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.

In vivo selection of intrabodies specifically targeting protein-protein interactions: a general platform for an "undruggable" class of disease targets.

Protein-protein interactions represent a major potential drug target for many human diseases, but these are unanimously considered undruggable with small chemical molecules. We have developed 3-SPLINT, a novel technology for the selection of antibodies that are intrinsically endowed with the ability to interfere with a given protein-protein interaction. The selection procedure exploits the recently described yeast SPLINT libraries of intrabodies, adapting them to a reverse-hybrid system, yielding the selection of recombinant antibodies that are able to disrupt a target protein-protein interaction in vivo. This class of antibodies should therefore perturb an individual protein-protein interaction, without perturbing the scaffolding function of the target protein in that complex, or other protein interactions of that same protein. We provide here a proof of concept of the technology, by the de novo selection of antibodies against two distinct interacting protein pairs: the GABARAP, which interact with the gamma2 subunit of GABA(A) receptor, and the p65 protein dimer, involved in the NF-kappaB-mediated signalling transduction pathway. Intrabodies selected against the latter were functionally validated in cells. Such antibodies, by interfering with the dimerization domain of p65, lead to an activation of the NF-kappaB-mediated transcriptional activity, which is normally inhibited by p65 knock-down RNAi. This provides a clear-cut demonstration that interfering with a protein interaction can be functionally very different from physically removing one of the interacting proteins. The 3-SPLINT approach provides a general and finer tool for the functional validation of selected protein interactions in protein networks, and is ideally applied to protein "hubs", displaying multiple distinct interactions. 3-SPLINT will therefore complement RNAi-based approaches, in the toolkit of target validation strategies, and is amenable to the systematic isolation of comprehensive sets of antibodies against most protein-protein interactions of a given protein network.

Gephyrin selective intrabodies as a new strategy for studying inhibitory receptor clustering.

The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner.

Intracellular antibodies for proteomics.

The intracellular antibody technology has many applications for proteomics studies. The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology. We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins. The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.