Modified Thrombin Binding Aptamer Treatment of CRC Metastasis

PROJECT SUMMARY Metastatic colorectal cancer (mCRC) remains highly lethal despite decades of optimizing 5-fluorouracil (5-FU)- based combination chemotherapy regimens that are central to treatment. The causes of treatment failure with 5-FU-based regimens include decreased metabolic conversion of 5-FU to Fluorodeoxyuridylate (FdUMP) in cancer cells, and overexpression of thymidylate synthase (TS), the primary molecular target. Sub-optimal biodistribution and pharmacokinetics of 5-FU likely also contribute to treatment failure with active metabolites not accumulating in metastatic lesions at levels sufficient to reduce tumor progression. To improve outcomes and overcome therapy resistance in mCRC, Deep Creek Pharma is partnering with Wake Forest University Health Sciences to develop a novel nanoscale material, TBAEFPREX, a modified form of the thrombin-binding aptamer (TBA). The TBA is a quadruplex DNA structure consisting of two G4-tetrads linked by three single-strand DNA loops. Research from the academic partner, WFUHS, confirmed that substitution of the 6 thymidine nucleotides in the 15mer TBA structure with 5-Fluoro-2’-deoxyuridine (FdU) did not disrupt the quadruplex DNA structure required for thrombin recognition but rendered the modified TBA highly cytotoxic to cancer cells. Thrombin, the target for TBA binding, is important for blood coagulation but also plays an important role in metastasis and is abundant in metastatic lesions. Deep Creek Pharma proposes to develop a modified TBA, TBAEFPREX, as a next-generation product for the oncology market with broad-spectrum anti- metastatic activity based on high binding affinity for thrombin that is abundant at metastatic sites. We hypothesize that TBAEFPREX will display broad-spectrum anti-metastatic activity in CRC and other malignancies through (1) thrombin-directed localization to metastatic sites, (2) nucleolin-mediated uptake into cancer cells, and (3) release of three drugs, FdU, EdU, and Prexasertib (PREX – A Chk1 inhibitor), to cause DNA damage and apoptosis in cancer cells. The resulting modified TBAF6, TBAEFPREX, will release of EdU, FdU, and PREX, display excellent nuclease stability, and retain thrombin-directed localization to metastatic sites. In Aim 1, we will optimize the modified nucleotide content in TBAEFPREX to maximize cytotoxicity and minimize exonuclease- mediated degradation while preserving thrombin binding. In Aim 2, we will test if TBAEFPREX is well tolerated in mice and if it displays potent antitumor and anti-metastatic activity in a CRC liver metastasis model and in a thrombin-dependent mouse colon cancer lung metastasis model. Completing these studies will provide a foundation for the advanced pre-clinical development of TBAEFPREX during Phase 2 of STTR funding and advancement to clinical studies. Ultimately, TBAEFPREX holds the promise to significantly increase long-term survival in mCRC patients and other malignancies, offering a bright beacon of hope in the fight against cancer.