Antitumor Efficacy of EGFR-Targeted Recombinant Immunotoxin in Human Head and Neck Squamous Cell Carcinoma

SIMPLE SUMMARY: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with more than 500,000 new cases diagnosed annually. Surgical resection, chemoradiotherapy, targeted therapy, and immunotherapy have been approved for HNSCC treatment. While a minority of patients experience dramatic long-lasting and favorable clinical responses, the majority of patients fail to achieve durable clinical responses. Thus, alternative options with improved beneficial response are urgently needed. In HNSCC, over 90% of tumors overexpress the cell surface epidermal growth factor receptor (EGFR). We previously produced a humanized recombinant immunotoxin, hDT806, targeting tumor-specific overexpressed EGFR and/or the EGFRvIII mutant. Here, we set out to explore the effects and mechanisms of hDT806 in treating HNSCC in both in vitro and in vivo settings. We found that hDT806 exhibits a significant cytotoxicity in HNSCC through disrupting EGFR signaling, transcription inhibition, and inducing apoptosis and DNA damage. ABSTRACT: Over 90% of head and neck squamous cell carcinoma (HNSCC) overexpresses the epidermal growth factor receptor (EGFR). However, the EGFR-targeted monotherapy response rate only achieves 10–30% in HNSCC. Recombinant immunotoxin (RIT) often consists of an antibody targeting a tumor antigen and a toxin (e.g., diphtheria toxin [DT]) that kills cancer cells. We produced a humanized RIT, designated as hDT806, targeting overexpressed EGFR and investigated its effects in HNSCC. Distinct from the EGFR-targeted tyrosine kinase inhibitor erlotinib or antibody cetuximab, hDT806 effectively suppressed cell proliferation in the four HNSCC lines tested (JHU-011, -013, -022, and -029). In JHU-029 mouse xenograft models, hDT806 substantially reduced tumor growth. hDT806 decreased EGFR protein levels and disrupted the EGFR signaling downstream effectors, including MAPK/ERK1/2 and AKT, while increased proapoptotic proteins, such as p53, caspase-9, caspase-3, and the cleaved PAPR. The hDT806-induced apoptosis of HNSCC cells was corroborated by flow cytometric analysis. Furthermore, hDT806 resulted in a drastic inhibition in RNA polymerase II carboxy-terminal domain phosphorylation critical for transcription and a significant increase in the γH2A.X level, a DNA damage marker. Thus, the direct disruption of EGFR signaling, transcription inhibition, DNA damage, as well as apoptosis induced by hDT806 may contribute to its antitumor efficacy in HNSCC.