Abstract 1769: Phage display selection of cancer cell-binding and -internalizing ligands using random peptide libraries constructed at N-terminus of P99 cephalosporinase

Abstract

Abstract The cancer researchers today are focused in developing new types of cancer treatments that use drugs or other substances to identify and kill cancer cells while doing very little or no damage to normal healthy cells. It is believed that an ability to home in on cancer cells makes chemotherapy both more effective and less likely to cause side effects. One of the strategies to achieve this goal depends on developing ligands that specifically bind/internalize to cancer cells and exert therapeutic effects of their own or guide a targeted delivery of a therapeutic agent (cytotoxic drug, gene, radionuclide, or enzyme) to the tumor cells. Phage display is one example of high throughput technologies that has been successfully utilized for the generation of massive ligand libraries and their selection against specific targets. We constructed novel phage-displayed random linear dodecapeptide (X12) and cysteine-constrained random decapeptide (CX10C) libraries at N-terminal end of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase: β-lactam hydrolase, E.C. # 3.5.2.6). The peptide-β-lactamase fusions were displayed at the amino terminus of the minor coat pIII protein of M13 filamentous phage. Several cancer cell-specific binding β-lactamase-peptide fusion ligands were isolated by selecting these libraries on live BT-474 human breast cancer cells. The identified ligands shared several significant motifs, which showed their selectivity and possible binding to some common cancer cell targets. β-Lactamase is a high turnover enzyme with several available chromogenic and fluorogenic substrates. The peptide fusion to this enzyme made the post-panning steps very fast and simple. The concentrations of β-lactamase-peptide fusions in phage samples, lysates and purified fusion proteins were normalized based on the β-lactamase activity. The clone screening was also based on assaying the residual cell-bound β-lactamase activity. Some of the cancer cell binding β-lactamase-peptide fusion proteins showed cell internalization by both immunofluorescence and β-lactamase activity-based procedures. The staining with FRET-based substrate CCF4 confirmed the functional integrity of the internalized β-lactamase, and demonstrated that at least a part of the internalized enzyme conjugate could escape the endosomal degradation pathway in intact form and reacted with the substrate present in the cytoplasm. The specificity of the selected ligands was assessed by studying their binding and internalization in 11 other cancer and non-cancer cell lines. The β-lactamase fusion to peptides not only accelerates the clone screening process but can also facilitate the tracking of ligand molecules in various biological studies. The cancer-specific affinity reagents selected from these libraries have a potential for their use in targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1769. doi:10.1158/1538-7445.AM2011-1769