High-throughput saturation mutagenesis generates a high-affinity antibody against SARS-CoV-2 variants using protein surface display assay on a human cell

Author summaryThe COVID-19 pandemic caused by SARS-CoV-2 has become a global health problem of wide concern due to the continuous emergence of mutants, so that human social and economic activities have suffered a serious blow. The development of potent neutralizing antibodies is critically important in the coming time. At present, the main way to obtain effective neutralizing antibodies against SARS-CoV-2 and its variants is to isolate the serum of convalescent patients or immunized humanized mice. However, this method has high requirements on samples, and the whole process is time-consuming and labor-intensive. We here report a single-cell screening assay that allows the rapid and high-throughput optimizing of a human antibody displayed on a mammalian cell surface. The process can improve the affinity of antibodies with EC50 below 5 ng/mL within 2 weeks. Our approach with short turnaround and significant enhancement in antibody affinity presents an effective strategy for fighting against the ever-evolving COVID variants. As new mutations continue to emerge, the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus to evade the human immune system and neutralizing antibodies remains a huge challenge for vaccine development and antibody research. The majority of neutralizing antibodies have reduced or lost activity against SARS-CoV-2 variants. In this study, we reported a novel protein surface display system on a mammalian cell for obtaining a higher-affinity antibody in high-throughput manner. Using a saturation mutagenesis strategy through integrating microarray-based oligonucleotide synthesis and single-cell screening assay, we generated a group of new antibodies against diverse prevalent SARS-CoV-2 variants through high-throughput screening the human antibody REGN10987 within 2 weeks. The affinity of those optimized antibodies to seven prevalent mutants was greatly improved, and the EC50 values were no higher than 5 ng/mL. These results demonstrate the robustness of our screening system in the rapid generation of an antibody with higher affinity against a new SARS-CoV-2 variant, and provides a potential application to other protein molecular interactions.

Automated summary

We report a single-cell screening assay that allows rapid and high-throughput optimization of a human antibody displayed on a mammalian cell surface. Our approach can improve antibody affinity with an EC50 below 5 ng/mL within 2 weeks. This provides an effective strategy for fighting against evolving COVID variants.