Selection and characteriazation of Anticalins specific for human glutamate carboxypeptidase II

Jakub Ptáček^1,2, Antonia Richter³, Arne Skerra³, Cyril Bařinka¹

¹Laboratory of Structural Biology, Institute of Biotechnology, AS CR, Prague

²Department of Biochemistry, Faculty of Science, Charles University, Prague

³Lehrstuhl für Biologische Chemie, Technische Universität München, München, Germany

Prostate cancer (PCa) is the most prevalent tumor disease in man worldwide and as such is the subject of an extensive biomedical research. Glutamate carboxypeptidase II (GCPII) is a metallopeptidase overexpressed on the surface of castrate-resistant prostate tumors and is therefore exploited as a biomarker for the PCa imaging and therapy. Currently, only antibody and small molecule-based agents are used clinically for imaging PCa, although with variable degrees of success. As a novel strategy we are developing Anticalin-based binders specifically targeting GCPII.

Anticalins belong to a new generation of artificial binding proteins and a part their sequence can be randomized to obtain binding surface functionally similar to CDR of antibodies. We have used a synthetic combinatorial DNA library together with the phage display methodology to select Anticalin clones specific for the extracellular part of GCPII with affinities between 10 and 50 nM. By the affinity maturation technique where error-prone PCR was exploited to insert random mutations into the best Anticalin variant, we improved the affinity of the lead Anticalin to 1 nM. Furthermore, by the FACS analysis we showed that the best Anticalin clone is able to specifically bind mammalian cells expressing GCPII. These data were confirmed by immunofluorescence confocal microscopy. The best Anticalins will be subjected to next rounds of affinity maturation, crystallography assisted design and aimed at in vivo experiments to validate their biomedical potential.