Data_Sheet_1_Generation of synthetic antibody fragments with optimal complementarity determining region lengths for Notch-1 recognition.docx

Synthetic antibodies have been engineered against a wide variety of antigens with desirable biophysical, biochemical, and pharmacological properties. Here, we describe the generation and characterization of synthetic antigen-binding fragments (Fabs) against Notch-1. Three single-framework synthetic Fab libraries, named S, F, and modified-F, were screened against the recombinant human Notch-1 extracellular domain using phage display. These libraries were built on a modified trastuzumab framework, containing two or four diversified complementarity-determining regions (CDRs) and different CDR diversity designs. In total, 12 Notch-1 Fabs were generated with 10 different CDRH3 lengths. These Fabs possessed a high affinity for Notch-1 (sub-nM to mid-nM KDapp values) and exhibited different binding profiles (mono-, bi-or tri-specific) toward Notch/Jagged receptors. Importantly, we showed that screening focused diversity libraries, implementing next-generation sequencing approaches, and fine-tuning the CDR length diversity provided improved binding solutions for Notch-1 recognition. These findings have implications for antibody library design and antibody phage display.

合成抗体已被设计针对多种具有理想生物物理、生化及药理特性的抗原。在本研究中，我们描述了针对Notch-1的合成抗原结合片段（Fab）的生成与表征。三种单框架合成Fab文库，分别命名为S、F和改良型F，通过噬菌体展示技术对重组人Notch-1细胞外结构域进行筛选。这些文库基于改良的曲妥珠单抗框架构建，包含两个或四个多样化的互补决定区（CDR）以及不同的CDR多样性设计。总计生成了12个Notch-1 Fab，其中包含10种不同的CDRH3长度。这些Fab对Notch-1展现出高亲和力（亚纳摩尔至中纳摩尔KDapp值）并呈现出不同的结合谱（单特异性、双特异性或三特异性）。值得注意的是，我们证明了针对多样性文库的筛选、实施下一代测序方法以及精细调整CDR长度多样性，为Notch-1识别提供了改进的结合解决方案。这些发现对抗体文库设计和抗体噬菌体展示具有重要意义。