| Homologues of RNase A isolated from oocytes and early embryos of frogs as well as one from bovine seminal plasma (BS-RNase) have been shown to exert selective toxicity to human cancer cells. In particular, OnconaseRTM (ONC), isolated from the Northern leopard frog, Rana pipiens, has recently been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma.;Mammalian ribonucleases such as RNase A and its human homologue, RNase 1, are not toxic to cancer cells as a result of their potent inhibition by the cytosolic ribonuclease inhibitor (RI). Nonetheless, they exhibit a number of properties that make them otherwise more desirable as therapeutics. The natural cytotoxic activity of ONC and BS-RNase is largely attributed to their inherent insensitivity to RI. Using a variety of protein engineering strategies, RNase A and RNase I have been endowed with cytotoxic activity by reducing their high affinity (femtomolar) for RI (reviewed in CHAPTER 1).;CHAPTER 2 describes the exploitation of an unbiased computational algorithm (FADE) to target for disruption those regions of greatest shape-complementarity within the RI-RNase A. Guided by this analysis, several variants of RNase A were created that are more potent cytotoxins than ONC in vitro.;BS-RNase derives its cytotoxic activity from its unique dimeric quaternary structure. Inspired by this design, CHAPTER 3 describes the production of dimers and trimers of mammalian ribonucleases resulting from the restrictive tethering of these enzymes to preclude RI-binding. These semi-synthetic conjugates are shown to reduce the growth of tumor cells both in vitro and in vivo.;The parenteral administration of therapeutic proteins poses unique challenges to the development of biologics. The covalent attachment of large, inert polymers such as poly(ethylene glycol) (PEG) to candidate protein therapeutics (a process termed PEGylation) is a strategy proven to overcome many of these hurdles. The next two chapters describe the site-specific PEGylation of RNase A (C HAPTER 4) and RNase 1 (CHAPTER 5) to both enhance RI-evasion and increase the persistence of these proteins in the circulation of animals. Finally, future research directions into ribonuclease-based cancer therapeutics are described in CHAPTER 6. |
