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Experimental Therapeutics, Preclinical Pharmacology |
1 Division of Viral Products and
2 Division of Cellular and Gene Therapies, Center for Biologics Evaluation Research, United States Food and Drug Administration, Bethesda, Maryland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetically altered bacterial DNA contains more unmethylated CpG motifs than mammalian DNA because of CpG methylation and CpG suppression in mammalian DNA (10) . Oligodeoxynucleotides containing the CpG motifs (CpG ODNs) activate B cells, natural killer (NK) cells, macrophages, and dendritic cells, resulting in robust innate immune activation (11 , 12) . These activities of CpG ODNs are being harnessed therapeutically in antitumor agents as well as in vaccine adjuvants and antiallergic agents (13, 14, 15, 16, 17) . The antitumor effects of bacterial DNA and CpG ODNs have been shown in human as well as in animal models (6 , 15) . The activity of CpG ODNs in vivo can be further enhanced by encapsulating CpG ODNs in sterically stabilized cationic liposomes (SSCLs; Ref. 18 ).
Bacterial toxins such as Pseudomonas exotoxin (PE) are among the bacterial components with strong antitumor activity (9 , 19) . Fusion of the PE to a molecule that targets tumor cells increases cytolytic activity and reduces nonspecific cytotoxicity (20 , 21) . One example is IL13-PE, modified Pseudomonas aeruginosa exotoxin-A fused with interleukin-13 (IL-13), which targets PE to tumor cells expressing IL-13 receptor, such as glioblastoma, head and neck cancer, AIDS-associated Kaposi’s sarcoma, renal cell carcinoma, and prostate cancer (22, 23, 24, 25) .
Although the immunostimulatory activities of CpG ODNs have been appreciated, it has not been shown whether CpG ODNs have antitumor effects on solid tumors, such as head and neck carcinoma, or whether CpG ODNs have any impact on IL13-PE antitumor therapy if coadministered. To determine whether a combination of these two unique approaches will produce potent antitumor activity, we chose a human squamous cell carcinoma head and neck tumor model in athymic mice in which we evaluated the contribution of innate immune activation by CpG ODNs to IL13-PE-induced antitumor activity. Our study demonstrates that the combination of these two modified "bacterial products" shows potent antitumor activity in vivo, inducing complete regression of the aggressive solid tumor in most treated mice. The mechanisms of this combination of treatments were further analyzed in vivo and in vitro.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells.
A human head and neck cancer cell line, KCCT873, was established in the Research Institute, Kanagawa Cancer Center (Yokohama, Japan; Ref. 25
). Cells were cultured in complete RPMI 1640 containing 10% fetal bovine serum, 1 mM HEPES, 1 mM L-glutamine, 100 µg/ml penicillin, and 100 µg/ml streptomycin.
Oligonucleotides.
Synthetic single-stranded ODNs with phosphorothioate linkages were synthesized at the Center for Biologics and Evaluation Research core facility (Bethesda, MD). The sequences of the ODNs were as follows: CpG ODN (1555), 5'-GCTAGACGTTAGCGT-3'; and control ODN (1612), 5'-GCTAGATGTTAGCGT-3' (12)
. Endotoxin levels in all DNA stock solutions were undetectable (<0.01 unit/ml) as determined by the Limulus amebocyte lysate assay (Bio-Whittaker, Walkersville, MD).
Preparation of SSCL.
ODNs encapsulated in sterically stabilized cationic liposomes were generated as described previously (18)
. Briefly, liposomes were generated by evaporating phospholipid mixtures in a round-bottomed flask on a rotary evaporator (Büchi, Flawil, Switzerland). The solvent-free dry lipid film was purged with argon. To generate empty multilamellar vesicles, 1 ml of PBS was added to 20 µmol of dried lipid film. The mixtures were sonicated at 4°C in a Vibra Cell Sonicator (Sonics and Materials, Danbury, CT). The small unilamellar vesicles were then mixed with 1 mg/ml ODN, frozen on dry ice, and freeze-dried overnight (Flexi-Dry; Kinetics Group, Santa Clara, CA). ODN encapsulation was achieved during rehydration. Sterile distilled H2O (100 µl) was added to the dehydrated liposome/ODN powder and vortexed for 30 min at room temperature. PBS (900 µl) was added to the mixture, yielding a final liposome concentration of 20 µmol of lipid/mg of DNA. Vesicles <150 nm in diameter were produced by 20–30 cycles of extrusion through polycarbonate filters in a Liposofast extruder (Avestin, Ottawa, Canada). Liposome formulations were stored at 4°C until use.
