Abstract
The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy.
Highlights
Prostate cancer (PCa) is the second leading cause of death in men in the United States and is the most diagnosed cancer in men
Recoveries of phage during panning A 15 amino acid fUSE5 phage display library was used as input phage to screen against poly-histidine tagged prostate-specific membrane antigen (PSMA) (PSMA-His6) immobilized in 6-well plates
After a depletion of the library of phage that bound non- to the selection components consisting of BSA and anti-His6-Mab, the phage display library of 15-mers, was panned against PSMA-His6 that was captured by immobilized anti-His6-Mab
Summary
Prostate cancer (PCa) is the second leading cause of death in men in the United States and is the most diagnosed cancer in men. PSA levels are not an accurate predictor of the aggressiveness of prostate cancer, with the PSA rate of change (PSA velocity) not able to discriminate between men with cancer and those with negative biopsies [3,4,5]. There are high frequencies of prostate-specific membrane antigen (PSMA) overexpression in all stages and grades of PCa patients. The overexpression of PSMA was significantly associated with tumor stage, where the higher Gleason graded tumors corresponded with higher expression levels of PSMA [6,7]. PSMA is an alternative to PSA as a diagnostic biomarker for detection of PCa [6]
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