Targeting of human fibroblast growth factor receptor 2 by a novel specific nanobody

Abstract

Inhibition of FGFR2 signaling is promising in targeted therapy of FGFR2-related tumors. In this study, anti-FGFR2 nanobodies (Nbs) were isolated through screening of an immune camelid phage display library. Four rounds of biopanning were carried out with commercial human FGFR2 antigen and enrichment was assessed by ELISA and phage titration. The gene of Nb was sub-cloned into the expression vector, and the recombinant vector was transformed into Escherichia coli WK6 cells. The recombinant protein was purified using Ni–NTA affinity chromatography. The anti-FGFR2 Nb (C13) was characterized by SDS-PAGE, western blotting, competitive inhibition ELISA, flow cytometry, MTT, and migration assay. C13 Nb recognized FGFR2 with high specificity and no cross-reactivity was observed with other tested antigens. The affinity of C13 Nb was calculated to be 1.5 × 10⁻⁹ M. Results of cytotoxicity showed that C13 Nb (10 µg/ml) inhibited 85% of the proliferation of T-47D cells (p < 0.001). In addition, C13 inhibited the migration of 68% of T-47D toward the source of the growth factor (p < 0.01). The flow cytometry showed that C13 Nb bound to the surface of FGFR2⁺ cells, T-47D cell line (96%). Results indicate the potential of anti-FGFR2 Nb for targeted therapy of FGFR2-overexpressing tumors after complementary investigations.

Keywords:

• Fibroblast growth factor receptor
• immunotherapy
• phage display
• single-domain antibodies

Ethical approval

IR.PII.REC.1398.035.

Authors’ contributions

MI: performed experiments and prepared the draft of the manuscript. MH-A: statistical analysis, project administration, funding acquisition, writing—review, and editing. MB: investigation, experiments. FK-L: conceptualization, supervision, project administration, funding acquisition, writing—review, and editing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data were included in the manuscript.

Additional information

Funding

This study was funded by the Pasteur Institute of Iran (grant number: 9611189).