Characterization of prostate-specific antigen binding peptides selected by phage display technology.

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.

Usefulness of the phage display technology for the identification of a hepatitis C virus NS4A epitope recognized early in the course of the disease.

A dodecapeptide phage-displayed library was screened with the mouse monoclonal antibody (mAb) 2E3C2 which competed with human antibodies for the binding to the HCV c100 recombinant protein. Four mimotopes shared a consensus motif with the HCV 1701-1707 sequence corresponding to the carboxyl-terminal domain of the non-structural protein NS4A. However, these mimotopes reacted with 2E3C2 only, whereas the corresponding NS4 epitope defined at the sequence 1698-1709 and displayed on phage was recognized by both 2E3C2 and sera from HCV infected patients. Using the Spot method of multiple peptide synthesis and alanine replacement analysis, the respective reactivities of mAb 2E3C2 and anti-NS4A human antibodies against NS4 were shown to be directed against two slightly different overlapping minimal linear sequences and to involve different critical residues. The phage clone displaying the NS4 epitope was used to study the specific recognition of this epitope by different individual HCV positive sera as well as by two seroconversion panels of sera from HCV infected patients. Compared with the detection by RIBA of the different HCV antigens and c100 particularly, these results indicated that the antibodies directed against the NS4 (1698-1709) epitope were produced early during the course of the disease and decreased later.