A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of one hundred billion randomized sequences. We applied CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike proteins and identified >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally tested families, 30 were true RBD binders and 11 inhibited SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increased nanobody binding affinity and yielded a virus neutralizer with picomolar IC50. Furthermore, the unique capability of CeVICA for comprehensive binder prediction allowed us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.