High-throughput synthesis and specificity characterization of natively paired antibodies using oPool⁺ display

Antibody discovery is crucial for developing therapeutics and vaccines as well as understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we presented oPool⁺ display, a high-throughput cell-free platform that combined oligo pool synthesis and mRNA display to rapidly construct and characterize many natively paired antibodies in parallel. As a proof-of-concept, we applied oPool⁺ display to probe the binding specificity of >300 uncommon influenza hemagglutinin (HA) antibodies against 9 HA variants through 16 different screens. Over 5,000 binding tests were performed in 3-5 days with further scaling potential. Follow-up structural analysis of two HA stem antibodies revealed the previously unknown versatility of IGHD3-3 gene segment in recognizing the HA stem. Overall, this study established an experimental platform that not only accelerate antibody characterization, but also enable unbiased discovery of antibody molecular signatures.