Intracellular and cell surface displayed single-chain diabodies

• Published in: Journal of immunological methods 1999-06, Vol.226 (1), p.179-188
• Main Authors: Kontermann, Roland E, Müller, Rolf
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Antibodies, Bispecific - biosynthesis; Antibodies, Bispecific - immunology; beta-Galactosidase - immunology; Biological and medical sciences; Bispecific recombinant antibody fragment; Carcinoembryonic Antigen - immunology; Cell Line; Cell Membrane - metabolism; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunoglobulin Fragments - biosynthesis; Immunoglobulin Fragments - immunology; Intrabodies; Intracellular Fluid - metabolism; Mice; Molecular immunology; Molecular Sequence Data; Single-chain diabodies; Subcellular Fractions; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00062-9

Intracellularly expressed antibody fragments have found various applications in therapy by virtue of their ability to inhibit the function of cellular proteins or interfere with subcellular trafficking. Bivalent antibody fragments might further improve this inhibitory potential by increasing the functional affinity and bispecific antibody fragments may also be useful for the intracellular retargeting of molecules. Here, we have evaluated the functional expression of intracellular diabodies. A previously constructed secreted bispecific single-chain diabody directed against carcinoembryonic antigen and Escherichia coli β-galactosidase was modified for subcellular targeting to the cell surface membrane, endoplasmic reticulum, mitochondria, cytoplasm, and nucleus. Subcellular localisation was analysed by immunofluorescence, and the assembly of functional antibodies was analysed by binding of β-galactosidase to the antibody fragment and subsequent substrate conversion. Bispecific single-chain diabodies could be directed to all subcellular compartments analysed. However, functional assembly was only observed for single-chain diabodies retained in the endoplasmic reticulum or displayed in the cell membrane while no antigen binding activity was seen with diabodies directed to the cytoplasm, nucleus, or mitochondria. The results demonstrate the functional expression of bispecific recombinant antibody fragments in the secretory pathway and integration into the plasma membrane of mammalian cells.