Anticalins Reveal High Plasticity in the Mode of Complex Formation with a Common Tumor Antigen

We describe the comparative X-ray structural analysis of three Anticalin proteins directed against the extra-domain B (ED-B) of oncofetal fibronectin (Fn), a validated marker of tumor neoangiogenesis. The Anticalins were engineered from the human lipocalin 2 (Lcn2) scaffold via targeted randomization of the structurally variable loop region and selection by phage display, resulting in 15–19 exchanged residues. While the four reshaped loops exhibit diverse conformations (with shifts in Cα positions up to 20.4 Å), the β-barrel core of the lipocalin remains strongly conserved, thus confirming the extraordinary robustness of this scaffold. All three Anticalins bind the cc' hairpin loop of ED-B, the most exposed motif in the context of its neighboring Fn domains, but reveal entirely different binding modes, with orientations differing by up to 180°. Hence, each Anticalin recognizes its molecular target in an individual manner, in line with the distinct epitope specificities previously seen in binding experiments. Anticalin proteins are engineered lipocalin-like molecules that, like antibodies, show specific binding ability for different molecular targets. Schiefner et al. analyze three distinct Anticalin complexes, bound to an oncogenic domain of fibronectin. These structures confirm the high paratope plasticity and scaffold robustness of engineered lipocalins.