Cloning, expression, purification of FGF7 and Identification of novel single chain variable fragment (scFv) antibodies against FGF7 using Phage Display technology

Abstract

Introduction: Fibroblast growth factor 7 (FGF7) is a member of the fibroblast growth factor (FGF) family of proteins. FGF7 is of stromal origin and produces a paracrine effect on epithelial cells. Receptor tyrosine kinase (RTK) inhibitors are widely used pharmaceuticals in cancer therapy. Fibroblast growth factor receptors (FGFRs) are members of RTK superfamily which are highly expressed on the surface of carcinoma associate fibroblasts (CAFs). The involvement of FGFRs in different types of cancer makes them promising target in cancer therapy and hence, the identification of novel FGFR inhibitors is of great interest. Purpose: In the current investigation we aimed to identify new single domain antibodies (sdAb) against FGF7 using phage display technology and to develop an alignment independent three dimensional quantitative structure-activity relationship (3D-QSAR) model for a set of 26 FGFR2 kinase inhibitors allowing the prediction of activity and identification of important structural features for these inhibitors. Method: Recombinant FGF7 protein was produced at high purity and used as target in panning experiment to identify specific sdAb. Pentacle software was used to calculate grid independent descriptors (GRIND) for the active conformers obtained by docking and structural similarity filtering methods. Significant descriptors were selected using fractional factorial design (FFD) for the generation of 3D-QSAR model. Results: The purity of the produced FGF7 was 92% with production yield of 3.5 mg/L of culture. The identified single domain antibodies showed significant affinity to FGF7. Six descriptors were identified as the most important variables with high impact on the biological activity of the compounds. Internal and external validations were lead to good statistical parameters (r2 values of 0.93 and 0.665, respectively). Conclusion: The selected sdAb can be used for developing novel tumor suppressing agents where inhibition of FGF7 is required. The developed 3D-QSAR model has good predictive power and may be used for designing novel FGFR2 kinase inhibitors.