Direct in Vivo Intracellular Selection of Conformation-sensitive Antibody Domains Targeting Alzheimer's Amyloid-β Oligomers

Abstract

The development of conformation-sensitive antibody domains targeting the misfolding beta amyloid (Aβ) peptide is of great interest for research into Alzheimer's disease (AD). We describe the direct selection, by the Intracellular Antibody Capture Technology (IACT), of a panel of anti-Aβ single chain Fv antibody fragments (scFvs), targeting pathologically relevant conformations of Aβ. A LexA-Aβ1-42 fusion protein was expressed in yeast cells, as the “intracellular antigen”. Two different scFv antibody libraries (Single Pot Libraries of Intracellular Antibodies, SPLINT) were used for the intracellular selections: (i) a naïve library, derived from a natural, non-immune, source of mouse antibody variable region (V) genes; and (ii) an immune library constructed from the repertoire of antibody V genes of Aβ-immunized mice. This led to the isolation of 18 different anti-Aβ scFvs, which bind Aβ both in the yeast cell, as well as in vitro, if used as purified recombinant proteins. Surprisingly, all the anti-Aβ scFvs isolated are conformation-sensitive, showing a high degree of specificity towards Aβ oligomers with respect to monomeric Aβ, while also displaying some degree of sequence-specificity, recognizing either the N-terminal or the C-terminal part of Aβ1-42; in particular, the scFvs selected from Aβ-immune SPLINT library show a relevant N-terminal epitope bias. Representative candidates from this panel of the anti-Aβ scFvs were shown to recognize in vivo-produced Aβ “deposits” in histological sections from human AD brains and to display good neutralization properties, significantly inhibiting Aβ oligomer-induced toxicity and synaptic binding of Aβ oligomers in neuronal cultured cells. The properties of these anti-Aβ antibody domains, as well as their direct availability for intra- or extra-cellular “genetic delivery” make them ideally suited for new experimental approaches to study and image the intracellular processing and trafficking of Aβ oligomers.