(2002). PRODUCTION AND CHARACTERIZATION OF AN ANTI-IDIOTYPIC SINGLE CHAIN FV THAT RECOGNIZES AN ANTI-DNA ANTIBODY. Immunological Investigations: Vol. 31, No. 3-4, pp. 205-218. doi: 10.1081/IMM-120016241

Targeting and modification of important intracellular proteins using efficient vehicles are invaluable in diagnostic and therapeutic fields. Cell-penetrating antibodies and their fragments can be utilized as vehicles for the delivery of modifiers into cells. In this study, we explored the applicability of variable heavy chain (VH) domain as delivery vehicles for mammalian cells. The characteristics of the recombinant VH domain produced from a cell-penetrating monoclonal anti-double stranded DNA antibody 2C10 were analyzed using flow cytometry, confocal microscopy, cell proliferation assay, and cell cycle analysis in various mammalian cell lines. The VH domain penetrated into various cell lines in a time- and dose-dependent manner, although the internalization efficiency varied. The domain was localized in the nuclei as well as the cytoplasm of living cells. It was also internalized into cells mainly through the clathrin-mediated endocytosis pathway. We tested further its efficiency in delivering specific biomolecule(s) using the conjugates of the single domain molecule and small interfering RNA (siRNA) for the testicular nuclear auto-antigenic sperm protein (tNASP). It was found that the siRNA was successfully delivered by the VH domain into cancer cells, and knockdown effects from the delivered tNASP-siRNA were observed. The levels of the RNA transcript and protein of tNASP were decreased and the down-regulated tNASP inhibited cell proliferation and caused G0G1 phase arrest of the cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as an efficient vehicle capable of delivering valuable biomolecule into the cytoplasm or cell nuclei for clinical uses.

A subset of anti-dsDNA autoantibodies (autoAbs), cell-penetrating Abs, may play a pathogenic role in lupus nephritis. However, the pathogenic role(s) of the Abs has not been well explored. In this study the pathological effects of a positively charged CDR3-VH-containing and cell-penetrating anti-dsDNA monoclonal mouse autoAb 2C10 immunoglobulin G (IgG) and its recombinant VH domain were investigated in a mouse mesangial cell line with respect to activation of signaling molecules and transcription of pro-inflammatory cytokines. The IgG and VH reduced cell viability in cytotoxicity assays and delayed cell cycle progress in flow cytometric analysis. Western blotting experiments showed that they activated p38, MAPKAPK-2, RSK-1, Bcl-2 and ATF-2 in the associated pathway; RSK-1 activation was regulated by p38; p38 also activated MAPKAPK-2 and ATF-2; MAPKAPK-2 regulated RSK-1 activation, and Bcl-2 was up-regulated by RSK-1. The IgG and VH remarkably stimulated the transcription of pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β And the transcription was regulated by p38 activation. These results indicate that the cell-penetrating autoAbs such as 2C10 may play a pathogenic role in mesangial cells mainly through activation of p38 signaling pathway in combination with the stimulation of pro-inflammatory cytokine production.