Disialoganglioside GD2-Targeted Near-Infrared Photoimmunotherapy (NIR-PIT) in Tumors of Neuroectodermal Origin

Abstract

Disialoganglioside (GD2) is a subtype of glycolipids that is highly expressed in tumors of neuroectodermal origins, such as neuroblastoma and osteosarcoma. Its limited expression in normal tissues makes GD2 a potential target for precision therapy. Several anti-GD2 monoclonal antibodies are currently in clinical use and have had moderate success. Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that arms antibodies with IRDye700DX (IR700) and then exposes this antibody-dye conjugate (ADC) to NIR light at a wavelength of 690 nm. NIR light irradiation induces a profound photochemical response in IR700, resulting in protein aggregates that lead to cell membrane damage and death. In this study, we examined the feasibility of GD2-targeted NIR-PIT. Although GD2, like other glycolipids, is only located in the outer leaflet of the cell membrane, the aggregates formation exerted sufficient physical force to disrupt the cell membrane and kill target cells in vitro. In in vivo studies, tumor growth was significantly inhibited after GD2-targeted NIR-PIT, resulting in prolonged survival. Following GD2-targeted NIR-PIT, activation of host immunity was observed. In conclusion, GD2-targeted NIR-PIT was similarly effective to the conventional protein-targeted NIR-PIT. This study demonstrates that membrane glycolipid can be a new target of NIR-PIT.

Keywords: IR700; ganglioside; glycosphingolipid; immunotherapy; neuroblastoma.

Figure 1

Characterization of αGD2-IR700 and cytotoxic effects of in vitro GD2-targeted NIR-PIT. (A) Validation of covalently bound IR700 to αGD2 antibody by SDS-PAGE (left: colloidal blue staining, right: IR700 channel). Unconjugated αGD2 antibody was used as a control. (B) SEC analysis of αGD2-IR700. The pink line points to the absorption at 690 nm, and the black line points to the absorption at 280 nm. (C) Expression analysis of GD2 in EL4-luc, LAN-1, and T98G cell lines using flow cytometry. Red, blue and orange histograms show unstained, αGD2-IR700 staining without blocking, and with blocking, respectively. (D) Differential interference contrast (DIC) images and fluorescence images before and after GD2-targeted NIR-PIT. Dead cells were stained with propidium iodide (PI). Scale bar = 50 μm. (E) Light-dose-dependent cell death in EL4-luc cells induced by GD2-targeted NIR-PIT. Cell viability was analyzed by flow cytometry after PI staining. Data are shown as mean ± SEM (n = 4, * p < 0.05, **** p < 0.0001 vs. no light exposure group, N.S. not significant). (F) Dose-dependent cell death in LAN-1 cells induced by GD2-targeted NIR-PIT. Cell viability was measured by PI staining. Data are shown as mean ± SEM (n = 4, **** p < 0.0001 vs. no light exposure group, N.S. not significant). (G) Dose-dependent cell death in T98G cells induced by GD2-targeted NIR-PIT. Cell viability was measured by PI staining. Data are shown as mean ± SEM (n = 4, **** p < 0.0001 vs. no light exposure group, N.S. not significant).

Figure 2

Biodistribution of αGD2-IR700 in EL4-luc tumor bearing mice. (A) In vivo serial fluorescence images of the EL4-luc tumor-bearing mice. Mice were intravenously administered with αGD2-IR700, and IR700 fluorescence images were obtained at the time points indicated. (B) Changes in IR700 fluorescence intensity in tumor and liver. Data are shown as mean ± SEM. Data were obtained from seven animals at each time point. (C) Changes in target-to-background ratios in tumor and liver. Data are shown as mean ± SEM. Data were obtained from seven animals at each time point.

Figure 3

In vivo tumor response of GD2-targeted NIR-PIT. (A) Treatment regimen and imaging study protocol. (B) Representative IR700 fluorescence images of mice with EL4-luc tumor before and after GD2-targeted NIR-PIT. (C) Representative bioluminescence images of mice with EL4-luc tumor. (D) Quantitative analysis of changes in luciferase activity in EL4-luc tumors (values before treatment were set to 100%; n = 10 animals per group; two-way repeated measures ANOVA followed by Tukey’s multiple comparison test; * p < 0.05, *** p < 0.001). (E) Tumor growth curves (n = 10 animals per group; two-way repeated measures ANOVA followed by Tukey’s multiple comparison test; *** p < 0.001). (F) Survival curves (n = 10; log-rank test followed by Bonferroni correction; * p < 0.05, *** p < 0.001).

Figure 4

Pathological analysis of EL4-luc tumor after GD2-targeted NIR-PIT. (A) Hematoxylin and eosin staining of tumors resected one day after NIR-PIT (White scale bars, 100 μm; black scale bars, 50 μm). (B,C) Immune cell response after GD2-targeted NIR-PIT was analyzed by flow cytometry 1 day after NIR-PIT. (B) Activation marker expression in CD8+ killer T cells in the regional lymph nodes. (n = 6; one-way ANOVA followed by Tukey’s test; * p < 0.05, **** p < 0.0001; N.S., not significant) (C) Number of NK and NKT cells in the regional lymph nodes. (n = 6; one-way ANOVA followed by Tukey’s test; ** p < 0.01, *** p < 0.001, **** p < 0.0001; N.S., not significant).