Targeting the Extracellular Domain of Fibroblast Growth Factor Receptor 3 with Human Single-Chain Fv Antibodies Inhibits Bladder Carcinoma Cell Line Proliferation

Abstract

Purpose: Previous gene expression studies have shown that fibroblast growth factor receptor 3 (FGFR3) is overexpressed in early stages of bladder cancer. To study the potential use of therapeutic antibodies against FGFR3, we have produced a collection of human single-chain Fv (scFv) antibody fragments by using phage display libraries.Experimental Design:Two ''naï l « ve''semi-synthetic human scFv libraries were used to select antibodies against the extracellular domain of FGFR3a(IIIc).The reactivity of the selected scFvs with a recombinant FGFR3 was characterized by an enzyme immunoassay and surface plasmon resonance analysis and with RT112 bladder carcinoma cells by a fluorescence-activated cell sorter. The capacity of the selected scFvs to block RT112 cell proliferation was determined. Results: We have isolated six human scFv antibody fragments directed against FGFR3. These human scFvs specifically bound FGFR3, but not the homologous molecule FGFR1. Biacore analysis was used to determine the affinity constants, which ranged from 12 to 40 nmol/L. Competition analysis showed that the FGF9 ligand was able to block the binding of two scFvs, 3C and 7D, to FGFR3, whereas FGF1 only blocked 7D. Immunoprecipitation and flow cytometric analysis confirmed the specificity of the antibodies to native membrane FGFR3. Two scFvs, 3C and 7D, gave an strong immunofluorescence staining of RT112 cells. Moreover, they recognized equally well wild-type and mutant FGFR3 containing the activating mutation S249C. Furthermore, they blocked proliferation of RT112 cells in a dose-and FGF-dependent manner. Conclusion: Our results suggest that these human anti-FGFR3 scFv antibodies may have potential applications as antitumoral agents in bladder cancer.The fibroblast growth factors (FGF) represent one of the largest families of polypeptide growth and differentiation factors for mesodermal and epithelial cells (1). FGFs have been implicated in multiple biological processes during embryo development, wound healing, hematopoiesis, and angiogenesis. In addition, FGFs have been shown to increase the invasiveness of a variety of tumors from prostate, bladder, kidney, breast, and pancreas (2-5).Although more than 20 FGFs with different effects on various cells have been reported, only five FGF receptors (FGFR) have been described (6, 7). These receptors share 55% to 72% homology at the protein level. The FGFR structure consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular kinase domain. The ligand-binding domain contains three different immunoglobulin-like domains (called immunoglobulin I, immunoglobulin II, and immunoglobulin III). The ligand domain is followed by a single transmembrane domain and a cytoplasmic split tyrosine kinase domain in the intracellular domain. Alternative splicing of the mRNAs corresponding to FGFR1-3 gives place to two isoforms, a and h. Only isoform a, which contains the three immunoglobulin domains, has been found for FGFR3. Alternative splicing of FGFR3 transcripts ...

Affinity maturation of antibodies assisted by in silico modeling

“…Elution, infection and production of phages for other rounds of panning were performed as described in ref. 42. The human scFvs were produced and isolated according to protocols established in refs.…”

Fibroblast growth factor signaling in skeletal development and disease

“…Subcutaneous injections of recombinant sFGFR3 in a mouse model for achondroplasia was found to decrease mortality and improve skeletal growth (Garcia et al 2013). Inhibitory antibodies that specifically target the FGFR3 extracellular domain have been developed as potential cancer therapeutics but have not been evaluated for treatment of achondroplasia (Martinez-Torrecuadrada et al 2005;Hadari and Schlessinger 2009;Qing et al 2009). A recent drug screen of induced pluripotent stem cell-derived chondrocytes from patients with achondroplasia and thanatophoric dysplasia identified statin drugs as effective in improving chondrogenic differentiation in vitro and improving the phenotype (limb and body length) of achondroplasia mice in vivo (Yamashita et al 2014).…”

Novel targeted agents for the treatment of bladder cancer: translating laboratory advances into clinical application

“…The stable expression of a small hairpin RNA (shRNA) against FGFR3 in bladder cancer cells demonstrates an inhibition of cancer cell growth, supporting the central cancer cell growth, supporting the central importance of this pathway in bladder cancer (23). Additionally, human single chain Fv antibody fragments uman single chain Fv antibody fragments that recognize the extracellular domain of FGFR3 have been isolated and characterized (24). This antibody inhibits ligand-binding by the wild-type receptor and has been shown to inhibit the growth of xenografts expressing FGFR3 in S249C bladder cancer cells (24).…”