Development and application of nbLIBRA-seq for high-throughput discovery of antigen-specific nanobodies

Nanobodies are of high interest in many fields of medicine and biotechnology, although nanobody discovery has been limited by laborious screening techniques. Here, we demonstrate the successful adaptation of linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) to immunized alpacas for the rapid identification of antigen-specific nanobodies, derived from heavy-chain antibodies. We validated LIBRA-seq for nanobody discovery (nbLIBRA-seq) in two different disease settings. First, we identified over 300 antigen-specific heavy-chain antibodies against human transferrin receptor (TfR1) from a single alpaca blood sample. Second, we tested the efficiency of nbLIBRA-seq with multiple antigens in the screening library. Using fusion glycoproteins from the related respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), 1,125 antigen-specific heavy-chain-expressing B cells were recovered. Our results illustrate the potential of nbLIBRA-seq to rapidly identify antigen-specific heavy-chain antibodies for a range of diverse targets, a capability that will be critical for the efficient development of nanobody-based therapeutics.