HCDR3 Diversity: Necessary, but Not Sufficient for Specific Antibody Binding

摘要

The immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is traditionally viewed as an antibody molecule's signature component for binding and specificity. HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs. In our in vitro antibody discovery platform we show that, in selected populations, multiple antibodies carry the same HCDR3 and they all recognize the intended target, within a significant range of affinities. In contrast, within unselected populations, the vast majority of antibodies with the same HCDR3 sequence do not bind the target. When a specific HCDR3 was examined, we found that all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 originated from many different gene rearrangements. Expanding on this observation, we show that in depth analysis of previously published in vivo dat asets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding. We have used this information at Specifica to build a bioinformatics selection analysis pipeline that takes into account more than the HCDR3 to characterize the diversity of binders in a selection output.