Targeting cancer cells by using an photoimmunosensitizer- 4D5scFv-KillerRed Kamil Tomaszewski Photodynamic therapy (PDT) is a promising approach tocancer treatment orbecause of the absence of systemic toxicityof the drug in the absence of light irradiation, the possibility toirradiate the tumor selectively, the opportunity of treatingmultiplelesionssimultaneously, Results Here isa fully genetically encoded immunophotosensitizerconsisting of a fusion of an anti-p185HER-2-ECD 4D5 singlechainFvfragment (scFv) and KillerRed. scFvantibodyfragmentsare ideal for use as targeting moieties in selective deliveryapplications and offer significant advantages over full-size antibodiesbecause of their smaller size and rational design. The resulting 55-kDa 4D5scFv-KillerRed protein was expressed in Escherichia coli strain SB536 and purified by a combination of IMAC and molecular size exclusion chromatography, with a final purification yield of 0.3–0.5 mg per L of bacterial culture. C N C N scFV scFV 4D5 VL 4D5 VH KillerRed ompAsignal linker hinge his5tag FluorescencespectroscopyshowedthatThe binding activity of 4D5scFv-KillerRed to thep185HER-2-ECD receptor was evaluated byfluorescence microscopy and flowcytometry. Purified4D5scFv-KillerRed effectively accumulated after 1-h incubation at 4 °C onthe surface of the ovarian carcinoma SKOV-3 cell line, which ischaracterized by high expression levels of p185HER-2-ECD.At the same time, no binding activitywas detected for the CHO cells that do not overexpress p185HER-2-ECD. Anti-p185HER-2-ECD binding specificity was further verified by flow cytometry experimentsand ELISA with purified p185HER-2-ECD. Binding of 4D5scFv-KillerRed was decreasedby 4-fold upon addition of parental 4D5scFv antibody at themolar ratio of 1:2 (fusion protein:parental antibody). To evaluate the specific cytotoxicity of 4D5scFv-KillerRed onliving cells we performed an (MTT)-basedcolorimetriccellproliferation assay on the SKOV-3 cell line. Decreasing concentrationsof 4D5scFv-KillerRed were added to the target cells,and proliferation of these cells after a 1-h incubation at 4 °C andirradiation with bright white light for 10 min (1 W/cm2, i.e.,0.2 W/cm2 of KillerRed-activating green light, within theefficient KillerRed absorption range) was determined comparedwithuntreatedcells. Thus, we canalsodiagnosethecenter of the development of cancer cells. Discussion 4D5scFv-KillerRedisa type I photosensitizer. Phototoxicityof 4D5scFv-KillerRed in D2O was even lower compared withwater. These data show that cytotoxicity of 4D5scFv-KillerRedis mediated by ROS other than singlet oxygen. Decreasedphototoxicity inD2O indicates that 4D5scFv-KillerRed photoreactioninvolves proton transfer, which is slowed down indeuteratedwater because of an isotopic effect. Therefore, the phototoxic effect observed could be attributed solelyto the KillerRedphototoxicity, not to the KillerRed and scFvsynergism. Combined treatment with conventional chemotherapeuticagent cisplatin and genetically encoded photosensitizer is likelyto employ different mechanisms of cell death, thus increasing theprobabilityof successful killing of a given tumor cell.