TY - JOUR
T1 - Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies
AU - Xiang, Yufei
AU - Sang, Zhe
AU - Bitton, Lirane
AU - Xu, Jianquan
AU - Liu, Yang
AU - Schneidman-Duhovny, Dina
AU - Shi, Yi
N1 - Funding Information: We thank J.L.Jia and Y.Jiao for the NGS experiments, Z.Shen and Z.Y.Xiao for assistance in the mutagenesis experiments, J.W. Ahn for the help on the biophysics experiments, P.W. Duplex for access to the SPR device, J.J.Wang (Rockefeller University) for providing a script for the preliminary database analysis, and Y.Liu, H.Maayan and D.Groebe for comments on the manuscript. This work was supported by the University of Pittsburgh School of Medicine (Y.S.) and a University of Pittsburgh Aging Institute pilot fund (Y.S.), NIH grant 1R35GM137905-01 (Y.S.), and ISF 1466/18, Israel ministry of Science and Technology and HUJI-CIDR (D.S.). Y.S. conceived the research. Y.S. and D.S. supervised the study. Y.X. performed all the benchwork. Y.X. and Y.S. performed the MS analysis. Z.S. D.S. Y.S. Y.X. and L.B. developed informatics tools and analyzed data. J.X. and Y.L. facilitated fluorescence imaging experiments. Y.S. drafted the manuscript. Y.S. D.S. X.Y. and Z.S. edited the manuscript. Funding Information: We thank J.L.Jia and Y.Jiao for the NGS experiments, Z.Shen and Z.Y.Xiao for assistance in the mutagenesis experiments, J.W. Ahn for the help on the biophysics experiments, P.W. Duplex for access to the SPR device, J.J.Wang (Rockefeller University) for providing a script for the preliminary database analysis, and Y.Liu, H.Maayan and D.Groebe for comments on the manuscript. This work was supported by the University of Pittsburgh School of Medicine (Y.S.) and a University of Pittsburgh Aging Institute pilot fund (Y.S.), NIH grant 1R35GM137905-01 (Y.S.), and ISF 1466/18 , Israel ministry of Science and Technology and HUJI-CIDR (D.S.). Publisher Copyright: © 2021 Elsevier Inc.
PY - 2021/3/17
Y1 - 2021/3/17
N2 - 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 VHH 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.
AB - 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 VHH 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.
KW - antibody immune response
KW - antibody technology
KW - nanobody
KW - structural modeling
KW - structural proteomics
UR - http://www.scopus.com/inward/record.url?scp=85101708862&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.cels.2021.01.003
DO - https://doi.org/10.1016/j.cels.2021.01.003
M3 - Article
C2 - 33592195
SN - 2405-4712
VL - 12
SP - 220
EP - 234
JO - Cell Systems
JF - Cell Systems
IS - 3
ER -