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Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity

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Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity Research Article Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity Bruce Acres,1 Murielle Gantzer,2 Christelle Remy,3 Nicolas Futin,3 Nathalie Accart,3 Olivier Chaloin,4 Johan Hoebeke,4 Jean-Marc Balloul,2 and Ste´phane Paul2 1Division of Medical and Regulatory Affairs, 2Molecular Immunology Laboratory, 3Histology and Animal Facilities Laboratory, Transgene SA; and 4Immunologie et Chimie The´rapeutiques,Centre National de la Recherche Scientifique/Institut de Biologie Mole´cullaireet Cellulaire, Strasbourg, France Abstract In many of these studies, the relative level of each cytokine was To redress the immune imbalances created by pathologies such important. Synergy studies between IL-12 and other cytokines for as cancer, it would be beneficial to create novel cytokine the generation of antitumor responses in mice showed mixed molecules, which combine desired cytokine activities with results. Whereas the addition of IL-12 in the presence of reduced toxicities. Due to their divergent but complementary suboptimal amounts of IL-2 led to synergy in the induction, g activities, it is of interest to combine interleukin-2 (IL-2) and IL- proliferation, cytolytic activity, and IFN induction, combinations 18 into one recombinant molecule for immunotherapy. Eva- of IL-2 and IL-12 using a higher dose of one cytokine were found to luation of a fusokine protein that combines murine IL-2/IL-18 be antagonistic and to enhance toxicity (14, 15). These results may shows that it is stable, maintains IL-2 and IL-18 bioactivities, reflect the inherent difficulty of combining two potentially has notably reduced IL-2 associated toxicities, and has a novel synergistic cytokines in vivo, especially when there is a need to lymphocyte-stimulating activity. An adeno-viral expression maintain a fixed ratio of activities of two components with system was used to explore the biology of this ‘‘fusokine’’. different pharmacologic properties, such as differences in their Inclusion of the IL-18 prosequence (proIL-18) increases the circulating half-life and biodistribution. expression, secretion, and potency of this fusokine. In vivo gene To reduce the difficulties associated with cytokine combinations, transfer experiments show that Ad-IL-2/proIL-18 dramatically we undertook the genetic fusion of cytokines in an effort to enhance outdoes Ad-IL-2, Ad-proIL-18, or the combination of both, by the immune response, with special focus on the combination of a inducing high rates of tumor rejection in several murine specific (adaptive) with a nonspecific (innate) immune response in a models. Both innate and adaptive effector mechanisms are re- host organism without increasing cytokine associated toxicities. The quired for this antitumor activity. (Cancer Res 2005; 65(20): 9536-46) resulting cytokine-mediated immune stimulation may be useful for reversing immunosuppression or anergy mechanisms induced by pathogens or cancer cells. In this report, we focus mainly on the Introduction synergizing cytokine combination IL-2/IL-18. IL-2 is an important Numerous lines of evidence indicate that tumors, as well as growth and survival factor for T lymphocytes but also sensitizes various pathogens, can escape immune detection and/or elimi- these cells to Fas-mediated activation-induced cell death (AICD; nation (1, 2). Down-regulation of MHC expression as well as the refs. 16, 17). It has been described recently that AICD limits effector activation of immunosuppressive cells and molecules can dampen function of CD4 tumor-specific T cells and decreases T-cell effector the vigor of immune responses to antigens or can activate apoptosis activity (18). IL-2 is also known to be critically required for the of immune effector cells (2, 3). Recent evidence suggests that T and activation of CD4+CD25+ T-cell suppressor function (16). Although natural killer (NK) cells from tumor-bearing patients exhibit IL-2 therapy has yielded encouraging results in the treatment of abnormalities in signal transduction that render them unresponsive certain types of cancer, its use is limited by dose-dependent toxicity to appropriate activation signals (2, 4). Some of these signaling characterized by weight gain, dyspnea, ascites, and pulmonary defects can be reversed by cytokines (5, 6). edema (16). These signs of toxicity result from increased capillary Recently, studies in mouse tumor models and in patients have leak, also known as vascular leak syndrome (19). Modifications of the shown the importance of cytokine combinations in the development IL-2 molecule that maintain its well-known IL-2 biological activities of optimal immune responses (5, 7, 8). A clear synergy between but which reduce toxicities would be beneficial. IL-18 is a more interleukin-2 (IL-2) and IL-12 was first described in a reportedly recently described cytokine with properties associated with poorly