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[Cancer Research 65, 9536-9546, October 15, 2005]
© 2005 American Association for Cancer Research

Immunology

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

Bruce Acres1, Murielle Gantzer2, Christelle Remy3, Nicolas Futin3, Nathalie Accart3, Olivier Chaloin4, Johan Hoebeke4, Jean-Marc Balloul2 and Stéphane Paul2

1 Division of Medical and Regulatory Affairs, 2 Molecular Immunology Laboratory, 3 Histology and Animal Facilities Laboratory, Transgene SA; and 4 Immunologie et Chimie Thérapeutiques, Centre National de la Recherche Scientifique/Institut de Biologie Molécullaire et Cellulaire, Strasbourg, France

Requests for reprints: Stephane Paul, Molecular Immunology Laboratory, Transgene SA, 11 rue de Holsheim, 67082, Strasbourg, France. E-mail: paul{at}transgene.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To redress the immune imbalances created by pathologies such as cancer, it would be beneficial to create novel cytokine molecules, which combine desired cytokine activities with reduced toxicities. Due to their divergent but complementary activities, it is of interest to combine interleukin-2 (IL-2) and IL-18 into one recombinant molecule for immunotherapy. Evaluation of a fusokine protein that combines murine IL-2/IL-18 shows that it is stable, maintains IL-2 and IL-18 bioactivities, has notably reduced IL-2 associated toxicities, and has a novel lymphocyte-stimulating activity. An adeno-viral expression system was used to explore the biology of this "fusokine". Inclusion of the IL-18 prosequence (proIL-18) increases the expression, secretion, and potency of this fusokine. In vivo gene transfer experiments show that Ad-IL-2/proIL-18 dramatically outdoes Ad-IL-2, Ad-proIL-18, or the combination of both, by inducing high rates of tumor rejection in several murine models. Both innate and adaptive effector mechanisms are required for this antitumor activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Numerous lines of evidence indicate that tumors, as well as various pathogens, can escape immune detection and/or elimination (1, 2). Down-regulation of MHC expression as well as the activation of immunosuppressive cells and molecules can dampen the vigor of immune responses to antigens or can activate apoptosis of immune effector cells (2, 3). Recent evidence suggests that T and natural killer (NK) cells from tumor-bearing patients exhibit abnormalities in signal transduction that render them unresponsive to appropriate activation signals (2, 4). Some of these signaling defects can be reversed by cytokines (5, 6).

Recently, studies in mouse tumor models and in patients have shown the importance of cytokine combinations in the development of optimal immune responses (5, 7, 8). A clear synergy between interleukin-2 (IL-2) and IL-12 was first described in a reportedly poorly immunogenic tumor (MCA205) after i.t. administration using adenoviral vectors (9). The combination of IL-2 or IL-12 and IL-18 synergistically enhanced IFN{gamma} production and NK cell activation both in vitro and in vivo and synergized in vivo to induce complete durable regression of well-established 3LL tumors in a large number of treated mice (10–13).

In many of these studies, the relative level of each cytokine was important. Synergy studies between IL-12 and other cytokines for the generation of antitumor responses in mice showed mixed results. Whereas the addition of IL-12 in the presence of suboptimal amounts of IL-2 led to synergy in the induction, proliferation, cytolytic activity, and IFN{gamma} induction, combinations of IL-2 and IL-12 using a higher dose of one cytokine were found to be antagonistic and to enhance toxicity (14, 15). These results may reflect the inherent difficulty of combining two potentially synergistic cytokines in vivo, especially when there is a need to maintain a fixed ratio of activities of two components with different pharmacologic properties, such as differences in their circulating half-life and biodistribution.

To reduce the difficulties associated with cytokine combinations, we undertook the genetic fusion of cytokines in an effort to enhance the immune response, with special focus on the combination of a specific (adaptive) with a nonspecific (innate) immune response in a host organism without increasing cytokine associated toxicities. The 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 synergizing cytokine combination IL-2/IL-18. IL-2 is an important growth and survival factor for T lymphocytes but also sensitizes these cells to Fas-mediated activation-induced cell death (AICD; refs. 16, 17). It has been described recently that AICD limits effector function of CD4 tumor-specific T cells and decreases T-cell effector activity (18). IL-2 is also known to be critically required for the activation of CD4+CD25+ T-cell suppressor function (16). Although IL-2 therapy has yielded encouraging results in the treatment of certain types of cancer, its use is limited by dose-dependent toxicity characterized by weight gain, dyspnea, ascites, and pulmonary edema (16). These signs of toxicity result from increased capillary leak, also known as vascular leak syndrome (19). Modifications of the IL-2 molecule that maintain its well-known IL-2 biological activities but which reduce toxicities would be beneficial. IL-18 is a more recently described cytokine with properties associated with activation of innate immunity and stimulation of IFN{gamma} secretion (20). Similar to IL-1, IL-18 is produced in a precursor form that is cleaved in the cell cytoplasm to mature IL-18 and is then secreted. We produced a series of fusion proteins linking IL-2 and IL-18 and expressed them in E1- and E3-deleted adenovirus-5 as a convenient 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) in which mIL-2 is fused to mproIL-18. This multifunctional cytokine induces a high rate of tumor rejection after i.t. delivery in various animal models, using an encoding adenoviral vector. Further evidence indicates immunostimulation and very limited toxicity both in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning and Construction of Fusokine cDNAs
Splenocytes from C57Bl6 mice were harvested and stimulated for 3 days with a mix of concanavalin A (ConA, 10 µg/mL, Sigma-Aldrich, Lyon, France) and mIL-2 (10 IU/mL, R&D Systems, Minneapolis, MN) or lipopolysaccharide (10 µg/mL, Sigma) and murine granulocyte macrophage colony-stimulating factor (GM-CSF, 50 IU/mL, R&D Systems, United Kingdom). mRNA from activated splenocytes were then extracted with RNA Now (Ozyme, Saint Quentin en Yvelines, France). mIL-2 and IL-18 cDNAs were amplified by reverse transcription-PCR (RT-PCR; Platinum Quantitative RT-PCR, Thermoscript one-step system, Invitrogen, Cergy Pontoise, France) using specific oligonucleotides. The mutated form of mIL-18 (K89A) was produced using QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA; ref. 21). Two forms of mIL-18 cDNA have been used for the fusion molecules, encoding the precursor proIL-18 and the mature mIL-18 (devoid of the prosequence).

