Abstract
Many tumour causing proteins, such as those expressed after chromosomal translocations or from point mutations, are intracellular and are not enzymes per se amenable to conventional drug targeting. We previously demonstrated an approach (Antibody-antigen Interaction Dependent Apoptosis (AIDA)) whereby a single anti-β-galactosidase intracellular single chain Fv antibody fragment, fused to inactive procaspase-3, induced auto-activation of caspase-3 after binding to the tetrameric β-galactosidase protein. We now demonstrate that co-expressing an anti-RAS heavy chain single VH domain, that binds to mutant RAS several thousand times more strongly than to wild type RAS, with a complementary light chain VL domain, caused programmed cell death (PCD) in mutant RAS expressing cells when each variable region is fused to procaspase-3. The effect requires binding of both anti-RAS variable region fragments and is RAS-specific, producing a tri-molecular complex that auto-activates the caspase pathway leading to cell death. AIDA can be generally applicable for any target protein inside cells by involving appropriate pairs of antigen-specific intracellular antibodies.
Highlights
While the search for small molecule drugs to intracellular targets continues, a variety of macromolecules have been developed that bind to intracellular target proteins and protein complexes to ablate function
Following optimization of intracellular expression of the domain antibodies and the subsequent development of complementary, antigen-specific VL screening methods, we found anti-RAS single light chains that could couple with a potent anti-RAS VH single domain, that binds to mutant RAS several thousand times better than to wild type RAS6, to form an Fv
We have used separated VH and VL from an anti-RAS single chain Fv (scFv), to show that complementary binding of intracellular VH and VL, each fused to procaspase-3, can kill cells expressing mutant RAS
Summary
While the search for small molecule drugs to intracellular targets continues, a variety of macromolecules have been developed (we collectively refer to macromolecules of this type as macrodrugs[1] to discriminate them from conventional drugs) that bind to intracellular target proteins and protein complexes to ablate function. While intracellular antibodies are very effective inhibitors of protein function and of protein-protein interactions (PPIs) either as single chain Fv (scFv) or single domain formats, they require prolonged expression to sustain an anti-tumour effect, such as shown for anti-RAS intracellular antibody fragments[6,8]. Intacellular protein multimerization will not occur, or will not occur in a way that causes scFv dimers to locate close enough to trigger procaspase auto-activation It was, necessary to evaluate if a trimeric antigen-antibody fragment protein-protein interaction (i.e. VH, VL and antigen) would elicit AIDA. The AIDA method can be adapted to kill cancer cells expressing any specific target antigen when suitable complementary pairs of variable region antibody fragments are selected
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