Journal of Neuroimmunology 106 (2000) 23–31 www.elsevier.com / locate / jneuroin Modulation of Alzheimer’s b-amyloid neurotoxicity by site-directed single- chain antibody * Dan Frenkel, Beka Solomon , Itai Benhar Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel- Aviv University, Ramat Aviv 69978, Tel- Aviv, Israel Received 7 July 1999; received in revised form 13 October 1999; accepted 14 October 1999 Abstract A single-chain antibody was constructed from variable regions of heavy and light genes of the parental anti-b-amyloid peptide IgM 508 antibody. This antibody exhibits anti-aggregating properties, leading to disaggregation of Alzheimer b-amyloid (bA) fibrils and prevents its toxic effect on cultured PC-12 cells. Sequencing of the small antibody, namely 508 (Fv), revealed that the V domain contained a L cysteine residue in the complementary determining region (CDR)3 (residue 96) which affects its solubility and stability. The cysteine codon was replaced using SOE PCR, and one of the mutants obtained, namely 508F(Fv) (containing phenylalanine instead of cysteine), showed an increased storage stability and higher affinity compared to the wild type. Antibody 508F(Fv) prevents the neurotoxicity of bA (90% cell viability) and disrupts the fibril structure of b-amyloid (62% decrease in ThT fluorescence). The ability of antibody 508F(Fv) to dissolve already-formed bA fibrils makes it a good candidate for intracellular expression and modulation of APP processing as the first step towards the production of therapeutic protection molecules for Alzheimer’s disease treatment.  2000 Published by Elsevier Science B.V. All rights reserved. Keywords: b-Amyloid modulation; Neurotoxicity; Engineered antibodies 1. Introduction toxicity has been linked to the aggregation and conforma- tional status of bAP (Lorenzo and Yankner, 1994). The One of the major pathological features of Alzheimer’s dependence of bAP polymerization on peptide–peptide disease (AD) is the abundant presence of amyloid plaques interactions to form a b-pleated sheet fibril and the in the brain tissues of affected individuals (Vines, 1993). stimulatory influence of other proteins on the reaction The plaques are predominantly comprised of a 40–42- suggest that amyloid formation may be subject to modula- residue-long b-amyloid peptide ( bAP) whose neurotoxici- tion (Ma et al., 1996; Cruz et al., 1997). While several ty is related to the aggregation process (Cruz et al., 1997). bAP domains may be associated with fibril formation, The source of b-amyloid peptide is a family of amyloid researchers found that only the N-terminal region of bAP precursor proteins (APP) that arises by alternative splicing remains outside the fibril core (Talafous et al., 1994; Lee of a primary APP gene transcript (Ashall and Goate, 1994; et al., 1995). The involvement of the N-terminal region in Golde, 1994). Once released by proteolytic cleavage of the conformational transition of bAP was previously APP the b-peptide may remain in solution either as a confirmed in studies using synthetic peptides bearing the random coil or as an a-helical structure (Barrow et al., bAP sequence under various experimental conditions 1992; Talafous et al., 1994). The transition of the a-helix (Lorenzo and Yankner, 1994; Lee et al., 1995; Soto et al., to b-sheet conformation with concomitant peptide aggrega- 1995). Recent in vitro studies showed that specific mono- tion is one of the proposed mechanisms of fibril formation clonal antibodies (mAbs) raised against the N-terminal in Alzheimer’s disease (Kirshenbaum and Daggett, 1995; region of bAP can disaggregate bA fibrils, maintain bAP Soto et al., 1995). The presence of fibrils of bAP is toxic solubility and prevent the neurotoxic effects on PC 12 cells to cultured neuronal cells, leading to their death. The (Solomon et al., 1996, 1997; Frenkel et al., 1998, 1999). Since the pathological effects of bA fibrils in AD patients are confined only to the central nervous system (CNS), it *Corresponding author. Tel.: 1972-3-640-9711; fax: 1972-3-640- is necessary to overcome the low permeability of the blood 9407. E-mail address: beka@ccsg.tau.ac.il (B. Solomon) brain barrier (BBB) for targeting ‘anti-aggregating’ mAbs 0165-5728 / 00 / $ – see front matter  2000 Published by Elsevier Science B.V. All rights reserved. PII: S0165-5728(99)00232-5