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 15mer (X15) or a constrained 7mer (X2CX7CX2). To anchor peptides to the surface of E. coli, an outer membrane protein was circularly permuted to allow for peptide fusions to an extracellular terminus. The bacteria also expressed 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 tumor cells and negative selections on normal human breast epithelial cells enriched bacterial clones binding specifically to tumor cells as monitored by FACS. Additionally, specific fluorescent bacteria clones effectively stained tissue sections of human xenograft breast tumors in mice models verifying epitope expression. The selected fluorescent bacteria resulting from these screens provide multivalent affinity reagents that are self-renewing and self-amplifying allowing for single step labeling of targets for cytometric, microscopic, and array analysis.

Extended Abstract Status: Not Uploaded