Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: Potential advantages over antibodies expressed within the intracellular environment (Intrabody)

Abstract

Over the past decade, there has been growing interest in the use of antibodies against intracellular targets. This is currently achieved through recombinant expression of the single chain variable fragment (scFv) antibody format within the cell, which is commonly referred to as an intrabody. This possesses a number of inherent advantages over RNA interference (iRNA). Firstly, the high specificity and affinity of intrabodies to target antigens is well-established, whereas iRNA has been frequently shown to exert multiple non-specific effects. Secondly, intrabodies being proteins possess a much longer active half-life compared to iRNA. Thirdly, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA would be slow to yield any effect, whereas the effects of intrabody expression would be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others. There is, however, various technical challenges faced with intrabody expression through the application of recombinant DNA technology. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment. Also, there are overwhelming safety concerns surrounding the application of transfected recombinant DNA in human clinical therapy, which is required to achieve intrabody expression within the cell. Of particular concern are the various viral-based vectors that are commonly-used in genetic manipulation. A novel approach around these problems would be to look at the possibility of fusing protein transduction domains (PTD) to scFv antibodies, to create a 'cell-permeable' antibody or 'Transbody'. PTD are short peptide sequences that enable proteins to translocate across the cell membrane and be internalized within the cytosol, through atypical secretory and internalization pathways. There are a number of distinct advantages that a 'Transbody' would possess over conventional intrabodies expressed within the cell. For a start, 'correct' conformational folding and disulfide bond formation can take place prior to introduction into the target cell. More importantly, the use of cell-permeable antibodies or 'Transbodies' would avoid the overwhelming safety and ethical concerns surrounding the direct application of recombinant DNA technology in human clinical therapy, which is required for intrabody expression within the cell. 'Transbodies' introduced into the cell would possess only a limited active half-life, without resulting in any permanent genetic alteration. This would allay any safety concerns with regards to their application in human clinical therapy.