Fibroblast growth factor receptor 2 (FGFR2), a member of the FGFR family of receptor tyrosine kinases, plays a central role in regulating cellular proliferation, survival, migration, and differentiation through activation of downstream signaling cascades such as the MAPK and PI3K-AKT pathways (Zhang et al., 2024). Aberrant FGFR2 expression or activation has been implicated in the pathogenesis of multiple cancers, including breast, endometrial, and gastric (Gatius et al., 2011; Hunter et al., 2007; Kim et al., 2019). In gastric cancer, FGFR2 gene amplification and protein overexpression have been frequently reported, particularly in poorly differentiated subtypes such as diffuse-type and scirrhous gastric carcinomas, and are strongly associated with aggressive clinical behavior and poor prognosis (Nagatsuma et al., 2015). These findings have positioned FGFR2 as a promising therapeutic target in gastric cancer. Several FGFR2-directed therapeutic strategies have been developed, including tyrosine kinase inhibitors and monoclonal antibodies (Du et al., 2023). Among them, bemarituzumab, a monoclonal antibody specifically targeting the FGFR2b isoform, has demonstrated clinical potential in early-phase trials (Catenacci et al., 2020). Nevertheless, challenges such as target heterogeneity, resistance mechanisms, and limitations in selective targeting of membrane-bound FGFR2 highlight the continued need to develop novel FGFR2-targeting biologics with improved functional specificity and therapeutic efficacy.

Phage display has become a powerful and widely adopted platform for discovering monoclonal antibodies with therapeutic potential (Smith, 1985). This technology enables the presentation of antibody fragments, such as single-chain variable fragments (scFvs), on the surface of filamentous phages, creating a direct linkage between genotype and phenotype that facilitates high-throughput screening (Barbas et al., 1991, Barbas et al., 1996). Through iterative rounds of biopanning, specific binders can be enriched from antibody libraries by exposing them to the target antigen under selective conditions. Although purified recombinant proteins are commonly used as bait antigens, they often fail to reproduce the native conformation, post-translational modifications, or membrane integration of cell surface receptors, which may limit the physiological relevance of selected clones. Cell-based panning strategies have been developed to address this issue in which intact live cells expressing the native form of the target are used during selection. This approach identifies antibodies that recognize conformational or glycan-dependent epitopes not present in recombinant formats (Alfaleh et al., 2017; Weber et al., 2021). In addition, incorporating negative selection steps using antigen-deficient or knockdown cells further improves the specificity of recovered clones by eliminating nonspecific binders during the panning process (Alfaleh et al., 2017).

Immunizing phylogenetically distant species such as chickens offers advantage for generating antibodies against highly conserved human targets. Due to the evolutionary divergence between avian and mammalian immune systems, chickens can elicit robust immune responses against poorly immunogenic epitopes in rodents or primates (Ching et al., 2018; Leon-Nunez et al., 2022). Chicken-derived antibodies, particularly in scFv format, have been shown to exhibit strong binding to conformational and cross-species epitopes that are otherwise difficult to target (Chang et al., 2023; Lee et al., 2017). However, to minimize immunogenicity and enable clinical application in humans, conversion to a humanized IgG framework is essential. Conventional humanization methods, such as CDR grafting onto human frameworks, can restore compatibility but often require additional adjustments. Framework modifications to recover or enhance binding and stability are frequently necessary, as successful humanization depends not only on preserving CDR sequences but also on selecting an appropriate scaffold (Choi et al., 2015). Indeed, substitutions within framework regions have been shown to markedly affect antibody binding and biophysical properties, underscoring the critical need to evaluate scaffold selection during humanization (Choi et al., 2015).

Our previous work established an immunized chicken-derived scFv library targeting FGFR2(Tsai et al., 2019). Building upon this foundation, we applied a refined cell-based panning strategy incorporating FGFR2-knockdown cells for the absorption process, facilitating the enrichment of scFv clones with improved specificity. Among the selected candidates, the lead clone was subjected to humanization using several distinct human scaffold frameworks. Comparative analyses of biological activity were then conducted to evaluate the impact of different scaffolds. Our findings suggest that scaffold selection during humanization may influence antibody performance. This study highlights considerations for the rational design of humanized antibodies derived from chicken immunization and supports the potential application of FGFR2-targeting antibodies in gastric cancer research.