immunogenic tumor (MCA205) after i.t. administration using activation of innate immunity and stimulation of IFNg secretion adenoviral vectors (9). The combination of IL-2 or IL-12 and IL-18 (20). Similar to IL-1, IL-18 is produced in a precursor form that is synergistically enhanced IFNg production and NK cell activation cleaved in the cell cytoplasm to mature IL-18 and is then secreted. both in vitro and in vivo and synergized in vivo to induce complete We produced a series of fusion proteins linking IL-2 and IL-18 and durable regression of well-established 3LL tumors in a large number expressed them in E1- and E3-deleted adenovirus-5 as a convenient of treated mice (10–13). expression system to explore the biological activities of fusion cytokines (fusokines) in vitro as well as in vivo. This report focuses on the fusokine murine IL-2 (mIL-2)/IL-18 prosequence (proIL-18) Competing interests statement. The other authors declare that they have no in which mIL-2 is fused to mproIL-18. This multifunctional cytokine competing financial interests. Requests for reprints: Stephane Paul, Molecular Immunology Laboratory, induces a high rate of tumor rejection after i.t. delivery in various Transgene SA, 11 rue de Holsheim, 67082, Strasbourg, France. E-mail: paul@ animal models, using an encoding adenoviral vector. Further transgene.fr. I2005 American Association for Cancer Research. evidence indicates immunostimulation and very limited toxicity doi:10.1158/0008-5472.CAN-05-0691 both in vitro and in vivo. Cancer Res 2005; 65: (20). October 15, 2005 9536 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Fusokine IL-2/IL-18 Immunostimulation Materials and Methods (TIB-239, ATCC), and the murine 2B4.11 T-cell hybridoma (26) were grown at 37jC in DMEM (Life Technologies, Gaithersburg, MD) supplemented Cloning and Construction of Fusokine cDNAs with 10% FCS and antibiotics. The 2B4.11 T-cell hybridoma was kindly Splenocytes from C57Bl6 mice were harvested and stimulated for 3 days provided by Prof. D. Ganea (Newark, NJ). with a mix of concanavalin A (ConA, 10 Ag/mL, Sigma-Aldrich, Lyon, P815 murine mastocytoma (DBA/2; FcR+,H2Dd, MHCI+, ICAM1+, France) and mIL-2 (10 IU/mL, R&D Systems, Minneapolis, MN) or lipopoly- and CD48+) and B16F10 murine melanoma (C57Bl/6; H2Db, MHCIÀ, saccharide (10 Ag/mL, Sigma) and murine granulocyte macrophage colony- MHCIIÀ, ICAM1À, and CD48À) were obtained from ATCC (TIB-64 and stimulating factor (GM-CSF, 50 IU/mL, R&D Systems, United Kingdom). CRL-6475, respectively). The TC1 murine tumor cell line cell line was mRNA from activated splenocytes were then extracted with RNA Now generously provided by Dr. T.C. Wu (The Johns Hopkins University School (Ozyme, Saint Quentin en Yvelines, France). mIL-2 and IL-18 cDNAs were of Medicine) has been described previously (27). All cell lines were tested amplified by reverse transcription-PCR (RT-PCR; Platinum Quantitative negative for Mycoplasma using Hoechst dye, cell culture, and PCR. All have RT-PCR, Thermoscript one-step system, Invitrogen, Cergy Pontoise, France) tested negative for murine pathogens and are acceptable for use in a using specific oligonucleotides. The mutated form of mIL-18 (K89A) was specific pathogen-free animal environment. produced using QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA; ref. 21). Two forms of mIL-18 cDNA have been used for the Antibodies and Cytokines fusion molecules, encoding the precursor proIL-18 and the mature mIL-18 Biotin-labeled anti-mIL-2 and anti-mIFNg were purchased from R&D (devoid of the prosequence). Systems (United Kingdom). Biotin-labeled anti-mIL-18 was purchased from As described in Fig. 1, once amplified by RT-PCR, the sequences encoding Peprotech, Inc. (Rocky Hill, NJ). Biotin-labeled anti-goat IgG or anti-rabbit the two cytokine moieties (X and Y) were cloned in frame by PCR with IgG were purchased from Amersham Biosciences (Orsay, France). 2 3 PerCP-CY5.5, FITC, or phycoerythrine-labeled rat anti-mouse CD4, CD8, a flexible linker (G4S) or (G4S) inserted between them (22). CD3, CD25, CD31, CD69, MAC1, CD11c, H-2Kb/Db,Iab, NK-1.1, and NK-T/ Adenovirus Production and Titration NK (CD3/NK1.1) cell antigen or unconjugated rat anti-mouse CD4 and CD8 The sequence encoding each fusion protein was inserted in an adenoviral were used as defined by the manufacturer (PharMingen, San Diego, CA). shuttle plasmid containing a cytomegalovirus (CMV)–driven expression Unconjugated rabbit anti-human CD3 (which cross-reacts with mouse cassette surrounded by adenoviral sequences (adenoviral nucleotides 1-458 CD3) or rabbit anti-rat IgG and peroxidase-labeled goat anti-rabbit were and nucleotides 3328-5788, respectively) to allow generation of the vector used at concentrations suggested by DakoCytomation (Glostrup, Denmark). genome by homologous recombination (23). The resulting adenoviral T cell apoptosis (AICD) was measured using the Annexin V-FITC apoptosis vectors are E3-deleted (nucleotides 28592-30470) and E1-deleted (nucleo- detection kit (PharMingen).
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