Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insights into B cell immunogenetics and paratope diversity. By analyzing large public NGS datasets, we map the landscape of non-canonical cysteines in human variable heavy-chain domains (V_Hs) at the repertoire level. We identify remarkable usage of non-canonical cysteines within the heavy-chain complementarity-determining region 3 (CDR-H3) and other CDRs and framework regions. Furthermore, our study reveals the diversity and location of non-canonical cysteines and their associated motifs in human V_Hs, which are reminiscent of and more complex than those found in other non-human species such as chicken, camel, llama, shark, and cow. These results explain how non-canonical cysteines strategically occur in the human antibodyome to expand its paratope space. This study will guide the design of human antibodies harboring disulfide-stabilized long CDR-H3s to access difficult-to-target epitopes and influence a paradigm shift in developability involving non-canonical cysteines.

通过下一代测序（NGS）捕获的人类抗体库数据使人们对B细胞免疫遗传学和对位抗原多样性有了更深入的了解。通过分析大型的公共NGS数据集，我们在曲目级别上绘制了人类可变重链域（V _H s）中非规范性半胱氨酸的景观。我们在重链互补决定区3（CDR-H3）和其他CDR和框架区域内确定非规范性半胱氨酸的显着用途。此外，我们的研究揭示了非规范性半胱氨酸及其在人V _H中的相关基序的多样性和位置比其他非人类物种（例如鸡，骆驼，美洲驼，鲨鱼和牛）中的那些让人想起并且更复杂。这些结果解释了非规范性半胱氨酸如何在人类抗体组中策略性地发生以扩展其互补位空间。这项研究将指导人抗体的设计，该抗体带有二硫键稳定的长CDR-H3，可进入难以靶向的表位，并影响涉及非规范性半胱氨酸的可开发性的范式转变。