As described in Fig. 1, once amplified by RT-PCR, the sequences encoding the two cytokine moieties (X and Y) were cloned in frame by PCR with a flexible linker (G4S)2 or (G4S)3 inserted between them (22).



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  		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 ß-globin/IgG intron; IRES, internal ribosomal entry site; pA, SV40 late polyadenylation signal. Fusokines were constructed by double PCR to incorporate, in frame, a synthetic flexible linker (G4S)3 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).

 
Adenovirus Production and Titration
The sequence encoding each fusion protein was inserted in an adenoviral shuttle plasmid containing a cytomegalovirus (CMV)–driven expression cassette surrounded by adenoviral sequences (adenoviral nucleotides 1-458 and nucleotides 3328-5788, respectively) to allow generation of the vector genome by homologous recombination (23). The resulting adenoviral vectors are E3-deleted (nucleotides 28592-30470) and E1-deleted (nucleotides 459-3327), with the E1 region replaced by the expression cassette containing, from 5' to 3', the CMV immediate-early enhancer/promoter, a chimeric human ß-globin/IgG intron, the sequence encoding the fusion protein, and the SV40 late polyadenylation signal. The recombinant adenoviruses were generated by transfecting the PacI-linearized viral genomes into the PER C6 complementation cell line (24). Virus propagation, purification, and titration were accomplished as described previously (25). Negative control virus, referred to as "empty vector," is the same virus as described above, with no transgene.

Cell Culture
Human pulmonary carcinoma cell line A549 (CCL-185; American Type Culture Collection, ATCC, Manassas, VA), the 2E8 murine lymphoblast (TIB-239, ATCC), and the murine 2B4.11 T-cell hybridoma (26) were grown at 37°C in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS and antibiotics. The 2B4.11 T-cell hybridoma was kindly provided by Prof. D. Ganea (Newark, NJ).

P815 murine mastocytoma (DBA/2; FcR+, H2Dd, MHCI+, ICAM1+, and CD48+) and B16F10 murine melanoma (C57Bl/6; H2Db, MHCI–, MHCII–, ICAM1–, and CD48–) were obtained from ATCC (TIB-64 and CRL-6475, respectively). The TC1 murine tumor cell line cell line was generously provided by Dr. T.C. Wu (The Johns Hopkins University School of Medicine) has been described previously (27). All cell lines were tested negative for Mycoplasma using Hoechst dye, cell culture, and PCR. All have tested negative for murine pathogens and are acceptable for use in a specific pathogen-free animal environment.

Antibodies and Cytokines
Biotin-labeled anti-mIL-2 and anti-mIFN{gamma} were purchased from R&D Systems (United Kingdom). Biotin-labeled anti-mIL-18 was purchased from Peprotech, Inc. (Rocky Hill, NJ). Biotin-labeled anti-goat IgG or anti-rabbit IgG were purchased from Amersham Biosciences (Orsay, France).

PerCP-CY5.5, FITC, or phycoerythrine-labeled rat anti-mouse CD4, CD8, CD3, CD25, CD31, CD69, MAC1, CD11c, H-2Kb/Db, Iab, NK-1.1, and NK-T/NK (CD3/NK1.1) cell antigen or unconjugated rat anti-mouse CD4 and CD8 were used as defined by the manufacturer (PharMingen, San Diego, CA). Unconjugated rabbit anti-human CD3 (which cross-reacts with mouse CD3) or rabbit anti-rat IgG and peroxidase-labeled goat anti-rabbit were used at concentrations suggested by DakoCytomation (Glostrup, Denmark). T cell apoptosis (AICD) was measured using the Annexin V-FITC apoptosis detection kit (PharMingen). Recombinant murine IFN{gamma} and IL-2 were purchased from R&D Systems (United Kingdom). Recombinant mIL-18 (rmIL-18) was purchased from Peprotech. ConA was purchased from Sigma and used at 1 µg/mL.

Analysis of Fusokine Expression
A549 cells were infected in suspension with adenoviral vectors, as previously described, at a multiplicity of infection (MOI) of 10 (30-minute incubation of cells with virus dilutions in 100 µL of PBS supplemented with 2% FCS, 1% cations; ref. 25). Cells were then cultured in complete medium containing 5% FCS for 72 hours. Supernatants and cell extracts were collected then analyzed by Western blot on 4% to 12% Nupage gel (Novex, Invitrogen). Expression of individual cytokines constituting each fusion protein was analyzed by Western blot according to the enhanced chemiluminescence Western blotting protocol provided by Amersham Life Sciences using specific anti-mouse cytokine antibodies.

Fusokine concentrations were estimated by ELISA immunoassay. Briefly, dilution of fusokine-containing supernatants were coated on a Maxisorp 96-well plate (Nalge Nunc, Rochester, NY) overnight at 4°C. Cytokines and 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 with a specific monoclonal anti-rabbit IgG conjugated with HRPO (The Jackson Laboratory, Bar Harbor, ME). Wells coated with serial dilutions of rmIL-2 or mIL-18, in tissue culture medium, were used as positive control (R&D Systems, Minneapolis, MN) to generate standard curves for the estimations of fusokine concentrations.

