Modern diagnostic and imaging approaches aimed at various tumors require protein target allowing a drug to distinguish between carcinoma and healthy cells. In the case of prostate cancer (PCa), which is by far the most prevalent tumor in men, prostate‑specific membrane antigen (PSMA) serves as such a target. Beside the PCa it is also expressed on the neovasculature of most solid tumors. Anticalins are a prominent members of a rapidly growing family of non-immunoglobulin protein scaffolds. Exploiting combinatorial libraries the Anticalins can be engineered to specifically bind virtually any target.
In presented work we have used DNA library based on human lipocalin gene sequence to select Anticalins specific for GCPII. Phage display together with ELISA screening were used to select first generation of GCPII binding Anticalins. The most promising variants were further subjected to affinity maturation, where new DNA library was obtained by random mutagenesis of selected clones and another selection procedure via phage and bacterial surface display was applied then. The binding properties were analysed by ELISA and SPR measurements which revealed improvement from nanomolar (about 10 nM dissociation constant) in the first generation clones) to subnanomolar (KD ~ 500 pM) affinities for GCPII in the third generation clones. Subsequent immunofluorescence microscopy using cell lines expressing PSMA on the surface showed specific binding of the best clones to PSMA positive cells. This observation was then confirmed by flow cytometry on live cells. Thus we have proved the usability of selected Anticalin clones for selective labelling cells expressing PSMA on the surface and their potential for in vivo bioimaging applications.