Antibody discovery from phage libraries lacking clear target-cell-selective enrichment through NGS-guided search space definition and cloning-free binding screening

Abstract Antibody discovery targeting primary cells or transient cellular states remains challenging because conventional cell-based phage display relies on clonal enrichment, which may fail when specific binders do not become dominant. Here, we present an antibody discovery workflow for phage libraries lacking clear target-cell-selective enrichment that integrates next-generation sequencing (NGS)-guided definition of a candidate search space with cloning-free antibody reconstruction and screening. Using disease-associated CD11c⁺F4/80⁺ macrophages isolated from a diet-induced MASH mouse model, we found that conventional panning did not produce clear target-cell-selective enrichment at the phage-pool level, even after selection stringency was increased. Although NGS analysis revealed reduced library diversity and subtle shifts in clone frequencies, no dominant clones emerged. Applying predefined selection criteria to the NGS data instead enabled the identification of a tractable set of candidate antibodies. The selected sequences were directly reconstructed and screened using a cloning-free workflow, yielding antibodies with measurable target-cell binding. Selected antibodies showed modest target-cell-preferential binding after IgG conversion, and two antibodies additionally stained MASH-associated inflammatory lesions. This study provides a practical NGS-guided framework for candidate prioritization and functional screening when clear pool-level enrichment is not observed.