N-Terminal Conjugation of a Near-Infrared Photosensitizer to an Antibody for Cancer Diagnosis and Treatment

We developed immunoprobes by conjugating fluorophores to the N-terminal region of an antibody to detect cancer markers via fluorescence and destruct cancer cells via antigen-dependent photosensitization. To fabricate an immunoprobe for detection, peptides conjugated with fluorophores, such as TAMRA or methylene blue (MB), were ligated at the N-terminus of the heavy chain of trastuzumab (Traz), which is an antibody that recognizes the cancer marker human epidermal growth factor receptor 2 (HER2). The fluorescence intensities of both immunoprobes were decreased upon the addition of HER2 in a dose-dependent manner, which is promising for cancer diagnosis. To achieve antigen-dependent ¹O₂ generation under photodynamic therapy, both MB as a photosensitizer and black hole quencher 3 were conjugated to the N-terminal region of the antibody, forming a quenching photoimmunoconjugate (Q-PIC). Upon HER2 addition, the Q-PIC exhibited increased fluorescence intensity of MB and ¹O₂ generation from MB after irradiation over time. When the cytotoxicity of Q-PIC against cancer cells was investigated under irradiation immediately after the addition of an immunoprobe, we found no significant effect on cell viability, highlighting its potential to reduce adverse effects. However, when irradiated after a prolonged incubation to allow sufficient binding of the antibody to HER2-positive cells, the Q-PIC showed an IC₅₀ value comparable to that of Traz-MB, which is Traz randomly labeled with MB, in HER2-overexpressing SK-BR-3 cells, maintaining its cytotoxicity against cancer. Altogether, we believe that the conjugation of the N-terminal region of fluorophores and photosensitizers to antibodies is a promising strategy for improving the reliability of cancer diagnosis and reducing the adverse effects of photodynamic therapy, thus establishing a basis for developing a theranostic agent for a wide variety of cancers.