Preparation of IL-13 Toxin.
The chimeric fusion gene encoding IL-13 cytotoxin (hIL13-PE38QQR; referred to as IL13-PE) was constructed by use of human IL-13 cDNA cloned from human peripheral blood mononuclear cells and the plasmid PE38QQR (pRKL438QQR) as described previously (26
, 27) . Endotoxin levels were <0.01 unit/mg in all preparations.
Human Head and Neck Cancer Xenografts, Treatments, and Evaluations.
Human head and neck tumors were established in nude or beige mice by s.c. injection of 5 x 106 KCCT873 cells in 150 µl of PBS plus 0.2% human serum albumin into the flank as described previously (25)
. Palpable tumors developed within 3–4 days. The mice then received injections of excipient (0.2% human serum albumin in PBS) or differing doses of IL13-PE by intratumoral injection (30 µl) with a 27-gauge needle. (CpG ODN)SSCL (50 µg in 20 µl) was injected in the same manner. At various time points, tumor growth was measured by Vernier calipers in a standard manner as described elsewhere (25)
. Tumor size was calculated by multiplying the length and width of the tumor on a given day. In some experiments, mice were rechallenged with the same number of KCCT873 cells. In some experiments, NK cells were depleted by pre- and posttreatment with rabbit anti asialo-GM1 antibody (Ab; 50 µg/injection; Wako, Osaka, Japan) at days -3, 4, 10, and 16 after tumor implantation, whereas the control group was treated with the same amount of normal rabbit IgG (R&D Systems, Minneapolis, MN).
Measurement of Cytotoxicity.
Mice (3 mice/group) with or without tumor implantation received intratumoral or s.c. injections of IL13-PE (50 µg/kg) with or without either (CpG ODN)SSCL or (Control ODN)SSCL (50 µg/mouse). Twenty-four h or 10 days after injection, mice were sacrificed, spleen cells were removed, and a single cell suspension was prepared. The cytotoxicities of these spleen cells against tumor cells were measured by a modified 51Cr release assay as described previously (28)
. Briefly, KCCT873 cells (1 x 106/ml) were labeled with 1 µCi of 51Cr (NEN, Boston, MA) for 18 h before the assay. Tumor cells were carefully harvested and incubated with spleen cells from treated mice as described above at various E:T ratio for 24 h. The released 51Cr in the supernatant was measured by gamma counting (Wallac Inc., Gaithersburg, MD).
Measurement of Cytokine Production.
Supernatants from the spleen cell culture described above were immediately analyzed by ELISA to measure cytokine concentrations, as described previously (29)
. Briefly, 96-well Immulon 2 plates were coated with antimouse IL-12 (BD PharMingen, San Diego, CA), or antimouse IFN
(Biosource, Camarillo, CA) in PBS (pH 7.2) for 4 h. After the plates were blocked and washed, supernatants from stimulated cells were added and incubated for 2 h at room temperature. The plates were then washed and treated with biotinylated anticytokine Ab [IL-12 (Genzyme, Cambridge, MA) or IFN (Endogen, Woburn, MA)] followed by phosphatase–streptavidin (BD PharMingen). The cytokine concentration was determined by comparison with purified recombinant mouse cytokines included in the same experiment.
Protein Synthesis Inhibition Assay.
The cytotoxic activity of IL13-PE was tested as described previously (25)
. Typically, 104 KCCT873 cells were cultured in leucine-free medium with or without various concentrations of IL13-PE38QQR for 20–22 h at 37°C, and then 1 µCi of [3
H]leucine (New England Nuclear Research Products, Boston, MA) was added to each well and incubated for an additional 4 h. Cells were harvested, and the radioactivity incorporated into the cells was measured by a beta plate counter (Wallac).
