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/cm² ) 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.

Keywords: drug delivery system; endosomal escape; immunotoxin; mono-l-aspartyl chlorin e6 (NPe6); photodynamic therapy.

FIGURE 1

Fluorescence microscopic analysis of cetuximab release into the cytosol by the photodynamic effect. (a) Fluorescence microscopy images of A431 cells treated in four different ways: (I) cetuximab‐fluorescein isothiocyanate (FITC), (II) cetuximab‐FITC + light irradiation, (III) cetuximab‐FITC + NPe6, and (IV) cetuximab‐FITC + NPe6 + light irradiation. (b) Mean fluorescence intensity (MFI) in the cells was quantitatively determined using ImageJ and is presented graphically. MFI was calculated as the average of the difference between the fluorescence intensity of the three intracellular areas (yellow arrows) and that of the extracellular area (white arrows). The fluorescence intensity values for each treatment are listed. The values of IV were significantly higher than that of the other conditions (AVOVA, *p < 0.05). Data are shown as the mean ± standard deviation.

FIGURE 2

The expression level of epidermal growth factor receptor (EGFR) in each of the cell lines. Analysis of EGFR expression level on the cell surface was performed by flow cytometry using QIFIKIT. Histograms showed staining of A431, A549, and MCF7 cells.

FIGURE 3

Cell viability assay for antitumor effect. We analyzed the cytotoxicity of IT‐cetuximab combined with NPe6‐PDT in A431 (a), A549 (b), and MCF7 (c) cells, and evaluated the half‐maximal inhibitory concentration (IC₅₀) of each cell line from the sigmoid curve. The cytotoxicity of IT‐cetuximab was enhanced by the combination of NPe6‐PDT. This effect was even observed in MCF7 cells with low EGFR expression (c). Data are shown as the mean ± standard deviation. IT‐cetuximab, saporin‐conjugated cetuximab; PDT, photodynamic therapy; EGFR, epidermal growth factor receptor

FIGURE 4

Cell viability assay of NPe6‐PDT for A549. We examined the cytotoxic effect of PDT with different concentrations of NPe6 and amounts of light irradiation. A549 cells were seeded at 2.5 × 10³ cells per well in 96‐well plates, cultured for 24 h, and then exposed to various concentrations (1–100 μM) of NPe6. After 21 h, cells were washed with 100 μl/well of PBS, and 3 h later, cells were irradiated with LED light for 5, 10, and 15 min (18.8, 37.6, and 56.4 J/cm²). Cell viability was determined 48 h after LED irradiation using cell counting kit‐8. The dose of PDT in the experiments shown in Figure 3(b) (30 μM NPe6 and 37.6 J/cm² light irradiation) did not exert cytotoxicity by itself. PDT, photodynamic therapy; PBS, phosphate buffered saline; LED, light‐emitting diode

FIGURE 5

Tumor growth curve of A549 tumor‐bearing mice after different treatments. (a) Pre‐experiments to determine the dose of IT‐cetuximab. Mice were injected intraperitoneally with IT‐cetuximab at several doses. Injection of IT‐cetuximab at 3 mg/kg significantly inhibited tumor growth compared to control (ANOVA, p < 0.01), but the tumors did not achieve complete response and showed a tendency for regrowth from the 6th day after injection. (b) In vivo tumor growth in mice treated with the following: (1) control, (2) IT‐cetuximab, (3) NPe6 + light irradiation (PDT), and (4) IT‐cetuximab and PDT (iTAP) (n = 3). Compared to controls, IT‐cetuximab alone or PDT alone did not show much enhancement of antitumor effect, but the iTAP method using low dose of IT‐cetuximab and NPe6 with laser irradiation showed the most enhanced antitumor effect (ANOVA, p < 0.01). The tumor volume is normalized based on the day 0 value. Data are shown as the mean ± standard deviation. PDT, photodynamic therapy; IT‐cetuximab, saporin‐conjugated cetuximab; iTAP, intelligent targeted antibody phototherapy

FIGURE 6

Schema of the action of mechanisms and comparison of three methods of phototherapy. (a) iTAP method, (b) PDT, and (c) PIT. Each of these methods requires multiple steps to achieve its antitumor effect. (a) Schema of the principles of the iTAP method. (1) Binding of antibodies to antigens expressed on the cell surface. (2) Endocytosis. (3) Endosome permeability increased by light irradiation. (4) Toxin translocation into the cytoplasm and cell death. (b) Schematic of the mechanism of action of PDT. (1) Accumulation of the PS to tumor cells. (2) On laser irradiation, the PS is transferred from its ground state to an excited state. (3) The excited‐state PS transfers energy to ground‐state oxygen, generating singlet oxygen. (4) The excited‐state PS reacts with a neighboring molecule, producing various ROS. (c) Schematic of the mechanism of action of PIT. (1) Binding of IR700‐conjugated antibodies to antigens expressed on the cell surface. (2) Red light irradiation releases a portion of IR700 from the antibody‐IR700 conjugate. (3) Physical changes in the antibody‐IR700 conjugate. (4) Physical stress on the local cell membrane and cell rupture. iTAP, intelligent targeted antibody phototherapy; PDT, photodynamic therapy; PIT, photoimmunotherapy; PS, photosensitizer; ROS, reactive oxygen species