Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice
Jing Qing, … , Christian Wiesmann, Avi Ashkenazi
Jing Qing, … , Christian Wiesmann, Avi Ashkenazi
Published April 20, 2009
Citation Information: J Clin Invest. 2009;119(5):1216-1229. https://doi.org/10.1172/JCI38017.
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Research Article

Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice

Abstract

Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14)-positive multiple myeloma. While FGFR3 is frequently overexpressed and/or activated through mutations in bladder cancer, the functional importance of FGFR3 and its potential as a specific therapeutic target in this disease have not been elucidated in vivo. Here we report that inducible knockdown of FGFR3 in human bladder carcinoma cells arrested cell-cycle progression in culture and markedly attenuated tumor progression in xenografted mice. Further, we developed a unique antibody (R3Mab) that inhibited not only WT FGFR3, but also various mutants of the receptor, including disulfide-linked cysteine mutants. Biochemical analysis and 2.1-Å resolution crystallography revealed that R3Mab bound to a specific FGFR3 epitope that simultaneously blocked ligand binding, prevented receptor dimerization, and induced substantial conformational changes in the receptor. R3Mab exerted potent antitumor activity against bladder carcinoma and t(4;14)-positive multiple myeloma xenografts in mice by antagonizing FGFR3 signaling and eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). These studies provide in vivo evidence demonstrating an oncogenic role of FGFR3 in bladder cancer and support antibody-based targeting of FGFR3 in hematologic and epithelial cancers driven by WT or mutant FGFR3.

Authors

Jing Qing, Xiangnan Du, Yongmei Chen, Pamela Chan, Hao Li, Ping Wu, Scot Marsters, Scott Stawicki, Janet Tien, Klara Totpal, Sarajane Ross, Susanna Stinson, David Dornan, Dorothy French, Qian-Rena Wang, Jean-Philippe Stephan, Yan Wu, Christian Wiesmann, Avi Ashkenazi

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Figure 4

Epitope mapping for R3Mab and crystal structure of the complex between R3Mab Fab fragment and IgD2–D3 of human FGFR3-IIIb.

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Epitope mapping for R3Mab and crystal structure of the complex between R...
(A) Epitope determined by the binding of 13 peptides spanning IgD2–D3 of human FGFR3 to R3Mab. Each biotinylated peptide was captured onto a streptavidin-coated microtiter well and incubated with R3Mab. Specifically bound R3Mab was detected using a goat anti-human IgG antibody. (B) Sequence alignment of human FGFR3 peptides 3 and 11 with extracellular segments of human FGFR1. FGFR1 residues engaged in the primary FGF2-FGFR1 interaction, heparin binding, and receptor-receptor association are shown in red, blue, and orange, respectively. Functional assignment of FGFR1 residues is based on the work of Plotnikov et al. (34). (C) Structure of R3Mab Fab (shown in ribbon-helix: light chain, yellow; heavy chain, green) in complex with human FGFR3 IgD2–D3 (shown in molecular surface, white). Receptor residues involved in ligand binding and dimerization are colored in violet and orange, respectively, based on the work of Plotnikov et al. (34). (D) The close-up of the crystal structure shows that CDR-H3 and -H2 from the Fab constitute the major interaction sites with IgD2 and IgD3 of FGFR3. (E) Superposition of the FGFR3-IIIc–FGF1 complex (Protein Data Bank code 1RY7) with the FGFR3-IIIb–Fab complex. FGFR3-IIIc and FGF1 are shown in cyan and violet, respectively. FGFR3-IIIb is shown in white, and the Fab is shown in yellow for light chain, green for heavy chain. IgD2 was used as the anchor for superposition. Note the well-superposed IgD2 from both structures and the new conformation adopted by IgD3 of FGFR3-IIIb when bound by R3Mab.