Generating the Engineered Form of a Nanobody Against Placenta Growth Factor with High Antiangiogenesis Potential

Antibody engineering is a dynamic field in antibody industry. Over 30% of novel monoclonal antibodies (mAbs) in R&D and clinical trials are engineered forms. Affinity enhancement contributes to the development of new binders that are not only effective in low dose and cost but also improve some drawbacks of antibody production. After previous successful work on in silico affinity maturation of nanobody against placenta growth factor and finding the best engineered nanobodies (Mut2:S31D and Mut4:R45E), according to bioinformatic parameters and molecular dynamics (MD) simulation results, in this study we focused on experimental confirmation of affinity enhancement of a mutant form of nanobody. So, we cloned and expressed two of four mutant forms in pHEN6c vector. Affinity binding was assayed by enzyme-linked immunosorbent assay on purified mutants, with results showing that 10-time enhancement in affinity compared with the native form associated with MD simulation results. We checked the effectiveness of these mutant nanobodies in angiogenesis inhibition by human umbilical vein endothelial cell proliferation and 3D capillary tube formation. EC50 of mut2, mut4, and native in proliferation assay was 110, 140, and 190 ng/mL, respectively, and that in 3D capillary tube formation was 80, 83, and 100 ng/mL. The results of functional studies revealed strong effectiveness of Mut2 followed by Mut4 compared with the native form. Our study confirmed that in silico approach could facilitate development of novel versions of mAb with better characteristics, which could save cost and time.