Chimeric Anti-Cancer Proteins Engineered for Delivery by Tumor-Targeted Salmonella

Salmonella str. VNP20009 selectively colonizes solid tumors and has been shown to reduce their growth in mouse models. Studies have demonstrated that Salmonella can induce apoptosis through direct contact with cells. However, this property limits the potential of the bacterial therapy by requiring physical association with tumor cells. In order to overcome this limitation, these bacteria can be engineered to express and secrete cytotoxins that are tumor cell specific, which may enhance tumor destruction while sparing healthy adjacent tissues. Epidermal growth factor receptor (EGFR) is overexpressed in many cancer cell lines including breast, prostate, colon, brain and others, and is a validated target in the clinical setting. In this thesis, EGFR-targeting toxins were engineered and assessed for their ability to be secreted or released into the extracellular milieu by VNP20009. VNP20009 was able to secrete or release three forms of a truncated Pseudomonas aeruginosa Exotoxin A (ToxA) cytotoxin that was fused to the EGFR-binding ligand, tumor growth factor-alpha (TGFα), which resulted in bacterial culture supernatants that were selectively cytotoxic to cells overexpressing EGFR. Additionally, the native ToxA C-terminal endoplasmic reticulum (ER) retention signal was replaced with the mammalian consensus sequence KDEL to enhance toxicity, and were co-expressed with the pColE3 lysis protein to facilitate secretion. These genetic manipulations resulted in a supernatant that is more active against EGFR positive cells than the secreted wild type ToxA, and may translate into a potent, targeted bacterial therapy.