Optimization Of Tumor Targeting Properties Of Adenovirus Fiber-modified Recombinant TRAIL Protein

Tumor necrosis facter-related poptosis-inducing ligand(TRAIL)is one of the TNF superfamily,which selectively induces by binding to death receptors(TRAIL-R1 and TRAIL-R2)Apoptosis of many tumor cells.TRAIL’s specific killing properties make it a potential drug for bio-targeted therapy of malignant tumors.TRAIL-based tumor treatment strategies include recombinant soluble TRAIL,TRAIL receptor agonists,and gene therapy,among which the research and application of recombinant TRAIL are the most.However,the activity of recombinant TRAIL depends on its homotrimeric form,which results in its poor stability and short half-life,which limits its clinical application.Therefore,obtaining a more stable trimeric recombinant TRAIL through bioengineering technology is very important for its further application and effective treatment of malignant tumors.The research team used sequence and structure design to modify soluble TRAIL(sTR)by using the tail and shaft domains of avian type 1 adenovirus(FAV1)fiber.Internal and external stability and antitumor activity in vitro,however,FA 1 FT has strong liver enrichment in vivo and thus limits its in vivo activity.In order to further improve the application prospect of FA1FT,this project designed to optimize FA1FT through two different strategies to reduce its liver enrichment and enhance tumor targeting capabilities,including:(1)analysis and identification of avian adenovirus type 1 Fiber Mutation or deletion of possible liver-targeting sites of the sequence to reduce liver enrichment;(2)Modification of pH-targeting peptides,relying on pH-targeting peptides to specifically target the acidic environment of tumors,and improve FA1FT’s tumor location Enrichment enhances its tumor targeting and thus reduces liver enrichment.In the first part of the design,after sequence alignment analysis and literature review,three consecutive non-classical HSPG binding sites with positively charged basic amino acids on the FAV1-tail were mutated to KAG by GAG,and the protein mut-FA1FT was obtained.It has good killing activity.The results of the protein-hepatocyte binding experiment showed that the mutation of KRK did not reduce the binding to hepatocytes,but it was verified from the heparin sodium blocking experiment that the binding of KRK to HSPG was related,which further explained that The presence of non-HSPG-related sequences affects liver enrichment.In order to investigate the non-HSPG-related sequences in FAV1-Fiber,the FAV1-Fiber was truncated after literature research and previous laboratory experience,and the final spiral structure of tail-free FAV1-shaft-sTR(FA 1ST)and tail+shaft was designed.Two proteins of FAV1-tail-last shaft-sTR(FA1TLST),both of which have good killing activity.In the hepatocyte binding experiment,FA1ST and FA1FT did not decrease the binding with hepatocytes but increased the tendency,Compared with FA1FT,FA1TLST significantly reduced the binding,but still 53%of the binding;in the heparin sodium binding experiment,the blocking of heparin sodium more effectively reduced the binding of FA1TLST to hepatocytes,so the HSPG binding site in FA1TLST was excluded as Focus on the next step.Based on the above results,the mutation of KRK in the tail of the FA1TLST sequence to GAG was obtained based on the FA1TLST to obtain the protein mut-FA1TLST.However,in vitro experiments,the binding rates of mut-FA1TLST and FA1TLST to liver cells were similar.In the second part of the design,pHLIP targeting peptide modification was used to improve tumor targeting and reduce liver enrichment.pHLIP(pH membrane insertion peptide),a short pH-responsive peptide,provides a new opportunity to target acidic tissues.It can insert its C-terminus into the cell membrane and form a transmembrane helix under acidic conditions,which can be achieved in tumors.Enrichment of therapeutic drugs and tumor targeting.Based on the structural and functional characteristics of TRAIL,this project designed two protein forms,FAlFT-pHLIP and FAlFT-linker-pHLIP.Because pHLIP is easy to form a-helix anchor on the membrane in the acidic environment,after prolonged induction during prokaryotic expression,the culture medium is in the acidic environment,which makes the protein more anchored on the membrane of E.coli and cannot be supernatant.To express the protein,we purified the protein in the inclusion body by dialysis.The purification efficiency can reach 40-50%.In the in vitro test,the biological activity of the recombinant protein was measured by MTT and apoptosis kits.It was found that FAlFT-linker-pHLIP can maintain the strong tumor apoptosis and inhibitory activity of FA1FT,while FA1FT-pHLIP decreased slightly.In order to verify the tumor tissue targeting of the modified protein,the protein was incubated with cells at 4 ℃ at pH 7.4 and 6.5,respectively.Confocal immunofluorescence and flow cytometry were used to detect FA1FT-linker under acidic conditions.-pHLIP can bind to tumor cells better than FA1FT-pHLIP.The following conclusions were obtained through the study of this paper:Through analysis of liver enrichment sequences in FA1FT through in vitro experiments,a novel recombinant TRAIL protein that can reduce liver enrichment and maintain its high tumor killing activity was obtained;pHLIP targeting peptide modification can effectively enhance FA1FT The tumor targeting ability of the protein has good antitumor prospects.Follow-up experiments on in vivo targeting and treatment will be conducted to further evaluate the effects of the above-mentioned modifications and modifications on tumor cells in vivo,which will lay a good foundation for future research on recombinant proteins.