使用连接IRDye700Dx的抗CEA抗体诱导靶向细胞毒性和免疫原性细胞死亡

Introduction: Near-infrared photoimmunotherapy (NIR-PIT) uses antibody-photosensitizer conjugates to induce spatially controlled and targeted cytotoxicity. The IRDye700DX phthalocyanine photosensitizer undergoes photochemical alterations upon activation with 690 nm light which results in targeted cell death via membrane disruption on cancer cells. This study evaluated the therapeutic efficacy and antigen specificity of a carcinoembryonic (CEA) antigen-targeted NIR-PIT using the humanized anti-CEA antibody (M5A) conjugated to IRDye700Dx (M5A-IR700) in a colon cancer model. Methods: M5A was conjugated to NHS-IRDye700DX and validated for binding to human colon cancer cell lines with high-CEA (LS174T), low-CEA (HCT116) by flow cytometry. In vitro, cells were exposed to graded NIR irradiation (690 nm), assessed for cell death (MTS) and evaluated for immunogenic cell death markers (ICD, extracellular ATP, HMGB1). In vivo efficacy was evaluated in athymic mice bearing subcutaneous LS174T xenografts. Mice received intravenous M5A-IR700 (75 μg) followed 24 hours later by a 690 nm laser treatment (150 mW/cm², 30 minutes). Results: Flow cytometry confirmed robust, antigen-dependent binding in high-CEA LS174T cells relative to the weak binding observed in low-CEA HCT116 cells (Figure 1A). NIR-PIT with M5A-IR700 resulted in light fluence-dependent and concentration-dependent cytotoxicity (Figure 1B). Maximal phototoxicity was observed in LS174T at 64 J/cm². In contrast, there was minimal response in low CEA expressing HCT116 cells, confirming antigen-density dependent cytotoxicity. Extracellular ATP and HMGB1 were significantly elevated in LS174T but not HCT116 cells (Figure 1C). Rapid release of extracellular ATP occurred immediately following NIR-PIT in LS174T cells and this response was attenuated in HCT116 cells. These findings indicate an antigen-restricted induction of ICD. Mice treated with M5A-IR700 and laser activation (M5A-IR700 PIT) demonstrate significant suppression of tumor volume over 30 days compared with untreated controls and mice receiving antibody alone (p<0.01) (Figure 1E). Conclusion: CEA-targeted NIR-PIT using M5A-IR700 effectively induced antigen-specific immunogenic cell death in vitro and robust tumor control in vivo. Differential responses between LS174T and HCT116 validate antigen density dependence which is essential for clinical translation. These findings provide strong rationale for clinical development of M5A-IR700-mediated NIR-PIT as targeted therapy for CEA-positive colorectal cancers.