Abstract
ABSTRACTApplication of antibodies in most therapeutic area is limited to extracellular or membranous targets because of their impermeability of membrane. For the purpose of biotechnological and therapeutic application, developing intracellular localizing antibody is the invaluable research field. A new recombinant single-chain variable fragment of an anti-dsDNA monoclonal antibody G2-6, IgG of which has been previously shown to have a cell-penetrating activity, was engineered and produced for the use as a delivery vehicle of biomolecule(s). The penetrating capacity of single-chain variable fragment in three mammalian cell lines, L929, NIH/3T3, and COS-7 was analyzed using flow cytometry and confocal microscopy. The results demonstrated that the single-chain variable fragment can effectively internalize all three cell lines, although the internalization level varied. It was also shown that the internalization was time- and concentration-dependent. Moreover, the single-chain variable fragment was located in nuclei as well as cytoplasm of L929 cells. Overall, the G2-6 single-chain variable fragment might be a candidate vehicle which could be used to deliver specific genes or biomolecules for therapy or diagnosis into the cytoplasm or cell nucleus.
Highlights
Therapeutic reagents have been explored to target cells for modifying diseases
We have reported that four anti-double stranded DNA (dsDNA) monoclonal Ab immunoglobulin G (IgG), including G2-6 IgG produced from MRL-lpr/lpr mice, could internalize viable mouse kidney mesangial (MES) cells with a minimal cytotoxicity (Im et al 2015)
G2-6 scFv retains the affinity of G2-6 IgG to dsDNA
Summary
Therapeutic reagents have been explored to target cells for modifying diseases. Use of antibodies (Abs) is a hopeful field in the therapy of diseases like cancer as well as biotechnology (Scott et al 2012). Expanding the use of therapeutic Abs to intracellular targets or antigen molecules can be an invaluable tool for modulating diseases through Ab-target interactions. The internalizing Abs can be engineered to small fragments such as variable region fragment (Fv) or single domain (Holliger and Hudson 2005) for binding to intracellular targets with high specificity, resulting in inhibiting the function of target molecules
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