Abstract
<jats:p>Polyspecificity has been widely regarded as a fundamental feature of germline antibodies that enables broad antigen recognition with the limited size of the germline antibody repertoire. However, accumulating evidence indicates that germline antibodies against carbohydrate antigens can exhibit high specificity, thereby challenging this traditional paradigm. To investigate whether the polyspecificity paradigm applies to haptens (single-epitope antigens), we inferred and identified 14 germline antibodies targeting distinct haptens; unexpectedly, all exhibited intrinsically high specificity. To explore the underlying mechanisms, we constructed a comprehensive sequence-structure database of germline antibodies recognizing haptens, peptides, and proteins. Our results reveal that hapten-binding germline antibodies are characterized by shorter CDRH3 regions, enrichment of aromatic residues in CDRs, and fewer CDR-localized surface patches—features that may underlie their intrinsically high specificity. Conversely, germline antibodies recognizing peptides and proteins display longer CDRH3 regions, increased enrichment of polar and charged residues, and denser CDR-localized surface patches, providing structural determinants of their greater polyspecificity. In addition, germline gene usage biases—particularly V-gene selection and IGHV–IGKV/IGLV pairing patterns—collectively shape the antigen-binding properties of germline antibodies. Together, these findings represent the first large-scale, systematic analysis of germline antibody polyspecificity and have broad implications for fundamental immunology, vaccine development, and antibody discovery.</jats:p>