Current Pharmaceutical Biotechnology - Volume 9, Issue 3, 2008

As the annual number of newly diagnosed cases of cancer is about 10 million worldwide, combat of cancer is one of the most outstanding challenges of recent medicine. The considerable undesired side effects of chemotherapy and/or radiotherapy caused by their little selectivity call for a specific targeting of the affected tissue. Exploiting the target specificity of antibodies, which selectively bind to tumor-associated antigens presented on the cell surface, reduces the systemic toxicity of the coupled compounds. All organisms contain numerous ribonucleolytic enzymes for RNA digestion, processing, and regulation etc., which provide a wide palette of potential cytotoxins. Several of these display a preference for transformed cells per se, others can be converted into tumor specific drugs by modification or fusion to respective targeting moieties. Hence, intensive research is carried out on the use of these enzymes as therapeutics. This issue of Current Pharmaceutical Biotechnology consists of a comprehensive compilation of reviews on the therapeutic potential of RNases, mainly from the RNase A (bovine pancreatic RNase) superfamily. The cytotoxicity of RNases was discovered back in the 1950s but cytotoxic effects were observed only when milligrams of enzyme were injected into solid tumors, while smaller doses of RNase A had no effect. In parallel, RNase A evolved as one of the most intensively studied model proteins in virtually all fields of biochemical research. Still, cytotoxicity has remained one of the most attractive characteristics of RNases because these enzymes could be used, alone or conjugated to ligands or antibodies, as therapeutic agents for cancer treatment. The subject is timely, as an important series of new discoveries and developments in this area has emerged over the past years. With Onconase® (Alfacell Corp., Somerset, NJ, U.S.A.), an RNase from the Northern Leopard frog Rana pipiens, an RNase has reached phase III clinical trials for treatment of non-small cell lung cancer and confirmatory phase IIIb for treatment of malignant mesothelioma. Furthermore, a recently generated fully human fusion protein composed of an anti-ErbB2 single-chain antibody fragment and the human pancreatic RNase (ERB-hRNase immunoRNase) has portended the development of RNase-based drugs from human-sourced building blocks. Thus, the promising medicinal applicability of RNases emphasizes the lasting topicality of these rather old enzymes. With techniques to follow molecules on the cellular and subcellular level, the understanding of the mechanisms and the interplay by which RNases exert cytotoxicity begins to become lucid. On their way from administration to the final intracellular target, the proteins face numerous obstacles and there are tremendous differences in how efficient the particular RNase can cope with the respective prerequisites. The reviews of this issue cover the different stages and underline the sophisticated requirements an RNase-based drug must meet. ACKNOWLEDGEMENTS The editor gratefully acknowledges the contributions of all authors of the reviews for this special issue of Current Pharmaceutical Biotechnology. In the following, the corresponding authors are listed in alphabetical order: Wojciech Ardelt, Ester Boix, Giuseppe D'Alessio, Junichiro Futami, Jurgen Krauss, Massimo Libonati, Alvaro Martinezdel- Pozo, Ronald T. Raines, Marc Ribo, Helene F. Rosenberg, and Susanna M. Rybak

The eosinophil-derived neurotoxin (EDN, also known as eosinophil protein-X) is best-known as one of the four major proteins found in the large specific granules of human eosinophilic leukocytes. Although it was named for its discovery and initial characterization as a neurotoxin, it is also expressed constitutively in human liver tissue and its expression can be induced in macrophages by proinflammatory stimuli. EDN and its divergent orthologs in rodents have ribonuclease activity, and are members of the extensive RNase A superfamily, although the relationship between the characterized physiologic functions and enzymatic activity remains poorly understood. Recent explorations into potential physiologic functions for EDN have provided us with some insights into its role in antiviral host defense, as a chemoattractant for human dendritic cells, and most recently, as an endogenous ligand for toll-like receptor (TLR)2.

The eosinophil cationic protein (ECP) is a secretory ribonuclease, which is found in the eosinophilic leukocyte and involved in the innate immune system. Its cytotoxic activity is effective against a wide range of pathogens, suggesting a relatively non-specific mechanism of action. We review here the specific antipathogen activities that have been characterized for ECP. Although eosinophils and ECP are primarily associated with the host defense against nonphagocytosable pathogens, such as helminthic parasites, ECP has also an antibacterial activity, which is not shared by the other, closelyrelated eosinophil ribonuclease, the eosinophil derived neurotoxin (EDN). Although there is no evidence for direct involvement in vivo of eosinophils in the host response to bacterial infection, ECP is active against both Gram-negative and Gram-positive bacterial strains and its mechanism depends on its action both at the bacterial cell wall and cytoplasmic membrane levels. Other antipathogen activities, including antihelminthic activity, are also discussed. Modulation of the protein activity by posttranslational modifications and the currently identified polymorphisms are reviewed. Antimicrobial RNases, as innate immune proteins with anti-infective and immunomodulatory properties, present substantial therapeutic potential in the drug development industry, both in the search of alternative antibiotics and for the treatment of inflammatory disorders.

Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin-ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of high-resolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the mid-term future.

By virtue of their RNA degrading catalytic activity, ribonucleases are potentially cytotoxic. For the application of these enzymes as therapeutics, however, they have to overcome several obstacles whose interplay is not yet fully understood. Ribonucleases with a basic pI are not only able to interact with the (negatively charged) cellular membrane but they are also distinctively selective for tumor cells. After the (endocytotic) uptake into the cell and release into the cytosol from the endosomes where they have to resist the attack by proteases, they face the cytosolic ribonuclease inhibitor. Only if they are able to evade the tight binding to the inhibitor (or if enough ribonuclease molecules enter the cell to neutralize the inhibitor protein) they are able to attack their target RNA, for which a sufficient ribonucleolytic activity is indispensable. Each of these steps can turn into an insurmountable hurdle spoiling the cytotoxic potential of these enzymes. In the present review I will summarize the status quo of the knowledge on the mechanisms and their interdependence as well as to develop strategies to overcome possible limitations.

In addition to their ribonucleolytic activity, several ribonucleases (RNases) play important roles in other specific biological activities, such as dendritic cell activation, certain pollen-induced allergies, blood vessel formation and defense against parasitic or microbial infections. Among these diverse actions, cytotoxic activity, which relies in most cases on ribonucleolytic activity, has attracted a considerable attention because of the potential for using RNases as therapeutic agents for the treatment of different malignancies. In addition to use naturally existing RNases, major efforts have been made in the development of engineered variants, which display more potent cytotoxic activity and greater selectivity for malignant cells. This review focuses on the molecular and cellular aspects of the internalization, intracellular trafficking and final sorting of cytotoxic RNases. Knowledge about the strategies used by these promising toxins provides us with essential information about the mechanisms that can be used to gain access to different subcellular compartments and intracellular sorting.

The cytotoxic properties of naturally occurring or engineered RNases correlate well with their efficiency of cellular internalization and digestion level of cellular RNA. Cationized RNases are considered to adsorb to the anionic cellular surface by Coulombic interactions, and then become efficiently internalized into cells by an endocytosis-like pathway. The design of cytotoxic RNases by chemical modification of surface carboxylic residues is one of the powerful strategies for enhancing cellular internalization and is accompanied with a decreased sensitivity for the cytoplasmic RNase inhibitor. Although chemically modified cationized RNases showed decreased ribonucleolytic activity, improved endocytosis and decreased affinity to the endogenous RNase inhibitor conclusively contribute to their ability to digest cellular RNA. Furthermore, the cytotoxicity of cationized RNases can be drastically enhanced by co-endocytosis with an endosomedestabilizing peptide. Since efficient cellular internalization of proteins into living cells is an important technology for biotechnology, studies concerning the design of cytotoxic RNases provided general perceptions for protein-based drug design.

Onconase® (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a ribonuclease. Investigations into the mechanism of ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most striking similarity between ONC and bovine seminal ribonuclease, another naturally cytotoxic ribonuclease, is their insensitivity to inhibition by the potent cytosolic ribonuclease inhibitor protein (RI). RI typically binds to its ribonuclease ligands with femtomolar affinity—an extraordinary feat considering the lack of sequence identity among the bound ribonucleases. Mammalian ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more desirable chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RI ribonuclease complexes and progress towards the development of mammalian ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.

After a short introduction with some examples of cytotoxic ribonucleases, the importance of natural or artificial dimerization (oligomerization) as a way for a ribonuclease to acquire novel functional properties has been pointed out. In particular, the role of the three dimensional domain swapping mechanism in bovine pancreatic ribonuclease A oligomerization, as well as its impact for the acquisition of novel biological functions (among which a remarkable antitumor action) by the enzyme protein in oligomeric form have been discussed. Finally, the structural and functional features that could explain why oligomeric ribonuclease A becomes able to display a cytotoxic activity, and the possible use and limits of the three dimensional domain-swapped oligomers of ribonuclease A as anticancer therapeutic agents have been described and discussed.

