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 "naïve" semi-synthetic human scFv libraries were used to select antibodies against the extracellular domain of FGFR3alpha(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.

• Authors: Jorge Martínez-Torrecuadrada, Gabriela Cifuentes, Paula López-Serra, Pilar Saenz, Antonio Martínez, J Ignacio Casal
• Journal: Clinical cancer research : an official journal of the American Association for Cancer Research (Clin Cancer Res) Vol. 11 Issue 17 Pg. 6280-90 (Sep 01 2005) ISSN: 1078-0432 [Print] United States
• PMID: 16144932 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Blocking
• Antibodies, Monoclonal
• Immunoglobulin Variable Region
• Peptide Library
• Receptors, Fibroblast Growth Factor
• Recombinant Proteins
• FGFR1 protein, human
• FGFR3 protein, human
• Protein-Tyrosine Kinases
• Receptor Protein-Tyrosine Kinases
• Receptor, Fibroblast Growth Factor, Type 1
• Receptor, Fibroblast Growth Factor, Type 3

Topics

• Amino Acid Sequence
• Antibodies, Blocking (therapeutic use)
• Antibodies, Monoclonal (immunology)
• Cell Proliferation
• Enzyme-Linked Immunosorbent Assay
• Flow Cytometry
• Humans
• Immunoglobulin Variable Region (genetics, immunology)
• Immunoprecipitation
• Molecular Sequence Data
• Peptide Library
• Protein-Tyrosine Kinases (genetics, immunology)
• Receptor Protein-Tyrosine Kinases (genetics, immunology)
• Receptor, Fibroblast Growth Factor, Type 1
• Receptor, Fibroblast Growth Factor, Type 3
• Receptors, Fibroblast Growth Factor (genetics, immunology)
• Recombinant Proteins (immunology)
• Sequence Homology, Amino Acid
• Surface Plasmon Resonance
• Urinary Bladder Neoplasms (immunology, pathology)