Abstract 720: Phage display selection of ligands against extracellular domain of ErbB2 receptor using novel β-lactamase-random peptide fusion libraries
Authors: Shukla Girja S. , Krag David N. 
Published: February 2011

Abstract
There are now unprecedented opportunities for increased throughput to develop novel molecules that target cancer. Phage display is one example of a high throughput method of generating massive libraries of molecules that have different structural properties. We constructed novel phage-display random peptide libraries in fusion to β-lactamase (β-lactam hydrolase, E.C. # 3.5.2.6) enzyme as a reporter. The linear dodecapeptide (X12) and cysteine-constrained decapeptide (CX10C) libraries were created at the N-terminal end of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase), between the signal peptide and the enzyme protein. Peptide-β-lactamase fusions were displayed at the amino terminus of the minor coat pIII protein of filamentous (M13) phage. The overall representations of different amino acids in the random regions of both libraries were very good. The amino acid diversity at each position in the random regions of both the libraries was also good. The libraries were selected against the extracellular domain of ErbB2 receptor (ErbB2-ECD). The target-selected clones were already conjugated to an enzyme reporter, therefore, did not require subcloning or any other post-panning modifications. β-Lactamase is known for high turnover of its enzyme activity with several commercially available chromogenic and fluorogenic substrates. This helped us to develop a very sensitive and efficient assay for measuring concentrations and target-binding of β-lactamase-peptide fusion proteins in their both phage-displayed and soluble forms. The β-lactamase enzyme assay protocol is a one-step process and unlike a typical phage-ELISA it does not require secondary proteins, several steps of lengthy incubations, or washings and can be finished in a few minutes instead of hours. The clone screening protocol can be adopted for a high throughput platform. The identities of peptide-β-lactase fusion clones that bound to purified ErbB2-ECD and ErbB2-overexpressing MCF-7/ErbB2 and SK-BR-3 cell lines were determined by DNA sequencing of the random inserts. The peptide sequences of the selected binding clones were found to share significant motifs with several rationally designed peptide mimetics and phage-display derived peptides that have been reported to bind ErbB2-ECD. Target-specific β-lactamase-linked affinity reagents selected by this procedure can be produced in bulk and purified. The synthesis and successful use of cephalosporin prodrugs of mechanistically diverse anticancer agents, such as doxorubicin, taxol, platinum complexes, phenylenediamine mustard, and vinblastine have been reported in experimental studies. Using an appropriate β-lactam-substrate, the cancer cell-specific ligands selected from these libraries can be used directly for the targeted enzyme prodrug therapy and/or in vivo imaging of cancers.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 720.