Head and neck cancer (HNC) is a heterogeneous and difficult-to-treat tumor that affects in various regions, including the oral cavity, nasopharynx, oropharynx, hypopharynx, larynx, paranasal sinuses, and salivary glands. Therefore, research and treatment adapted to these specific characteristics are necessary. Overexpression of the epidermal growth factor receptor (EGFR) has been identified in various type of cancers, including HNC. EGFR mediated Signaling is pivotal in cell proliferation, differentiation, invasion, and the prevention of apoptosis. Cetuximab (CTX), an FDA-approved EGFR-targeted antibody has been used in the treatment of HNC. However, the therapeutic effect demonstrated by EGFR inhibition was not satisfactory and was further limited due to the rapid development of drug resistance. To enhance the efficacy of targeted therapy, we developed CTX-LR-LO10, an EGFR-targeting antibody-drug conjugate (ADC) containing clinically validated anti- IgG (CTX) conjugate with a toxin fragment derived from Pseudomonas exotoxin A (LR-LO10). The CTX-LR-LO10 exhibited EGFR-specific binding and inhibition through the CTX part, displaying IgG characteristics such as stability, interaction with immune cell receptors, and favorable pharmacokinetic properties. Additionally, the LR-LO10 part inhibited protein synthesis in cancer cells We demonstrate that CTX-LR-LO10 induces significant apoptosis in EGFR-positive HNC cells at very low dose. It also inhibits cancer cell migration and invasion, which are associated with cancer metastasis and poor prognosis. The therapeutic efficacy of CTX-LR- LO10 was confirmed in the CTX sensitive FaDu and CTX resistant Detroit562 xenograft mouse models. CTX-LR-LO10 exhibited significantly superior tumor suppression effects compared to CTX, independent of CTX sensitivity, with no apparent side effects. Immunofluorescence data using mouse tumor tissue samples demonstrated that cleaved caspase 3 and TUNEL staining were more effectively induced in the CTX-LR-LO10 treated group compared to the CTX or PBS control groups, indicating an increase in apoptotic cell death. And PCNA and Ki67 were reduced in the CTX-LR-LO10 treated group indicating inhibition of cell proliferation. These results demonstrate that CTX-LR-LO10 suppresses EGFR-positive HNC through the dual mechanisms of EGFR signaling inhibition and protein synthesis inhibition, regardless of CTX sensitivity and EGFR expression levels. HNC expresses not only EGFR but also PD-L1. Immune checkpoint blocker (ICB) has been proven to be effective as a first-line therapy for PD-L1 expressing head and neck squamous cell carcinoma (HNSCC), however the mechanisms underlying these effects are not well understood. In this study, we successfully established syngeneic mouse models that reflect the characteristics of human HNSCC and are suitable for studying the tumor microenvironment in relation to immunotherapy. Using this model, we confirmed that immunotherapy alone provides sufficient tumor- suppressive effects while also promoting vascular normalization and enhancing the infiltration of CD8 T cells, thereby altering the tumor microenvironment. The HNSCC model also showed that type I interferon plays an essential role in this proces. These findings helped to elucidate the mechanisms by which immunotherapy exerts its effects. This study demonstrates that by exploring various approaches to analyzing targeted therapy for HNC with diverse characteristics, this foundational study contributes to the development and optimization of effective therapies for heterogeneous HNC. Keywords EGFR, epidermal growth factor receptor; ADC, antibody drug conjugate; PD-L1, programmed death-ligand 1; Targeted therapy; TME, tumor microenvironment; Tumor regression; HNC, head and neck cancer
