Enhanced Antitumor Efficacy of a Combination of Immunotoxin and Photosensitizer Under Illumination in Xenograft Mice

<b>Background/Objectives</b>: Head and neck squamous cell carcinoma (HNSCC) affects over 600,000 individuals worldwide each year, and its incidence continues to rise. There is a growing need for novel therapeutic strategies that achieve high antitumor efficacy while minimizing functional impairment. We developed a novel approach to enhance intracellular delivery of immunotoxins (ITs) by combining a photosensitizer under illumination. This method, termed intelligent Targeted Anti-body Phototherapy (iTAP), utilizes light as a spatiotemporal trigger to promote the cytoplasmic release of toxins. In the present study, we investigated the in vivo therapeutic efficacy of iTAP using an EGFR-targeted IT composed of cetuximab conjugated to saporin (IT-Cmab), administered in combination with the clinically used photodynamic therapy (PDT) photosensitizer NPe6, in a xenograft mouse model. <b>Methods</b>: Sa3 cells were implanted subcutaneously into the right hind limb of nude mice. Mice were randomized into four groups (n = 5): (i) iTAP (IT-Cmab plus NPe6), (ii) IT-Cmab alone, (iii) NPe6 alone, and (iv) saline control. Treatment was initiated once tumors exceeded 40 mm³. Mice received intraperitoneal IT-Cmab (0.5 mg/kg), followed 72 h later by intravenous NPe6 (5 mg/kg). Tumors were irradiated 3-4 h later using a custom LED device (670 nm, 262 mW/cm², 30 J/cm²). Tumor volume and body weight were monitored over time, and antitumor effects were analyzed using a linear mixed-effects model. <b>Results</b>: iTAP treatment produced the earliest and most pronounced inhibition of tumor growth among the four groups. Significant suppression was observed from day 9 and persisted throughout the study. IT-Cmab alone showed a moderate but sustained antitumor effect with a later onset, whereas NPe6-mediated PDT exhibited only a delayed and weaker response. On the final day, median tumor volumes showed substantial reductions relative to the Control group (601%), with decreases of 41% in PDT (357%), 55% in IT-Cmab (271%), and 70% in iTAP (178%). Overall, iTAP demonstrated the strongest and most durable therapeutic efficacy in vivo. <b>Conclusions</b>: These findings indicate that iTAP represents a promising therapeutic strategy for HNSCC.