Yii, Shyi-ChunShyi-ChunYiiLin, Tsai-YuTsai-YuLinChang, Fu-LingFu-LingChangTsai, Keng-ChangKeng-ChangTsaiFU-SHAN JAWLin, Yun-ShihYun-ShihLinLee, Yu-ChingYu-ChingLee2025-12-042025-12-042026-0101681656https://www.scopus.com/record/display.uri?eid=2-s2.0-105020851929&origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/734308Fibroblast growth factor receptor 2 (FGFR2) is frequently overexpressed in gastric cancer and represents a promising therapeutic target. We developed FGFR2-specific single-chain variable fragments (scFvs) using a chicken-derived immune library and a refined cell-based panning strategy incorporating FGFR2-knockdown cells for negative selection. The lead clone, scFv R21, exhibited high binding affinity and strong tumor-inhibitory effects in vitro. To enable therapeutic application, R21 was humanized using three distinct framework strategies: CDR grafting, light chain replacement, and structure-guided back-mutation. Structural modeling by AlphaFold3 indicated that the hR21-Bf variant preserved CDR conformation and overall stability. Despite lower expression, hR21-Bf retained moderate FGFR2 binding affinity ( K D) of 38 nM, while other variants showed markedly reduced reactivity. The hR21-Bf construct was further reformatted as a full-length human IgG1 and evaluated in a gastric cancer xenograft model. Mice treated with IgG hR21-Bf showed significant tumor growth inhibition without observable toxicity. Immunohistochemical and biochemical analyses of resected tumors confirmed reduced Ki-67 expression and downregulation of FGFR2-mediated signaling. Our study highlights the impact of scaffold selection on antibody structure and function, supporting a rational approach to antibody humanization using avian-derived libraries for cancer therapy.falseAlphaFold3Cell-based panningFibroblast growth factor receptor 2Gastric cancerHumanizationSingle-chain variable fragments[SDGs]SDG3journal article10.1016/j.jbiotec.2025.10.0052-s2.0-105020851929