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

Research Article

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

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). Recombinant murine IFNg and IL-2 were tides 459-3327), with the E1 region replaced by the expression cassette purchased from R&D Systems (United Kingdom). Recombinant mIL-18 containing, from 5V to 3V, the CMV immediate-early enhancer/promoter, a (rmIL-18) was purchased from Peprotech. ConA was purchased from Sigma chimeric human h-globin/IgG intron, the sequence encoding the fusion and used at 1 Ag/mL. protein, and the SV40 late polyadenylation signal. The recombinant adenoviruses were generated by transfecting the PacI-linearized viral Analysis of Fusokine Expression A549 cells were infected in suspension with adenoviral vectors, as genomes into the PER C6 complementation cell line (24). Virus propagation, previously described, at a multiplicity of infection (MOI) of 10 (30-minute purification, and titration were accomplished as described previously (25). incubation of cells with virus dilutions in 100 AL of PBS supplemented with Negative control virus, referred to as ‘‘empty vector,’’ is the same virus as 2% FCS, 1% cations; ref. 25). Cells were then cultured in complete medium described above, with no transgene. containing 5% FCS for 72 hours. Supernatants and cell extracts were Cell Culture collected then analyzed by Western blot on 4% to 12% Nupage gel (Novex, Human pulmonary carcinoma cell line A549 (CCL-185; American Type Invitrogen). Expression of individual cytokines constituting each fusion Culture Collection, ATCC, Manassas, VA), the 2E8 murine lymphoblast protein was analyzed by Western blot according to the enhanced

Figure 1. Construction and expression of murine fusokines. A, schematic representation of recombinant adenovirus. All vectors are derived from human adenovirus type 5 deleted in the E1 region (nucleotides 459-3327) and in the E3 region (nucleotides 28, 592-30, 470). The E1 region is replaced by an expression cassette containing the different transgenes. Abbreviations: CMVpro, CMV promoter; IVS, chimeric human h-globin/IgG intron; IRES, internal ribosomal entry site; pA, SV40 late polyadenylation signal. Fusokines were constructed by double PCR to incorporate, 3 in frame, a synthetic flexible linker (G4S) between the two cytokines. Determination of the fusokine concentration. B, ELISA quantification of mIL-2 and mIL-18 on 72-hour supernatants of A549 infected cells based on (C) standard curves of ELISA analysis of IL-2/proIL-18* with rabbit anti-mIL-2 (C.1) and rabbit anti-mIL-18 (C.2).

www.aacrjournals.org 9537 Cancer Res 2005; 65: (20). October 15, 2005

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Cancer Research chemiluminescence Western blotting protocol provided by Amersham Life 120 days. In vivo depletion of CD4, CD8, and NK cells were made as Sciences using specific anti-mouse cytokine antibodies. previously described (32). The statistical difference in in vivo survival Fusokine concentrations were estimated by ELISA immunoassay. Briefly, experiment between the different groups was assessed using Fischer exact dilution of fusokine-containing supernatants were coated on a Maxisorp application (Statistica 5.1 software, Statsoft, Inc., Tulsa, OK) of the Kaplan- 96-well plate (Nalge Nunc, Rochester, NY) overnight at 4jC. Cytokines and Meier survival curves. P V 0.05 is considered statistically significant. fusokine were then revealed with purified polyclonal rabbit anti-mouse IL-2 or IL-18 (Biovision Inc., Mountain View, CA). Rabbit IgG was then revealed Immunohistochemistry, Immunofluorimetry, and Flow with a specific monoclonal anti-rabbit IgG conjugated with HRPO (The Cytometry Analysis Jackson Laboratory, Bar Harbor, ME). Wells coated with serial dilutions of Tumors were established and injected with the various viruses as rmIL-2 or mIL-18, in tissue culture medium, were used as positive control described above for in vivo experiments. On day 13, tumors were measured (R&D Systems, Minneapolis, MN) to generate standard curves for the and excised. Tumor draining lymph nodes were also removed at the same estimations of fusokine concentrations. time. For flow cytometry analysis, tumors were disrupted into a single-cell suspension using collagenase (Sigma) digestion, cells were stained with the In vitro Biological Activity of Fusokine mIL-2/proIL-18* indicated antibodies, and analyzed by flow cytometry as previously T-cell proliferation assay. Mouse spleen cell proliferation was assessed described (33). 3 by the uptake of [ H]-thymidine. Splenocytes were coactivated with (20 ng/ P815 tumor tissues were removed and directly embedded in ornithine mL) murine CD3-specific antibody (145-2C11, PharMingen) as previously carbamyl transferase compound on isopentane cooled on dried ice. Five- described (28). Splenocytes were mixed with the fusokine-containing micrometer sections were used for H&E staining (structural observations A549 supernatants and anti-CD3 antibody. As a positive control, spleen by light microscopy) or for immunohistochemistry. Infiltrating cells and 4 cells (5 Â 10 cells per well) were stimulated in complete medium with blood vessels detection were done on methanol/acetone-fixed cryosec- either ConA (10 Ag/mL) or 100 ng/mL rmIL-2. After 96 hours, the cells were tions using the following antibodies: rat anti-mouse CD4 (PharMingen), 3 pulsed with 1 ACi per well [ H]-thymidine (Amersham Biosciences). rat anti-mouse CD8 (PharMingen), rabbit anti-human CD3 (DAKO), 3 Incorporation of [ H]-thymidine into the DNA of proliferating T cells was hamster anti-mouse CD11c (PharMingen), rat anti-mouse Ia-Ie (PharMin- measured by harvesting cellular DNA onto glass filter