Immunotoxins are chimeric molecules that specifically target tumor cells, as they are made up of toxins linked to an antibody directed to a specific, cell-surface tumor-associated-antigen (TAA). When the immune moiety is internalized by the tumor cell, it will carry the conjugated toxin into the cell, so that the cell will be selectively killed in a way postulated more than a hundred years ago by Paul Ehrlich, the first author to use the term magic bullet. To date, toxicity and immunogenicity have complicated the clinical use of most immunotoxins. More recently, based on the immunotoxin principle, immunoRNases have been proposed, in which the toxin moiety of immunotoxins is replaced by a non-toxic RNase. An immunoRNase (IR) is in fact an immuno-pro-toxin, as it can travel in the bloodstream without any damages to cells devoid of the targeted TAA, while magically selecting the cells targeted by the immune moiety. Once internalized, the RNase moiety will exert its RNA degrading activity, which will readily lead to the death of the targeted cell. By choosing a human RNase, and a human antibody fragment as immune moiety, an IR would be not only non-toxic, but also non-immunogenic. As for the possible inhibitory action of the cytosolic RNase inhibitor, exerted on all non-toxic vertebrate RNases, it can be opposed by flooding the cytosol with high levels of IR, which will neutralize the RNase inhibitor, or by using RNases resistant to the inhibitor.

Rana pipiens oocytes contain two homologues of pancreatic ribonuclease A that are cytostatic and cytotoxic to human cancer cells. Extensively studied Onconase is in advanced Phase IIIb clinical trials against malignant mesothelioma, while Amphinase is a novel enzyme in pre-clinical development. Onconase is the smallest (104 amino acid residues) member of the ribonuclease A superfamily while Amphinase (114 residues) is the largest among amphibian ribonucleases. Both enzymes share the characteristic frog ribonucleases C-terminal disulfide bond but another signature of this group, the N-terminal pyroglutamate, an integral part of Onconase active site is not conserved in Amphinase. Although Onconase and Amphinase are weak catalysts their enzymatic activities are required for cytostatic and cytotoxic activity. While it was postulated that tRNA is the primary substrate of Onconase in vivo there is also extensive indirect evidence that suggests other RNA species, in particular micro RNAs, may actually be the critical target of these ribonucleases. The cytostatic effects of Onconase and Amphinase are manifested as cell arrest in the G1 cell cycle phase. Apoptosis then follows involving activation of endonucleases(s), caspases, serine proteases and transglutaminase. Onconase was shown to be strongly synergistic when combined with numerous other antitumor modalities. Onconase and Amphinase are highly cationic molecules and their preferential toxicity towards cancer cells (having distinctly higher negative charge compared to normal cells) may depend on increased binding efficiency to the cell surface by electrostatic interactions. Here we will discuss the structures of Onconase and Amphinase and the molecular basis for their enzymatic and anticancer functions.

Onconase, a member of the pancreatic ribonuclease A superfamily, is currently in Phase III clinical trials for treatment of unresectable malignant mesothelioma. The anticancer effect of onconase may relate to its intracellular target, a non-coding RNA. Some non- coding RNAs are aberrantly expressed in cancer cells. This discovery is creating new interest in drugs that target RNA. Conjugating onconase to agents that recognize tumor associated molecules further increases its potency and specificity. Analysis of onconase activity when directed to two different internalizing and one noninternalizing receptor reveals that the ideal targeting agents would rapidly enter lysosomal compartments before onconase escaped to the cytosol. Antibody-onconase conjugates are effective in preclinical models, cause little non-specific toxicities in mice and have favorable formulation properties. Understanding the reason for their potency coupled with understanding novel RNA-based mechanisms of tumor cell death will lead to improved variations of targeted onconase.

Ribonucleases (RNases) of the superfamily A exhibit potent antineoplastic activity yet do not mediate appreciable immunogenicity or non-specific toxicity in both animal models and cancer patients. Ranpirnase (Onconase®), the first ribonuclease being evaluated as a therapeutic in humans, has progressed to phase III clinical trials in patients with unresectable mesothelioma. Conjugation of RNases to internalizing tumor-targeting monoclonal antibodies was shown to enhance specific cell killing by several orders of magnitude both in vitro and in animal models. In this review we describe the development and current status of genetically engineered 2nd generation immunoRNases as promising novel anticancer therapeutics.