Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies

The antibody immune response is essential for the survival of mammals. However, we still lack a systematic understanding of the antibody repertoire. Here, we developed a proteomic strategy to survey, at an unprecedented scale, the landscape of antigen-engaged, circulating camelid heavy-chain antibodies, whose minimal binding fragments are called V_HH antibodies or nanobodies. The sensitivity and robustness of this approach were validated with three antigens spanning orders of magnitude in immune responses; thousands of distinct, high-affinity nanobody families were reliably identified and quantified. Using high-throughput structural modeling, cross-linking mass spectrometry, mutagenesis, and deep learning, we mapped and analyzed the epitopes of >100,000 antigen-nanobody complexes. Our results revealed a surprising diversity of ultrahigh-affinity camelid nanobodies for specific antigen binding on various dominant epitope clusters. Nanobodies utilize both shape and charge complementarity to enable highly selective antigen binding. Interestingly, we found that nanobody-antigen binding can mimic conserved intracellular protein-protein interactions. A record of this paper's Transparent Peer Review process is included in the Supplemental information.