Intradiabodies, bispecific, tetravalent antibodies for the simultaneous functional knockout of two cell surface receptors.
The specific and high affinity binding properties of intracellular antibodies (intrabodies), combined with their ability to be stably expressed in defined organelles, provides powerful tools with a wide range of applications in the field of functional genomics and gene therapy. Intrabodies have been used to specifically target intracellular proteins, manipulate biological processes, and contribute to the understanding of their functions as well as for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. In order to study the biological consequences of knocking down two receptor-tyrosine kinases, we developed a novel intrabody-based strategy. Here we describe the design, engineering, and characterization of a bispecific, tetravalent endoplasmic reticulum (ER)-targeted intradiabody for simultaneous surface depletion of two endothelial transmembrane receptors, Tie-2 and vascular endothelial growth factor receptor 2 (VEGF-R2). Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment (scFv) intrabodies demonstrated that the intradiabody is significantly more efficient with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2. In vitro endothelial cell tube formation assays suggest that the bispecific intradiabody exhibits strong antiangiogenic activity, whereas the effect of the monospecific scFv intrabodies was weaker. These findings suggest that simultaneous interference with the VEGF and the Tie-2 receptor pathways results in at least additive antiangiogenic effects, which may have implications for future drug developments. In conclusion, we have identified a highly effective ER-targeted intrabody format for the simultaneous functional knockout of two cell surface receptors.
Jendreyko
Nina
N
Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Popkov
Mikhail
M
Beerli
Roger R
RR
Chung
Junho
J
McGavern
Dorian B
DB
Rader
Christoph
C
Barbas
Carlos F
CF
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry; D042787:Receptor, TIE-2 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D020794:Receptor Protein-Tyrosine Kinases; D042787:Receptor, TIE-2; D040301:Vascular Endothelial Growth Factor Receptor-2