Characterization of antilytic peptide antibody: application for the detection of lytic-based hybrid peptide in serum...

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)-lytic peptide, made by chemical conjugation of targeted binding peptide and cell-killing, lytic-peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR-lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic-peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic-based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA-MB-231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15-30 min of i.v. administration of EGFR-lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic-based peptides to investigate their in vivo stability and pharmacokinetics.