Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface display technology.

The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.

Engineered high-affinity affibody molecules targeting platelet-derived growth factor receptor ß in vivo.

Platelet-derived growth factor receptor (PDGFR) ß is a marker of stromal pericytes and fibroblasts and represents an interesting target for both diagnosis and therapy of solid tumors. A receptor-specific imaging agent would be a useful tool for further understanding the prognostic role of this receptor in vivo. Affibody molecules constitute a class of very small binding proteins that are highly suited for in vivo imaging applications and that can be selected to specifically recognize a desired target protein. Here we describe the isolation of PDGFRß-specific Affibody molecules with subnanomolar affinity. First-generation Affibody molecules were generated from a large naive library using phage display selection. Subsequently, sequences from binders having a desired selectivity profile and competing with the natural ligand for binding were used in the design of an affinity maturation library, which was created using a single partially randomized oligonucleotide. From this second-generation library, Affibody molecules with a 10-fold improvement in affinity (K(d)=0.4-0.5 nM) for human PDGFRß and a 4-fold improvement in affinity (K(d)=6-7 nM) for murine PDGFRß were isolated and characterized. Complete reversible folding after heating to 90 °C, as demonstrated by circular dichroism analysis, supports tolerance to labeling conditions for molecular imaging. The binders were highly specific, as verified by dot blot showing staining reactivity only with human and murine PDGFRß, but not with human PDGFRα, or a panel of control proteins including 16 abundant human serum proteins. The final binder recognized the native conformation of PDGFRß expressed in murine NIH-3T3 fibroblasts and human AU565 cells, and inhibited ligand-induced receptor phosphorylation in PDGFRß-transfected porcine aortic endothelial cells. The PDGFRß-specific Affibody molecule also accumulated around tumoral blood vessels in a model of spontaneous insulinoma, confirming a potential for in vivo targeting.