This study explores the possibility of isolating high-affinity human antibodies directly from large and diverse synthetic phage antibody repertoires. The authors created a highly diverse repertoire of heavy and light chains in vitro using human V gene segments, and then combined these repertoires in bacteria to generate a large (6.5×10^10) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire, they isolated Fab fragments that bound to various antigens and haptens with affinities comparable to those of antibodies from a secondary immune response in mice (up to 4 nM). The VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, but there were also significant differences in segment usage patterns. These differences may help to dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo. The study also analyzed the distribution of CDR3 lengths and the pairings of heavy and light chains, providing insights into the relationship between affinity and repertoire size, as well as the structures of antigen-binding sites and V, D, and J segment usage. Overall, the results demonstrate that high-affinity human antibodies can be derived directly from large and diverse synthetic phage antibody repertoires, with affinities typical of somatically mutated mouse antibodies produced in vivo.This study explores the possibility of isolating high-affinity human antibodies directly from large and diverse synthetic phage antibody repertoires. The authors created a highly diverse repertoire of heavy and light chains in vitro using human V gene segments, and then combined these repertoires in bacteria to generate a large (6.5×10^10) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire, they isolated Fab fragments that bound to various antigens and haptens with affinities comparable to those of antibodies from a secondary immune response in mice (up to 4 nM). The VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, but there were also significant differences in segment usage patterns. These differences may help to dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo. The study also analyzed the distribution of CDR3 lengths and the pairings of heavy and light chains, providing insights into the relationship between affinity and repertoire size, as well as the structures of antigen-binding sites and V, D, and J segment usage. Overall, the results demonstrate that high-affinity human antibodies can be derived directly from large and diverse synthetic phage antibody repertoires, with affinities typical of somatically mutated mouse antibodies produced in vivo.