In vitro Biological Activity of Fusokine mIL-2/proIL-18*
T-cell proliferation assay. Mouse spleen cell proliferation was assessed by the uptake of [3H]-thymidine. Splenocytes were coactivated with (20 ng/mL) murine CD3-specific antibody (145-2C11, PharMingen) as previously described (28). Splenocytes were mixed with the fusokine-containing A549 supernatants and anti-CD3 antibody. As a positive control, spleen cells (5 x 104 cells per well) were stimulated in complete medium with either ConA (10 µg/mL) or 100 ng/mL rmIL-2. After 96 hours, the cells were pulsed with 1 µCi per well [3H]-thymidine (Amersham Biosciences). Incorporation of [3H]-thymidine into the DNA of proliferating T cells was measured by harvesting cellular DNA onto glass filter paper (PHD Harvester, Cambridge Technology, Watertown, MA) after 4 hours and by counting the radioactivity in a liquid scintillation counter (Beckman Coulter, Roissy CDG, France). All measurements were in triplicate.

IFN{gamma} secretion assay. The relative bioactivity of mIL-18 was determined by the ability of the fusokine contained in supernatants from Ad-cytokine or Ad-fusokine infected A549 cells to augment IFN{gamma} production in vitro (29). In brief, mouse splenocytes (106 cells/mL) were cocultured with or without ConA (1.25 µg/mL) in 24-well plated for 48 hours. Ad-fusokine supernatants were added to cell suspensions of ConA-primed, or unprimed, splenocytes in 96-well plates for 24 hours. The supernatants were collected and assayed by ELISA to detect IFN{gamma} production (Quantikine, R&D Systems, Minneapolis, MN).

CTL and natural killer cell cytotoxicity assays. Activities of the fusokine were also assayed for CTL and NK cytotoxicity as previously described (30). Mouse C57Bl/6 splenocytes were cocultured with Ad-fusokine- or Ad-cytokine-containing supernatants obtained from A549-infected cells for 8 days. The cytotoxic activities of primed splenocytes were measured on P815-CTL target or YAC-NK target using the EuDTPA cytotoxicity assay (Wallac Lab, Turku, Finland; ref. 31).

Mouse cytokine antibody array. Mouse C57Bl/6 splenocytes (106 cells/mL) were cultured with 20 ng/mL of mIL-2/proIL-18* fusokine for 72 hours. Mouse cytokine antibody array III was purchased from RayBiotech, Inc. (Norcross, GA) and used according to the manufacturer's instructions to detect supernatant cytokine content.

Immunostimulation in vitro. To analyze the in vitro effect of the fusokine, bone marrow–derived dendritic cells or splenocytes were incubated with fusokine-containing supernatants for 3 to 7 days. Phenotypic markers of maturation and/or activation of dendritic cells, other antigen-presenting cells (APC), B, T (CD4 and CD8), NK, and NKT cells were analyzed using mouse-specific antibodies by flow cytometry analysis (FACScan, BD Biosciences, San Jose, CA).

Statistical analyses were done using the Student's t test. P < 0.05 was considered statistically significant. All experiments were run in triplicate and the results are represented as the mean ± SD of triplicate determinations or, where indicated, representative data of three independent experiments.

In vivo Experiments
Murine P815, B16F10, and TC1 tumor cells were trypsinized, washed, and resuspended in PBS at 3 x 106 cells/mL. One hundred microliters of the cell suspension were then injected s.c. into the right flank of 6- to 7-week-old immunocompetent B6D2 mice. At days 7, 8, and 9 after injection, when tumors were palpable, the mice received three i.t. injections of 5 x 108 IU of Ad fusion or Ad controls diluted in 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L MgCl2. Tumors size and survival rate were then evaluated for the following 120 days. In vivo depletion of CD4, CD8, and NK cells were made as previously described (32). The statistical difference in in vivo survival experiment between the different groups was assessed using Fischer exact application (Statistica 5.1 software, Statsoft, Inc., Tulsa, OK) of the Kaplan-Meier survival curves. P ≤ 0.05 is considered statistically significant.

Immunohistochemistry, Immunofluorimetry, and Flow Cytometry Analysis
Tumors were established and injected with the various viruses as described above for in vivo experiments. On day 13, tumors were measured and excised. Tumor draining lymph nodes were also removed at the same time. For flow cytometry analysis, tumors were disrupted into a single-cell suspension using collagenase (Sigma) digestion, cells were stained with the indicated antibodies, and analyzed by flow cytometry as previously described (33).

P815 tumor tissues were removed and directly embedded in ornithine carbamyl transferase compound on isopentane cooled on dried ice. Five-micrometer sections were used for H&E staining (structural observations by light microscopy) or for immunohistochemistry. Infiltrating cells and blood vessels detection were done on methanol/acetone-fixed cryosections using the following antibodies: rat anti-mouse CD4 (PharMingen), rat anti-mouse CD8 (PharMingen), rabbit anti-human CD3 (DAKO), hamster anti-mouse CD11c (PharMingen), rat anti-mouse Ia-Ie (PharMingen), rat anti-mouse CD25-FITC (PharMingen), goat anti-mouse IL18-R (R&D Systems), and rabbit anti-human von Willebrand factor (DAKO). Primary antibodies were revealed by specific secondary antibodies rabbit anti-rat immunoglobulin (DAKO), rabbit anti-hamster immunoglobulin (Rockland, Gilbertsville, PA), horse anti-goat biotinylated 0.5% (Vectastain Elite PK6200, Vector Labs, Burlingame, CA), or rabbit anti-FITC HRP (P0404, DAKO) coupled. Horseradish peroxidase (HRP)–labeled polymer conjugated with the second rabbit antibody (EnVision + System, DAKO) or Streptavidin-HRP (Vector Labs) was applied, and 3,3'-diaminobenzidine was used as substrate. All slides were counterstained with hematoxylin.