paper (PHD gen), rat anti-mouse CD25-FITC (PharMingen), goat anti-mouse IL18-R Harvester, Cambridge Technology, Watertown, MA) after 4 hours and by (R&D Systems), and rabbit anti-human von Willebrand factor (DAKO). counting the radioactivity in a liquid scintillation counter (Beckman Primary antibodies were revealed by specific secondary antibodies rabbit Coulter, Roissy CDG, France). All measurements were in triplicate. anti-rat immunoglobulin (DAKO), rabbit anti-hamster immunoglobulin IFN; secretion assay. The relative bioactivity of mIL-18 was determined (Rockland, Gilbertsville, PA), horse anti-goat biotinylated 0.5% (Vectastain by the ability of the fusokine contained in supernatants from Ad-cytokine or Elite PK6200, Vector Labs, Burlingame, CA), or rabbit anti-FITC HRP Ad-fusokine infected A549 cells to augment IFNg production in vitro (29). In (P0404, DAKO) coupled. Horseradish peroxidase (HRP)–labeled polymer 6 brief, mouse splenocytes (10 cells/mL) were cocultured with or without conjugated with the second rabbit antibody (EnVision + System, DAKO) ConA (1.25 Ag/mL) in 24-well plated for 48 hours. Ad-fusokine supernatants or Streptavidin-HRP (Vector Labs) was applied, and 3,3V-diaminobenzidine were added to cell suspensions of ConA-primed, or unprimed, splenocytes in was used as substrate. All slides were counterstained with hematoxylin. 96-well plates for 24 hours. The supernatants were collected and assayed by ELISA to detect IFNg production (Quantikine, R&D Systems, Minneapolis, MN). Activation-Induced Cell Death Assay CTL and natural killer cell cytotoxicity assays. Activities of the AICD, in which signals normally associated with lymphocyte stimulation fusokine were also assayed for CTL and NK cytotoxicity as previously instead result in the demise of the cell, has been proposed as a mechanism described (30). Mouse C57Bl/6 splenocytes were cocultured with Ad- of deletion of antigen-specific lymphocytes. T cells can be sensitive or fusokine- or Ad-cytokine-containing supernatants obtained from A549- resistant to AICD, and IL-2 can regulate the susceptibility of T cells to AICD infected cells for 8 days. The cytotoxic activities of primed splenocytes were (16–18). AICD can be characterized by the de novo synthesis of Annexin V, measured on P815-CTL target or YAC-NK target using the EuDTPA Fas (CD95), and its ligand (FasL; refs. 16–18). AICD was measured in vitro cytotoxicity assay (Wallac Lab, Turku, Finland; ref. 31). on 2B4.11 murine T-cell hybridoma and in vivo after s.c. injection of Mouse cytokine antibody array. Mouse C57Bl/6 splenocytes (106 cells/ adenoviruses encoding fusokine. In brief, C57BL/6 mice were injected s.c. mL) were cultured with 20 ng/mL of mIL-2/proIL-18* fusokine for 72 hours. once with 2 Â 108 IU of Ad-fusokine (or as a control with an adenovirus Mouse cytokine antibody array III was purchased from RayBiotech, Inc. encoding mIL-2 or an empty adenovirus). Draining lymph nodes were then (Norcross, GA) and used according to the manufacturer’s instructions to removed at 8 hours after injection. AICD was measured by flow cytometry detect supernatant cytokine content. analysis using a phycoerythrin-labeled mouse anti-mouse FasL-specific Immunostimulation in vitro. To analyze the in vitro effect of the antibody (PharMingen) and an FITC-labeled Annexin V Apoptosis fusokine, bone marrow–derived dendritic cells or splenocytes were Detection kit (PharMingen). incubated with fusokine-containing supernatants for 3 to 7 days. Phenotypic markers of maturation and/or activation of dendritic cells, other antigen- Quantification of Vascular Leak Syndrome Assay presenting cells (APC), B, T (CD4 and CD8), NK, and NKT cells were Vascular leak was studied by measuring the extravasation of Evans blue analyzed using mouse-specific antibodies by flow cytometry analysis which, when given i.v., binds to plasma proteins, particularly albumin, and following extravasation can be detected in various organs as described (19, 34). (FACScan, BD Biosciences, San Jose, CA). Â 9 Statistical analyses were done using the Student’s t test. P < 0.05 was Vascular leak was induced by injecting s.c. 2 10 IU of mIL-2-encoding considered statistically significant. All experiments were run in triplicate adenoviral vector once per day for 3 days. Groups of five C57Bl/6 mice were injected s.c. with PBS, empty adenovirus, Ad-mIL-2, Ad-mIL-2 + Ad-mproIL- and the results are represented as the mean F SD of triplicate determinations or, where indicated, representative data of three indepen- 18, or Ad-fusokine mIL-2/proIL-18*. On day 4, mice were injected i.v. with dent experiments. 0.1 mL of 1% Evans blue in PBS. After 2 hours, the mice were bled to death under anesthesia, and the heart was perfused with heparin in PBS. The In vivo Experiments lungs and liver, where maximum extravasation is known to occur, were Murine P815, B16F10, and TC1 tumor cells were trypsinized, washed, and harvested, and placed in formamide at 37jC overnight. The Evans blue in resuspended in PBS at 3 Â 106 cells/mL. One hundred microliters of the cell the organs was quantified by measuring the absorbance of the supernatant suspension were then injected s.c. into the right flank of 6- to 7-week-old at 650 nm. The vascular leak syndrome seen in Ad-cytokine-treated mice immunocompetent B6D2 mice. At days 7, 8, and 9 after injection, when was expressed as the percent increase in extravasation compared with that tumors were palpable, the mice received three i.t. injections of 5 Â 108 IU of in PBS-treated controls. For histopathologic studies, groups of five separate Ad fusion or Ad controls diluted in 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L mice were injected with empty Ad or PBS, Ad-mIL-2, Ad-mIL-2/proIL-18*,

