Studies on the efficient intracellular delivery vehicle using variable region domain of a cell-penetrating anti-DNA monoclonal antibody

Abstract

The cell-penetrating antibodies and antibody fragments can be utilized as a tool for the medical diagnosis and treatment. In this study, we have produced the protein of recombinant VH domain of a monoclonal anti-DNA antibody 2C10, IgG of which has been previously shown to have cell-penetrating activities for studying the applicability of the domain as a cellular delivery vehicle. To evaluate the cell-penetrating property of 2C10 VH domain in various mammalian cell lines, the flow cytometric and confocal microscopic analysis and cell proliferation assay were used. The VH domain penetrated into all cell lines we tested in time- and dose-dependent manner, although the internalization efficiency was varied. We also found that the VH domain was localized in nuclei as well as cytoplasm of living cells and it was mainly internalized by the clathrin-mediated endocytosis pathway. The VH domain did not change the cell cycle and significantly affect the cell proliferation in various mammalian cell lines at the concentration that we used for functional assays. We tested further the delivering activity of a valuable molecule by the VH domain using the conjugates of VH and siRNA for testicular Nuclear Auto-antigenic Sperm Protein (tNASP). It was found that the siRNA was successfully transferred by 2C10 VH domain into Caki and HeLa cancer cell lines, and the knockdown effects of tNASP-siRNA were observed in Caki cells, in where the level of tNASP expression is known to be high; the levels of RNA transcripts and proteins of tNASP were decreased by the delivered tNASP-siRNA; the down-regulated tNASP effectively inhibited cell proliferation and caused G1 phase arrest of cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as a valuable vehicle which can deliver specific biomolecule(s) into the cytoplasm or cell nuclei for therapy and diagnosis of diseases.