Activation-Induced Cell Death Assay
AICD, in which signals normally associated with lymphocyte stimulation instead result in the demise of the cell, has been proposed as a mechanism of deletion of antigen-specific lymphocytes. T cells can be sensitive or resistant to AICD, and IL-2 can regulate the susceptibility of T cells to AICD (16–18). AICD can be characterized by the de novo synthesis of Annexin V, Fas (CD95), and its ligand (FasL; refs. 16–18). AICD was measured in vitro on 2B4.11 murine T-cell hybridoma and in vivo after s.c. injection of adenoviruses encoding fusokine. In brief, C57BL/6 mice were injected s.c. once with 2 x 108 IU of Ad-fusokine (or as a control with an adenovirus encoding mIL-2 or an empty adenovirus). Draining lymph nodes were then removed at 8 hours after injection. AICD was measured by flow cytometry analysis using a phycoerythrin-labeled mouse anti-mouse FasL-specific antibody (PharMingen) and an FITC-labeled Annexin V Apoptosis Detection kit (PharMingen).

Quantification of Vascular Leak Syndrome Assay
Vascular leak was studied by measuring the extravasation of Evans blue which, when given i.v., binds to plasma proteins, particularly albumin, and following extravasation can be detected in various organs as described (19, 34). Vascular leak was induced by injecting s.c. 2 x 109 IU of mIL-2-encoding 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-18, or Ad-fusokine mIL-2/proIL-18*. On day 4, mice were injected i.v. with 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 lungs and liver, where maximum extravasation is known to occur, were harvested, and placed in formamide at 37°C overnight. The Evans blue in the organs was quantified by measuring the absorbance of the supernatant at 650 nm. The vascular leak syndrome seen in Ad-cytokine-treated mice was expressed as the percent increase in extravasation compared with that in PBS-treated controls. For histopathologic studies, groups of five separate mice were injected with empty Ad or PBS, Ad-mIL-2, Ad-mIL-2/proIL-18*, and Ad-mIL-2/IL-18* as described earlier, and on day 4, lungs and liver were fixed in 10% formalin solution. The organs were embedded in paraffin, sectioned, and stained with H&E. Perivascular infiltration was scaled by counting the number of lymphocytes infiltrating the vessel and averaging the minimum and maximum range for each group. Sera from injected mice were also collected for ASAT and ALAT measurement.

Biosensor Experiments
The BIACORE 3000 system, sensor chip CM5, surfactant P20, amine-coupling kit containing N-hydroxysuccinimide and N-ethyl-N'-dimethylaminopropyl carbodiimide, were from BIACORE AB (Uppsala, Sweden). All biosensor assays were done with HBS-EP as running buffer [10 mmol/L HEPES, 150 mmol/L sodium chloride, 3 mmol/L EDTA, 0.005% surfactant P20 (pH 7.4)]. Flow cells were precoated with a goat anti-human IgG (Fc{gamma} fragment specific, Jackson ImmunoResearch, West Grove, PA) using amine coupling at 30 µg/mL in acetate buffer (10 mmol/L, pH 5.5) according to manufacturer conditions. The chip was then blocked with 1 mol/L ethanolamine hydrochloride (pH 8.5, BIACORE) and washed abundantly to eliminate excess of not covalently bound antibody. We obtained about 15,000 RU of IgG after immobilization. Capture of soluble recombinant human CD40 (rhCD40)-muIg fusion protein (used as control, gift from Dr. Schneider P., Lausanne, Switzerland), rhIL-2 R{alpha}/Fc chimera (R&D Systems, Minneapolis, MN), and rmIL-18 BPd/Fc chimera (R&D Systems, Minneapolis, MN) was done on individual flow cells at a flow rate of 5 µL/min.

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 in the running buffer. All the binding experiments were carried out at 25°C with a constant flow rate of 20 µL/min. For the experiments, different concentrations of cytokine or fusokine were injected for 2 minutes and 30 seconds followed by a 5-minute dissociation phase. The sensor chip surface was regenerated after each experiment with a 30-second flush with 50 mmol/L HCl. The kinetic variables were calculated using the BIAeval 3.1 software. Analysis was done using the binding with drifting baseline model. The specific binding profiles were obtained after subtracting the response signal from the channel control (CD40) and from the running buffer. The fitting to each model was judged by the {chi}2 value and randomness of residue distribution compared with the theoretical model.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction and expression of murine fusokine IL-2/proIL-18*. A series of fusion proteins linking IL-2 and IL-18 were constructed and expressed in E1- and E3-deleted adenovirus-5 as outlined in Fig. 1A and in Materials and Methods. The DNA segment encoding each of these multifunctional cytokines was cloned in an adenovirus shuttle plasmid and used to generate E1- and E3-deleted adenovirus vectors. Recombinant adenovirus-expressing individual cytokines were also generated (Ad-mIL-2, Ad-mproIL-18, and Ad-mproIL-18*; * denotes the K89A mutation; ref. 21). A549 cells were infected with each of these constructs and the culture supernatants assessed for cytokine content. Amounts of secreted recombinant proteins were estimated using a specific ELISA assay. Using this assay, the concentrations of cytokines or fusion protein were determined using half maximum value on the ELISA standard curves (Fig. 1C). "Fusokine" mIL-2/proIL-18* was expressed in approximately half the quantity as that obtained with Ad-mIL-2 alone but was 10-fold greater than that of mIL-2/IL-18*, which lacks the prosequence (Fig. 1B). Accurate quantification by commercial ELISA kits was not feasible, because monoclonal antibodies were found not to recognize the fusion cytokine protein.

