(490c) Characterization of Peptides Isolated from Bacterial Display Libraries Binding to Tumor Cell Surface Receptors | AIChE

(490c) Characterization of Peptides Isolated from Bacterial Display Libraries Binding to Tumor Cell Surface Receptors



Receptors known to be overexpressed on the surface of human breast cancer cells, such as Her2/neu, have stimulated the development of successful, targeted therapies like the antibody HerceptinTM. However, application is limited to only one-fourth of patients who express the receptor. The isolation and characterization of multiple, novel overexpressed and specific breast cancer receptors would greatly improve applicability for patients and allow for combination therapies. To discover novel receptors, a methodology was developed to isolate peptide sequences that bind with high specificity to several breast carcinoma cell types through alternating positive selections on tumor cells and negative selections on normal human breast epithelial cells. E. coli expressing both an intracellular fluorescent protein and an outer member protein capable of displaying peptides on the cell surface were used to screen peptide libraries. Selections were performed by co-sedimentation of binding bacteria with tumor cells followed by quantitative fluorescence activated cell sorting, which discriminated unlabeled tumor cells from those with bound fluorescent bacteria. Following peptide characterization and cross-reactivity analysis using whole cell binding assays, a FRET-based screening method was used to interrogate a library of tumor cell cDNA clones containing putative peptide receptors. Interacting partners were screened by genetically tethering a specific binding peptide to the C-terminus of CyPet, a cyan fluorescent protein optimized for FRET. Similarly, the tumor cell cDNA library was tethered to YPet, the yellow fluorescent protein FRET partner. Receptor-peptide interactions isolated with this FRET-based screen provide novel insights for the profiling of biomarkers on tumor cell surfaces.