MgCl2. Tumors size and survival rate were then evaluated for the following and Ad-mIL-2/IL-18* as described earlier, and on day 4, lungs and liver were

Cancer Res 2005; 65: (20). October 15, 2005 9538 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Fusokine IL-2/IL-18 Immunostimulation fixed in 10% formalin solution. The organs were embedded in paraffin, mproIL-18*) or with an adenovirus expressing the fusokine mIL-2/ sectioned, and stained with H&E. Perivascular infiltration was scaled by proIL-18* (Fig. 2A). Supernatants obtained after 72 hours of counting the number of lymphocytes infiltrating the vessel and averaging adenoviral infection were analyzed. Unexpectedly, IL-2 was the minimum and maximum range for each group. Sera from injected mice observed to be more stable when expressed as the mIL2/proIL- were also collected for ASAT and ALAT measurement. 18* fusokine compared with the cytokine alone or to the Biosensor Experiments combination of the two cytokines (Fig. 2A). This could, at least The BIACORE 3000 system, sensor chip CM5, surfactant P20, amine- in part, explain the accumulation of higher levels of prosequence- coupling kit containing N-hydroxysuccinimide and N-ethyl-NV-dimethyla- containing fusokines in supernatants as measured by ELISA and minopropyl carbodiimide, were from BIACORE AB (Uppsala, Sweden). shown in Fig. 1. More of the mproIL-18* portion was also observed All biosensor assays were done with HBS-EP as running buffer [10 mmol/L after infection with Ad-mIL-2/proIL-18* (Fig. 2B) than with the HEPES, 150 mmol/L sodium chloride, 3 mmol/L EDTA, 0.005% surfactant Ad-mproIL-18* construct. As expected, infection with Ad-fusokine P20 (pH 7.4)]. Flow cells were precoated with a goat anti-human IgG (Fcg fragment specific, Jackson ImmunoResearch, West Grove, PA) using amine maintains a fixed ratio of both mIL-2 and mproIL-18* in contrast to coupling at 30 Ag/mL in acetate buffer (10 mmol/L, pH 5.5) according the combination of Ad-mIL-2 + Ad-mproIL-18* where relative to manufacturer conditions. The chip was then blocked with 1 mol/L expression was variable. A549 cells were used for these in vitro ethanolamine hydrochloride (pH 8.5, BIACORE) and washed abundantly to experiments, because these cells represent a convenient and well- eliminate excess of not covalently bound antibody. We obtained about characterized model for infection with adenovirus and high level 15,000 RU of IgG after immobilization. Capture of soluble recombinant of gene expression. human CD40 (rhCD40)-muIg fusion protein (used as control, gift from Dr. In vivo analysis of fusokine mIL-2/proIL-18* expression was Schneider P., Lausanne, Switzerland), rhIL-2 Ra/Fc chimera (R&D Systems, measured by RT-PCR (Fig. 2C). Immunocompetent B6D2 mice Minneapolis, MN), and rmIL-18 BPd/Fc chimera (R&D Systems, Minneap- bearing palpable P815 tumors were injected once with 5 Â 108 IU olis, MN) was done on individual flow cells at a flow rate of 5 AL/min. of empty Ad, Ad-mIL-2+Ad-mproIL-18*, and Ad-mIL-2/proIL-18*. Quantified supernatant from Ad-mIL-2 or Ad-MIL-2/proIL-18* A549- infected cells and rmIL-18 (R&D Systems, Minneapolis, MN) were dissolved Tumors were removed 72 hours after injection and mRNA was in the running buffer. All the binding experiments were carried out at 25jC extracted. RT-PCR was carried out using oligonucleotide probes with a constant flow rate of 20 AL/min. For the experiments, different specific for mIL-2, mproIL-18*, or mIL-2/linker (specific for the concentrations of cytokine or fusokine were injected for 2 minutes and fusokine). The fusokine was well expressed in vivo at a level 30 seconds followed by a 5-minute dissociation phase. The sensor chip comparable with the combination of the two individual cytokine surface was regenerated after each experiment with a 30-second flush with constructs. As expected, injection of Ad-fusokine results in the 50 mmol/L HCl. The kinetic variables were calculated using the BIAeval maintenance of a fixed ratio of both mIL-2 and mproIL-18* in 3.1 software. Analysis was done using the binding with drifting baseline contrast to the combination of Ad-mIL-2+Ad-mproIL-18* when model. The specific binding profiles were obtained after subtracting the probed with oligonucleotides specific for mIL-2 or mproIL-18*. response signal from the channel control (CD40) and from the running Fusokine IL-2/proIL-18* activates T, natural killer, and buffer. The fitting to each model was judged by the m2 value and randomness of residue distribution compared with the theoretical model. dendritic cells. Immunologic activities of this fusokine were next assessed. First, effects on CTL and NK cytotoxic activities were determined. Murine splenocytes were cultured for 7 days with Results supernatants from A549 cells infected with Ad-fusokines. Super- Construction and expression of murine fusokine IL-2/proIL- natant concentrations were adjusted to have equivalent (20 ng/mL) 18*. A series of fusion proteins linking IL-2 and IL-18 were content of total cytokine or fusokine. As shown in Fig. 3, constructed and expressed in E1- and E3-deleted adenovirus-5 as supernatants containing mIL-2/proIL-18* induced splenic cytotox- outlined in Fig. 1A and in Materials and Methods. The DNA ic activity as assessed on both P815 and YAC target cells (Fig. 3A segment encoding each of these multifunctional cytokines was and B). The lytic activity by splenocytes cultured with fusokine cloned in an adenovirus shuttle plasmid and used to generate mIL-2/proIL-18* was equal to, if not greater than, that observed by E1- and E3-deleted adenovirus vectors. Recombinant adenovirus- splenocytes cultured with supernatants containing individual expressing individual cytokines were also generated (Ad-mIL-2, cytokines or cytokine mixtures. Ad-mproIL-18, and Ad-mproIL-18*; * denotes the K89A mutation; The effect of this fusokine on T-cell proliferation was also ref. 21). A549 cells were infected with each of these constructs and analyzed (Fig. 3C) by assessing the proliferation of murine the culture supernatants assessed for cytokine content. Amounts splenocytes when incubated with anti-CD3 plus Ad-fusokine of secreted recombinant proteins were estimated using a specific supernatants. Supernatant concentrations were adjusted to have ELISA assay. Using this assay, the concentrations of cytokines or equivalent (20 ng/mL) content of total cytokine or fusokine. As fusion protein were determined using half maximum value on the illustrated in Fig. 3C, a higher proliferation rate was obtained with ELISA standard curves (Fig. 1C). ‘‘Fusokine’’ mIL-2/proIL-18* Ad-mIL-2/proIL-18* supernatants than with supernatants contain- was expressed in approximately half the quantity as that obtained ing Ad-mIL-2, Ad-mproIL-18*, or the combination of the two. No with Ad-mIL-2 alone but was 10-fold greater than that of mIL-2/ proliferation was observed with supernatant from cells infected IL-18*, which lacks the prosequence (Fig. 1B). Accurate quantifi- with an empty virus. cation by commercial ELISA kits was not feasible, because Splenocytes activated with fusokine mIL-2/proIL-18* (20 ng/mL) monoclonal antibodies were found not to recognize the fusion were assessed for cytokine/chemokine profile using a comm- cytokine protein. ercial mouse cytokine array (Fig. 3D). A profile associated with Increased expression and stability with fusokine IL-2/proIL- chemoattraction, stimulation, and/or function of T cells (IFN-g, 18*. In vitro production and stability of this recombinant fusokine IL-2, SDF-1a, CTACK, and TCA-3), dendritic cells (GM-CSF, IL-4, was assessed by Western blot. A549 cells were infected with Ad- PF-4, and MIP-3a/h), and NK cells (IL-12, TNF-a, TPO, TIMP-1, and mIL-2 or Ad-mproIL-18* alone; the combination of two vectors SCF) was detected. This cytokine profile correlates with biological each encoding one of the cytokines (denoted as Ad-mIL-2 + Ad- activities observed with this fusokine. www.aacrjournals.org 9539 Cancer Res 2005; 65: (20). October 15, 2005

Figure 2. In vitro and in vivo analysis of fusokine expression. A-B, analysis of protein expression by Western blot of 72-hour supernatants of empty adenovirus, combination of Ad-mIL-2 + Ad-mproIL-18*, and Ad-mIL-2/proIL-18*. A549 cells were infected at an MOI of 50. Supernatants were assessed after 24, 48, and 72 hours of infection. Blots were probed with (A) rabbit anti-mouse IL-2 antibody or (B) rabbit anti-mouse IL-18 antibody. As positive control, recombinant mIL-2 (100 ng) and mIL-18 (100 ng) were used. C, measurement of the fusokine gene expression in vivo by RT-PCR. P815 tumors growing in B6D2 mice were injected with an empty adenovirus (tumor mRNA from two mice), the combination of Ad-mIL-2 + Ad-mproIL-18* (three mice), or the Ad-mIL-2/proIL-18* (three mice). Tumors were removed 24 hours after i.t. injection and analyzed by RT-PCR for the presence of mIL-2, mIL-18, and mIL-2/proIL-18* mRNA (as indicated) using specific oligonucleotides. Fusokine-specific sequence was probed with a 5V oligonucleotide in the mIL-2 sequence and a 3V oligonucleotide in the linker sequence. Fusokine plasmid DNA was used as positive control where indicated.

