Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Abstract

Deposition of beta-amyloid (A beta) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of A beta levels by various therapeutic approaches is actively being pursued. A potentially non-inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze A beta at its alpha-secretase site. We have previously identified a light chain fragment, mk18, with alpha-secretase-like catalytic activity, producing the 1-16 and 17-40 amino acid fragments of A beta 40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking alpha-secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for A beta. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the alpha-secretase site of A beta. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3- and 6-fold increase in catalytic activity (k(cat)/K(M)) toward the synthetic A beta substrate compared to the original scFv primarily due to an expected decrease in K(M) rather than an increase in k(cat). This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for A beta. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble A beta levels in vivo.

Inhibiting β-Secretase Activity in Alzheimer's Disease Cell Models with Single-Chain Antibodies Specifically Targeting APP

“…We have shown that a proteolytic scFv with α-secretase-like activity can promote non-amyloidogenic processing of APP and reduce toxicity in 7PA2 cells. 44 A bispecific antibody construct combining iBSEC1, which inhibits BACE activity and amyloidogenic processing of APP, with an α-secretase-like catalytic scFv, which promotes non-amyloidogenic processing of APP substrates, would allow a single therapeutic molecule to process multiple APP substrates through the nonamyloidogenic pathway (Fig. 1).…”

Targeted Hydrolysis of Beta-Amyloid with Engineered Antibody Fragments

“…The specificity constant, (k cat /K M ), toward the A substrate for ASec-1A is 5.6-fold greater and for ASec-1B is 2.8-fold greater than the parent scFv. The increase in activity against A for both scFvs is due to decreases in K M compared to the c23.5 scFv, rather than increases in k cat [67] . The A fragments generated by proteolytic cleavage have a decreased tendency to aggregate and induce cytotoxicity compared to the full length A [57].…”

“…Following bio-panning, isolated single clones were screened for proteolytic activity against a fluorogenic A substrate. Two clones (Asec-1A and Asec-1B) with improved proteolytic activity compared to the parent clone were identified [67]. The cleavage products of A 40 substrate with both scFvs were similar to those of the parent mk18 light chain antibody as determined by mass spectrometry, with A 1-16 as the predominant product (Fig.…”

“…2a) [67]. Since the catalytic residues of c23.5 are located in the V L domain and additional binding specificity is contained in the V H domain, randomizing the heavy chain CDR3 residues should affect antigen binding specificity without affecting catalytic groups [68].…”

Immunotherapy for Alzheimer’s disease: past, present and future

“…We found the addition of the LDLR binding domain of ApoB, in addition to improving BBB transport, facilitated neuronal and glial cell uptake through the LDLR receptor leading to endosomal sorting complex required for transport (ESCRT) mediated endocytosis and autophagosome degradation thus bypassing the Fc receptor signaling cascade that could lead to cytotoxicity (Spencer et al, unpublished). scFV antibodies targeted to Aβ could be used with this approach to target AD (Liu et al, 2004; Fukuchi et al, 2006; Kasturirangan et al, 2009; Kasturirangan and Sierks, 2010). …”