Dose-dependent effect of dinutuximab-IR700 photoimmunotherapy on GD2-expressing tumors.

Abstract

e14556 Background: Near-infrared photoimmunotherapy (PIT) is a novel, molecularly targeted cancer therapy. It combines tumor-directed immunotherapy agents with a phthalocyanine dye IR700 to induce rapid cell death upon light activation, while sparing adjacent normal tissue. GD2 is a targetable tumor-associated antigen expressed in neuroblastoma, osteosarcoma and glioblastoma. Dinutuximab, an anti-GD2 monoclonal antibody, has demonstrated clinical efficacy in neuroblastoma immunotherapy. We evaluated the efficacy of Dinutuximab-IR700 as a targeted PIT agent in in vitro models of GD2-expressing tumors. Methods: We synthesized a Dinutuximab-IR700 antibody dye conjugate (ADC) and tested it on human-derived SK-N-BE(2) and NMB6 (neuroblastoma), LM7 and 143B (osteosarcoma), U87 (glioblastoma) and mouse-derived GL261 and SB28 (glioblastoma) cell lines. For PIT, cells were incubated with ADC (15 μg/mL), followed by activation with light at 10-100 J/cm 2 at 150 mW/cm 2 . Controls included untreated, light-only and ADC-only groups, along with apoptosis (camptothecin) and necrosis (H 2 O 2 ) positive controls. Additional ADC doses of 5, 10 and 20 μg/mL were evaluated in neuroblastoma. Cell death was evaluated by Annexin V/Propidium Iodide (PI) flow cytometry, and brightfield/fluorescence microscopy at 24 h and 48 h post-PIT. GD2 expression per cell for each cell line was also quantified by flow cytometry to correlate with PIT response. Results: Dinutuximab-IR700 PIT markedly reduced cancer cell viability in both neuroblastoma cell lines by up to 78%. Light and ADC had dose-dependent effects on cell death, plateauing at 50 J/cm 2 and 15 μg/mL, respectively. PIT efficacy on osteosarcoma cell lines was heterogeneous, with up to 14% cell death observed only in LM7. PIT was not effective in U87, GL261 and SB28 glioblastoma cells, which showed minimal ADC binding on fluorescence microscopy. Cell death by PIT strongly correlated with GD2 expression per cell across tumor cell lines. Light-only and ADC-only groups matched untreated controls. PIT efficacy was similar with different degrees of labeling (2 vs 3 IR700 molecules per Dinutuximab molecule). Annexin V/PI staining indicated apoptosis over necrosis as the primary mode of cell death, supported by microscopy. Dinutuximab-IR800, studied for intraoperative molecular imaging, was ineffective under identical conditions, demonstrating IR700’s unique utility for PIT. Conclusions: GD2-targeted Dinutuximab-IR700 PIT induces profound dose-dependent cytotoxicity across neuroblastoma cell lines, with variable activity in osteosarcoma and none in glioblastoma. The precise mechanism of action of PIT and reasons for its heterogeneous efficacy in different GD2-expressing tumors remain to be elucidated. This represents a novel precision medicine approach to neuroblastoma treatment, particularly for residual disease after surgical resection.