IL-18 is described as a strong inducer of IFNg secretion both culture with cytokines or fusokines. Incubation of immature in vitro and in vivo. To evaluate this biological activity of IL-18- dendritic cells with Ad-mIL-2/proIL-18* was shown to up-regulate containing fusokines, secretion of murine IFNg by ConA-primed the CD80, CD86, and MHCII markers, reflecting maturation of splenocytes was quantified in vitro as described in Materials and murine dendritic cells (data not shown). Methods. As illustrated in Fig. 4A, ConA plus supernatants, Immunotherapy with Ad-mIL-2/proIL-18* induces high containing 20 ng/mL each, of mIL-2 + proIL-18*, mIL-2/proIL- rates of tumor rejection. The antitumor activity of the Ad- 18*, or mIL2/IL-18* induce similar levels of IFNg secretion (f100 fusokines was investigated in three tumor models: P815, B16F10, ng/mL/106 cells) with a tendency towards a greater effect by the and TC1. Tumors were established in B6D2 mice and injected with fusokines. Unexpectedly, unprimed splenocytes (with no ConA Ad-cytokines or Ad-fusokines (5 Â 108 IU), then tumor growth and costimulation) were stimulated to induce high levels of IFNg mouse survival were followed over a 120-day time period. Figure 5A secretion upon culture with mIL-2/proIL-18* or mIL-2/IL-18* describes a typical result obtained after i.t. injection of palpable fusokines but not with individual cytokines or the mixture of IL-2 + P815 tumors with Ad-mIL-2/proIL-18*. In this experiment, proIL-18* (Fig. 4A). This suggests a novel activity associated with injection of Ad-mIL-2/proIL-18* induced tumor rejection in 70% the fusokine, which is not seen with individual cytokines or a of the animals. This is significantly more than that observed with mixture of the two. To ensure that the combination of individual the combination of Ad-mIL-2 + Ad-mproIL-18*. As described in cytokines does not show this activity at higher concentrations, a Fig. 5B, Ad-mIL-2/proIL-18* is therapeutically effective in various dose-response curve was generated (Fig. 4B). Only the fusokine tumor models, in particular murine mastocytomas (P815), murine mIL-2/proIL-18* was able to induce this activity, whereas the melanoma (B16F10), and human papillomavirus-transformed combination of individual cytokines is unable to do so at all tumors (TC1; Fig. 5B). More importantly, the therapeutic benefit concentrations tested. Subsequently described experiments focus observed with this fusokine is significantly higher than that on the fusokine mIL-2/proIL-18* (in which the IL-18 portion conferred by administration of a vector encoding the individual contains both the prosequence and the K89A mutation). cytokines (see Ad-mIL-2 or Ad-mproIL-18*) or by the coadminis- The capacity of the fusion cytokines to induce proliferation of tration of vectors encoding these cytokines separately (Ad-mIL-2 + both innate and adaptive immune effector cells was also evaluated Ad-mproIL-18*), at least in the P815, B16F10, and TC1 tumor (Fig. 4B). For this purpose, the percentage of splenic CD8 T models. Tumor rejection was associated with immune memory in lymphocytes, NK, and NK/NKT cells were quantified by flow all models tested, because rechallenge with the same tumor was cytometry following 1 week of culture with Ad-fusokine super- consistently rejected (data not shown). natants. Incubation of murine splenocytes with mIL-2/proIL-18* Innate and adaptive immunity are required for tumor (Fig. 4C, IV) induces a dramatic increase in the percentage of CD8+ rejection. The antitumor effects of Ad-mIL-2/proIL-18* was (50%), NK+ (18%), and NK+/NKT+ (51%) cells in comparison with dependent on CD8 and NK cell activity as shown by the survival splenocytes cultured with empty Ad-generated (Fig. 4B, I), Ad-mIL- data generated using the in vivo depletion of CD8, NK, and 2-generated (Fig. 4B, II), or Ad-mproIL-18*-generated (Fig. 4B, III) CD4 cells (Fig. 5C). Interestingly, CD4 depletion increased the supernatants. in vivo activity of i.t. administration of the Ad-fusokine. This is Maturation of murine dendritic cells (35) was determined by not surprising in light of data supporting the immunosuppressive + + quantifying activation markers CD80, CD86, and MHC II after role of CD4 CD25 Treg cells in tumor-bearing animals. Importantly,

Cancer Res 2005; 65: (20). October 15, 2005 9540 www.aacrjournals.org

Figure 3. In vitro functionality of mediated expression of recombinant fusokine. A-B, splenocytes were cultured for 7 days with A549 supernatants containing 20 ng/mL of mproIL-18*, mIL-2, the combination of mIL-2 + mproIL-18*, and fusokine mIL-2/ proIL-18*. Supernatants of A549-infected cells with an empty adenoviral vector were used as negative control. Cultured splenocytes were then assessed for the ability to lyse (A) P815 target cells or (B) YAC target cells. Measurement means F SD. **, P < 0.005. C, in vitro T-cell proliferation. B6D2 splenocytes were cultured with A549 supernatants containing 20 ng/mL of mproIL-18*, mproIL-18, mIL-2, the combination of mIL-2 + mproIL-18*, mIL-2/IL-18, mIL-2/IL-18*, mIL-2/proIL-18, or mIL-2/proIL-18*. As positive control, splenocytes were stimulated with 10 Ag/mL ConA or with an anti-CD3 (10 Ag/mL) monoclonal antibody + rmIL-2 (100 ng/mL). As negative control, supernatant of control virus–infected A549 cells was used. T-cell proliferation was assessed by [3H]-thymidine incorporation after 4 days in culture. Columns, means; bars, SD. **, P < 0.005. Experiments A, B, C were repeated three times with similar results. D, mouse C57Bl/6 splenocytes (106 cells per mL) were cultured with 20 ng/mL of mIL-2/ proIL-18* fusokine for 72 hours. Culture supernatant was subsequently assessed for cytokine profile using the mouse cytokine array III (RayBiotech Inc., Norcross, GA). no immune response against the fusion cytokine was observed Methods. Histologic examination revealed significant necrosis in vivo in the serum of treated mice (data not shown). associated with i.t. injection of Ad-mIL-2/proIL-18* (data not The in vivo antitumor effects of the fusion cytokines was also shown). Moreover, immunohistology shows pronounced increases assessed by analysis of tumor cellular infiltrates. Proximal in CD8+/CD25+ activated T cells (Fig. 6B), CD4+ T cells, and APC activation of both innate and adaptive immune effector cells (in numbers (data not shown). In addition, injected tumors clearly the draining lymph nodes) was observed by immunohistochemistry show up-regulation of IL-18 receptor (Fig. 6A). These changes are or flow cytometry in the P815 model, as described in Materials and also observed in P815 tumors injected with Ad-mIL-2, although at a

Figure 4. In vitro activation of splenocytes. A, analysis of IFNg secretion induce on ConA-primed (10 Ag/mL) or unprimed splenocytes by A549 supernatants containing 20 ng/mL of mproIL-18*, the combination of mIL-2 + mproIL-18*, mIL-2/IL-18, mIL-2/IL-18*, mIL-2/proIL-18, and mIL-2/proIL-18*. As negative control, supernatant of control virus–infected A549 cells were used. Columns, means F SD. P < 0.005. B, the same assay system as in (A) comparing titrations of mIL-2/ proIL-18* and mIL-2 + mproIL-18* in the absence of ConA. Experiments were repeated three times with similar results. C, flow cytometry analysis of the immune cell population obtained from splenocytes after a 7-day culture period with supernatants of A549 cells infected with control virus–infected (I), Ad-mIL-2 (II), Ad-mproIL-18* (III), and Ad-mIL-2/ proIL-18* (IV). All supernatants were adjusted to contain 20 ng/mL of cytokine or fusokine. Percentages of CD8+,NK+, or NK/NKT+ cells were reported in the table (C). Numbers represent means of three independent experiments F SD.

