Phage peptide display is a powerful technique for discovery of various target-specific ligands. However, target-unrelated peptides can often be obtained and cause ambiguous results. Peptide PB-TUP has been isolated repeatedly in our laboratory on different targets and we conducted a research on PB-TUP phage to investigate their binding properties and rate of propagation. ELISA and phage recovery assay demonstrated that PB-TUP phage had a significant superior affinity to polystyrene solid surface compared with control phage clones. In this study, some incidental bindings are excluded like blocking agents and non-specific binding of secondary antibodies. Propagation rate assays of the selected phage clones showed that the growth rate of PB-TUP phage was not superior to the control phages. Furthermore, the binding of PB-TUB to polystyrene was concentration dependent and varied with solution pH. Molecular modeling revealed that stable structures of α-helix and β-turn may contribute to the binding of PB-TUP to polystyrene plate. The PB-TUP sequence was fused to the N-terminus of peptide P2 and the fusion peptide significantly increased the binding affinity to polystyrene. The fusion peptide also enhanced the cell adhesion ability of peptide P2 with human umbilical vein endothelial cell (HUVEC). The addition of the polystyrene binding peptide provided a convenient method for peptide immobilization.

The expression level of folate receptor alpha (FRα) is located highly rate in ovarian cancer though it is remained absent in normal tissues. This highly tumor restricted expression profile makes FRα a promising target for tumor therapy and diagnosis. In this research we report a FRα binding peptide C7(Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser) discovered by phage display and this peptide showed specific binding to FRα expressing cells by cell ELISA and flow cytometry. Tumor targeting ability of C7 was proved in vivo by both phage homing experiment and fluorescence imaging. C7 can be internalized by SKOV3 cells and its affinity to FRα was determined by MST. The molecular recognition was revealed by structure modeling, suggesting its binding mode with FRα.

Folate receptor α (FRα) is overexpressed in a variety of solid tumors and has become an attractive target antigen for immunotherapy purposes. A DNA vaccine was generated by ligation of FRα cDNA into the eukaryotic vector pcDNA3.1. Expression of FRα was confirmed in transiently transfected B16 cells. B16 cell lines that stably express FRα were set up by G418 selection. A total of 100 µg purified plasmid DNA alone or in combination with CpG oligodeoxynucleotides (CpG ODN) was injected intramuscularly in C57BL/6 mice four times at one week intervals. ELISA analysis confirmed that high titers of antibodies against FRα existed in the sera of the experimental animals. Specific cytotoxic T lymphocyte activity against FRα-expressing B16 cells was found and FRα specific lymphocyte proliferation was detected. Coinjection of CpG ODN increased both humoral and cellular immune responses. In the protective model, in which C57BL/6 mice were immunized with the FRα DNA vaccine four weeks before tumor cell inoculation, the growth of tumor was significantly inhibited, and the presence of CpG ODN further increased the inhibitory effect. FRα DNA vaccine alone did not show a significant inhibitory effect in the therapeutic model, in which the DNA vaccine was immediately injected after tumor inoculation. However, FRα DNA vaccine plus CpG ODN showed a significant inhibitory effect in tumor growth. Survival curves for both animal experiments confirmed that mice immunized with pcDNA3.1/FRα plus CpG ODN had a significantly prolonged survival period than that of the pcDNA3.1 control group, the CpG ODN group or the pcDNA3.1/FRα group. The above showed that human FRα based DNA vaccination with CpG ODN as an adjuvant was effective in growth inhibition of a FRα expressing tumor in mice and deserves further evaluation as a possible immunotherapy.