Immunohistochemistry.
Frozen sections of implanted KCCT873 tumors were prepared as described previously (30)
. Samples were fixed and washed with ice-cold PBS and then incubated with the following Abs diluted 1:100 in PBS containing 10% rat serum for 1 h in the dark at 4°C: anti-asialo-GM1 Ab, FITC-labeled antimouse DX-5 (pan-NK; BD PharMingen) and phycoerythrin-labeled antimouse Gr-1 or CD11b (BD PharMingen). The sample stained with anti-asialo-GM Ab was washed with PBS and incubated by FITC-labeled antirabbit IgG at 1:1000 dilution for 1 h at 4°C. Samples were washed five times with PBS and mounted with the Prolong antifade kit (Molecular Probes, Eugene, OR) as recommended by the manufacturer. Photographs of the samples were taken by fluorescence microscope (Olympus America, Melville, NY).
Statistical Analysis.
Statistical analysis was performed with the paired Student’s t test. P < 0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. KCCT873 cells express the IL-13 receptor 
2 and thus are sensitive to treatment with IL13-PE (31
, 32)
. Consistent with previous reports, intratumoral injection of 1 µg of IL13-PE into these mice (50 µg/kg) on days 4, 6, and 8 reduced tumor growth by >75% (P < 0.0005; Fig. 1A
). By comparison, treating these mice with 50 µg of immunostimulatory CpG ODNs encapsulated in SSCL [(CpG ODN)SSCL] reduced tumor growth by >50% (P < 0.004; Fig. 1A
).
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and Table 1
, the combination of (CpG ODN)SSCL plus low-dose IL13-PE not only reduced average tumor size by >90%, but led to a complete regression of tumors in 23% of mice (P < 0.0005; Table 1
). Tumors did not recur in these animals after >40 days of follow-up (data not shown).
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; Fig. 1C
). It is of note that in this tumor model the antitumor effect of IL13-PE alone was not as dose dependent as the combination treatment with (CpG ODN)SSCL, indicating that IL13-PE potentiates the antitumor activity of (CpG ODN)SSCL. Although these tumor-free mice did not have any recurrence for as long as we observed (40 days), they accepted rechallenge of the same tumor with neither regression nor delayed growth (data not shown), suggesting that the antitumor effect of IL13-PE and (CpG ODN)SSCL was solely attributable to enhanced innate immunity.
Histological Analysis of Tumors Treated with (CpG ODN)SSCL Plus IL13-PE.
To identify the cell types associated with tumor regression, we treated KCCT873 tumors on day 4 and removed them for histological analysis 5 and 10 days later. As seen in Fig. 2
, there were considerably more CD11b granulocytes and macrophages infiltrating tumors that had been treated with IL13-PE plus (CpG ODN)SSCL than those treated with IL13-PE alone. NK cells (expressing asialo-GM1) were also present in tumors treated with combined therapy on day 5, and the number of such cells increased dramatically by day 10. In contrast, NK cells were rarely detected in untreated tumors or in tumors treated with IL13-PE alone (Fig. 2)
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Antitumor Activity of NK Cells in Mice Treated with IL13-PE Plus (CpG ODN)SSCL.
The above findings suggested that immune cells from mice treated with IL13-PE plus (CpG ODN)SSCL facilitated the elimination of tumor cells in vivo. To determine whether cell-mediated tumor lysis was involved, we isolated spleen cells 10 days after the last treatment and incubated them with 51Cr-labeled KCCT873 cells. Cells from untreated mice with tumors produced only low-level cytotoxicity (<20%; Fig. 3A
). The spleen cells from mice treated with IL13-PE plus (CpG ODN)SSCL boosted tumor-specific cytotoxicity to >60% (P < 0.001; Fig. 3A
), which was significantly higher than the cytotoxicities obtained with the controls, IL13-PE alone, or IL13-PE plus (control ODN)SSCL (P < 0.01). These data strongly suggest that (CpG ODN)SSCL increased the systemic activity of NK cells in vivo, which are then able to kill the implanted tumor.
