Intradiabodies, Bispecific, Tetravalent Antibodies for the Simultaneous Functional Knockout of Two Cell Surface Receptors

Abstract

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 receptortyrosine 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.Antibodies can bind almost any molecule with high specificity and affinity, providing powerful biotechnological tools for diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation of the antibody genes, so that design, cloning, expression, and use of single-chain antibodies have become routine procedures in protein engineering. The potential of single-chain antibody fragments (scFv) 1 for intracellular applications, termed "intrabodies," has been exploited in a number of laboratories (1-8). To date, intrabodies have been utilized for targeting proteins in a singular fashion. Bispecific and tetravalent antibody fragments could improve and expand the inhibitory potential of intrabodies by exhibiting increased apparent affinity for their antigen and by being more efficient at inhibiting protein function or intracellular trafficking (9). Intrabodies present a potent alternative to methods of gene inactivation that target at the level of DNA or mRNA, such as antisense (10), zinc finger proteins (11), targeted gene disruption, or the relatively new RNA interference (12). Operating at the posttranslational level, intrabodies can be directed to relevant...

Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesisin vivo

“…Interference with the VEGF͞VEGF receptor system by means of retroviral expression of dominant-negative VEGF receptor variants (8), VEGF-neutralizing antibodies (9,10), or recombinant VEGF-neutralizing receptor variants (11,12), or interference with the Tie-2 receptor pathway by means of soluble dominant-negative receptors (13,14), intrabodies (15), or oligonucleotide-based strategies including RNA aptamers and RNA interference (16), resulted in reduced tumor growth and tumor vascularization. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent mediators of in vivo angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects on tumor growth (14,17). To date, no studies have reported the effect of simultaneous VEGF-R2͞Tie-2 antagonism in vivo.…”

“…The assembly of intradiabody constructs, the generation of adenoviral plasmids by homologous recombination, and the generation of high titer viral stocks was done essentially as described in ref. 17.…”

“…Cells were analyzed for Tie-2 and VEGF-R2 surface expression 3 days after infection by flow cytometry essentially as described in ref. 17. Formulas used for calculations of surface inhibition, after subtraction of the background of normal-goat IgG stained cells were: % of inhibition VEGF-R2 ϭ (VEGF-R2 T2V2 Ϫ VEGF-R2 intrabody )͞VEGF-R2 T2V2 ϫ 100 or % of inhibition Tie-2 ϭ (Tie-2 T2V2 Ϫ Tie-2 intrabody )͞Tie-2 T2V2 ϫ 100.…”

“…Finally, both constructs were combined by using the SacI sites and confirmed by DNA sequence analysis. The T2-C H 2-C H 3-V2 control construct is described elsewhere (17).…”

“…Bispecific and tetravalent antibody fragments should improve and expand the inhibitory potential of single-chain, monospecific intrabodies by increasing affinity for antigen and more efficiently inhibiting protein function or intracellular trafficking. Recently, we reported the development and in vitro characterization of a human reactive tetravalent endoplasmic reticulum (ER)-targeted intradiabody for the simultaneous surface depletion of two endothelial transmembrane receptors, VEGF-R2 and Tie-2 (17).…”