Multivalent ScFv Against TRAIL Receptor For Tumor Therapy

Single chain variable fragment (scFv) antibody has relativly small molecular size and is easy to penetrate into solid tumor. However, due to its low affinity and rapid clearance in blood, the usage of scFv is limited. In this study, we utilized COMP48, a coiled coil domain of human cartilage oligomerization protein, to pentamerize a group of agonistic scFv to TRAIL receptors DR4and DR5. As expected, the affinity of scFv increased, half life extended, and finally, efficacy to kill tumor greatly improved.After fusion with COMP48, pentavelant scFv has been successfully formed. The pentavalent scFvs (combody),4ccomp and8fcomp have great improvement in affinity in BIAcore assay. The KD value of4ccomp and8fcomp is about10-10M, while that of the monomeric scFv,4cmono and8fmono is about10-M. ELISA data showed that4ccomp and8fcomp specifically bind to respective receptor without showing apparent cross reactivity. In apoptosis and cell inhibition assays it is indicated that pentavalent scFvs have stronger toxicity on tumor cell lines, such as A549, colo205and HCT-116. In the xenograft tumor model,8fcomp showed best efficacy, but4cmono was better than4ccomp in therapy, which demonstrates that both affinity and penetration efficiency are important for a protein agonist drug against tumor.