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; data not shown). The group treated with control Ab had no change in tumor size compared with that of mice receiving IL13-PE and (CpG ODN)SSCL (Fig. 3B)
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We also tested the effect of NK cells on the antitumor effect of (CpG ODN)SSCL plus IL13-PE, using beige mice lacking NK cells. KCCT873 tumor growth in beige mice was similar to growth in nude mice (Fig. 3, B and C)
. Treatment of the tumor with IL13-PE reduced tumor size by 43%; however, (CpG ODN)SSCL did not alter the tumor size and the number of animals showing complete remission when used alone or with IL13-PE, respectively (Fig. 3C)
. These data suggest that the additive effect of (CpG ODN)SSCL on IL13-PE-induced antitumor activity is dependent on NK activity.
IL13-PE Synergizes Cytotoxicity and Cytokine Production Induced by (CpG ODN)SSCL in Vivo.
Studies were conducted to clarify the mechanism(s) involved in the antitumor effect of (CpG ODN)SSCL plus IL13-PE. Spleen cells were isolated from nude mice 1 day after treatment and tested for cytotoxic activity against 51Cr-labeled KCCT873 cells (Fig. 4A)
. Of interest, cells from mice treated with (CpG ODN)SSCL lysed KCCT873 cells significantly more efficiently than cells from control mice or mice treated with IL13-PE alone (P < 0.05). Cells from animals treated with the combination of (CpG ODN)SSCL plus IL13-PE were even more active than those from animal treated with (CpG ODN)SSCL alone (P < 0.01), suggesting that IL13-PE might synergistically increase the ability of (CpG ODN)SSCL to activate NK cells.
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production by spleen cells from these animals was monitored ex vivo. Consistent with the increased NK activity observed above, cells from CpG ODN-treated mice produced significantly more of both cytokines than those from control or IL13-PE-treated animals (P < 0.05). The results for cells from animals treated with CpG ODN plus IL13-PE were 2–3-fold higher than those for cells from ODN-treated mice (Fig. 4B
; P < 0.01). These results support the conclusion that IL13-PE synergizes with CpG ODN to activate cytotoxic and cytokine-secreting cells in vivo. On the other hand, CpG ODN had no direct effect on the viability as well as the protein synthesis of KCCT873 tumor cells in vitro, as confirmed by a protein synthesis assay (data not shown). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Fig. 1
).
There has been considerable interest in the use of modified bacterial toxins to lyse tumor cells (20
, 21)
. Genetic modification of these "cytotoxins" improves their specificity and reduces toxicity (20
, 21)
. IL13-PE efficiently targets the KCCT873 tumor, which expresses IL-13 receptors (25
, 27)
. Uptake of the cytokine–toxin combination is mediated via endocytosis. The resultant inhibition of protein synthesis leads to tumor cell death by both necrosis and apoptosis (21
, 34)
. Although these events lead to accumulation of macrophages and granulocytes in the tumor, they are not enough to clear all tumor cells and thus lead to complete regression (Fig. 2)
. Limiting the utility of such immunotoxins is their toxicity (particularly to the liver and kidney) and the induction of neutralizing host Abs that lower the efficacy of subsequent treatments (20
, 21)
. We therefore focused on local (intratumoral) administration of IL13-PE to reduce possible side effects by increasing the local concentration.
As seen in Figs. 3
and 4
, IL13-PE did not induce the production of inflammatory cytokines or increase NK cell activity. By comparison, CpG motifs in bacterial DNA stimulate innate immune responses (11, 12, 13)
. CpG DNA stimulates the production of proinflammatory and Th1 cytokines (including IL-12 and IFN) and boosts NK cell activity (Refs. 12
, 35
; Fig. 4
). Previous studies suggested that by improving the activity of the innate immune system, CpG DNA may improve immune surveillance and facilitate the elimination of tumor cells via increased NK activity and IFN production (Fig. 4
; Refs. 3
, 36
). Local (intratumoral) administration of (CpG ODN)SSCL together with IL13-PE was superior to systemic administration of IL13-PE alone in inducing complete remission (data not shown).
