Development of a new minimally invasive phototherapy for lung cancer using antibody–toxin conjugate

Abstract

Background Photodynamic therapy (PDT) is a cancer‐targeted treatment that uses a photosensitizer (PS) and laser irradiation. The effectiveness of current PDT using red light for advanced cancers is limited, because red light can only reach depths within a few millimeters. To enhance the antitumor effect for lung cancers, we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin and a PS, mono‐L‐aspartyl chlorin e6 (NPe6). Methods We examined whether cetuximab encapsulated in endosomes was released into the cytosol by PS in PDT under light irradiation. A431 cells were treated with fluorescein isothiocyanate‐labeled cetuximab, NPe6, and light irradiation and were observed with fluorescence microscopy. We analyzed the cytotoxicity of saporin‐conjugated cetuximab (IT‐cetuximab) in A431, A549, and MCF7 cells and the antitumor effect in model A549‐bearing mice in vivo using the iTAP method. Results Fluorescent microscopy analysis showed that the photodynamic effect of NPe6 (20 μM) and light irradiation (37.6 J/cm 2 ) caused the release of cetuximab from the endosome into the cytosol. In vitro analysis demonstrated that the iTAP method enhanced the cytotoxicity of IT‐cetuximab by the photodynamic effect. In in vivo experiments, compared with IT‐cetuximab alone or PDT alone, the iTAP method using a low dose of IT‐cetuximab showed the greatest enhancement of the antitumor effect. Conclusions Our study is the first report of the iTAP method using NPe6 for lung cancer cells. The iTAP method may become a new, minimally invasive treatment superior to current PDT methods.

Cetuximab–Toxin Conjugate and NPe6 with Light Enhanced Cytotoxic Effects in Head and Neck Squamous Cell Carcinoma In Vitro

“…In other words, NPe6 and the specific wavelength enhanced the cytotoxic effect of IT-Cmab by internalizing it into the cytoplasm via endosomal escape. It was inferred that saporin accumulated at high concentrations in cancer cells with high expression of EGFR, and saporin exerted its enzymatic activity only in specific wavelength to NPe6-irradiated cells in this approach [9]. Similarly, in this study, we presumed that iTAP exerted a cytotoxic effect by internalizing IT-Cmab into the cytoplasm.…”

“…A saporin-conjugated anti-EGFR antibody (Cmab), hereafter called IT-Cmab, was prepared as follows: biotinylated Cmab was purified by mixing cetuximab with EZ-LINK sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA, USA) at a 1:20 molar ratio using PD-10 (GE Healthcare Life Sciences, Piscataway, NJ, USA), as previously reported [3,4,6]. Next, biotinylated Cmab and streptavidin-saporin (Biotin-Z Internalization Kit [KIT-27-Z]) (Advanced Targeting Systems, Carlsbad, CA, USA) were mixed in approximately equivalent amounts and allowed to react at room temperature for 30 min to obtain IT-Cmab, as previously reported [9].…”

“…[7,8]. We have developed a novel method using a PS for PDT, mono-L-aspartylchlorine6 (NPe6), and named this therapeutic method "intelligent targeted antibody phototherapy" (iTAP) [9]. We first reported saporin-conjugated antiepidermal growth factor receptor (EGFR) antibody target EGFR (cetuximab) (IT-Cmab), NPe6, and light (664 nm) irradiation (i.e., iTAP) in lung cancer cells in 2022.…”

“…Both of them are then endocytosed to the endosome. During subsequent irradiation with the specific wavelength of PS, the photodynamic effect causes the endosome to rupture, enabling the endosomal escape of IT (i.e., iTAP) [7][8][9]. It was inferred that saporin accumulates at high concentrations in the cytoplasm of cancer cells with high expression of EGFR, and specifically leads to cell death [7][8][9].…”

“…Therefore, iTAP has a higher antitumor effect than PDT, antibody, or IT alone [7][8][9]. To examine the possibility of clinical application of iTAP, we used an anti-epidermal growth factor receptor (EGFR) antibody immunotoxin (IT-cetuximab, IT-Cmab) that targets EGFR, which is widely used for the treatment of HNSCC.…”