Laboratory Investigation - Human/Animal Tissue

Journal of Neuro-Oncology

, Volume 103, Issue 2, pp 255-266

A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature

  • Alexander K. TsaiAffiliated withDepartment of Therapeutic Radiology-Radiation Oncology, Section on Molecular Cancer Therapeutics, University of Minnesota Masonic Cancer Center
  • , Seunguk OhAffiliated withDepartment of Therapeutic Radiology-Radiation Oncology, Section on Molecular Cancer Therapeutics, University of Minnesota Masonic Cancer Center
  • , Hua ChenAffiliated withDepartment of Therapeutic Radiology-Radiation Oncology, Section on Molecular Cancer Therapeutics, University of Minnesota Masonic Cancer Center
  • , Yanqun ShuAffiliated withDepartment of Therapeutic Radiology-Radiation Oncology, Section on Molecular Cancer Therapeutics, University of Minnesota Masonic Cancer Center
  • , John R. OhlfestAffiliated withDepartment of Pediatrics, University of Minnesota Masonic Cancer Center
  • , Daniel A. ValleraAffiliated withDepartment of Therapeutic Radiology-Radiation Oncology, Section on Molecular Cancer Therapeutics, University of Minnesota Masonic Cancer Center Email author 

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Abstract

A bispecific ligand-directed toxin (BLT), called EGFATFKDEL, consisting of human epidermal growth factor, a fragment of urokinase, and truncated pseudomonas exotoxin (PE38) was assembled in order to target human glioblastoma. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGFATFKDEL 7mut. In vitro, the drug selectively killed several human glioblastoma cell lines. EGFATFKDEL is our first BLT designed to simultaneously target EGFR on solid tumors and uPAR on the tumor neovasculature. In vitro assays revealed that the agent is effective against glioblastoma cell lines as well as human umbilical vein endothelial cells (HUVEC). Additionally, the bispecific drug displayed enhanced binding to overexpressed epidermal growth factor receptor and urokinase receptor when compared to similar monospecific drugs, EGFKDEL and ATFKDEL. In vivo, an aggressive human glioblastoma cell line was genetically marked with a firefly luciferase reporter gene and administered to the flanks of nude mice. Treatment with intratumoral injections of EGFATFKDEL 7mut eradicated small tumors in over half of the treated mice, which survived with tumor free status at least 100 days post tumor inoculation. ATFKDEL, which primarily targets the tumor neovasculature, prevented tumor growth but did not result in tumor-free mice in most cases. Specificity was shown by treating with an irrelevant BLT control which did not protect mice. Finally, immunization experiments in immunocompetent mice revealed significantly reduced anti-toxin production in EGFATFKDEL 7mut treated groups. Thus, EGFATFKDEL 7mut is an effective drug for glioblastoma therapy in this murine model and warrants further study.

Keywords

Immunotoxin Pseudomonas exotoxin Glioblastoma Xenograft model EGF