Our results show that IFN and NK activities induced by (CpG ODN)SSCL were further enhanced by IL13-PE (Fig. 4)
. When IL13-PE kills and induces massive death of tumor cells, it may produce a large amount of "danger signals" from tumor cells, such as heat shock proteins and genomic DNA, which are shown to induce dendritic cell maturation as well as cytokine production (29
, 37) . Tumor cells undergoing necrosis, but not live tumor cells, stimulate spleen cells to produce large amounts of IFN.3
It is also possible that IL13-PE acts as inhibitor of endogenous IL-13, leading to more Th1-type inflammatory responses; in the present study CpG ODN-induced IL-12 and IFN were inhibited by the addition of IL-13 and enhanced by the common IL-13 inhibitor IL-13 R1/Fc chimera (data not shown). It is also of note that IL13-PE did not alter the expression level of toll-like receptor 9 in spleen cells in vitro (data not shown).
CpG ODNs plus high-dose IL13-PE induced long-term and complete tumor regression in nude mice. Because these athymic animals lack functional T cells, the efficacy of combination therapy can be primarily attributed to tumor lysis plus enhanced activity of the innate immune system. Whether additional, tumor antigen-specific immunity can be induced by this combination of agents in animals with an intact immune system is the subject of an ongoing investigation. CpG ODNs have strong adjuvant-like activities and have been shown to boost the adaptive immune response to coadministered antigens (13 , 14) .
Recently, it has been shown that CpG ODN-induced antitumor activity can be further enhanced by other antitumor therapies, such as anti-IL-10 Ab, tumor antigen-pulsed dendritic cells in a syngeneic mouse colon carcinoma model (38 , 39) . Decker et al. (40) demonstrated that CpG ODNs efficiently sensitize human B-CLL cells to anti-CD25 immunotoxin by up-regulation of its target, CD25, in vitro. Our data support these observations and demonstrate that a combination of CpG ODNs and a receptor-targeted cytotoxin is superior compared to either single treatment. Thus, a combination therapy, with CpG ODNs, tumor-specific antigens, and immunotoxins could be most effective for the eradication of head and neck cancers that are resistant to conventional chemo- and/or radiation therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Some of these studies were conducted as part of a collaboration between the Food and Drug Administration and NeoPharm (Lake Forest, IL) under a cooperative Research and Development Agreement.
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.
Requests for reprints: Ken J. Ishii, Group Leader, Department of Host Defense, Research Institute for Microbial Diseases, Osaka University; 3-1 Yomadaoka, Suita, Osaka, Japan 565-0871. Phone and Fax: 81-6-6879-8301; E-mail: kenishii{at}biken.osaka-u.ac.jp or Raj K. Puri, Acting Director, Division of Cellular and Gene Therapies, Center For Biologics Evaluation and Research, United States Food and Drug Administration, NIH Building 29B, Room 2NN10, 29 Lincoln Drive, Bethesda, MD 20892.
The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Food and Drug Administration at large.
Drs. Ishii and Kawakami contributed equally to this work.
3 K. J. Ishii et al., unpublished data. 
Received 7/ 8/03; revised 8/25/03; accepted 8/26/03.
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REFERENCES |
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. Proc. Natl. Acad. Sci. USA, 93: 2879-2883, 1996.2 chain inhibits tumorigenicity of human breast and pancreatic tumors in immunodeficient mice. J. Exp. Med., 194: 1743-1754, 2001.2 chain. J. Biol. Chem., 276: 25114-251120, 2001.2 chain: an essential component for binding and internalization but not for interleukin-13-induced signal transduction through the STAT6 pathway. Blood, 97: 2673-2679, 2001. and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature (Lond.), 410: 1107-1111, 2001.[CrossRef][Medline]
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, control (PBS); 
, (control ODN)SSCL; 
, (CpG ODN)SSCL; 
, IL13-PE; 
, IL13-PE + (control ODN)SSCL; •, IL13-PE + (CpG ODN)SSCL. B and C, dose-dependent synergistic effect of IL13-PE (100 or 250 µg/kg) with (CpG ODN)SSCL (50 µg). 
