A diverse semisynthetic humanized scFv phage display library for anti-CXCL16 antibodies

Phage display libraries of human single-chain variable fragments (scFvs) serve as a valuable resource for generating fully human antibodies for scientific and clinical applications. In this study, we designed and constructed a highly diverse semisynthetic humanized scFv phage display library using an optimized Kunkel mutagenesis approach. Our optimizations eliminated residual template, enhancing mutagenesis efficiency and expanding library diversity with a reservoir capacity exceeding 10¹⁰. For this semisynthetic library, the complementarity-determining region 3 was structurally designed to mimic the natural human antibody repertoires, encompassing comprehensive sequence variability and length distributions. To functionally characterize this antibody library, we performed targeted screening against CXC motif chemokine ligand 16 (CXCL16), a crucial chemokine involved in inflammatory pathogenesis and tumor microenvironment regulation. We successfully identified multiple CXCL16 specific antibodies, one of which demonstrated strong blocking activity for the CXCL16-CXCR6 axis. Structural modeling and docking analyses further elucidated the key binding sites of CXCL16 and the antibody, offering insights for future antibody optimization. These results highlight the potential of this humanized scFv library as a robust platform for therapeutic antibody discovery, with broad applications in diagnostics and biomedical research.