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

Nat Chem. 2017 Aug;9(8):762-771. doi: 10.1038/nchem.2811. Epub 2017 Jul 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • 3T3 Cells
  • Animals
  • Antigens / immunology
  • Antigens / metabolism
  • Biological Transport
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Cell Nucleolus / metabolism
  • Cell-Penetrating Peptides / chemical synthesis
  • Cell-Penetrating Peptides / chemistry*
  • Drug Carriers / chemical synthesis
  • Drug Carriers / chemistry*
  • Green Fluorescent Proteins / immunology
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Methyl-CpG-Binding Protein 2 / pharmacokinetics
  • Mice
  • Proliferating Cell Nuclear Antigen / metabolism
  • Proto-Oncogene Proteins c-mdm2 / metabolism
  • Single-Domain Antibodies / chemistry
  • Single-Domain Antibodies / immunology
  • Single-Domain Antibodies / metabolism*
  • Tumor Suppressor Protein p53 / metabolism


  • Antigens
  • Cell-Penetrating Peptides
  • Drug Carriers
  • MECP2 protein, human
  • Methyl-CpG-Binding Protein 2
  • PCNA protein, human
  • Proliferating Cell Nuclear Antigen
  • Single-Domain Antibodies
  • Tumor Suppressor Protein p53
  • Green Fluorescent Proteins
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2