Many pharmaceutical therapies are based on the use of small molecules to target intracellular sites because cells are impervious to large molecules such as proteins. However, small molecule inhibitors are prone to have undesirable side effects as a result of binding unintended targets. By contrast, antibodies have excellent binding specificity, but most do not penetrate living cells. Thus, the current use of therapeutic antibodies is limited to targeting molecules that are secreted or located on the cell membrane.
Intracellular antibodies can be generated by gene therapy, but the potential dangers have not justified its use. Cell-penetrating peptides (CPPs) also referred to as protein transduction domains (PTDs) are currently used to transport proteins into cells but an important limitation of these intracellular transporters is that they may be targeted to endosomes through lipid rafts. In addition, some are highly cationic peptides that have been shown to be toxic to normal cells.
Previously researchers identified a unique monoclonal anti-DNA antibody, mAb 3E10 which penetrates living cells and localizes in the nucleus without apparent harm. In contrast to CCPs, mAb 3E10 and its single-chain Fv fragment (scFv) are internalized through hENT2, an equilibrative nucleoside salvage pathway. hENT2 is expressed in most cells, but its expression is increased in muscle and cancer cells.
On the basis of these findings, Department of Veterans Affairs scientists developed bispecific antibodies having Fv fragments with a cell-penetrating determinant (3E10 Fv) and a second Fv fragment with an intracellular target-binding determinant (3G5 Fv). The intracellular target-binding determinant can target an oncoprotein such as myc or ras, DNA repair proteins such as RAD52, ATM, CHK2, CHK1 and BCL2 as well as BRCA1, MDC1, 53BP1, p53, and p21. The 3E10 bispecific antibodies may be joined or attached to localizing signals so as to direct the scFvs to intracellular compartments such as endoplasmic reticulum and mitochondria.
The technology demonstrates the feasibility of transporting antibodies into cells for therapeutic regulation of intracellular targets and the possibility for enhanced or synergistic inhibition of the growth of tumor cells when multiple components of a regulatory pathway are targeted with more than one therapeutic agent. It further provides novel reagents for treatment of tumors, cancers, diseases, and disregulated processes along with a rationale for their combined use in targeting a regulatory pathway disregulated in tumor cells, or alternatively, components of any number of pathways that might be disregulated within tumors, cancers, diseases or conditions.
- Antigen-binding specificity of antibodies delivered into intracellular compartments may result in improved therapeutic indices by avoiding off-target binding responsible for toxic side effects of small-molecule inhibitor
- Businesses can turn the research into a treatment product by licensing US patent 9,283,272 from the VA
- License fees paid to the VA are negotiable
- Businesses that acquire a patent license could potentially collaborate with the inventors
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