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.
Highlights
More than thirty years after their discovery [1], antibody fragments derived from heavy-chain only antibodies of camelids, termed as variable heavy chain of heavy-chain only antibodies (VHH) or nanobodies (Nbs), have emerged as highly potent and versatile binding molecules for biomedical research, diagnostics and therapy [2]
Either complete libraries or selected high affinity binders initially identified from conventional screenings such as phage display were site directly mutated to cysteine-free derivates [11]
ScFv-derived intrabodies were selected by FACS analysis of impaired degranulation as a selection marker in rat leukemic cells [29], or intracellular binding of scFvs fused to the cytoplasmic domain of a receptor tyrosine kinase to its homo-oligomeric antigen became detectable by growth signal induction in a B-cell line [30]
Summary
More than thirty years after their discovery [1], antibody fragments derived from heavy-chain only antibodies of camelids, termed as variable heavy chain of heavy-chain only antibodies (VHH) or nanobodies (Nbs), have emerged as highly potent and versatile binding molecules for biomedical research, diagnostics and therapy [2]. Having the ability to visualize and/or modulate endogenous targets within living cells, intrabodies provide distinct advantages compared to other technologies such as fluorescent fusion proteins, conditional gene expression, RNA interference and chemical or genetic knockouts When it comes to intracellular applications it has to be considered that Nbs, like any other antibody-derived binding molecule, comprise evolutionary conserved disulfide bonds. As the screening is performed in bacteria, Biomolecules 2020, 10, 1701 functionality and specificity of B2H-selected intrabodies has to be further validated in eukaryotic systems e.g., by employing the mammalian cell-based fluorescent-2-hybrid (F2H) approach [27] This limitation was elegantly overcome by Schmidt et al using a lentiviral screening technology to select Nbs that are stably producible in mammalian cells and simultaneously neutralize influenza virus or vesicular stomatitis virus. As shortly outlined in this chapter, different screening approaches became available which support accelerated identification of suitable intrabodies based on Nbs
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