Large Diversity of Functional Nanobodies from a Camelid Immune Library Revealed by an Alternative Analysis of Next-Generation Sequencing Data
Large Diversity of Functional Nanobodies from a Camelid Immune Library Revealed by an Alternative Analysis of Next-Generation Sequencing Data
复制标题
DOI：
10.3389/fimmu.2017.00420
复制
发表时间：
2017-04-10
影响因子：
7.3
通讯作者：
Dombrecht, Bruno
中科院分区：
文献类型：
--
作者：
Deschaght, Pieter;Vintem, Ana Paula;Dombrecht, Bruno
下一代测序（NGS）已成功应用于治疗性抗体发现领域，通常优于倾向于仅鉴定更丰富的选择性抗体序列的常规筛选活动。我们使用NGS从针对受体d'origine nantais（罗恩）受体激酶的噬菌体展示骆驼免疫文库中挖掘功能性纳米抗体库。NGS应用的挑战包括准确去除读取错误，正确识别相关序列，以及为感兴趣的序列建立有意义的纳入标准。为此，定义了序列同一性阈值，以通过探索大量不同的公开可用的纳米抗体序列来分离不相关的全长序列簇。当与多数规则一致性建立相结合时，将这种优雅的聚类方法应用于NGS数据集揭示了丰富的> 5，000个富集的候选罗恩结合物。通过一组随机选择的候选物探索了NGS方法预测的巨大结合潜力：90%被证实为罗恩结合剂，其中50%在ERK磷酸化测定中功能性阻断罗恩。另外的验证来自对所有35种罗恩结合纳米抗体的正确预测，所述纳米抗体通过相同免疫文库的常规筛选活动鉴定。对罗恩结合剂亚组的更详细表征揭示了优异的功能效力和有希望的表位多样性。总之，我们的方法揭示了远交骆驼科动物仅重链免疫应答的功能多样性和质量，并证实了NGS鉴定大量有前途的纳米抗体的能力。
Next-generation sequencing (NGS) has been applied successfully to the field of therapeutic antibody discovery, often outperforming conventional screening campaigns which tend to identify only the more abundant selective antibody sequences. We used NGS to mine the functional nanobody repertoire from a phage-displayed camelid immune library directed to the recepteur d'origine nantais (RON) receptor kinase. Challenges to this application of NGS include accurate removal of read errors, correct identification of related sequences, and establishing meaningful inclusion criteria for sequences-ofinterest. To this end, a sequence identity threshold was defined to separate unrelated full-length sequence clusters by exploring a large diverse set of publicly available nanobody sequences. When combined with majority-rule consensus building, applying this elegant clustering approach to the NGS data set revealed a wealth of >5,000-enriched candidate RON binders. The huge binding potential predicted by the NGS approach was explored through a set of randomly selected candidates: 90% were confirmed as RON binders, 50% of which functionally blocked RON in an ERK phosphorylation assay. Additional validation came from the correct prediction of all 35 RON binding nanobodies which were identified by a conventional screening campaign of the same immune library. More detailed characterization of a subset of RON binders revealed excellent functional potencies and a promising epitope diversity. In summary, our approach exposes the functional diversity and quality of the outbred camelid heavy chain-only immune response and confirms the power of NGS to identify large numbers of promising nanobodies.