Isolation, characterization, and recovery of small peptide phage display epitopes selected against viable malignant glioma cells

Phage display techniques rely on nearly random oligonucleotide sequences in serted into the protein III filament binding protein of an Escherichia coli filamentous phage M13 to generate a library of phage that express more tha n 10(7) different peptides. Phage that expresses a sequence having high aff inity for a specific molecule, cell, or tissue can then be isolated through selective binding and recovery. Selected phage cannot only be used as gene transfer vectors in themselves, but the small peptide epitopes can be sequ enced and potentially recombined into the attachment proteins of viral vect ors, or used by themselves to target other therapeutic agents and diagnosti c imaging radiolabels. Most phage display selections are carried out agains t purified and/or fixed protein targets, raising concerns as to the relevan ce of the selected epitopes. We have selected phage from the CMTI library a gainst viable U87-MG human malignant glioma cells using a derivation of bio panning. The library, which initially contained phage expressing 2x10(7) di fferent epitope sequences, collapsed after four rounds of selection such th at 42% of recovered clones expressed a consensus sequence. Selective bindin g to viable adherent U87-MG cells was subsequently demonstrated under physi ologic conditions at 167% (+/- 27%) unselected phage using a novel, viable enzyme-linked immunosorbent assay technique. In comparison, there was no di fference in binding to control 9L rat gliosarcoma, PANC-1 human pancreatic adenocarcinoma, T98-MG human malignant glioma, or AST-4 human malignant gli oma cells of selected compared to unselected phage. Using polymerase chain reaction, the epitope was recovered with flanking unique restriction sites for recombination into a herpes simplex virus type-1 vector. This study dem onstrates and discusses optimized methodologies for using phage display to target viable cells.