Misfolding and aggregation of amyloid-β (Aβ) is an important early event in the pathogenesis of Alzheimer's disease. Aβ is produced by sequential proteolysis of the amyloid-β protein precursor (AβPP) by β- and γ-secretases. A third protease, α-secretase, cleaves AβPP in the middle of the Aβ sequence precluding formation of Aβ. The levels of Aβ generated from AβPP can therefore be controlled by tailoring activity of these proteases toward AβPP. We previously showed that β-secretase proteolysis of AβPP could be selectively inhibited using the single chain antibody fragment (scFv) iBSEC1, which blocks the cleavage site on AβPP, and α-secretase proteolysis of AβPP could be selectively enhanced using a proteolytic scFv (Asec1A) engineered to have α-secretase-like activity. Here we show that DIA10D, a novel tandem bispecific scFv combining iBSEC1 with the ASec1A can control amyloidogenic processing of AβPP by simultaneously inhibiting β-secretase and increasing α-secretase processing of AβPP. When expressed in H4 (neuroglioma) cells overexpressing AβPP, DIA10D potently reduces levels of extracellular Aβ by around 50% while also increasing levels of neuroprotective sAβPPα. DIA10D activity has been designed to selectively target AβPP, so this modulation of AβPP processing should not affect endogenous activity of α-and β-secretases towards other substrates.
