Supplementary Methods, Supplementary Figures 1 to 9, and Supplementary Tables 1 to 3 from Enhancement of the Tumor Penetration of Monoclonal Antibody by Fusion of a Neuropilin-Targeting Peptide Improves the Antitumor Efficacy
PDF - 2613K, Figure S1: Structural analysis of NRP1, NRP2, and Sema3A, and their interactions to facilitate the understanding of the interactions between Sema3A and NRP1/2. Figure S2: SDS-PAGE analyses of the purified proteins showing that NRP1/2-b1b2 domains, Fc-A22, and Fc-A22p are well prepared. Figure S3: FACS and confocal fluorescence data showing that Fc-A22p, but not Fc, specifically interacts with cell-surface expressed NRP1/2 undergoing cellular internalization. Figure S4: Cell viability data showing that Fc-A22p does not cause significant cytotoxicity on HUVECs and tumor cells. Figure S5: Tumor tissue analyzing data showing that Fc-A22p homes to tumor, induces vascular permeability, and downregulates VE-cadherin in endothelial cells and E-cadherin in tumor cells. Figure S6: Confocal fluorescence data showing that Fc-A22p causes downregulation of VE-/E-cadherin and their dissociation from F-actin cytoskeleton resulting in disruption of cell-cell contacts. Figure S7: Data showing the expression and biochemical characterization of mAb-A22p antibodies, cetuximab-A22p and trastuzumab-A22p, compared with the parent mAbs. Figure S8: Cell proliferation and Western blotting data showing that cetuximab-A22p exerts in vitro biological activities comparable to those of cetuximab in A431 and FaDu tumor cells. Figure S9: Human tumor xenografted mouse data showing that mAb-A22p antibodies show increased tumor homing and penetration and thus superior in vivo anti-tumor efficacy, compared with the parent mAbs. Table S1: SPR data showing the kinetic binding parameters for the interactions of Fc-A22, Fc-A22p, VEGF165, and Sema3A with soluble NRP1-b1b2 and NRP2-b1b2 fragments. Table S2: Purification yields of Fc proteins and antibodies obtained from transient expression in HEK293F cells. Table S3: SPR data showing the kinetic binding parameters for the interactions of mAbs and mAb-A22p antibodies with the indicated proteins. Supplementary Materials and Methods: Detailed description of Reagents; Recombinant protein expression and purification; Construction, expression, and purification of antibodies; Binding analysis by ELISA; Surface plasmon resonance (SPR); Flow cytometric analysis; Cell proliferation assay; Western blotting and immunoprecipitation; Co-localization studies by confocal immunofluorescence microscopy; RNA Interference; Ex vivo tumor penetration assays.