Photoactive immunoconjugates for targeted photodynamic therapy of cancer.

Photodynamic therapy (PDT) has been used as an alternative or as a complement of conventional approaches for cancer treatment. In PDT, the reactive oxygen species (ROS) produced from the interaction between the photosensitizer (PS), visible light and molecular oxygen, kill malignant cells by triggering a cascade of cytotoxic reactions. In this process, the PS plays an extremely important role in the effectiveness of the therapy. In the present work, a new photoimmunoconjugate (PIC), based on cetuximab and the known third generation PS-glycophthalocyanine ZnPcGal was synthesized via reductive amination. The rationale behind this was the simultaneous cancer-associated specific targeting of PIC and photosensitization of targeted receptor positive cells. Varied reaction parameters and photodynamic conditions, such as PS concentrations and both type and intensities of light, were optimized. ZnPcGal showed significant photoactivity against EGFR expressing A431, EGFR-transfected HCT116 and HT29 cells when irradiated with white light of stronger intensity (38 mW/cm). Similarly, the synthesized PICs-T1 and T2 also demonstrated photoactivity with high intensity white light. The best optimized PIC: sample 28 showed no precipitation and aggregation when inspected visually and analyzed through SE-HPLC. Fluorescence excitation of sample 28 and I-sample 28 radioconjugate (I-PIC, I-radiolabeling yield ≥95%, determined with ITLC) at 660 nm showed presence of appended ZnPcGal. In addition, simultaneous fluorescence and radioactivity detection of the I-PIC in serum and PBS (pH 7.4) for the longest incubated time point of 72 h, respectively, and superimposed signals thereof demonstrated ≥99% of loading and/or labeling yield, assuring overall stability of the PIC and corresponding PIC-radioconjugate w.r.t. both the appended ZnPcGal and bound-I. Moreover, real-time binding analyses on EGFR-transfected HCT116 cells showed specific binding of I-PIC, suggesting no alternation in the binding kinetics of the mAb after appending it with ZnPcGal. These results suggest dual potential applications of synthesized PICs both for PDT and radio-immunotherapy of cancer.