www.aacrjournals.org 9541 Cancer Res 2005; 65: (20). October 15, 2005

Figure 5. In vivo functionality of adenoviral vectors encoding mIL-2/proIL-18*. A, antitumor activity of different adenoviral vectors in the P815 tumor model. Palpable P815 tumors growing in B6D2 mice (groups of 10) were injected thrice, on days 0, 2, and 4 with 5 Â 108 IU of control adenovirus Ad-null, Ad-mIL-2, Ad-mproIL-18*, or Ad-mIL-2/proIL-18* or with a combination of 2.5 Â 108 IU each of Ad-mIL2 and AdmproIL18*. Tumor volumes (mm3) and survival of mice (%) were monitored. Significant differences between the mean survival of animals treated with control viruses or specific viruses are indicated. Statistical values were calculated as described in Materials and Methods and are indicated in (B). Percentages of tumor-free mice at day 120 obtained in different murine tumor models are summarized in the table. Number, means of three independent experiments F SD. C, antitumor activity of i.t. injection of Ad-mIL-2/proIL-18* in CD4-, CD8-, and NK-depleted mice bearing P815 tumors (groups of five mice).

lower level than those observed with Ad-mIL-2/proIL-18*. Surpris- increase in the total numbers of cells (Â30 to Â40) in the lymph ingly, tumors injected with Ad-mIL-2/proIL-18* are highly positive nodes was observed (Fig. 6C, gray histograms), whereas a lower for small blood vessels expressing the von Willebrand factor, number of cells was detected in the lymph nodes of mice treated suggesting neovascularization (Fig. 6A). with Ad-mIL-2 (Fig. 6C). This augmentation correlates with a Similar results were observed in the P815 tumor-draining lymph dramatic decrease of the absolute number of tumor cells (Fig. 6C, nodes (Fig. 6C-D). In mice treated with Ad-mIL-2/proIL-18*, an white histograms) at the site of tumor growth. This shows clearly

Figure 6. Immunohistochemistry and flow cytometry analysis of in vivo immunostimulation by Ad-fusokine. A, tumor immune cell infiltration induced by injection of an empty adenovirus (I), an adenoviral vector encoding mIL-2 (II), or mIL-2/proIL-18* (III) in P815 tumors. Tissue sections were prepared and stained as described (Materials and Methods). B, immunofluorimetry analysis of infiltrated effector cells in single-cell suspensions prepared from Ad-mIL-2/proIL-18*-injected tumors and stained as described (Materials and Methods). Labeled cells were centrifuged on cytospin slides before analysis of CD8+/CD25+, CD4+/CD25+, and NK/NKT+ cells. C, enumeration of the total number of all cells present in the tumor (white histogram) or in the draining lymph nodes (gray histogram) 24 hours after a typical i.t. administration of an empty Ad, Ad-mIL-2, or Ad-mIL-2/ proIL-18*. Columns, means of three experiments; bars, SD. **, P < 0.05; *, P < 0.1. D, FACScan analysis of single cell suspensions prepared from the draining lymph nodes and stained as described (Materials and Methods). Percentages represent the number of positive cells in 105 total viable cell events. Number, means of three experiments F SD. **, P < 0.05.

Cancer Res 2005; 65: (20). October 15, 2005 9542 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Fusokine IL-2/IL-18 Immunostimulation the inverse correlation between the total number of cells in the process that leads to the elimination of self-reactive T cells (36) and tumor and the total number of cells in the draining lymph node. which may be involved in maintaining tumor-induced immune The immune effector cells present in the lymph nodes following anergy to tumor-associated antigens. AICD may also play a role in i.t. injection of Ad-mIL-2/proIL-18* are primarily activated CD8+ T limiting T-cell responses to cancer vaccines that contain IL-2. For lymphocytes (CD3+/CD69+ and CD8+/CD25+) and activated APCs these reasons, the T-cell toxicity of the fusokine was compared with such as mature dendritic cells (CD11c+/MHCII+; Fig. 6D). The total that associated with IL-2. The percentages of two apoptotic number (Fig. 6C) and percentage (Fig. 6D) of these effector cells markers (Annexin and FasL) were evaluated in AICD assays both is higher following injection with Ad-mIL-2/proIL-18* than with in vitro on the T-cell hybridoma 2B4.11 (data not shown) and Ad-mIL-2 or the combination of Ad-mIL-2 + Ad-mproIL-18*. in vivo, as described in Materials and Methods. Briefly, AICD was Fusion of proIL-18* with interleukin-2 reduces interleukin- evaluated in vivo using the draining lymph nodes, 8 hours after s.c. 2-associated toxicity. To assess the toxicity of the fusokine, injection of Ad-fusokine or Ad-mIL-2. As illustrated in the table in groups of healthy C57Bl/6 mice were treated, by s.c. injection, with Fig. 7B, flow cytometric analysis of the cells contained in the lymph high doses of empty adenovirus or adenoviral vectors encoding nodes shows that injection of Ad-mIL-2 induces a strong AICD mIL-2, mproIL-18*, the combination of mIL-2 + mproIL-18* or the in vivo (48% Annexin V+). In marked contrast, mIL-2/proIL-18* (9% fusokine mIL-2/proIL-18*. The data as presented in Fig. 7A show Annexin V+) protects T cells from IL-2-induced AICD (Fig. 7B). that the fusokine mIL-2/proIL-18* induce much less vascular leak Moreover, similar results were obtained by FasL staining under the than does mIL-2 or the combination of mIL-2 + mproIL-18*. These same conditions (data not shown). Fusokine mIL-2/proIL-18* also data show that the genetic fusion of IL-2 and proIL-18* protected 2B4.11 cells from IL-2-induced apoptosis in vitro (data dramatically affects cytokine toxicity by decreasing IL-2-associated not shown). vasopermeability. Quantification of ASAT or ALAT in injected mice Reduced affinity of mIL-2/proIL-18* fusokine for interleu- sera shows the absence of hepatic toxicity after treatment with kin-2 receptor A and IL-18 binding protein. In an effort to Ad-mIL-2 or Ad-Fusokine (data not shown). Some mice were quantify the binding affinity of fusokine for the IL-2 and IL-18 injected i.v. with either the Ad-mIL-2/proIL-18* or the combination receptors, surface plasmon resonance (SPR) was used. Human of Ad-mIL-2 and Ad-mproIL-18*. Although the numbers of mice is IL-2Ra (hIL-2Ra) chain/Fc chimera and mIL-18 BPd/Fc chimera small, it is of interest to note that three of three mice injected were used, because they are the only commercially available with the combination of the two adenoviral constructs died within receptors for this type of experiment. BIACORE analysis (Fig. 8A) 7 days, whereas the three mice injected with the Ad-fusokine shows that mIL-2 binds to the hIL-2Ra with a fast association and survived with no apparent ill effects (data not shown). a slow dissociation rate constant (7.38 Â 105 (mol/L)À1 sÀ1 and In addition to its role in the initial activation of T and NK cells, 3.51 Â 10À3 sÀ1). Binding is specific, because no binding to the IL-2 has a critical role in the maintenance of peripheral tolerance IL-18 BP or mCD40/Ig by mIL-2 was observed (data not shown). (36). In this respect, IL-2 has a central importance in AICD, a The dissociation K (Kd; Fig. 8, Table) indicates a high binding

