Photodynamic therapy (PDT) is a promising cancer treatment due to its minimal invasiveness, repeatability, negligible drug resistance, and high spatiotemporal precision. Photosensitizers generate reactive oxygen species (ROS) upon light irradiation, leading to cancer cell death and tumor ablation. However, challenges remain, including off-target toxicity, tumor hypoxia, and inadequate photosensitizer accumulation or retention at the tumor site. Antibody-drug conjugates (ADCs) offer a targeting approach, but monoclonal antibodies (mAbs) have limitations in specific conjugation and high production costs. Nanobodies (Nbs), smaller antigen-binding fragments, offer advantages in accessibility and ease of production. However, their rapid *in vivo* clearance restricts accumulation at the tumor site. This study aims to address this limitation by developing a nanobody conjugate for precise and sustainable PDT, particularly in large-volume tumors. The epidermal growth factor receptor (EGFR), overexpressed in many tumor cells, serves as the target. The study focuses on creating a conjugate of the anti-EGFR nanobody 7D12 and the type I photosensitizer MNB-Pyra, designed for ROS-mediated release and fluorescence-based self-reporting.

Antibody-drug conjugates (ADCs), particularly those using monoclonal antibodies (mAbs), have shown promise in targeted cancer therapy. Examples include the conjugation of cetuximab mAb and IRdye700DX for recurrent head and neck cancer. However, challenges persist with mAb-based ADCs, including specific conjugation and high production costs. Nanobodies (Nbs), offering advantages in size, accessibility, and ease of production, have emerged as an attractive alternative. Early work demonstrated the potential of nanobody-targeted PDT. Nevertheless, the rapid clearance of Nbs in vivo remains a significant hurdle, necessitating strategies to improve tumor retention. The need for sustainable, precise PDT, especially for large-volume tumors, requires addressing both the rapid clearance and potential phototoxic side effects of prolonged photosensitizer retention. This work aims to improve upon previous research by developing a conjugate that addresses both these issues.

The study involves synthesizing the MNB-Pyra photosensitizer, characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The MNB-Pyra dimer, characterized by absorption and fluorescence spectroscopy, exhibits fluorescence quenching due to π-π stacking. Computational methods (GFNn-xTB and B97-3c) were used to analyze the dimer's structure and quenching mechanism. ROS generation capacity was assessed using fluorescent probes (DPBF, ABDA, DHE, DHR 123) and electron paramagnetic resonance (EPR) spectroscopy. The MNB-Pyra was site-specifically conjugated to the 7D12 nanobody (modified to 7D12-fGly) via a Knoevenagel-Michael tandem reaction, creating the MNB-Pyra Nbs conjugate, characterized by SDS-PAGE, HPLC-HRMS, and dynamic light scattering (DLS). Binding affinity to EGFR was determined using biolayer interferometry (BLI). *In vitro* studies involved cell viability assays (MTT), confocal laser scanning microscopy (CLSM), flow cytometry, ROS detection (DCFH-DA, DHE), apoptosis assays (Annexin V-FITC/PI), and wound healing assays. *In vivo* studies utilized A431 tumor-bearing nude mice for biodistribution analysis using fluorescence imaging and HPLC. Large-volume tumor models were employed to evaluate therapeutic efficacy, with assessments using tumor volume measurements, tumor weight analysis, TUNEL staining, H&E staining, and IHC analysis of Ki-67.

The MNB-Pyra dimer exhibited fluorescence quenching due to π-π stacking, which was reversed upon light irradiation and ROS-mediated cleavage. The MNB-Pyra Nbs conjugate showed high conjugation efficiency (>95%), maintaining good water solubility and EGFR binding affinity. *In vitro*, MNB-Pyra Nbs demonstrated high selectivity for EGFR-positive cells, effective ROS generation under both normoxia and hypoxia, and significant phototoxicity, inducing apoptosis. *In vivo*, MNB-Pyra Nbs efficiently targeted tumors, exhibiting rapid clearance (24 h) without illumination and prolonged retention (5 days) of released photosensitizer after light irradiation. In large-volume tumor models, a single dose of MNB-Pyra Nbs followed by three rounds of PDT resulted in >95% tumor growth inhibition, with minimal observed toxicity. Similar results were obtained using a Hela tumor model.

The MNB-Pyra Nbs conjugate successfully addresses the limitations of both traditional photosensitizers and rapidly cleared nanobodies in PDT. The self-reporting feature allows for real-time monitoring of PDT efficacy. The combination of a rapidly cleared nanobody and a long-term retention photosensitizer, connected by a ROS-cleavable linker, is crucial for achieving targeted and sustained release at the tumor site, reducing off-target effects. The high efficacy against large-volume tumors demonstrates the potential of this approach for improving PDT outcomes.

This study demonstrates the successful development and application of a self-reporting photodynamic nanobody conjugate (MNB-Pyra Nbs) for highly efficient and safe large-volume tumor treatment. The strategy of combining a rapidly cleared nanobody for targeted delivery with a long-term retention photosensitizer released by ROS at the tumor site shows great potential for future applications in PDT. Future research could focus on exploring different tumor models and investigating the underlying mechanisms of action in greater detail.

The study primarily focuses on A431 and Hela cell lines and corresponding xenograft models. While the results are promising, further investigation in other tumor types and with diverse genetic backgrounds would strengthen the generalizability of the findings. The long-term effects and potential for drug resistance remain to be thoroughly explored.