Immunogenetic Analysis Reveals a Novel Landscape of Non-Canonical Cysteines in Human V <sub>H</sub> Repertoire

Abstract

Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insight into the B-cell immunogenetics and paratope diversity and it has potential to shed light on structural, functional and evolutionary perspectives on human antibodies. By analyzing large NGS datasets that were only recently published, we mapped for the first time at the repertoire level, the huge landscape of non-canonical cysteines in human heavy chain variable (VH) domains. Notably, we identified (i) non-canonical cysteine that can range in number between 1 and 8, creating diverse cysteine motifs and patterns, within heavy chain complementarity determine region 3 (CDR-H3) (ii) increase in their frequency of up to 30% in CDR-H3s of lengths 16 to 39 amino acids, and (iii) occurrence of non-canonical cysteines in other CDRs and framework regions. The location and nature of non-canonical cysteine in human VHs appears to be unique and more complex than those found in the other species such as chicken, camel, llama, shark and cow, for which antibody paratopes are primarily diversified through cysteines. These results explain how non-canonical cysteines strategically occur in human VHs to expand its special paratope space, which was hitherto unknown. The knowledge gained from this study will be a resource for designing the next-generation human antibody libraries to access difficult to access epitopes. It can also change the paradigm about understanding of non-canonical cysteines and their status in antibody developability.