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

Abstract

Targeted toxins (TTs) are a class of therapeutic molecules directed against human cancer. By directing TTs toward cancer specific targets using tumor reactive ligands, they can be designed to be selective against numerous cancer types. Because TTs are designed to specifically destroy cancerous cells without damaging healthy cells, their potential outweighs most of the non-specific therapies such as chemotherapy and radiotherapy currently used in cancer patients. Recently, the potential of TTs has grown in the field of cancer research through continuous improvement using genetic engineering. Thorough preclinical studies of TTs are important for the characterization of TT biology, identification of possible solutions to TT drawbacks, and the development of novel TTs. Studies have led to several clinical trials, some of which have displayed promising results and confirmed the potential of TTs in cancer therapy. To date, most TTs attack cancer using a single targeting molecule. Two novel drugs, DTEGFATF and EGFATFKDEL that are the subject of this thesis, are unique in that they are bispecific ligand-directed toxins (BLTs), and are designed to simultaneously target both solid tumors and their associated neovasculature. DTEGFATF is a diphtheria toxin (DT) containing BLT, while EGFATFKDEL is a pseudomonas exotoxin (PE) containing BLT. Both BLTs target two receptors, epidermal growth factor receptor (EGFR) and urokinase plasminogen activator receptor (uPAR), which are commonly overexpressed on the cell surface of several different cancers. uPAR-targeting was used because it is overexpressed not only on solid tumors, but also on the neovasculature. We observed that the two BLTs possessed similar in vitro biological properties and activities because DT and PE have identical mechanisms of action. Additionally, by modifying certain amino acids on the PE molecule in EGFATFKDEL, we were able to produce a novel third agent, EGFATFKDEL 7mut, which possesses significantly reduced immunogenicity while maintaining activity. The efficacy of a TT is dependent on the ability to give multiple courses of treatment, and the production of neutralizing antibodies against TTs has historically been a major limitation in TT clinical trials. By modifying PE and targeting dual markers, we were able to produce a novel TT with impressive antitumor activity against glioblastoma in vitro and in vivo.