Increased expression and stability with fusokine IL-2/proIL-18*. In vitro production and stability of this recombinant fusokine was assessed by Western blot. A549 cells were infected with Ad-mIL-2 or Ad-mproIL-18* alone; the combination of two vectors each encoding one of the cytokines (denoted as Ad-mIL-2 + Ad-mproIL-18*) or with an adenovirus expressing the fusokine mIL-2/proIL-18* (Fig. 2A). Supernatants obtained after 72 hours of adenoviral infection were analyzed. Unexpectedly, IL-2 was observed to be more stable when expressed as the mIL2/proIL-18* fusokine compared with the cytokine alone or to the combination of the two cytokines (Fig. 2A). This could, at least in part, explain the accumulation of higher levels of prosequence-containing fusokines in supernatants as measured by ELISA and shown in Fig. 1. More of the mproIL-18* portion was also observed after infection with Ad-mIL-2/proIL-18* (Fig. 2B) than with the Ad-mproIL-18* construct. As expected, infection with Ad-fusokine maintains a fixed ratio of both mIL-2 and mproIL-18* in contrast to the combination of Ad-mIL-2 + Ad-mproIL-18* where relative expression was variable. A549 cells were used for these in vitro experiments, because these cells represent a convenient and well-characterized model for infection with adenovirus and high level of gene expression.



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  		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 5' oligonucleotide in the mIL-2 sequence and a 3' oligonucleotide in the linker sequence. Fusokine plasmid DNA was used as positive control where indicated.

 
In vivo analysis of fusokine mIL-2/proIL-18* expression was measured by RT-PCR (Fig. 2C). Immunocompetent B6D2 mice bearing palpable P815 tumors were injected once with 5 x 108 IU of empty Ad, Ad-mIL-2+Ad-mproIL-18*, and Ad-mIL-2/proIL-18*. Tumors were removed 72 hours after injection and mRNA was extracted. RT-PCR was carried out using oligonucleotide probes specific for mIL-2, mproIL-18*, or mIL-2/linker (specific for the fusokine). The fusokine was well expressed in vivo at a level comparable with the combination of the two individual cytokine constructs. As expected, injection of Ad-fusokine results in the maintenance of a fixed ratio of both mIL-2 and mproIL-18* in contrast to the combination of Ad-mIL-2+Ad-mproIL-18* when probed with oligonucleotides specific for mIL-2 or mproIL-18*.

Fusokine IL-2/proIL-18* activates T, natural killer, and 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 supernatants from A549 cells infected with Ad-fusokines. Supernatant concentrations were adjusted to have equivalent (20 ng/mL) content of total cytokine or fusokine. As shown in Fig. 3, supernatants containing mIL-2/proIL-18* induced splenic cytotoxic activity as assessed on both P815 and YAC target cells (Fig. 3A and B). The lytic activity by splenocytes cultured with fusokine mIL-2/proIL-18* was equal to, if not greater than, that observed by splenocytes cultured with supernatants containing individual cytokines or cytokine mixtures.



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  		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 ± 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 µg/mL ConA or with an anti-CD3 (10 µg/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).

 
The effect of this fusokine on T-cell proliferation was also analyzed (Fig. 3C) by assessing the proliferation of murine splenocytes when incubated with anti-CD3 plus Ad-fusokine supernatants. Supernatant concentrations were adjusted to have equivalent (20 ng/mL) content of total cytokine or fusokine. As illustrated in Fig. 3C, a higher proliferation rate was obtained with Ad-mIL-2/proIL-18* supernatants than with supernatants containing Ad-mIL-2, Ad-mproIL-18*, or the combination of the two. No proliferation was observed with supernatant from cells infected with an empty virus.

Splenocytes activated with fusokine mIL-2/proIL-18* (20 ng/mL) were assessed for cytokine/chemokine profile using a commercial mouse cytokine array (Fig. 3D). A profile associated with chemoattraction, stimulation, and/or function of T cells (IFN-{gamma}, IL-2, SDF-1{alpha}, CTACK, and TCA-3), dendritic cells (GM-CSF, IL-4, PF-4, and MIP-3{alpha}/ß), and NK cells (IL-12, TNF-{alpha}, TPO, TIMP-1, and SCF) was detected. This cytokine profile correlates with biological activities observed with this fusokine.

IL-18 is described as a strong inducer of IFN{gamma} secretion both in vitro and in vivo. To evaluate this biological activity of IL-18-containing fusokines, secretion of murine IFN{gamma} by ConA-primed splenocytes was quantified in vitro as described in Materials and Methods. As illustrated in Fig. 4A, ConA plus supernatants, containing 20 ng/mL each, of mIL-2 + proIL-18*, mIL-2/proIL-18*, or mIL2/IL-18* induce similar levels of IFN{gamma} secretion (~100 ng/mL/106 cells) with a tendency towards a greater effect by the fusokines. Unexpectedly, unprimed splenocytes (with no ConA costimulation) were stimulated to induce high levels of IFN{gamma} secretion upon culture with mIL-2/proIL-18* or mIL-2/IL-18* fusokines but not with individual cytokines or the mixture of IL-2 + proIL-18* (Fig. 4A). This suggests a novel activity associated with the fusokine, which is not seen with individual cytokines or a mixture of the two. To ensure that the combination of individual cytokines does not show this activity at higher concentrations, a dose-response curve was generated (Fig. 4B). Only the fusokine mIL-2/proIL-18* was able to induce this activity, whereas the combination of individual cytokines is unable to do so at all concentrations tested. Subsequently described experiments focus on the fusokine mIL-2/proIL-18* (in which the IL-18 portion contains both the prosequence and the K89A mutation).



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  		Figure 4. In vitro activation of splenocytes. A, analysis of IFN{gamma} secretion induce on ConA-primed (10 µg/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 ± 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 ± SD.

 
The capacity of the fusion cytokines to induce proliferation of both innate and adaptive immune effector cells was also evaluated (Fig. 4B). For this purpose, the percentage of splenic CD8 T lymphocytes, NK, and NK/NKT cells were quantified by flow cytometry following 1 week of culture with Ad-fusokine supernatants. Incubation of murine splenocytes with mIL-2/proIL-18* (Fig. 4C, IV) induces a dramatic increase in the percentage of CD8+ (50%), NK+ (18%), and NK+/NKT+ (51%) cells in comparison with splenocytes cultured with empty Ad-generated (Fig. 4B, I), Ad-mIL-2-generated (Fig. 4B, II), or Ad-mproIL-18*-generated (Fig. 4B, III) supernatants.

