Designing and Development of a Tandem Bivalent Nanobody against VEGF165

Background: inhibition of angiogenesis using monoclonal antibodies is an effectivestrategy in cancer therapy. however, they could not penetrate sufficiently into solidtumors. antibody fragments have solved this issue. however, they suffer from short invivo half-life. in the current study, a tandem bivalent strategy was used to enhancethe pharmacokinetic parameters of an anti-vegf165 nanobody.methods: homology modeling and md simulation were used to check the stability ofprotein. the cdna was cloned into phen6c vector and the expression was investigatedin wk6 escherichia coli (e. coli) cells by sds-page and western blot. after purification,the size distribution of tandem bivalent nanobody was investigated by dynamiclight scattering. moreover, in vitro antiproliferative activity and pharmacokineticstudy were studied in huvecs and balb/c mice, respectively.results: rmsd analysis revealed the tandem bivalent nanobody had good structuralstability after 50 ns of simulation. a hinge region of llama igg2 was used to fuse thedomains. the expression was induced by 1 mm iptg at 25°c for overnight. a 30 kdaband in 12% polyacrylamide gel and nitrocellulose paper has confirmed the expression.the protein was successfully purified using metal affinity chromatography. mttassay revealed there is no significant difference between the antiproliferative activityof tandem bivalent nanobody and the native protein. the hydrodynamic radius andterminal half-life of tandem bivalent nanobody increased approximately 2-fold bymultivalency compared to the native protein.conclusion: our data revealed that the physicochemical as well as in vivo pharmacokineticparameters of tandem bivalent nanobody was significantly improved.