Abstract

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein–protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

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Acknowledgements

We thank K. K. Hassanin and A. Lehmkuhl for excellent technical assistance. We are grateful to J. Hewing and A. Krella for the generation and characterization of MaSat fusion and PCNA expression constructs, respectively, and R. Kühne for providing the pGEX4T1eGFP plasmid. Furthermore, we thank J. Helma for his great support during the nanobody cloning and expression. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP1623) to M.C.C. (CA 198/8-2), C.P.R.H. (HA 4468/9-1) and H.L. (LE 721/13-2), the Einstein Foundation Berlin (Leibniz-Humboldt Professorship) and the Boehringer-Ingelheim Foundation (Plus 3 award) to C.P.R.H., the Fonds der Chemischen Industrie to C.P.R.H. and to D.S. (Kekulé fellowship) and A.F.L.S (Chemiefonds fellowship) and the Nanosystems Initiative Munich to H.L.

Author information

Author notes

    • Henry D. Herce
    •  & Dominik Schumacher

    These authors contributed equally

    • Marion Fillies

    Present addresses: Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.

Affiliations

  1. Department of Biology, Technische Universität Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt, Germany

    • Henry D. Herce
    • , Anne K. Ludwig
    •  & M. Cristina Cardoso
  2. Chemical Biology Department, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany

    • Dominik Schumacher
    • , Anselm F. L. Schneider
    • , Florian A. Mann
    • , Marc-André Kasper
    • , Stefan Reinke
    • , Eberhard Krause
    •  & Christian P. R. Hackenberger
  3. Department of Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany

    • Dominik Schumacher
    • , Anselm F. L. Schneider
    • , Florian A. Mann
    • , Marc-André Kasper
    •  & Christian P. R. Hackenberger
  4. Max Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Strasse 10, 13125 Berlin, Germany

    • Marion Fillies
  5. Department of Biology II, and Center for Integrated Protein Science Munich, Ludwig Maximilians Universität München, Großhadenerstrasse 2, 82152 Martinsried, Germany

    • Heinrich Leonhardt

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Contributions

H.D.H. and D.S. contributed equally. M.C.C., H.L. and C.P.R.H. designed and conceived the project. H.D.H. conceived and performed the cellular uptake experiments, the relocalization-based visualization assay, the uptake of recombinant GFP and Mecp2–GFP, PCNA relocalization and the modified F3H assay and microscale thermophoresis measurements to determine the binding constant of functionalized nanobodies. D.S. designed and optimized the cell-permeable nanobody synthesis, cloned and expressed GBP–intein–CBD fusions, established the refolding protocol, performed the EPL and analysed all the constructs (MS, CD, binding to GFP), synthesized the linear, cyclic and cleavable CPPs, generated double-functionalized nanobodies and performed eGFP expression and purification. A.F.L.S. generated the GBP11-117A3–intein–CBD fusion, established a purification strategy, performed EPLs and synthesized cCPPs. A.K.L. purified recombinant proteins and performed some cellular uptake experiments as well as RNA isolation and RNA-binding assays. F.A.M. optimized the EPL conditions and synthesized cCPPs. M.F. generated and characterized the cell lines with the permanent expression of GFP and its fusions. M.-A.K. synthesized Cy5. S.R. performed the cloning and initial testing of the GBP–intein–CBD fusions. E.K. contributed to the matrix-assisted laser desorption ionization measurements. H.L. provided the nanobodies. H.D.H. and D.S. wrote the manuscript supported by M.C.C., C.P.R.H., F.A.M., A.F.L.S. and A.K.L.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Henry D. Herce or Dominik Schumacher or M. Cristina Cardoso or Christian P. R. Hackenberger.

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DOI

https://doi.org/10.1038/nchem.2811