Figure 7. In vivo analysis of mIL-2/proIL-18* systemic and cellular toxicity. A, assessment of vascular leak syndrome (VLS) induced by i.v. treatment of mice with 2 Â 109 IU of an empty adenovirus (a), Ad-mIL-2 (b), Ad-mproIL-18* (c), the combination of Ad-mIL-2 + Ad-mproIL-18* (b + c), Ad-mIL-2/IL-18* (d), or Ad-mIL-2/proIL-18* (e). Columns, means of three experiments (two mice per group each); bars, SD. **, P < 0.05. Immunohistochemistry analysis of perivascular infiltration in the lungs is represented on the top of Ad-mIL-2, and Ad-mIL-2/proIL-18* column. Arrow, lymphocytic infiltration around the lung blood vessels. B, measurement of AICD in vivo after s.c. injection of adenoviruses encoding mIL-2 (b)or Ad-mIL-2/proIL-18* (e). Draining lymph nodes were removed and assessed 8 hours after injection. AICD was measured by flow cytometry analysis using an anti-mouse Annexin V-FITC–specific antibody. Percentages of apoptotic cells in the lymph nodes are summarized in the table (B). Numbers represent means of two experiments (two mice per group each) F SD. **, P < 0.05; *, P < 0.1.

www.aacrjournals.org 9543 Cancer Res 2005; 65: (20). October 15, 2005

Figure 8. A-B, sensorgrams obtained from the kinetic analysis of mIL-2 or mIL-2/ proIL-18* on hIL-2 Ra/Fc chimera-coated CM5 sensorchip. C-D, sensorgram obtained for the kinetic analysis of mIL-18 or mIL-2/proIL-18* on mIL-18 BP/Fc chimera-coated CM5 sensorchip. Table summarizes the binding affinities (K d) obtained. The kinetic variables were calculated using the BIAeval 3.1 software. Analysis was done using the binding with drifting baseline model. The fit to each model was verified by the m2 value and randomness of residue distribution compared with the theoretical model.

affinity between IL-2 and the hIL-2Ra (4.75 nmol/L). The results for to produce a single molecule that would keep intact their biological mIL-18 (Fig. 8C) show that IL-18 binds to the IL-18 binding protein properties and keep a constant molar ratio of the individual 5 À1 À1 with a fast association and a slow Kd [4.56 Â 10 (mol/L) s and proteins. It was found that the order IL-2/IL-18 was required to 2.92 Â 10À4 sÀ1]. Binding was specific, because IL-18 did not bind maintain secretion and activity. The constructs mproIL-18/IL-2 to the control receptors, IL-2a, or IL-18 BP (data not shown). The and mIL-18/IL-2 were nonfunctional (data not shown). Kd value indicates a high binding affinity between IL-18 and Several constructs of mIL-2/IL-18 fusokine were produced and IL-18BP (0.64 nmol/L). Affinity measurement indicates that IL-2/ assessed. We observed vastly different levels of secretion of IL-18 proIL-18* binds to IL-2 and IL-18 receptor but with significantly when fused with IL-2. IL-18, like IL-1, is produced in a precursor lower binding affinities. For the binding of fusokine to the hIL-2Ra form (proIL-18) initially. This form is not secreted as such but (Fig. 8B), values of 1.57 Â 105 (mol/L)À1 sÀ1 and 3.82 Â 10À3 sÀ1 rather is first cleaved by the enzyme caspase-3/IL-1h-converting were observed for the association and the dissociation rates and enzyme (ICE; ref. 37). The mature IL-18 is then secreted not 24.3 nmol/L for the Kd. For the mIL-18 BP (Fig. 8D), the values of through the endoplasmic reticulum but via an alternate pathway 1.05 Â 106 (mol/L)À1 sÀ1 and 2.74 Â 10À3 sÀ1 for the association (37). It is important to note that in some cases the expression of and the dissociation rate constants and 2.69 nmol/L for the Kd mature IL-18 in a transfected cell results in a molecule with no or value were observed. The decrease in affinity of the chimeric little IL-18 activity, possibly due to problems with protein folding protein for the IL-2 receptor is probably due to steric hindrance as (38). It is also known that expression of proIL-18 in cells that do reflected in the slower association rate constant. The lower affinity not also produce ICE (most cells do not) results in a nonsecreted for the IL-18 receptor is likely due to the formation of a less stable proIL-18 (37, 39). It was found that the fusion molecule mIL-2/ receptor-ligand complex as reflected in the faster dissociation rate proIL-18 is better secreted and more active than is mIL-2/IL-18. constant. This suggests that the fusokine-incorporating proIL-18 is correctly folded and is secreted, presumably, as a result of the IL-2- associated signal sequences. Fusion with IL-2 may also force the Discussion secretion of the fusokine molecule through the ER secretion The availability of recombinant cytokines has enabled research pathway, which is unusual for IL-18. It is noteworthy that whereas into cytokine biology as well as their application in a clinical the inclusion of the prosequence conferred a secretion advantage setting. One aspect which is becoming clear is that the systemic on the fusokine, the fusion molecules that do not include the injection of large doses of cytokines is associated with considerable prosequence had similar biological activity in vitro when equal toxicity, usually due to, or accompanied by, vascular leak syndrome. concentrations were used. Nevertheless, the fusokine IL-2/IL-18 A series of cytokine fusion proteins was produced in an effort not without the prosequence is inactive in vivo in tumor rejection only to reduce cytokine-associated toxicity but also to combine experiments (data not shown). It has recently been shown that the biological effects of cytokines that stimulate the innate immune mutation of the IL-18 molecule at K89A augments the biological system with those that promote an adaptive immune response. In activity of IL-18 (21). The mutated fusokine construct, described in this report is described one such fusion molecule that satisfies all of this report, was found to be more active than the nonmutated these criteria. A fusion protein combining IL-2 and IL-18 was made forms (data not shown).

