Bispecific Antibody Fragment Targeting APP and Inducing α-Site Cleavage Restores Neuronal Health in an Alzheimer’s Mouse Model Ping He 1 & Wei Xin 1 & Philip Schulz 1 & Michael R. Sierks 1 Received: 6 August 2018 /Accepted: 2 April 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract The amyloid β (Aβ) peptide, correlated with development of Alzheimer’ s disease (AD), is produced by sequential proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Alternative proteolytic cleavage of APP by α-secretase prevents formation of Aβ peptide and produces a neuroprotective protein, a soluble fragment of APPα (sAPPα). We previously generated a single-chain variable domain antibody fragment (scFv) that binds APP at the β-secretase cleavage site and blocks cleavage of APP (iBsec1), and a second scFv which has been engineered to have α-secretase-like activity that increases α- secretase cleavage of APP (Asec1a) and showed that a bispecific antibody (Diab) combining both iBsec1 and Asec1a constructs protects mammalian cells from oxidative stress. Here, we show that the diabody is an effective therapeutic agent in a mouse model of AD. An apolipoprotein B (ApoB) binding domain peptide was genetically added to the diabody to facilitate transfer across the blood-brain barrier, and a recombinant human adeno-associated virus 2/8 (rAAV2/8) was used as a vector to express the gene constructs in a APP/PS1 mouse model of AD. The diabody increased levels of sAPPα, decreased Aβ deposits and levels of oligomeric Aβ, increased neuronal health as indicated by MAP2 and synaptophysin staining, increased hippocampal neurogenesis, and most importantly dramatically increased survival rates compared with untreated mice or mice treated only with the β-secretase inhibitor. These results indicate that altering APP processing to inhibit β-site activity while simultaneously promoting α-secretase processing provides substantially increased neuronal benefits compared with inhibition of β-secretase processing alone and represents a promising new therapeutic approach for treating AD. Keywords Single-chain antibody . α-Secretase . β-Secretase . Amyloid precursor protein . Neuron . Alzheimer ’ s disease . Transgenic mice Introduction Amyloid β (Aβ) accumulation has been strongly correlated with AD. Therefore, both inhibiting Aβ production and facili- tating its clearance represent promising therapeutic strategies for treating AD. Aβ peptide is produced by proteolytic cleavage of the amyloid precursor protein (APP) [1]. Three proteases control the processing of APP into Aβ: α-secretase cleaves APP be- tween what would be residues 16(Lys) and 17(Leu) of Aβ, releasing a soluble α fragment of APP (sAPPα) and leaving a non-amyloidogenic membrane-bound protein; alternatively, β- secretase can cleave APP to form the amino terminal of Aβ, releasing a slightly shorter soluble β fragment of APP molecule (sAPPβ) and leaving a potentially amyloidogenic membrane- bound protein; γ-secretase cleaves the membrane-bound frag- ment at the C-terminal of Aβ to release the amyloidogenic Aβ protein. The β-site APP cleaving enzyme-1 (BACE-1) is the predominant enzyme involved in β-secretase processing of APP and is a primary therapeutic target for treatment of AD. Inhibitors of β- or γ-secretase are currently being pursued as potential therapeutics for AD (reviewed in [2–5]). Decreasing Aβ production by inhibiting β- or γ-secretase activity is a promising therapeutic approach though each of these enzymes also has other biological functions than pro- cessing APP. A recent clinical trial to inhibit γ-secretase ac- tivity using semagacestat was halted as it caused a decrease in cognitive ability and an increase in skin cancer risk [6]. Inhibiting BACE-1 activity is also a promising avenue, and numerous small-molecule inhibitors such as OM99-2 have Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12035-019-1597-z) contains supplementary material, which is available to authorized users. * Michael R. Sierks sierks@asu.edu 1 Department of Chemical Engineering, Arizona State University, ECG301- 501 Tyler Mall, Tempe, AZ 85281-6106, USA Molecular Neurobiology https://doi.org/10.1007/s12035-019-1597-z