Use of photoimmunoconjugates to characterize ABCB1 in cancer cells

Abstract

Abstract 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. Photoimmunoconjugates 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.