Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

Nature Chemistry volume 9, pages 762–771 (2017)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Strategy to visualize the successful transduction of functional cell-permeable nanobodies.
Figure 2: Synthesis of cell-permeable antigen-binding proteins.
Figure 3: Nanobodies efficiently transduce into living cells when coupled to cR10 (3), and remain functional and able to bind and relocalize intracellular GFP to the nucleolus.
Figure 4: The cell-permeable GFP-binding antibody 2C targets and relocalizes GFP-labelled functional proteins along with other interacting proteins, which facilitates the detection of protein–protein interactions in living cells.
Figure 5: The cell-permeable antibody is able to bind its antigen and transport it into living cells.
Figure 6: Cleavable cell-permeable nanobodies for immunostaining in living cells.

References

  1. Chames, P ., Van Regenmortel, M ., Weiss, E. &, Baty, D. Therapeutic antibodies: successes, limitations and hopes for the future. Br. J. Pharmacol. 157, 220–233 (2009).

    Article  CAS  Google Scholar 

  2. Leavy, O. Therapeutic antibodies: past, present and future. Nat. Rev. Immunol. 10, 297 (2010).

    Article  CAS  Google Scholar 

  3. Herce, H. D., Deng, W., Helma, J., Leonhardt, H. & Cardoso, M. C. Visualization and targeted disruption of protein interactions in living cells. Nat. Commun. 4, 2660 (2013).

    Article  Google Scholar 

  4. Courtete, J. et al. Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA. Mol. Cancer. Ther. 6, 1728–1735 (2007).

    Article  CAS  Google Scholar 

  5. Smith, K. G. & Clatworthy, M. R. FcγRIIB in autoimmunity and infection: evolutionary and therapeutic implications. Nat. Rev. Immunol. 10, 328–343 (2010).

    Article  CAS  Google Scholar 

  6. Freund, G. et al. Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells. MAbs 5, 518–522 (2013).

    Article  Google Scholar 

  7. Kimura, H., Hayashi-Takanaka, Y., Stasevich, T. J. & Sato, Y. Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo. Histochem. Cell. Biol. 144, 101–109 (2015).

    Article  CAS  Google Scholar 

  8. Marschall, A. L., Frenzel, A., Schirrmann, T., Schungel, M. & Dubel, S. Targeting antibodies to the cytoplasm. MAbs 3, 3–16 (2011).

    Article  Google Scholar 

  9. Biocca, S., Ruberti, F., Tafani, M., Pierandrei-Amaldi, P. & Cattaneo, A. Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria. Biotechnology 13, 1110–1115 (1995).

    Article  CAS  Google Scholar 

  10. Desplancq, D. et al. Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells. Exp. Cell. Res. 342, 145–158 (2016).

    Article  CAS  Google Scholar 

  11. Marschall, A. L., Dubel, S. & Boldicke, T. Specific in vivo knockdown of protein function by intrabodies. MAbs 7, 1010–1035 (2015).

    Article  CAS  Google Scholar 

  12. Sato, Y. et al. Genetically encoded system to track histone modification in vivo. Sci. Rep. 3, 2436 (2013).

    Article  Google Scholar 

  13. Worn, A. & Pluckthun, A. Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989–1010 (2001).

    Article  CAS  Google Scholar 

  14. Heng, B. C. & Cao, T. Making cell-permeable antibodies (transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: potential advantages over antibodies expressed within the intracellular environment (intrabody). Med. Hypotheses 64, 1105–1108 (2005).

    Article  CAS  Google Scholar 

  15. Gu, Z., Biswas, A., Zhao, M. & Tang, Y. Tailoring nanocarriers for intracellular protein delivery. Chem. Soc. Rev. 40, 3638–3655 (2011).

    Article  CAS  Google Scholar 

  16. Kamaly, N., Xiao, Z., Valencia, P. M., Radovic-Moreno, A. F. & Farokhzad, O. C. Targeted polymeric therapeutic nanoparticles: design, development and clinical translation. Chem. Soc. Rev. 41, 2971–3010 (2012).

    Article  CAS  Google Scholar 

  17. McNaughton, B. R., Cronican, J. J., Thompson, D. B. & Liu, D. R. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proc. Natl Acad. Sci. USA 106, 6111–6116 (2009).

    Article  CAS  Google Scholar 

  18. Fuchs, S. M. & Raines, R. T. Arginine grafting to endow cell permeability. ACS Chem. Biol. 2, 167–170 (2007).

    Article  CAS  Google Scholar 

  19. Cronican, J. J. et al. Potent delivery of functional proteins into mammalian cells in vitro and in vivo using a supercharged protein. ACS Chem. Biol. 5, 747–752 (2010).

    Article  CAS  Google Scholar 

  20. Zelphati, O. et al. Intracellular delivery of proteins with a new lipid-mediated delivery system. J. Biol. Chem. 276, 35103–35110 (2001).

    Article  CAS  Google Scholar 

  21. Liao, X., Rabideau, A. E. & Pentelute, B. L. Delivery of antibody mimics into mammalian cells via anthrax toxin protective antigen. ChemBioChem 15, 2458–2466 (2014).

