Direct in vivo intracellular selection of conformation-sensitive antibody domains targeting Alzheimer's amyloid-beta oligomers.
The development of conformation-sensitive antibody domains targeting the misfolding beta amyloid (Abeta) 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-Abeta single chain Fv antibody fragments (scFvs), targeting pathologically relevant conformations of Abeta. A LexA-Abeta1-42 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 Abeta-immunized mice. This led to the isolation of 18 different anti-Abeta scFvs, which bind Abeta both in the yeast cell, as well as in vitro, if used as purified recombinant proteins. Surprisingly, all the anti-Abeta scFvs isolated are conformation-sensitive, showing a high degree of specificity towards Abeta oligomers with respect to monomeric Abeta, while also displaying some degree of sequence-specificity, recognizing either the N-terminal or the C-terminal part of Abeta1-42; in particular, the scFvs selected from Abeta-immune SPLINT library show a relevant N-terminal epitope bias. Representative candidates from this panel of the anti-Abeta scFvs were shown to recognize in vivo-produced Abeta "deposits" in histological sections from human AD brains and to display good neutralization properties, significantly inhibiting Abeta oligomer-induced toxicity and synaptic binding of Abeta oligomers in neuronal cultured cells. The properties of these anti-Abeta 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 Abeta oligomers.
Meli
Giovanni
G
European Brain Research Institute, Rome, Italy.
Visintin
Michela
M
Cannistraci
Isabella
I
Cattaneo
Antonino
A
D000544:Alzheimer Disease / Q000276:immunology* / Q000473:pathology; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D013569:Synapses / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D016229:Amyloid beta-Peptides; D000906:Antibodies; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins