Anchoring Cytokines to Tumor Cells for the Preparation of Anticancer Vaccines Without Gene Transfection in Mice *Philippe Nizard, †David Alexandre Gross, *Aurélie Babon, *Alexandre Chenal, ‡Bruno Beaumelle, †Konstadinos Kosmatopoulos, and *Daniel Gillet *Département d’Ingénierie et d’Etudes des Protéines, CEA-Saclay, †INSERM U487, Institut Gustave Roussy; and ‡UMR 5539 CNRS, Université Montpellier II, France. Summary: The authors have investigated a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This method may offer an alternative to gene therapy approaches. It consists in anchoring recombinant cytokines to the cell membrane. Attachment is mediated by the transmembrane domain of diphtheria toxin (T) genetically fused to the cytokine and is triggered by an acid pH pulse. The authors found that the fusion protein T-hIL-2 anchored to the surface of tumor cells retained its IL-2 activity while remaining exposed for several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective antitumor immunity medi- ated by tumor-specific cytotoxic T lymphocytes. This procedure presents several ad- vantages as compared with the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and the selection of transfected clones, it eliminates the safety problems connected with viral vectors, and it allows the control of the amount of cytokines delivered with the vaccine. Key Words: Cancer vaccine—Interleukin-2—Diphtheria toxin—Membrane anchor—Transmembrane domain. One of the main strategies developed for the prepara- tion of anticancer vaccines is based on the use of trans- fected tumor cells expressing cytokines or costimulatory molecules (1). Their injection in animals gives highly promising results, and clinical trials in humans have be- gun (2–6). However, the production of genetically modi- fied tumor cells in a clinical context suffers from several drawbacks; for example, some tumor cells are difficult to transfect, the selection of stable transfectants requires several days of costly cell culture procedures incompat- ible with a routine treatment of many patients, the use of viruses for gene transfer may still be hazardous at the moment (7–9), and the amount of recombinant proteins produced by the transfected tumor cells varies dramati- cally from cell to cell, requiring the selection of clones or the use of tunable promotors for proper dosage of the cytokine delivered with the vaccine (1,10). In an attempt to overcome these problems, we have investigated a new way of combining cytokines with tumor cells for the production of antitumor vaccines. This method takes advantage from the membrane bind- ing properties of the diphtheria toxin transmembrane do- main (T). T is a nontoxic 22kD protein domain (11), which is soluble at neutral pH and which penetrates into lipid membranes at pH 5 (12). Within the whole toxin, the function of the T domain is to assist the translocation of the catalytic domain through the membrane of the early endosome after the toxin has been internalized by receptor mediated endocytosis (13). We have shown pre- viously that the T domain could be used as a pH- triggered membrane anchor for soluble proteins geneti- cally fused at its N- or C-terminus (14–16). We now have studied whether this anchoring system could be used to attach recombinant cytokines to tumor Received May 28, 2002; accepted September 16, 2002. Address correspondence and reprint requests to Daniel Gillet, Dé- partement d’Ingénierie et d’Etudes des Protéines, bat 152, CEA-Saclay, 91191 Gif sur Yvette cedex, France; e-mail: daniel.gillet@cea.fr Journal of Immunotherapy 26(1):63–71 © 2003 Lippincott Williams & Wilkins, Inc., Philadelphia 63