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Fig. 6 Lead variant potently inhibits HUVEC angiogenesis. A-D) Representative images of HUVECs after treatments. (A) HUVECs do not form tubular network in absence of VEGFA, (B) 1 nM VEGFA induces formation of a HUVEC tubular network. Addition of 2 nM (C) ranibizumab or (D) scFv1 inhibits tubular network formation. (E) Plot of angiogenesis parameters. scFv1 inhibits tubular network formation more efficiently than ranibizumab. Total length, total branching length, total segment length and total branch length were calculated by 2-way ANOVA followed by Dunett’s multiple comparison test, error bars represent SD. ns: non-significant, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. (F) Bar graph showing binding affinities of ranibizumab, brolucizumab and scFv1 to human VEGFs, VEGF isoforms and VEGFA orthologs in model organisms.