Maturation of murine dendritic cells (35) was determined by quantifying activation markers CD80, CD86, and MHC II after culture with cytokines or fusokines. Incubation of immature dendritic cells with Ad-mIL-2/proIL-18* was shown to up-regulate the CD80, CD86, and MHCII markers, reflecting maturation of murine dendritic cells (data not shown).

Immunotherapy with Ad-mIL-2/proIL-18* induces high rates of tumor rejection. The antitumor activity of the Ad-fusokines was investigated in three tumor models: P815, B16F10, and TC1. Tumors were established in B6D2 mice and injected with Ad-cytokines or Ad-fusokines (5 x 108 IU), then tumor growth and mouse survival were followed over a 120-day time period. Figure 5A describes a typical result obtained after i.t. injection of palpable P815 tumors with Ad-mIL-2/proIL-18*. In this experiment, injection of Ad-mIL-2/proIL-18* induced tumor rejection in 70% of the animals. This is significantly more than that observed with the combination of Ad-mIL-2 + Ad-mproIL-18*. As described in Fig. 5B, Ad-mIL-2/proIL-18* is therapeutically effective in various tumor models, in particular murine mastocytomas (P815), murine melanoma (B16F10), and human papillomavirus-transformed tumors (TC1; Fig. 5B). More importantly, the therapeutic benefit observed with this fusokine is significantly higher than that conferred by administration of a vector encoding the individual cytokines (see Ad-mIL-2 or Ad-mproIL-18*) or by the coadministration of vectors encoding these cytokines separately (Ad-mIL-2 + Ad-mproIL-18*), at least in the P815, B16F10, and TC1 tumor models. Tumor rejection was associated with immune memory in all models tested, because rechallenge with the same tumor was consistently rejected (data not shown).



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  		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 x 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 x 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 ± 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).

 
Innate and adaptive immunity are required for tumor rejection. The antitumor effects of Ad-mIL-2/proIL-18* was dependent on CD8 and NK cell activity as shown by the survival data generated using the in vivo depletion of CD8, NK, and CD4 cells (Fig. 5C). Interestingly, CD4 depletion increased the in vivo activity of i.t. administration of the Ad-fusokine. This is not surprising in light of data supporting the immunosuppressive role of CD4+CD25+ Treg cells in tumor-bearing animals. Importantly, no immune response against the fusion cytokine was observed in vivo in the serum of treated mice (data not shown).

The in vivo antitumor effects of the fusion cytokines was also assessed by analysis of tumor cellular infiltrates. Proximal activation of both innate and adaptive immune effector cells (in the draining lymph nodes) was observed by immunohistochemistry or flow cytometry in the P815 model, as described in Materials and Methods. Histologic examination revealed significant necrosis associated with i.t. injection of Ad-mIL-2/proIL-18* (data not shown). Moreover, immunohistology shows pronounced increases in CD8+/CD25+ activated T cells (Fig. 6B), CD4+ T cells, and APC numbers (data not shown). In addition, injected tumors clearly show up-regulation of IL-18 receptor (Fig. 6A). These changes are also observed in P815 tumors injected with Ad-mIL-2, although at a lower level than those observed with Ad-mIL-2/proIL-18*. Surprisingly, tumors injected with Ad-mIL-2/proIL-18* are highly positive for small blood vessels expressing the von Willebrand factor, suggesting neovascularization (Fig. 6A).



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  		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 ± SD. **, P < 0.05.

 
Similar results were observed in the P815 tumor-draining lymph nodes (Fig. 6C-D). In mice treated with Ad-mIL-2/proIL-18*, an increase in the total numbers of cells (x30 to x40) in the lymph nodes was observed (Fig. 6C, gray histograms), whereas a lower number of cells was detected in the lymph nodes of mice treated with Ad-mIL-2 (Fig. 6C). This augmentation correlates with a dramatic decrease of the absolute number of tumor cells (Fig. 6C, white histograms) at the site of tumor growth. This shows clearly the inverse correlation between the total number of cells in the tumor and the total number of cells in the draining lymph node. The immune effector cells present in the lymph nodes following i.t. injection of Ad-mIL-2/proIL-18* are primarily activated CD8+ T lymphocytes (CD3+/CD69+ and CD8+/CD25+) and activated APCs such as mature dendritic cells (CD11c+/MHCII+; Fig. 6D). The total number (Fig. 6C) and percentage (Fig. 6D) of these effector cells is higher following injection with Ad-mIL-2/proIL-18* than with Ad-mIL-2 or the combination of Ad-mIL-2 + Ad-mproIL-18*.

Fusion of proIL-18* with interleukin-2 reduces interleukin-2-associated toxicity. To assess the toxicity of the fusokine, groups of healthy C57Bl/6 mice were treated, by s.c. injection, with high doses of empty adenovirus or adenoviral vectors encoding mIL-2, mproIL-18*, the combination of mIL-2 + mproIL-18* or the fusokine mIL-2/proIL-18*. The data as presented in Fig. 7A show that the fusokine mIL-2/proIL-18* induce much less vascular leak than does mIL-2 or the combination of mIL-2 + mproIL-18*. These data show that the genetic fusion of IL-2 and proIL-18* dramatically affects cytokine toxicity by decreasing IL-2-associated vasopermeability. Quantification of ASAT or ALAT in injected mice sera shows the absence of hepatic toxicity after treatment with Ad-mIL-2 or Ad-Fusokine (data not shown). Some mice were injected i.v. with either the Ad-mIL-2/proIL-18* or the combination of Ad-mIL-2 and Ad-mproIL-18*. Although the numbers of mice is small, it is of interest to note that three of three mice injected with the combination of the two adenoviral constructs died within 7 days, whereas the three mice injected with the Ad-fusokine survived with no apparent ill effects (data not shown).



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  		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 x 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) ± SD. **, P < 0.05; *, P < 0.1.

