Study On The Preparation, Anti-tumor Activity And Mechanism Of A New Powerful TRAIL Fusion Protein

The conventional chemotherapy and radiotherapy are effective in the treatment of tumors,but normal tissues are also damaged due to their lack of specificity,which leads to the poor prognosis and decreased life quality for patients.Thus,drugs specifically target only neoplastic cells are urgently required.Tumor necrosis factor related apoptosis-inducing ligand(Apo2L/TRAIL)belongs to the TNF superfamily.It induces apoptosis selectively in tumor cells by interacting with its agonistic receptors(DR4 and DR5)while sparing normal cells,which makes it an attractive cancer therapeutic agent.Recombinant TRAIL and agonistic antibodies against the TRAIL receptors have been shown to be safe in clinical trials.Unfortunately,such drugs as single agents or combined with other chemotherapeutics have failed to show significant clinical efficacy.This leads to numerous studies in modifying TRAIL to maximize its antitumor activity,among which constructing TRAIL fusion proteins is one of the common methods.According to different strategies,we designed four fusion schemes and eonstructed eight new TRAIL fusions.And to the best of our knowledge,they were novel and original TRAIL fusion proteins.The cytotoxicity of these fusions was assessed by MTT assay for screening out better anti-cancer agents.It turned out that the "dual-target"TRAIL by fusing with TGF3L,an EGFR affinity peptide,was the most toxic to cancer cells while leaving normal cells unharmed.The greatest enhancements were in MDA-MB-468 and Colo205,with 994-and 3043-fold decreases in the LC50,respectively.Thus,the investigation in the mechanisms of enhanced cytotoxicity for TGF3L-TRAIL was carried out.Cell binding assay,antibody blockage assay,apoptosis assay and caspases enzymatic assay were implemented to investigate the EGFR-binding and apoptosis induction capacity of TGF3L-TRAIL.Surprisingly,TGF3L-TRAIL was found lack of EGFR binding eapability.It activated DR4 and DR5 in a way different from TRAIL,while activated caspase-8/3 and induced apoptosis at much lower doses than TRAIL.We found that TGF3L-TRAIL assembled into stable polymers spontaneously with molecular mass ranging 0.8～5×106 Da,which was proved responsible for its enhanced cytotoxicity.The formation of TRAIL polymer was attributed to the specific interactions among His-tag,TGF3L peptide and metal ions.In the Colo205 xenograft model,TGF3L-TRAIL inhibited the tumor growth in nude mice more efficiently than TRAIL did.Self-assembled TRAIL polymers are attractive death receptor agonists for possible cancer therapy.We tried to substitute the His-tag in the N-terminus of TGF3L-TRAIL with NCTR25,a metal affinity peptide truncated from human copper transporter 1.The new fusions NCTR25-TGF3L-TRAIL and NCTR25-TRAIL were able to assemble into polymers with molecular mass as 107-108 Da relying on the metal ions.They possessed similar cytotoxicity(10000-20000 folds higher than TRAIL)and in vivo antitumor activity to His-TGF3L-TRAIL.NCTR25-TRAIL showed exactly the same polymer form and bioactivity as NCTR25-TGF3L-TRAIL,indicating the dispensability of TGF3L Peptide.They provided valuable tools for the assessment of the mechanism of receptor activation and signal transduction for multi-valent TRAIL agonists.The polymers induced cytotoxicity through selective activation of DR4 and DR5,without the involvement of other TNFRSF receptors.The selectivity of TRAIL polymers on receptor subtypes was different from TRAIL and probably varied depending on the cell types.The multi-valent TRAIL showed higher potency and similar efficacy than TRAIL in activating caspases and inducing apoptosis in tumor cells.While in the NF-κB signaling pathway,both the higher potency,and the greater efficacy for the first time,were observed for the multi-valent TRAIL,comparing to TRAIL.TRAIL and TRAIL polymer activated NF-κB mainly through DR5,that was different from the previous reports.In conclusion,we have successfully constructed several novel TRAIL fusion proteins and three of them are capable of assembling into polymers with extremely high cytotoxicity.These polymeric TRAILS selectively activate DR4 and DR5 and trigger signaling pathways with higher potency than TRAIL,as well as higher efficacy than TRAIL which was observed here for the first time.This work also provides a mechanistic explanation of enhanced bioactivity of TRAIL polymers,which would contribute to the development of these TRAIL fusions for cancer therapy.A novel fusion strategy by fusing with a native human-derived metal affinity peptide is thus presented for improving the activity of TRAIL.