Fig. 1

Conjugation of tetravalent engineered antibody (T-Fc) with a cytotoxic payload. a Hypothetical model of the effect of FGFR1 clustering on the receptor endocytosis. FGFR1 dimerization via FGF1 binding induces receptor activation and clathrin-mediated endocytosis. Clustering of FGFR1 into large structures on the plasma membrane with tetravalent T-Fc largely improves the cellular uptake of FGFR1-antibody complexes. Furthermore, FGFR1 clustering changes the mechanism of the receptor endocytosis by engaging dynamin-2-dependent CIE pathways. b The chemical structure of monomethyl auristatin E bearing the valine-citrulline linker (vcMMAE). c The schematic representation of the conjugation of T-Fc with the cytotoxic compound MMAE. The Fc region of IgG (CH2 and CH3 domains) is labeled in gray, and anti-FGFR1 scFv proteins (VH and VL fusions) are marked in blue. Antibody regions recognizing epitopes within FGFR1 are marked in orange. Thiol groups of reduced cysteines are marked in yellow and attached cytotoxic payloads are marked in red. d, e The efficiency of the conjugation and purity of T-Fc-vcMMAE were analyzed with SDS/PAGE (d) and western blotting (e) with antibodies recognizing the Fc fragment. f The spectroscopic analysis of DAR parameter for T-Fc-vcMMAE. DAR was calculated through the absorbance measurement for T-Fc and T-Fc-vcMMAE at 248 nm and 280 nm wavelengths according to (Chen 2013).