Using in vivo biopanning for the development of radiation-guided drug delivery systems

This chapter illustrates our protocol for in vivo biopanning using T7 bacteriophage libraries for the purpose of selecting recombinant peptides for the tumor-specific delivery of radiosensitizers to radiation-inducible antigens within tumor neovasculature. Our goal is to discover peptides binding within tumor vascular endothelium of irradiated tumors. We have previously demonstrated that tumor irradiation increases the spectrum of antigenic targets for drug delivery. To identify candidate peptides with the ability to bind radiation-induced antigens, we inject the phage peptide library intravenously into mice bearing irradiated GL261 and Lewis lung carcinoma (LLC) hind limb tumors. Phage are recovered from excised tumors, amplified, and readministered to mouse-bearing tumors for six total rounds. At least 50 bacterial colonies are selected from each of the tumor types, and prioritized. This prioritization is based on their relative concentrations in tumor versus normal tissues, and then assessment of dominant phage present in both tumor types. These phage are amplified, and the gene sequences determined to deduce the recombinant peptide product. Further prioritization is performed by fluorescence labeling of the selected phage, and injection into irradiated and mock-irradiated tumor-bearing mice for evaluation of in vivo targeting of the candidate phage/peptides.