 
In addition to its role in the initial activation of T and NK cells, IL-2 has a critical role in the maintenance of peripheral tolerance (36). In this respect, IL-2 has a central importance in AICD, a process that leads to the elimination of self-reactive T cells (36) and which may be involved in maintaining tumor-induced immune anergy to tumor-associated antigens. AICD may also play a role in limiting T-cell responses to cancer vaccines that contain IL-2. For these reasons, the T-cell toxicity of the fusokine was compared with that associated with IL-2. The percentages of two apoptotic markers (Annexin and FasL) were evaluated in AICD assays both in vitro on the T-cell hybridoma 2B4.11 (data not shown) and in vivo, as described in Materials and Methods. Briefly, AICD was evaluated in vivo using the draining lymph nodes, 8 hours after s.c. injection of Ad-fusokine or Ad-mIL-2. As illustrated in the table in Fig. 7B, flow cytometric analysis of the cells contained in the lymph nodes shows that injection of Ad-mIL-2 induces a strong AICD in vivo (48% Annexin V+). In marked contrast, mIL-2/proIL-18* (9% Annexin V+) protects T cells from IL-2-induced AICD (Fig. 7B). Moreover, similar results were obtained by FasL staining under the same conditions (data not shown). Fusokine mIL-2/proIL-18* also protected 2B4.11 cells from IL-2-induced apoptosis in vitro (data not shown).

Reduced affinity of mIL-2/proIL-18* fusokine for interleukin-2 receptor {alpha} and IL-18 binding protein. In an effort to quantify the binding affinity of fusokine for the IL-2 and IL-18 receptors, surface plasmon resonance (SPR) was used. Human IL-2R{alpha} (hIL-2R{alpha}) chain/Fc chimera and mIL-18 BPd/Fc chimera were used, because they are the only commercially available receptors for this type of experiment. BIACORE analysis (Fig. 8A) shows that mIL-2 binds to the hIL-2R{alpha} with a fast association and a slow dissociation rate constant (7.38 x 105 (mol/L)–1 s–1 and 3.51 x 10–3 s–1). Binding is specific, because no binding to the IL-18 BP or mCD40/Ig by mIL-2 was observed (data not shown). The dissociation K (Kd; Fig. 8, Table) indicates a high binding affinity between IL-2 and the hIL-2R{alpha} (4.75 nmol/L). The results for mIL-18 (Fig. 8C) show that IL-18 binds to the IL-18 binding protein with a fast association and a slow Kd [4.56 x 105 (mol/L)–1 s–1 and 2.92 x 10–4 s–1]. Binding was specific, because IL-18 did not bind to the control receptors, IL-2a, or IL-18 BP (data not shown). The Kd value indicates a high binding affinity between IL-18 and IL-18BP (0.64 nmol/L). Affinity measurement indicates that IL-2/proIL-18* binds to IL-2 and IL-18 receptor but with significantly lower binding affinities. For the binding of fusokine to the hIL-2R{alpha} (Fig. 8B), values of 1.57 x 105 (mol/L)–1 s–1 and 3.82 x 10–3 s–1 were observed for the association and the dissociation rates and 24.3 nmol/L for the Kd. For the mIL-18 BP (Fig. 8D), the values of 1.05 x 106 (mol/L)–1 s–1 and 2.74 x 10–3 s–1 for the association and the dissociation rate constants and 2.69 nmol/L for the Kd value were observed. The decrease in affinity of the chimeric protein for the IL-2 receptor is probably due to steric hindrance as reflected in the slower association rate constant. The lower affinity for the IL-18 receptor is likely due to the formation of a less stable receptor-ligand complex as reflected in the faster dissociation rate constant.



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  		Figure 8. A-B, sensorgrams obtained from the kinetic analysis of mIL-2 or mIL-2/proIL-18* on hIL-2 R{alpha}/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 (Kd) 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 {chi}2 value and randomness of residue distribution compared with the theoretical model.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The availability of recombinant cytokines has enabled research into cytokine biology as well as their application in a clinical setting. One aspect which is becoming clear is that the systemic injection of large doses of cytokines is associated with considerable toxicity, usually due to, or accompanied by, vascular leak syndrome. A series of cytokine fusion proteins was produced in an effort not only to reduce cytokine-associated toxicity but also to combine biological effects of cytokines that stimulate the innate immune system with those that promote an adaptive immune response. In this report is described one such fusion molecule that satisfies all of these criteria. A fusion protein combining IL-2 and IL-18 was made to produce a single molecule that would keep intact their biological properties and keep a constant molar ratio of the individual proteins. It was found that the order IL-2/IL-18 was required to maintain secretion and activity. The constructs mproIL-18/IL-2 and mIL-18/IL-2 were nonfunctional (data not shown).

Several constructs of mIL-2/IL-18 fusokine were produced and assessed. We observed vastly different levels of secretion of IL-18 when fused with IL-2. IL-18, like IL-1, is produced in a precursor form (proIL-18) initially. This form is not secreted as such but rather is first cleaved by the enzyme caspase-3/IL-1ß-converting enzyme (ICE; ref. 37). The mature IL-18 is then secreted not through the endoplasmic reticulum but via an alternate pathway (37). It is important to note that in some cases the expression of mature IL-18 in a transfected cell results in a molecule with no or little IL-18 activity, possibly due to problems with protein folding (38). It is also known that expression of proIL-18 in cells that do not also produce ICE (most cells do not) results in a nonsecreted proIL-18 (37, 39). It was found that the fusion molecule mIL-2/proIL-18 is better secreted and more active than is mIL-2/IL-18. 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 secretion of the fusokine molecule through the ER secretion pathway, which is unusual for IL-18. It is noteworthy that whereas the inclusion of the prosequence conferred a secretion advantage on the fusokine, the fusion molecules that do not include the prosequence had similar biological activity in vitro when equal concentrations were used. Nevertheless, the fusokine IL-2/IL-18 without the prosequence is inactive in vivo in tumor rejection experiments (data not shown). It has recently been shown that the mutation of the IL-18 molecule at K89A augments the biological activity of IL-18 (21). The mutated fusokine construct, described in this report, was found to be more active than the nonmutated forms (data not shown).

