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A robust pipeline for rapid production of versatile nanobody repertoires

Abstract

Nanobodies are single-domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show promise as high-affinity reagents for research, diagnostics and therapeutics owing to their high specificity, small size (15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Our approach generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the subnanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultrahigh-affinity dimeric nanobodies with Kd values as low as 30 pM. The approach presented here is well suited for the routine production of high-affinity capture reagents for various biomedical applications.

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Figure 1: Overview of nanobody identification and production pipeline.
Figure 2: Characterization of VHH IgG and recombinant nanobodies.
Figure 3: Efficacy of LaG and LaM nanobodies in immunofluorescence microscopy.
Figure 4: Nanobody fluorescent protein binding.
Figure 5: Mapping of nanobody binding epitopes on GFP by NMR.

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Acknowledgements

We acknowledge support from US National Institutes of Health grants U54 GM103511 and P41 GM109824 (M.P.R. and B.T.C.), P41 GM103314 (B.T.C.), and AI072529-08 and AI037526-20A1 (M.C.N.) and support from the Howard Hughes Medical Institute (M.C.N.). M.O. was supported by the Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research and Fonds de recherches du Québec - Santé. We thank A. North for assistance with immunofluorescence microscopy; A. Viale and C. Zhao for support with high-throughput sequencing; A. Luz for assistance with SPR; S. Reed-Paske and the other members of Capralogics, Inc., for advice and animal husbandry; and members of the Rout and Chait laboratories, past and present, for helpful discussions and technical assistance, particularly A. Ferguson, K. Wei, H. Jiang and S. Obado.

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Contributions

The project was conceived of by M.P.R., B.T.C., P.C.F., Y.L., D.F. and M.C.N. Experiments relating to immunization, sample collection and processing were performed by P.C.F., M.K.T. and M.O.; J.F.S. assisted with bone marrow processing. MS was carried out by Y.L. Experimental work was supervised by M.P.R. and B.T.C. Bioinformatic analysis was performed by Y.L., S.K. and D.F. Production and characterization of recombinant nanobodies was performed by P.C.F. and M.K.T. NMR analyses were performed by I.N. The manuscript was cowritten by P.C.F., Y.L., I.N., M.P.R. and B.T.C., with contributions from all authors. M.P.R. communed with llamas in the Atacama desert.

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Correspondence to David Fenyö, Brian T Chait or Michael P Rout.

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B.T.C. and M.P.R. are inventors on a US patent application encompassing the method described in this manuscript.

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Supplementary Figures 1–17, Supplementary Tables 1 and 2 and Supplementary Protocol (PDF 18018 kb)

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Fridy, P., Li, Y., Keegan, S. et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nat Methods 11, 1253–1260 (2014). https://doi.org/10.1038/nmeth.3170

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