Selection and characterization of Anticalin-based binders targeting human glutamate carboxypeptidase II

Jakub Ptacek, Antonia Richter, Arne Skerra, Cyril Barinka

Laboratory of Structural Biology, Institute of Biotechnology AS CR, v. v. i.,
Videnska 1083, 142 20 Prague 4
Czech Republic

Anticalins are members of growing family of designed non-immunoglobulin proteins with binding properties similar to antibodies. Glutamate carboxypeptidase II (GCPII) is a membrane bound metallopeptidase highly upregulated on the surface of prostatic carcinomas and the neovasculature of solid tumors and it thus represents an excellent diagnostic target for detection of most solid cancers. Here we report our initial experiments aimed at the selection and characterization of Anticalin-based binders targeting GCPII. A randomized library of approximately 10¹⁰ different combinatorial variants derived from human lipocalin Lcn2 was panned against FLAG-tagged human GCPII and variants tested for GCPII specificity and affinity following the four rounds of selection. Best binders exhibit nanomolar affinity for GCPII in the ELISA setup and can be easily expressed and purified in milligram quantities from the prokaryotic expression system. These candidates will be further subjected to directed evolution to optimize their biophysical properties.