Isolation of specific tumor targeting peptides from fluorescent bacterial display libraries

Methods for the discovery of cell-specific ligands, including unique tumor targeting epitopes, would benefit from a more rapid and cost effective isolation procedure. To address this need, a novel bacterial surface display methodology was developed to discover peptide sequences that bind with high specificity to human breast carcinoma cells. Libraries were constructed by creating a collection of bacteria that each independently express a unique peptide, either a fully random 15-mer (X15) or a constrained 7-mer (X2CX7CX2). To anchor peptides to the surface of E. coli, native outer membrane proteins were engineered for use as display scaffolds. One scaffold allowed peptides to be displayed as sandwich fusions, while another was circularly permuted to allow for peptide fusions to an extracellular terminus as a means to potentially reduce peptide-scaffold dependence. The bacteria were also engineered to express an intracellular fluorescent protein allowing for facile screening of peptide libraries against tumor cells through the use of quantitative fluorescence activated cell sorting (FACS). Alternating positive selections on breast cancer cells and negative selections on normal human breast epithelial cells enriched bacterial clones binding specifically to tumor cells as monitored by FACS. These specific bacteria were used to profile several breast tumor types, providing a receptor fingerprint and differentiating basal and luminal subtypes. Furthermore, several peptide candidates were selected as targeting ligands for binding assays as soluble peptides tethered to microparticles, and one retained binding specificity in this multivalent context. In addition to isolating binding peptides, strategies were developed to allow for the selection of peptides that mediate invasion into tumor cells or induce apoptosis. The methodologies developed herein for isolating highly specific breast tumor cell binding ligands may provide an efficient route for tumor subtype classification, biomarker identification, and aid in the development of targeted diagnostics and therapeutics.