title: Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells.
abstract: In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany; Department of Pharmaceutical Biotechnology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
authors.forename: Philipp D
authors.initials: PD
authors.lastname: Kaiser
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany; Department of Pharmaceutical Biotechnology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Maier
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany; Department of Pharmaceutical Biotechnology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
authors.forename: Bjoern
authors.initials: B
authors.lastname: Traenkle
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany; Department of Pharmaceutical Biotechnology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Emele
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany; Department of Pharmaceutical Biotechnology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany. Electronic address: ulrich.rothbauer@uni-tuebingen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
keywords: Antibody-scaffold; Chromobody; Functional assay; Intrabody; Live cell imaging; Protein transduction