Molecular Cancer Therapeutics Landon Prizes for Basic and Translational Cancer Research Tumor Immunology: New Perspectives
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Mol Cancer Ther. 2006;5:1585-1592
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

Chemotherapy and chemosensitization of non–small cell lung cancer with a novel immunomodulatory oligonucleotide targeting Toll-like receptor 9

Hui Wang1,2, Elizabeth R. Rayburn1, Wei Wang1, Ekambar R. Kandimalla4, Sudhir Agrawal4 and Ruiwen Zhang1,2,3

1 Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, 2 Comprehensive Cancer Center, and 3 Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama and 4 Idera Pharmaceuticals, Inc., Cambridge, Massachusetts

Requests for reprints: Ruiwen Zhang, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Volker Hall Room 113, 1670 University Boulevard, Birmingham, AL 35294-0019. Phone: 205-934-8558; Fax: 205-975-9330. E-mail: ruiwen.zhang{at}ccc.uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is a leading cause of death world-wide and the long-term survival rate for lung cancer patients is one of the lowest for any cancer. New therapies are urgently needed. The present study was designed to evaluate an immunomodulatory oligonucleotide as a novel type of therapy for lung cancer. The in vivo effects of the immunomodulatory oligonucleotides were determined in four tumor models derived from human non–small cell lung cancer (NSCLC) cell lines (A549, H1299, H358, and H520), administered alone or in combination with conventional chemotherapeutic agents used to treat lung cancer. The in vitro effects of the immunomodulatory oligonucleotide on the growth, apoptosis, and proliferation of NSCLC cells were also determined. We also examined NSCLC cells for expression of Toll-like receptor 9 (TLR9), the receptor for the immunomodulatory oligonucleotide. We showed several important findings: (a) treatment with the immunomodulatory oligonucleotide led to potent antitumor effects, inhibiting tumor growth by at least 60% in all four in vivo models; (b) combination with the immunomodulatory oligonucleotide led to enhanced effects following treatment with gemcitabine or Alimta; (c) the immunomodulatory oligonucleotide increased apoptosis, decreased proliferation, and decreased survival in A549 cells in vitro; and (d) both TLR9 mRNA and protein were expressed in NSCLC cells. The immunomodulatory oligonucleotide has potent antitumor effects as monotherapy and in combination with conventional chemotherapeutic agents, and may act directly on NSCLC cells via TLR9. The present study provides a rationale for developing the immunomodulatory oligonucleotide for lung cancer therapy. [Mol Cancer Ther 2006;5(6):1585–92]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is the leading cause of cancer death in both men and women in the United States and one of the leading causes of death worldwide (1). More than 170,000 people were diagnosed with lung cancer in the United States in 2005, and more than 160,000 will have died from the disease or complications from treatment (1). Non–small cell lung cancer (NSCLC) represents ~80% of all types of lung cancer and includes squamous cell carcinomas, adenocarcinomas, and large-cell carcinomas. For NSCLC patients with early stage disease (stages I and II), surgical resection offers substantial cure rates (2, 3). However, most patients present with locally or regionally advanced (stage III) or metastatic (stage IV) cancers (2, 3). Current treatment strategies for advanced lung cancer include surgical resection, radiation, cytotoxic chemotherapy, and, more recently, photodynamic therapy (4). Although a combination of chemotherapy and radiation can improve survival, most patients die of disease progression, often resulting from acquired or intrinsic resistance to chemotherapeutic drugs (5). In addition, in almost two thirds of cases, the cancer has already spread beyond localized disease at the time of diagnosis (2, 3, 6), limiting the options for therapy.

Many patients receive chemotherapy or radiation before surgical resection to shrink the tumor and may continue to receive treatment following surgery, depending on the pathology of the tumor. There are several chemotherapeutic drugs being used to treat lung cancer, including platinum-based agents, taxanes, topoisomerase inhibitors, and gemcitabine (7, 8); more recently, Iressa (Gefitinib), an inhibitor of epidermal growth factor receptor signaling, has been used to treat lung cancer patients (9). Because chemotherapy decreases the quality of life for patients and is often responsible for serious and sometimes life-threatening complications, rationally designed tumor-specific drugs are especially needed.

Immunotherapy is one such novel approach that is gaining recognition. There are antibodies targeting oncogenes (i.e., trastuzumab), antibodies targeting angiogenic molecules (bevacizumab), and immune system adjuvants being used to protect immune cells from the toxic effects of chemotherapy (filgrastim), but there are other modes of immunotherapy that have not reached their full potential. One of these is immunostimulatory oligonucleotides. Identified less than a decade ago, oligonucleotides containing certain CpG sequence motifs can stimulate the innate and adaptive immune response and have been under investigation for treating infectious diseases, allergies, asthma, and cancer (10–15). Through the Toll-like receptor 9 (TLR9) signaling pathway, immunostimulatory oligonucleotides activate a complex cascade that leads to stimulation of an immune response and increased production of proinflammatory cytokines and chemokines (16, 17). It is generally believed that it is this stimulation of the immune system that leads to antitumor effects of the CpG oligonucleotides (10, 16–19).

