Nanobodies Right in the Middle: Intrabodies as Toolbox to Visualize and Modulate Antigens in the Living Cell

Abstract

In biomedical research, there is an ongoing demand for new technologies to elucidate disease mechanisms and develop novel therapeutics. This requires comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, post-translational modifications and dynamic interactions of cellular components. Traceable intracellular binding molecules provide new opportunities for real-time cellular diagnostics. Most prominently, intrabodies derived from antibody fragments of heavy-chain only antibodies of camelids (nanobodies) have emerged as highly versatile and attractive probes to study and manipulate antigens within the context of living cells. In this review, we provide an overview on the selection, delivery and usage of intrabodies to visualize and monitor cellular antigens in living cells and organisms. Additionally, we summarize recent advances in the development of intrabodies as cellular biosensors and their application to manipulate disease-related cellular processes. Finally, we highlight switchable intrabodies, which open entirely new possibilities for real-time cell-based diagnostics including live-cell imaging, target validation and generation of precisely controllable binding reagents for future therapeutic applications.

Keywords: biosensors; intrabody; live-cell imaging; nanobody; phage display; target validation.

Figure 1

Intrabodies for visualization. (a) Schematic depiction of nano- and chromobodies derived from heavy-chain antibodies of Camelidae (b) Illustration of different labelling strategies for visualization of the protein of interest (POI) using intrabodies coupled to a detectable moiety and summary of their characteristics: POI genetically coupled to GFP recognized by a GFP intrabody, POI genetically coupled to a short peptide-tag recognized by the tag-specific intrabody and POI directly recognized by a specific intrabody. Model protein structures adapted from PDBs 3OGO, 5H88, 1EMA and 5LEW. Representative images of living Hela cells transiently expressing PNCA^GFP and the GFP-specific chromobody (tagRFP), PNCA^PEP-tag and the PEP-tag-specific chromobody (tagRFP) or PCNA-specific chromobody (tagRFP) as examples for the described labeling strategies are shown. Scale bar: 25 µm.

Figure 2

Intrabodies as intracellular modulators. Graphical representation how intrabodies can address their POI (protein of interest) for functional modulation. Shown are models of blocking and activating intrabodies, intrabodies inducing degradation and switchable intrabody systems. Additionally, commonly applied testing systems and potential clinical applications of intrabodies are listed. Model protein structures adapted from PDB 3OGO, 1EMA, 5LEW, 2EPE and parts of schematic art pieces are freely available from Servier Medical Art (https://smart.servier.com; Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License).