    Article  CAS  Google Scholar 

  22. Khalil, I. A., Kogure, K., Akita, H. & Harashima, H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol. Rev. 58, 32–45 (2006).

    Article  CAS  Google Scholar 

  23. Krantz, B. A., Trivedi, A. D., Cunningham, K., Christensen, K. A. & Collier, R. J. Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin. J. Mol. Biol. 344, 739–756 (2004).

    Article  CAS  Google Scholar 

  24. Brock, R. The uptake of arginine-rich cell-penetrating peptides: putting the puzzle together. Bioconjug. Chem. 25, 863–868 (2014).

    Article  CAS  Google Scholar 

  25. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R. & Brock, R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8, 848–866 (2007).

    Article  CAS  Google Scholar 

  26. Herce, H. D., Garcia, A. E. & Cardoso, M. C. Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules. J. Am. Chem. Soc. 136, 17459–17467 (2014).

    Article  CAS  Google Scholar 

  27. Ter-Avetisyan, G. et al. Cell entry of arginine-rich peptides is independent of endocytosis. J. Biol. Chem. 284, 3370–3378 (2009).

    Article  CAS  Google Scholar 

  28. Tunnemann, G. et al. Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J. 20, 1775–1784 (2006).

    Article  Google Scholar 

  29. Lattig-Tunnemann, G. et al. Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides. Nat. Commun. 2, 453 (2011).

    Article  Google Scholar 

  30. Nischan, N. et al. Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability. Angew. Chem. Int. Ed. 54, 1950–1953 (2015).

    Article  CAS  Google Scholar 

  31. Helma, J., Cardoso, M. C., Muyldermans, S. & Leonhardt, H. Nanobodies and recombinant binders in cell biology. J. Cell. Biol. 209, 633–644 (2015).

    Article  CAS  Google Scholar 

  32. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).

    Article  CAS  Google Scholar 

  33. Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).

    Article  CAS  Google Scholar 

  34. Schumacher, D. et al. Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase. Angew. Chem. Int. Ed. 54, 13787–123791 (2015).

    Article  CAS  Google Scholar 

  35. Martin, R. M. et al. Principles of protein targeting to the nucleolus. Nucleus 6, 314–325 (2015).

    Article  CAS  Google Scholar 

  36. Herce, H. D., Rajan, M., Lattig-Tunnemann, G., Fillies, M. & Cardoso, M. C. A novel cell permeable DNA replication and repair marker. Nucleus 5, 590–600 (2014).

    Article  Google Scholar 

  37. Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).

    Article  CAS  Google Scholar 

  38. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  CAS  Google Scholar 

  39. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998).

    Article  CAS  Google Scholar 

  40. Hackenberger, C. P. & Schwarzer, D. Chemoselective ligation and modification strategies for peptides and proteins. Angew. Chem. Int. Ed. 47, 10030–10074 (2008).

    Article  CAS  Google Scholar 

  41. Muir, T. W. Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 72, 249–289 (2003).

    Article  CAS  Google Scholar 

  42. Chagin, V. O. et al. 4D visualization of replication foci in mammalian cells corresponding to individual replicons. Nat. Commun. 7, 11231 (2016).

    Article  CAS  Google Scholar 

  43. Leonhardt, H. et al. Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271–280 (2000).

    Article  CAS  Google Scholar 

  44. Amir, R. E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 23, 185–188 (1999).

    Article  CAS  Google Scholar 

  45. Rett, A. On an until now unknown disease of a congenital metabolic disorder. Krankenschwester 19, 121–122 (1966).

    CAS  PubMed  Google Scholar 

  46. Guy, J., Gan, J., Selfridge, J., Cobb, S. & Bird, A. Reversal of neurological defects in a mouse model of Rett syndrome. Science 315, 1143–1147 (2007).

    Article  CAS  Google Scholar 

  47. Thomas, C. E., Ehrhardt, A. & Kay, M. A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346–358 (2003).

    Article  CAS  Google Scholar 

  48. Brero, A. et al. Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation. J. Cell. Biol. 169, 733–743 (2005).

    Article  CAS  Google Scholar 

Download references

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

  1. Marion Fillies

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

  2. Henry D. Herce and Dominik Schumacher: These authors contributed equally

Authors and Affiliations

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

    Henry D. Herce, Anne K. Ludwig & M. Cristina Cardoso

  2. Chemical Biology Department, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, Berlin,, 13125, 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, Berlin, 12489, 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, Berlin, 13125, Germany

    Marion Fillies

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

    Heinrich Leonhardt

Authors
  1. Henry D. Herce
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dominik Schumacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anselm F. L. Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anne K. Ludwig
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Florian A. Mann
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marion Fillies
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marc-André Kasper
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stefan Reinke
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eberhard Krause
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Heinrich Leonhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. M. Cristina Cardoso
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Christian P. R. Hackenberger
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 6612 kb)

Supplementary movie

Supplementary movie 1 (MP4 7845 kb)

Supplementary movie

Supplementary movie 2 (MOV 615 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Herce, H., Schumacher, D., Schneider, A. et al. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nature Chem 9, 762–771 (2017). https://doi.org/10.1038/nchem.2811

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research