PEGylationand Pharmacological Characterization Of Arginine Deiminase

Arginine deiminase (ADI), catalyzing the conversion of L-arginine into L-citrulline andammonia, could inhibit the growth of arginine-auxotrophic tumor cells. PEGylation is one ofthe most promising methods to formulate a bioconjugated protein with extended physicalhalf-life and reduced immunogenicity in therapeutic applications. In this study, polyethyleneglycol (PEG) formulation of recombinant ADI was studied to improve its short serum half-life(4h) and high immunogenicity in vivo.In previous studies, based on ADI from Pseudomonas plecoglossicida, an excellent ADIvariant M13-3with enhanced activity and properties was obtained by protein engineering.Response surface methodology was used to optimize the fermentation medium for theproduction of ADI M13-3from recombinant E. coli on the basis of single factor optimization.Using the optimized medium, ADI activity reached15.17U mL-1broth, representing3.51-fold of that using LB medium in a3-L bioreactor.ADI protein was purified from cell-free extract by anion-exchange chromatography andgel filtration chromatography. Seven PEG reagents with succinimidyl ester groups, varying insize and linkers, were used in PEGylation of purified ADI. Three PEGylated products withhigh yield were further characterized, named ADI-SS-PEG20kDa, ADI-SC-PEG20kDa, andADI-SPA-PEG20kDa. Furthermore, stability analysis confirmed that the catalytic activity ofabove bioconjugates were well-preserved.ADI-SPA-PEG20kDawas selected for pharmacodynamic/pharmacokinetic studies. Aremarkable improvement in circulating half-life and efficacy compared with native ADI wasobserved. Our results demonstrated that single ADI-SPA administration of5U/mouse viaintravenous injection could maintain serum arginine at an undetectable level for5days with ahalf-life of53.2h, representing11-folds that of native ADI.In our in vivo evaluations in mice implanted with H22tumors, ADI-SPA injection of5Udosage/5days via intramuscular showed an inhibition rate of95.02%on tumor growth during15-day treatments, which is similar to chemotherapy group (98.34%). In contrast, ADIinjection of1U dosage/1day only showed87.19%inhibition rate. Therefore, it suggests thatPEGylated ADI (such as ADI-SPA) could become a potential anti-cancer agent for controllingarginine-auxotrophic cancers.