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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

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posted on 2023-04-03, 13:52 authored by Hye-Ji Choi, Ye-Jin Kim, Sangho Lee, Yong-Sung Kim

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.

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ARTICLE ABSTRACT

Heterodimeric Fc designed by engineering the CH3 homodimeric interface of immunoglobulin G1 serves as an attractive scaffold for the generation of bispecific antibodies (bsAb) due to the favorable properties of the Fc region. In this study, we describe a heterodimeric Fc generated by substituting the conserved electrostatic interactions at the CH3 core interface with asymmetric hydrophobic interactions and introducing asymmetric, long-range electrostatic interactions at the rim of the CH3 interface. Coexpression of Fc proteins carrying the combined CH3 variant pairs in HEK293F cells produced the heterodimer, which was purified with more than 90% yield, and retained wild-type Fc biophysical properties. The heterodimeric Fc was exploited to generate a bsAb simultaneously targeting both the Met receptor tyrosine kinase and the VEGF receptor 2 (VEGFR-2), with two respective antigen-specific, single-chain variable fragments (scFv) into the N-terminus. The Met × VEGFR-2 bsAb bound concurrently to the two target antigens, efficiently inhibited the downstream signaling and tube formation stimulated by the two receptors in human endothelial cells, and exhibited more potent antitumor efficacy in MKN45 human gastric cancer xenograft models than both the parent monospecific antibody alone. Collectively, based on the newly designed heterodimeric Fc-based bsAb, our results provide the therapeutic potential of bsAb targeting both Met and VEGFR-2 simultaneously for the treatment of human cancers. Mol Cancer Ther; 12(12); 2748–59. ©2013 AACR.