Generation of high‐affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. A generic procedure is established for the humanization of chicken‐derived antibodies. To this end, high‐affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high‐affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high‐affinity binders in the scFv and Fab formats. Next‐generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high‐throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance.

中文翻译：

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使用酵母表面显示和NGS数据挖掘对鸡源性scFv进行人性化

通过鸡免疫产生高亲和力单克隆抗体是一种有价值的策略，特别是对于获得针对哺乳动物中保守的表位的抗体而言。建立了一种通用程序，可对鸡源抗体进行人源化。为此，使用酵母表面展示技术从免疫鸡中分离了表皮生长因子受体胞外域的高亲和力结合剂。将两种高亲和力结合剂的互补决定区（CDR）嫁接到人类受体框架上。同时，负责空间CDR排列的游标区残基被部分随机化。筛选了一个包含约300,000个变体的酵母表面展示文库，以查找scFv和Fab格式的高亲和力结合剂。下一代测序揭示了与亲本鸡抗体相比具有恢复的亲和力和改善的蛋白质特性的人源化抗体变体。此外，测序数据为人源化过程中使用的抗体形式的重要性提供了新的见解。从免疫鸡的抗体库开始，这项工作采用有效且快速的高通量方法来生成具有潜在治疗意义的多种人源化抗体。