Supplementary Figures S1-S5; Supplementary Tables S1-S4; and Supplementary Materials & Methods from A Heterodimeric Fc-Based Bispecific Antibody Simultaneously Targeting VEGFR-2 and Met Exhibits Potent Antitumor Activity

PDF file - 596K, Figure S1: Sequence alignment of CH3 domain of human IgG isotypes to compare the mutation sites introduced for heterodimeric Fc variants between our study and other approaches. Figure S2 : The vector maps of the scFv-Fc/Fc expression system and western blotting analysis of the purified scFv-Fc/Fc proteins carrying the indicated CH3 variant pair. Figure S3 : SPR data showing the kinetic interactions of msMet, tanibirumab, and bsVeMet antibodies with the antigens, Met and VEGFR-2. Figure S4: FACS data showing binding profiles of the msMet, tanibirumab and bsVeMet antibodies to HUVECs and MKN45 cells and effects of the antibodies on the cellular proliferation and signaling of the endothelial HUVECs stimulated by single ligand HGF or VEGF alone. Figure S5: Effects of the msMet, tanibirumab and bsVeMet antibodies on the tube formation of HUVECs stimulated by treatments of HGF and/or VEGF. Table S1: The list of CH3 variant pairs constructed in this study. Table S2: Purification yields of Fc-WT, Fc-EW-RVT, and Fc-KiH proteins obtained from transient expression in HEK293F cells. Table S3: The binding parameters of Fc-WT, Fc-EW-RVT, and Fc-KiH proteins with FcRn and FcγR proteins, determined by SPR analyses. Table S4: The binding parameters of the msMet, tanibirumab and bsVeMet antibodies with the antigens, Met or VEGFR-2, determined by SPR analyses. Supplementary Materials and Methods : Detailed description of Reagents; design and evaluation of heterodimeric Fc; construction, expression, and purification of antibodies; SEC analysis; far-UV CD spectroscopy; DSC experiments; SPR analysis; sandwich ELISA; flow cytometry; cell proliferation assay; western blotting; animal experiments; immunofluorescence of tumor tissues.