Adenovirus vector has proven to be a convenient expression system both in vitro and in vivo (40). Therefore, an E1- and E3-deleted adenovirus was used to explore the in vitro and in vivo biology of this fusokine molecule. Nevertheless, the purification of human and mouse IL-2/proIL-18* fusokines are in progress and will be tested for activity in mice and on human cells then possibly considered for clinical testing.

It is well reported that for either IL-2 or IL-18 to stimulate T cells to produce IFN{gamma} requires the preactivation of splenic T cells by ConA (39). The fusokine described in this report does not require prestimulation of T cells for this activity. Thus, not only are known biological activities maintained and cytokine-related toxicity reduced, the mIL-2/proIL-18 fusion protein seems to have a novel activity which either cytokine is unable to exert alone or in combination. To determine whether this activity could be associated with the linkage of the two cytokines, recombinant IL-2 and IL-18 were tethered separately or both together to protein A-Sepharose beads. IFN{gamma} secretion was stimulated only by beads coated with both IL-2 and IL-18 (data not shown). This suggests that the close proximity of both cytokines is required for this activity.

In vivo experiments show that the fusokine mIL-2/proIL-18* is immunotherapeutically effective in various tumor models, including the difficult B16 model, when compared with either Ad-cytokine injection alone or as a combination of two individual constructs. Depletion experiments show clearly that both the innate (NK cells) and the adaptive (CD8+ T cell) immune response are involved in this therapeutic effect. It is now well established that CD4+ lymphocytes can exert an immunosuppressive function (41) and that the administration of anti-CD4 antibody can significantly increase the antitumor effects by monoclonal antibody therapy, radiation therapy, and chemotherapy in various tumor models. Our data are consistent with these results in that CD4+CD25+ Treg cell depletion may play a role in fusokine-induced remission of solid tumors.

Immunohistologic analysis of the injected tumors indicates that the therapeutic effects of i.t. injections of Ad-mIL-2/proIL-18* are associated with infiltration by activated T cells and APCs. This would be expected. Unexpected, however, are signs of increased von Willebrand factor, suggesting increased small vessel neovascularization. Whereas vascularization of tumors is normally associated with poor prognosis, in this case, it may be associated with the increased infiltration by immune effector cells. The histologic examination of lungs of healthy mice injected with either Ad-mIL-2 or Ad-mIL-2/proIL-18* showed a similar pattern. That is, lungs of mice injected with Ad-mIL-2 showed areas of necrosis and fluid build-up, whereas the lungs of mice injected with Ad-mIL-2/proIL-18* showed no necrosis but some areas of neovascularization (data not shown).

Fusion proteins comprising cytokines or a cytokine plus another molecule have been produced in the past. Some of these have been tested clinically. The construct PIXY-321 is a fusion of GM-CSF and IL-3, which is aimed more at hemopoiesis than at immune responsiveness (42). Fusion proteins combining a cytokine/chemokine and an antibody (immunocytokine) or between a cytokine and an antigen have also been evaluated (22, 43, 44).

The therapeutic value of IL-2 is limited by its short half-life and systemic toxicity. One approach to overcome these problems has been to fuse this protein to an antibody, a protein with a long half-life, which targets the fusion to a unique antigen within the body (22, 43). Competition studies showed that IL-2 immunocytokine binds the intermediate affinity form of the IL-2R, consisting of the ß and {gamma} subunits, with an affinity slightly less than that of rhIL-2. In contrast, IL-2-containing immunocytokine showed a greater affinity for the high-affinity IL-2R, consisting of {alpha}, ß, and {gamma} subunits, than does rhIL-2 (22). Here, we have compared the binding affinity of the fusokine mIL-2/proIL-18* to mIL-2 and mIL-18 by SPR analysis. The only commercially available IL-2R available is the human {alpha} chain. As reported elsewhere mIL-2 will bind to the {alpha} chain of the hIL-2R (45). We find a binding affinity of 4.75 x 10–9 for mouse IL-2. In contrast, the binding affinity for the fusokine is 20-fold lower (2.4 x 10–8). These data suggest that the fusion of IL-2 to IL-18 has introduced some structural modification that results, probably due to steric hindrance, in a reduced affinity of mIL-2/proIL-18* fusokine for the {alpha} chain, at least, of the IL-2R. A corresponding reduction in the affinity of IL-18 for the mIL-18-binding protein was also observed. These lowered affinities may explain, at least in part, the decreased toxicities observed with fusokine. The COOH terminus of the IL-2 portion of the fusokine molecule may be masked by the addition of the linker/proIL-18 part, which may result in the lowering of binding affinity for the IL-2R or favor interaction with one type (low, intermediate, or high) of IL-2R complex. It is also possible that this murine fusokine activates a specific population of IL-2R-expressing effector cells thus reducing the apparent toxicity of recombinant IL-2 (44). Moreover, mIL-2/proIL-18* fusokine has a reduced AICD activity, a property that seems crucial for the induction of tumor-specific T cells (18). The observed reduction in AICD could be explained as above or it could be the result of an immunologic synapse between IL-2R-expressing cells and IL-18-expressing cells causing costimulation of T cells rather than apoptosis, which often accompanies stimulation by IL-2 alone. Sequestration of IL-2 by attachment to IL-18-bearing cells may also play a role in the observed lower toxicity, as well as increased bioactivity, of fusokine when compared with IL-2.

Fusokine IL-2/proIL-18* is a stable and potent new immunostimulatory molecule that maintains the biological activities of IL-2 and IL-18 with a novel lymphocyte stimulating property but has dramatically reduced toxicities.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Competing interests statement. The other authors declare that they have no competing financial interests.

Received 2/28/05. Revised 6/17/05. Accepted 7/26/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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