Use of photoimmunoconjugates to characterize ABCB1 in cancer cells.

Accurate detection of ATP-binding cassette drug transporter ABCB1 expression is imperative for precise identification of drug-resistant tumors. Existing detection methods fail to provide the necessary molecular details regarding the functional state of the transporter. Photo-immunoconjugates are a unique class of antibody-dye conjugates for molecular diagnosis and therapeutic treatment. However, conjugating hydrophobic photosensitizers to hydrophilic antibodies is quite challenging. Here, we devise a photoimmunoconjugate that combines a clinically approved benzoporphyrin derivative (BPD) photosensitizer and the conformational-sensitive UIC2 monoclonal antibody to target functionally active human ABCB1 (i.e., ABCB1 in the inward-open conformation). We show that PEGylation of UIC2 enhances the BPD conjugation efficiency and reduces the amount of non-covalently conjugated BPD molecules by 17%. Size exclusion chromatography effectively separates the different molecular weight species found in the UIC2-BPD sample. The binding of UIC2-BPD to ABCB1 was demonstrated in lipidic nanodiscs and ABCB1-overexpressing triple negative breast cancer (TNBC) cells. UIC2-BPD was found to retain the conformation sensitivity of UIC2, as the addition of ABCB1 modulators increases the antibody reactivity in vitro . Thus, the inherent fluorescence capability of BPD can be used to label ABCB1-overexpressing TNBC cells using UIC2-BPD. Our findings provide insight into conjugation of hydrophobic photosensitizers to conformation-sensitive antibodies to target proteins expressed on the surface of cancer cells.