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

Srinath Kasturirangan, Dan Brune, Michael Sierks

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Deposition of beta-amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non-inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α-secretase site. We have previously identified a light chain fragment, mk18, with α-secretase-like catalytic activity, producing the 1-16 and 17-40 amino acid fragments of Aβ40 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 α-secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. 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 α-secretase site of Aβ. 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 Aβ 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 Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Ab levels in vivo.

Original languageEnglish (US)
Pages (from-to)1054-1063
Number of pages10
JournalBiotechnology Progress
Volume25
Issue number4
DOIs
StatePublished - Jul 1 2009

Keywords

  • Affinity maturation
  • Alpha-secretase
  • Beta-amyloid
  • Proteolytic antibody
  • Single chain antibody

ASJC Scopus subject areas

  • Biotechnology

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