrow ARS model.Next,we determined the CBLB502 neutralization antibody level from 44 normal individuals and set a cut-off value for screening the candidates in clinical trials(phase ?);developed a CBLB502 specific monoclonal antibody used for clinical pharmacokinetics study.In Con A-induced acute liver injury model,we found the protective effects of CBLB502-mediated mechanism in immune factor induced hepatic damage for the first time.Prophylactic treatment of CBLB502 exerted marvelous hepatoprotection efficacy including increased survival rates,ameliorated liver necrosis or apoptosis,reduced aminotransferase and inflammatory cytokines production,impaired lymphocytes infiltration.Mechanistic studies revealed that CBLB502 as a negative regulator limiting T/NKT cell activity and cytokine production in Con A induced liver injure model.In addition,we provided evidences that CBLB502 suppresses ?-Galcer-induced NKT-cell-dependent inflammatory liver injury.Bone marrow transplantation experiments showed that TLR5 in bone marrow–derived cells contributed to CBLB502 against Con A-induced liver injure.Moreover,IL-6 elevation induced by CBLB502 is demonstrated as one of important component of protection against Con A-induced liver injury as passive blocking of endogenous IL-6 significantly impaired CBLB502's hepatoprotection efficacy.Lastly,we found that endogenous TLR5 signaling deficiency resulted in an enhanced susceptibility to Con A challenge including reduced survival rates,increased inflammatory necrosis,elevated toxic cytokines production as well as immune cells activation and infiltration.The above findings reveal that CBLB502-mediated immune mechanism is capable of inhibiting T cell-activation induced hepatitis and and may be exploited for therapeutic treatment of inflammatory liver diseases.