Cancer Res 2005; 65: (20). October 15, 2005 9544 www.aacrjournals.org

Adenovirus vector has proven to be a convenient expression chemokine and an antibody (immunocytokine) or between a system both in vitro and in vivo (40). Therefore, an E1- and E3- cytokine and an antigen have also been evaluated (22, 43, 44). deleted adenovirus was used to explore the in vitro and in vivo The therapeutic value of IL-2 is limited by its short half-life and biology of this fusokine molecule. Nevertheless, the purification of systemic toxicity. One approach to overcome these problems has human and mouse IL-2/proIL-18* fusokines are in progress and been to fuse this protein to an antibody, a protein with a long will be tested for activity in mice and on human cells then possibly half-life, which targets the fusion to a unique antigen within the considered for clinical testing. body (22, 43). Competition studies showed that IL-2 immunocy- It is well reported that for either IL-2 or IL-18 to stimulate T cells tokine binds the intermediate affinity form of the IL-2R, to produce IFNg requires the preactivation of splenic T cells by consisting of the h and g subunits, with an affinity slightly less ConA (39). The fusokine described in this report does not require than that of rhIL-2. In contrast, IL-2-containing immunocytokine prestimulation of T cells for this activity. Thus, not only are known showed a greater affinity for the high-affinity IL-2R, consisting of biological activities maintained and cytokine-related toxicity a, h, and g subunits, than does rhIL-2 (22). Here, we have reduced, the mIL-2/proIL-18 fusion protein seems to have a novel compared the binding affinity of the fusokine mIL-2/proIL-18* to activity which either cytokine is unable to exert alone or in combi- mIL-2 and mIL-18 by SPR analysis. The only commercially nation. To determine whether this activity could be associated with available IL-2R available is the human a chain. As reported the linkage of the two cytokines, recombinant IL-2 and IL-18 were elsewhere mIL-2 will bind to the a chain of the hIL-2R (45). We tethered separately or both together to protein A-Sepharose beads. find a binding affinity of 4.75 Â 10À9 for mouse IL-2. In contrast, IFNg secretion was stimulated only by beads coated with both IL-2 the binding affinity for the fusokine is 20-fold lower (2.4 Â 10À8). and IL-18 (data not shown). This suggests that the close proximity These data suggest that the fusion of IL-2 to IL-18 has introduced of both cytokines is required for this activity. some structural modification that results, probably due to steric In vivo experiments show that the fusokine mIL-2/proIL-18* is hindrance, in a reduced affinity of mIL-2/proIL-18* fusokine for immunotherapeutically effective in various tumor models, including the a chain, at least, of the IL-2R. A corresponding reduction in the difficult B16 model, when compared with either Ad-cytokine the affinity of IL-18 for the mIL-18-binding protein was also injection alone or as a combination of two individual constructs. observed. These lowered affinities may explain, at least in part, Depletion experiments show clearly that both the innate (NK cells) the decreased toxicities observed with fusokine. The COOH and the adaptive (CD8+ T cell) immune response are involved in this terminus of the IL-2 portion of the fusokine molecule may be therapeutic effect. It is now well established that CD4+ lymphocytes masked by the addition of the linker/proIL-18 part, which may can exert an immunosuppressive function (41) and that the result in the lowering of binding affinity for the IL-2R or favor administration of anti-CD4 antibody can significantly increase the interaction with one type (low, intermediate, or high) of IL-2R antitumor effects by monoclonal antibody therapy, radiation complex. It is also possible that this murine fusokine activates therapy, and chemotherapy in various tumor models. Our data are a specific population of IL-2R-expressing effector cells thus + + consistent with these results in that CD4 CD25 Treg cell depletion reducing the apparent toxicity of recombinant IL-2 (44). Moreover, may play a role in fusokine-induced remission of solid tumors. mIL-2/proIL-18* fusokine has a reduced AICD activity, a property Immunohistologic analysis of the injected tumors indicates that that seems crucial for the induction of tumor-specific T cells (18). the therapeutic effects of i.t. injections of Ad-mIL-2/proIL-18* are The observed reduction in AICD could be explained as above or associated with infiltration by activated T cells and APCs. This it could be the result of an immunologic synapse between IL-2R- would be expected. Unexpected, however, are signs of increased expressing cells and IL-18-expressing cells causing costimulation von Willebrand factor, suggesting increased small vessel neo- of T cells rather than apoptosis, which often accompanies vascularization. Whereas vascularization of tumors is normally stimulation by IL-2 alone. Sequestration of IL-2 by attachment associated with poor prognosis, in this case, it may be associated to IL-18-bearing cells may also play a role in the observed lower with the increased infiltration by immune effector cells. The toxicity, as well as increased bioactivity, of fusokine when histologic examination of lungs of healthy mice injected with either compared with IL-2. Ad-mIL-2 or Ad-mIL-2/proIL-18* showed a similar pattern. That is, Fusokine IL-2/proIL-18* is a stable and potent new immunos- lungs of mice injected with Ad-mIL-2 showed areas of necrosis and timulatory molecule that maintains the biological activities of fluid build-up, whereas the lungs of mice injected with Ad-mIL-2/ IL-2 and IL-18 with a novel lymphocyte stimulating property but proIL-18* showed no necrosis but some areas of neovascularization has dramatically reduced toxicities. (data not shown). Fusion proteins comprising cytokines or a cytokine plus another Acknowledgments molecule have been produced in the past. Some of these have been tested clinically. The construct PIXY-321 is a fusion of Received 2/28/2005; revised 6/17/2005; accepted 7/26/2005. The costs of publication of this article were defrayed in part by the payment of page GM-CSF and IL-3, which is aimed more at hemopoiesis than at charges. This article must therefore be hereby marked advertisement in accordance immune responsiveness (42). Fusion proteins combining a cytokine/ with 18 U.S.C. Section 1734 solely to indicate this fact.

References 3. Khong HT, Restifo NP. Natural selection of tumor 6. Blattman JN, Greenberg PD. Cancer immunotherapy: a variants in the generation of ‘‘tumor escape’’ pheno- treatment for the masses. Science 2004;305:200–5. 1. Goulder PJ, Watkins DI. HIV and SIV CTL escape: impli- types. Nat Immunol 2002;3:999–1005. 7. Narvaiza I, Mazzolini G, Barajas M, et al. Intratumoral cations for vaccine design. Nat Rev Immunol 2004;4:630–40. 4. Pardoll D. Does the immune system see tumors as coinjection of two adenoviruses, one encoding the 2. Dunn GP, Old LJ, Schreiber RD. The immunobiology of foreign or self? Annu Rev Immunol 2003;21:807–39. chemokine IFN-g-inducible protein-10 and another cancer immunosurveillance and immunoediting. Immu- 5. Rosenberg SA. Shedding light on immunotherapy for encoding IL-12, results in marked antitumoral synergy. nity 2004;21:137–48. cancer. N Engl J Med 2004;350:1461–3. J Immunol 2000;164:3112–22. www.aacrjournals.org 9545 Cancer Res 2005; 65: (20). October 15, 2005

8. Zhu M, Terasawa H, Gulley J, et al. Enhanced mutations in the mature form of human IL-18 with ing the human IL-2 cDNA: induction of CD8(+) T-cell activation of human T cells via avipox vector-mediated enhanced biological activity and decreased neutraliza- immunity and NK activity. Cancer Gene Ther 2001; hyperexpression of a triad of costimulatory molecules in tion by IL-18 binding protein. Proc Natl Acad Sci U S A 8:321–32. human dendritic cells. Cancer Res 2001;61:3725–34. 2001;98:3304–9. 33. Paul S, Regulier E, Rooke R, et al. Tumor gene therapy 9. Addison CL, Bramson JL, Hitt MM, Muller WJ, Gauldie 22. Gillies SD, Lan Y, Brunkhorst B, Wong WK, Li Y, by MVA-mediated expression of T-cell-stimulating anti- J, Graham FL. Intratumoral coinjection of adenoviral Lo KM. Bi-functional cytokine fusion proteins for gene bodies. Cancer Gene Ther 2002;9:470–7. vectors expressing IL-2 and IL-12 results in enhanced therapy and antibody-targeted treatment of cancer. 34. Rafi-Janajreh AQ, Chen D, Schmits R, et al. Evidence frequency of regression of injected and untreated distal Cancer Immunol Immunother 2002;51:449–60. for the involvement of CD44 in endothelial cell injury tumors. Gene Ther 1998;5:1400–9. 23. Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani and induction of vascular leak syndrome by IL-2. 10. Johansson SE, Hall H, Bjorklund J, Hoglund P. A, Mehtali M. Efficient generation of recombinant J Immunol 1999;163:1619–27. Broadly impaired NK cell function in non-obese diabetic adenovirus vectors by homologous recombination in 35. Fields RC, Osterholzer JJ, Fuller JA, Thomas EK, mice is partially restored by NK cell activation in vivo Escherichia coli. J Virol 1996;70:4805–10. Geraghty PJ, Mule JJ. Comparative analysis of murine and by IL-12/IL-18 in vitro. Int Immunol 2004;16:1–11. 24. Fallaux FJ, Bout A, van der Velde I, et al. New helper dendritic cells derived from spleen and bone marrow. 11. Son YI, Dallal RM, Lotze MT. Combined treatment cells and matched early region 1-deleted adenovirus J Immunother 1998;21:323–39. with interleukin-18 and low-dose interleukin-2 induced vectors prevent generation of replication-competent 36. Lenardo MJ. Fas and the art of lymphocyte regression of a murine sarcoma and memory response. adenoviruses. Human Gene Ther 1998;9:1909–17. maintenance. J Exp Med 1996;183:721–4. J Immunother 2003;26:234–40. 25. Erbs P, Regulier E, Kintz J, et al. In vivo cancer gene 37. Dinarello CA. Interleukin-18. Methods 1999;19:121–32. 12. Baxevanis CN, Gritzapis AD, Papamichail M. In vivo therapy by adenovirus-mediated transfer of a bifunc- 38. Liu B, Novick D, Kim SH, Rubinstein M. Production antitumor activity of NKT cells activated by the tional yeast cytosine deaminase/uracil phosphoribosyl- of a biologically active human interleukin 18 requires combination of IL-12 and IL-18. J Immunol 2003; transferase fusion gene. Cancer Res 2000;60:3813–22. its prior synthesis as PRO-IL-18. Cytokine 2000;12: 171:2953–9. 26. Delgado M, Ganea D. Vasoactive intestinal peptide 1519–25. 13. Wigginton JM, Lee JK, Wiltrout TA, et al. Synergistic and pituitary adenylate cyclase-activating polypeptide 39. Hwang KS, Cho WK, Yoo J, Seong YR, Kim BK, Im DS. engagement of an ineffective endogenous anti-tumor inhibit expression of Fas ligand in activated T Adenovirus-mediated interleukin-18 mutant in vivo immune response and induction of IFN-g and Fas- lymphocytes by regulating c-Myc, NF-n B, NF-AT, gene transfer inhibits tumor growth through the ligand-dependent tumor eradication by combined and early growth factors 2/3. J Immunol 2001;166: induction of T cell immunity and activation of natural administration of IL-18 and IL-2. J Immunol 2002; 1028–40. killer cell cytotoxicity. Cancer Gene Ther 2004;11: 169:4467–74. 27. Lin KY, Guarnieri FG, Staveley-O’Carroll KF, et al. 397–407. 14. Mehrotra PT, Grant AJ, Siegel JP. Synergistic effects of Treatment of established tumors with a novel vaccine 40. Dummer R, Hassel JC, Fellenberg F, et al. Adenovirus- IL-7 and IL-12 on human T cell activation. J Immunol that enhances major histocompatibility class II presen- mediated intralesional interferon-(g)genetransfer 1995;154:5093–102. tation of tumor antigen. Cancer Res 1996;56:21–6. induces tumor regressions in cutaneous lymphomas. 15. Mehrotra PT, Wu D, Crim JA, Mostowski HS, Siegel 28. Ting CC, Hargrove ME, Yun YS. Augmentation by Blood 2004;104:1631–8. JP. Effects of IL-12 on the generation of cytotoxic anti-T3 antibody of the lymphokine-activated killer cell- 41. Hill JO, Awwad M, North RJ. Elimination of CD4+ activity in human CD8+ T lymphocytes. J Immunol 1993; mediated cytotoxicity. J Immunol 1988;141:741–8. suppressor T cells from susceptible BALB/c mice 151:2444–52. 29. Oshikawa K, Shi F, Rakhmilevich AL, Sondel PM, releases CD8+ T lymphocytes to mediate protective 16. Nelson BH. IL-2, regulatory T cells, and tolerance. Mahvi DM, Yang NS. Synergistic inhibition of tumor immunity against Leishmania. J Exp Med 1989;169: J Immunol 2004;172:3983–8. growth in a murine mammary adenocarcinoma model 1819–27. 17. Van Parijs L, Refaeli Y, Lord JD, Nelson BH, Abbas AK, by combinational gene therapy using IL-12, pro-IL-18, 42. Curtis BM, Williams DE, Broxmeyer HE, et al. Baltimore D. Uncoupling IL-2 signals that regulate T cell and IL-1h converting enzyme cDNA. Proc Natl Acad Sci Enhanced hematopoietic activity of a human granulo- proliferation, survival, and Fas-mediated activation- U S A 1999;96:13351–6. cyte/macrophage colony-stimulating factor-interleukin induced cell death. Immunity 1999;11:281–8. 30. Paul S, Bizouarne N, Dott K, et al. Redirected cellular 3 fusion protein. Proc Natl Acad Sci U S A 1991;88: 18. Saff RR, Spanjaard ES, Hohlbaum AM, Marshak- cytotoxicity by infection of effector cells with a 5809–13. Rothstein A. Activation-induced cell death limits effector recombinant vaccinia virus encoding a tumor-specific 43. Hu P, Mizokami M, Ruoff G, Khawli LA, Epstein AL. function of CD4 tumor-specific T cells. J Immunol 2004; monoclonal antibody. Cancer Gene Ther 2000;7:615–23. Generation of low-toxicity interleukin-2 fusion proteins 172:6598–606. 31. Blomberg K, Granberg C, Hemmila I, Lovgren T. devoid of vasopermeability activity. Blood 2003;101: 19. Assier E, Jillien V, Lefort J, et al. NK cells and Europium-labelled target cells in an assay of natural 4853–61. polymorphonuclear neutrophils are both critical for killer cell activity. I. A novel non-radioactive method 44. Bensinger SJ, Walsh PT, Zhang J, et al. Distinct IL-2 IL-2-induced pulmonary vascular leak syndrome. based on time-resolved fluorescence. J Immunol receptor signaling pattern in CD4+CD25+ regulatory T J Immunol 2004;172:7661–8. Methods 1986;86:225–9. cells. J Immunol 2004;172:5287–96. 20. Gracie JA, Robertson SE, McInnes IB. Interleukin-18. 32. Slos P, De Meyer M, Leroy P, Rousseau C, Acres B. 45. Collins MK. Species specificity of interleukin-2 J Leukoc Biol 2003;73:213–24. Immunotherapy of established tumors in mice by binding to individual receptor components. Eur J 21. Kim SH, Azam T, Yoon DY, et al. Site-specific intratumoral injection of an adenovirus vector harbor- Immunol 1989;19:1517–20.

Cancer Res 2005; 65: (20). October 15, 2005 9546 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity

Bruce Acres, Murielle Gantzer, Christelle Remy, et al.

Cancer Res 2005;65:9536-9546.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/20/9536

Cited articles This article cites 43 articles, 24 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/20/9536.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/20/9536. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.