Structural basis of Alzheimer ß-amyloid peptide recognition by engineered lipocalin proteins with aggregation-blocking activity.

We describe the structural analysis of two Anticalin® proteins that tightly bind Aß40, a peptide involved in the pathophysiology of Alzheimer's disease. These anticalins, US7 and H1GA, were engineered on the basis of the human lipocalin 2, thus yielding compact single-domain binding proteins as an alternative to antibodies. Albeit selected under different conditions and mutually deviating in 13 amino acid positions within the binding pocket (of 17 mutated residues in total), both crystallised anticalins recognize the same epitope in the middle of the ß-amyloid peptide. In the two complexes with the Aß40 peptide, its central part comprising residues LysP16 to LysP28 shows well defined electron density whereas the flanking regions appear structurally disordered. The compact zigzag-bend conformation which is seen in both structures may indicate a role during conversion of the soluble monomeric form into pathogenic Aß state(s) and, thus, explain the aggregation-inhibiting effect of the anticalins. In contrast to solanezumab, which targets the same Aß region in a different conformation, the anticalin H1GA does not show cross-reactivity with sequence-related human plasma proteins. Consequently, anticalins offer promising reagents to prevent oligomerization of Aß peptides to neurotoxic species in vivo and their small size may enable new routes for brain delivery.

High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer ß-amyloid peptide.

Amyloid beta (Aß) peptides, in particular Aß42 and Aß40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target for treatments of Alzheimer's disease (AD). We describe the engineering of cognate Anticalins as a novel type of neutralizing protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 (Lcn2) with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulted in three different Anticalins. Biochemical characterization of the purified proteins produced by periplasmic secretion in Escherichia coli revealed high folding stability in a monomeric state, with Tm values ranging from 53.4°C to 74.5°C, as well as high affinities for Aß40, between 95 pM and 563 pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Aß sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability-with varying extent-to inhibit Aß aggregation in vitro according to the thioflavin-T fluorescence assay and, furthermore, they abolished Aß42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of AD but they also show potential as alternative drug candidates compared with antibodies.

Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.

Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease.

Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools-necessary to understand disease processes and to identify subclinical infection-are much sought after. Here, we describe the production of single-chain antibodies with defined specificity for M. avium subsp. paratuberculosis surface proteins. Single-chain antibodies (scFv) were generated from sheep with Johne's disease by cloning heavy-chain and lambda light-chain variable regions and expressing these in fusion with gene III of filamentous phages. Two scFv clones (designated SurfS1.2 and SurfS2.2) were shown to be immunoreactive against M. avium subsp. paratuberculosis surface targets by flow cytometry, and immunoblotting identified specificity for a 34-kDa proteinase-susceptible determinant. Both antibodies were cross-reactive against Mycobacterium avium subsp. avium but nonreactive against Mycobacterium bovis or Mycobacterium phlei cells and were shown to be capable of enriching M. avium subsp. paratuberculosis cells by a factor of approximately 10(6)-fold when employed in magnetic bead separation of mixed Mycobacterium sp. cultures. Further, magnetic bead separation using SurfS1.2 and SurfS2.2 was capable of isolating as few as 10(3) M. avium subsp. paratuberculosis cells from ovine fecal samples, indicating the diagnostic potential of these reagents. Finally, inclusion of SurfS1.2 or SurfS2.2 in in vitro broth culture with M. avium subsp. paratuberculosis indicated that surface binding activity did not impede bacterial growth, although colony clumping was prevented. These results are discussed in terms of the potential use of single-chain phage display monoclonal antibodies as novel diagnostic reagents.