Selection and characteriazation of Anticalins specific for human glutamate carboxypeptidase II
Jakub Ptáček1,2,
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.