Recovery of IR700 Fluorescence After Near-Infrared Photoimmunotherapy: Discovery and Mechanistic Insights

<b>Background/Objectives</b>: Near-infrared photoimmunotherapy (NIR-PIT) is a molecularly targeted cancer therapy that employs antibody-photoabsorber conjugates (APCs) comprising the photosensitizer IRDye700DX (IR700) and tumor-specific antibodies. Following near-infrared (NIR) light irradiation, IR700 undergoes structural modification, inducing selective and rapid necrotic cell death. In mouse tumor models, we observed that IR700 fluorescence decreased during irradiation but recovered immediately afterward. This study aimed to characterize this novel phenomenon, named "early fluorescence recovery," and explore its therapeutic implications. <b>Methods</b>: Cetuximab-IR700 (Cet-IR700) was synthesized and administered to A431 and FaDu-Luc2 xenograft female BALB/c-nu/nu mouse models. In vivo fluorescence imaging was conducted using LIGHTVISION during and after NIR irradiation (690 nm, 50 J/cm²). Reactive oxygen species involvement was examined via intraperitoneal administration of L-sodium ascorbate. Tumor blood flow changes were assessed via indocyanine green (ICG) imaging, and therapeutic efficacy was compared between single and divided irradiation protocols. <b>Results</b>: Tumor fluorescence markedly decreased during NIR-PIT but rapidly recovered within 10 min after irradiation. This recovery was significantly inhibited by L-sodium ascorbate (<i>p</i> < 0.01) and accompanied by increased ICG fluorescence (<i>p</i> < 0.01), suggesting enhanced tumor perfusion. Divided irradiation performed after fluorescence recovery tended to yield greater tumor suppression than did single irradiation, although the difference was not statistically significant. <b>Conclusions</b>: Early fluorescence recovery after NIR-PIT reflects transient reactivation of photoactive APCs through oxygen-dependent molecular and vascular mechanisms. Exploiting this brief recovery window with divided irradiation may improve therapeutic efficacy and guide optimization of NIR-PIT protocols.