DEVELOPMENT OF SUICIDE ENZYME-BASED  THERAPEUTICS FOR CANCER THERAPY

Stacy Emilie Martin

Cancer biologists, oncologists, and therapists are not aiming to treat cancer—they aim to cure it. By advancing current therapeutics and therapeutic strategies, and improving the current standard of care, this goal can be achieved. Suicide gene therapy is a novel cancer treatment strategy in which suicide genes capable of metabolizing non-toxic prodrugs into toxic compounds are targeted specifically to cancer cells. However, suicide gene therapy has historically been limited by the lack of efficient gene delivery and therapeutically relevant gene expression. The work described here aims to overcome these limitations. To improve the yeast cytosine deaminase-triple (yCDtriple)/5-fluorocytosine (5-FC) suicide gene therapy system, we targeted the C-terminal region of yCDtriple for regio-specific random mutagenesis. We hypothesized that regio-specific random mutagenesis of amino acids E147-E158 would generate mutants that display improved kinetic parameters towards 5-FC. An additional alanine-scanning mutagenesis strategy of these C-terminal amino acids demonstrated that this region was important for overall catalytic activity, and that specific residues were integral for enzymatic function. Herein the pathogenesis of advanced breast and prostate cancers are discussed, and the potential molecular targets that can be exploited for the delivery of suicide enzymes to these cancers are reviewed. We evaluated azide-alkyne cycloaddition to couple yCDtriple to a small molecule prostate-specific membrane antigen (PSMA) inhibitor, to generate therapeutics for use in “inhibitor-directed enzyme-prodrug therapy (IDEPT)” for prostate cancer. For breast cancer therapy, we fused yCDtriple to a human epidermal growth factor receptor 2 (HER2) targeting affibody, resulting in the generation of “affibody-directed enzyme-prodrug therapy (AfDEPT).” Significant prostate and breast cancer cell killing was observed after treating PSMA-positive or HER2-positive cancer cells with the IDEPT or AfDEPT agents and 5-FC, respectively. Together, the work described in this dissertation demonstrates the adaptability of yCDtriple, click chemistry, and affibody fusion proteins for therapeutic applications. By integrating suicide gene therapy, IDEPT, and AfDEPT with current cancer treatment modalities, we can improve the treatment options for advanced cancer patients, contribute to personalized medicine, and increase the arsenal against cancer.

DEVELOPMENT OF SUICIDE ENZYME-BASED THERAPEUTICS FOR CANCER THERAPY

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