Sea anemone cytolysins as toxic components of immunotoxins Mayra Tejuca a, * , Gregor Anderluh b , Mauro Dalla Serra c a Centro de Estudios de Proteı ´nas y Departamento de Bioquı ´mica, Facultad de Biologia, Universidad de La Habana, Calle 25 #455 e/ J e I, Vedado, Ciudad de La Habana, Cuba b Department of Biology, Biotechnical Faculty, University of Ljubljana, Vec ˇna pot 111, 1000 Ljubljana, Slovenia c Fondazione Bruno Kessler-Consiglio Nazionale delle Ricerche-Istituto di Biofisica, Via alla Cascata 56/C, 38100 Povo (TN), Italy article info Article history: Available online 5 March 2009 Keywords: Pore-forming toxin Sea anemone cytolysin Actinoporin Immunotoxin Cancer abstract The use of membrane active toxins as toxic moieties in the construction of immunotoxins (ITs) is an attractive alternative to overcome some of the problems of classical ITs since these new conjugates are based in the use of a different mechanism of killing undesired cells. Pore-forming cytolysins from sea anemones were used in the construction of ITs targeted to different cell types including tumour cell lines and the parasite Giardia duo- denalis. The results obtained support the feasibility of directing these cytolysins to the surface of the cancer cells or the parasite through their conjugation to monoclonal anti- bodies recognizing tumour-associated or parasite antigens, respectively. However the main problem with the IT constructed in this fashion is the lack of specificity associated with the toxin moiety. An approach designed to overcome this limitation was the construction of inactive cytolysin with built-in biological ‘‘trigger’’ that renders the toxin active in the presence of tumour-specific proteinases. This construction is considered as a proof of concept to demonstrate the feasibility of such activation systems in the construction of ITs based on pore-forming cytolysins from sea anemones with reduced unspecific activity. The future prospects of the use of the N-terminal region of actinoporins for construction of IT is also described. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Immunotoxins (ITs) are chimeric molecules in which a cell binding ligand, such as a monoclonal antibody (mAb) or a growth factor are coupled to a killer toxin in order to address its activity towards a specific undesired cell. The target can be tumour cells, virally infected cells, subsets of normal cells or parasite cells. In fact the use of ITs in cancer therapy, graft-vs-host disease, autoimmune diseases, and AIDS has been ongoing for the past three decades. Most of the ITs currently in use or under investigation contain a toxic component, which acts intracellularly to inactivate protein synthesis. Once in the cytosol, a single molecule is capable of killing a cell, making ITs some of the most potent known killing agents. Among the most active clinically are those targeted to tumours, specifically those directed against haematological tumours. Obstacles to successful treatment of solid tumours include poor penetration into tumour masses, toxicity and the immune response to the toxin component. Exploring other classes of toxin to create new ITs is underway to improve the treatment of cancer. Relatively few membrane-acting toxins have been investi- gated for their use in immunoconjugates, in spite of some inherent advantages to their use. Pore-forming cytolysins from sea anemones, actino- porins, are one of the most potent groups of pore-forming toxins (PFTs), and they constitute an attractive target for exploitation in the construction of IT. In this review we summarize the main results obtained in the use of sea anemone PFTs in the construction of ITs. We also discuss the biochemical and crystallographic studies that have provided new information on the structure and function of * Corresponding author. Tel.: þ53 7 8321321; fax: þ53 7 8735774. E-mail address: tejuca@fbio.uh.cu (M. Tejuca). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.02.025 Toxicon 54 (2009) 1206–1214