Humanized EGFR and EGFRvlll-bispecific Immunotoxin for HNSCC Therapy

Shan, Liang; Gu, Xinbin

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with ~37,000 new cases diagnosed every year in the United States. Despite therapeutic advances, the 5-year survival rate has remained 55-65% since the mid-1970s. Novel targeted therapeutic agents that can be effectively integrated into the current treatment paradigms are urgently needed. Epidermal growth factor receptor (EGFR) is a membrane-bound tyrosine kinase receptor regulating cell proliferation and migration. Up to 90% of HNSCC have been found to overexpress EGFR, 10-58% have EGFR gene amplification, and up to 42% possess the cancer-specific mutation variant, EGFRvIII. Furthermore, the EGFR abnormality in HNSCC is closely associated with decreased patients' survival, resistance to radiotherapy, loco-regional treatment failure, and increased metastasis rate. The high frequency of EGFR abnormality and its significance in HNSCC progression have sparked development of monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) for EGFR-targeted therapy. In 2006, the recombinant chimeric mAb, Cetuximab, has been approved by FDA for HNSCC therapy in combination with radiation or chemotherapy. Despite the demonstrated benefits, continued development of EGFR-targeted therapy is facing formidable challenges including: 1) innate and acquired resistance due to EGFR mutation, persistent activation of multiple interacting pathways, etc.; 2) dose-limiting side effects induced by binding of the agents with EGFR expressed in normal tissues; and 3) suboptimal efficacy imposed by the large molecular size and poor tumor-penetrating capability of mAbs. Recombinant immunotoxin (RIT) represents a novel strategy of EGFR-targeted therapy of HNSCC and is characterized by high specificity, extraordinary potency, and lack of drug resistance. Taking advantage of the unique specificity of murine mAb806 to the EGFR and EGFRvIII overexpressed in cancer, we generated a bivalent RIT, designated as MuBiscFv-DT390, by fusing an engineered diphtheria toxin fragment (DT390) with a bivalent single-chain variable fragment (biscFv) via amino acid linkers. Encouraged by the promising results and to further leverage the mAb806 humanization-derived benefits, we further generated a humanized biscFv RIT, designated as HuBiscFv-DT390. Both bivalent RITs were expressed in a high yield and purity with a diphtheria toxin-resistant Pichia system invented by our collaborators (Patent No.: US7892786). Similar to the MuBiscFv-DT390, the HuBiscFv-DT390 also exhibited extraordinary potency and specificity against cancer cells with overexpressed EGFR only or with co-expressed EGFR and EGFRvIII. The desirable properties of the HuBiscFv-DT390 make it extremely attractive as an EGFR- targeted agent for HNSCC therapy. We hypothesize that HuBiscFv-DT390 is feasible and efficacious for targeted therapy of HNSCC with high sensitivity and specificity against the disease with EGFR overexpression only or with EGFR and EGFRvIII co-expression. Use of humanized biscFv and DT390 in the construction of HuBiscFv-DT390 will minimize its immunogenicity and potential side effects. Because of the unique cancer cell-killing mechanism, we further hypothesize that HuBiscFv-DT390 is also active to the disease with innate or acquired resistance to other EGFR-targeted therapies, and will achieve synergistic anti-tumor effect when integrated into the current therapeutic regimens. To address our hypotheses, we will first establish the anti-tumor activity profile of HuBiscFv-DT390 in animal models of HNSCC tumor xenografts by determining its anti-tumor efficacy, tumor uptake dynamics and pharmacokinetics. We will then establish its safety and toxicity profile in animals. Lastly, we wil predict the clinical outcome of HuBiscFv-DT390 by testing its efficacy against personalized tumorgrafts that are established in mice with fresh fragmented HNSCC tissue from patients. Completion of the proposed studies will enable us to obtain the critical information to further refine the HuBiscFv-DT390 and translate it to clinical studies. Also important to produce the future cadre of highly talented and innovative researchers, this work will provide opportunity for the minority students at Howard University College of Dentistry (HUCD) to involve in hands-on scientific research by conducting sub-research projects under direct supervision of the principal investigators and co-investigators at HUCD and Johns Hopkins University.

Public Health Relevance

Taking advantage of the high frequency of epidermal growth factor receptor (EGFR) abnormality, the unique specificity of antibody mAb806 to the EGFR and EGFRvIII overexpressed in cancers, and the mAb806 humanization-derived benefits, we generated a humanized bivalent single-chain variable fragment-derived, diphtheria toxin (DT390)-based recombinant immunotoxin (RIT), designated as HuBiscFv-DT390, for EGFR-targeted therapy of head and neck squamous cell carcinomas (HNSCC). In the proposed studies, we will determine its anti-tumor efficacy against HNSCC tumor xenografts, establish its safety and toxicity profile, and predict its clinical outcome by testing its efficacy against personalized tumorgrafts of HNSCC. The results will enable us to further refine the HuBiscFv-DT390 and translate it to clinical studies. This work will also provide opportunity for students to involve in hands-on research at both Howard University College of Dentistry and Johns Hopkins University to produce the future cadre of highly talented and innovative researchers.