American Association for Cancer Research
15357163mct130748-sup-mct-13-0748_meth_fig_1_to_9_tab_1_to_3.pdf (2.55 MB)

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

Download (2.55 MB)
journal contribution
posted on 2023-04-03, 14:04 authored by Tae-Hwan Shin, Eun-Sil Sung, Ye-Jin Kim, Ki-Su Kim, Se-Ho Kim, Seok-Ki Kim, Young-Don Lee, Yong-Sung Kim

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.



The limited localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. Here, we describe a solid tumor–targeting antibody with enhanced tumor penetration activity. We designed a 22-residue peptide (A22p), which was extracted from the C-terminal basic region of semaphorin 3A (Sema3A) but modified to have higher affinity with neuropilin receptors (NRP), and genetically fused it to the C-terminus of Fc of human immunoglobulin G1 via a 15-residue (G4S)3 linker, generating Fc-A22p, for the bivalent binding to NRPs. In contrast to Fc or the monovalent A22p peptide alone, Fc-A22p homed to tumor vessels and induced vascular permeability through VE-cadherin downregulation and penetrated tumor tissues by interacting with NRPs in mice bearing human tumor xenografts. We extended the Fc-A22p platform by generating mAb-A22p antibodies of two clinically approved solid tumor–targeting mAbs, the anti-EGF receptor mAb cetuximab (erbitux), and the anti-Her2 mAb trastuzumab (herceptin). The mAb-A22p antibodies retained the intrinsic antigen binding, natural Fc-like biophysical properties, and productivity in mammalian cell cultures, comparable with those of the parent mAbs. In mouse xenograft tumor models, the mAb-A22p antibodies more efficiently homed to tumor vessels and spread into the extravascular tumor parenchyma, which significantly enhanced antitumor efficacy compared with the parent mAbs. Our results suggest that mAb-A22p is a superior format for solid tumor–targeting antibodies due to its enhanced tumor tissue penetration and greater antitumor efficacy compared with conventional mAbs. Mol Cancer Ther; 13(3); 651–61. ©2014 AACR.