The intracellular protein BAK plays a crucial role in the mitochondrial pathway of apoptotic cell death. Recently, an antibody 7D10 that directly binds and triggers BAK activation and mitochondrial pore formation was reported by our group (Iyer et al., 2016), creating the opportunity to directly target the BAK protein to kill cancer cells.
We are using three approaches to obtain proof-of-concept that 7D10 when delivered into cancer cells can induce apoptosis in vivo. For receptor-mediated delivery, 7D10 is being conjugated to an anti-epidermal growth factor receptor (EGFR) affibody - linear polyethylenimine (LPEI) complex. For direct plasma membrane penetration, 7D10 is being conjugated to a cell-penetrating peptide. Our third delivery approach involves the expression of 7D10 as an “intrabody” inside the target cancer cell itself. For this purpose, 7D10 complementarity determining regions (CDRs) have been grafted onto single chain variable fragments (scFv) found to express stably inside the reducing environment (cytosol) of cells. Progress with the three delivery methods will be presented. If effective in BAK activation and subsequent cell death both in vitro and in vivo, 7D10 could be developed as a “cargo” in a range of novel anti-cancer therapeutics.
To also discover improved antibodies for activating BAK, we have used two antibody selection techniques: B cell cloning and nanobody phage library panning. We have modified the two methods to select for antibodies that bind either linear or conformational epitopes, as our initial BAK-activating antibody 7D10 targets a loop region.