There are several immunostimulatory oligonucleotides in clinical or preclinical development and antisense oligonucleotides unintentionally containing stimulatory sequences have been in clinical trials for more than a decade (10, 19). However, there have been stability, delivery, safety, and efficacy issues with these immunostimulatory drugs. We have developed a novel class of TLR9 agonists employing chemically modified nucleotides and a different structure allowing for increased stability, increased efficacy, and a more regulated stimulation of the immune response (20–27). This new class, called immunomodulatory oligonucleotides, also overcomes another problem with traditional immunostimulatory oligonucleotides. The traditional oligonucleotides are species specific; the optimally active sequence in mice is not the same for humans or other animals, making it difficult to translate the effects of the oligonucleotides in animal models to human trials (28, 29). In contrast, the novel modifications made to the immunomodulatory oligonucleotides allow them to stimulate the immune system of numerous animals, as well as purified and cultured human immune cells, and to be more effective than traditional CpG oligonucleotides (30–32). To our knowledge, the immunomodulatory oligonucleotides are the first TLR9 agonists designed using advanced chemistry and the results of structure-activity studies. Several immunomodulatory oligonucleotides have been evaluated in models of human cancer (33–35) and the most effective immunomodulatory oligonucleotide tested in the present study is currently in phase II clinical trials in cancer patients. This present study was designed to examine the most effective immunomodulatory oligonucleotide for its potential use as a therapeutic agent for NSCLC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Immunomodulatory Oligonucleotides
All chemicals and solvents were of the highest analytic grade available. Cell culture supplies were obtained from the Comprehensive Cancer Center Media Preparation Shared Facility at the University of Alabama at Birmingham. Gemcitabine (Gemzar, clinical grade) and pemetrexed (Alimta, clinical grade) were purchased from Eli Lily and Company (Indianapolis, IN). Matrigel basement membrane matrix was obtained from Becton Dickinson Labware (Bedford, MA). The TLR9 antibody was purchased from Calbiochem/EMD Biosciences (La Jolla, CA). The phosphorothioate-modified immunomodulatory oligonucleotide, 5'-TCTGTCRTTCT-X-TCTTRCTGTCT-5' (where R and X stand for 2'-deoxy-7-deazaguanosine and a glycerol linker, respectively), was synthesized, purified, and analyzed as previously reported (26, 33).

Animals
The animal use and care protocol was approved by the Institutional Animal Use and Care Committee of the University of Alabama at Birmingham. Female and male athymic pathogen-free nude mice (nu/nu, 4–6 weeks old) were purchased from Frederick Cancer Research and Development Center (Frederick, MD). All animals were fed with commercial diet and provided water ad libitum.

Cell Lines and Culture
Four human lung cancer cell lines were used: A549, H1299, H358, and H520. H520, H358, and H1299 cells were grown in RPMI 1640 supplemented with 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mmol/L HEPES buffer, 1 mmol/L sodium pyruvate, and 2 mmol/L L-glutamine. A549 cells were grown in Ham's F12K medium supplemented with 2 mmol/L L-glutamine and 1.5 g/L sodium bicarbonate. All media contained 10% fetal bovine serum and 1% penicillin/streptomycin.

Animal Xenograft Tumor Models
Human lung cancer xenograft models (A549, H1299, H358, and H520) were established using previously reported methods (36, 37). Briefly, male (H358 and H520) and female (A549 and H1299) athymic nude mice (4–6 weeks old) were injected s.c. with cultured cells suspended in serum-free medium/Matrigel basement membrane matrix (Becton Dickinson Labware) at a ratio of 3:1 into the left inguinal area. The animals were monitored for changes in body weight, behavior, and tumor growth. Growth was monitored by caliper measurement of two perpendicular diameters of the implanted tumor. Tumor mass (weight in grams) was calculated by the formula (1/2)(a x b2), where a is the long diameter (cm) and b is the short diameter (cm).

In vivo Treatment with Immunomodulatory Oligonucleotide Alone or in Combination with Cancer Chemotherapeutic Agents
Animals bearing human cancer xenografts were randomly divided into treatment groups and a control group (5–10 mice per group) when tumors reached at least 45 mg. The untreated control group received sterile physiologic saline (0.9% NaCl) only. The immunomodulatory oligonucleotide and control oligonucleotide were dissolved in physiologic saline (0.9% NaCl) and administered by s.c. injection at doses of 0.5 or 1 mg/kg/d, three doses per week. Gemcitabine was given by i.p. injection at 160 mg/kg on days 4 and 11 to the H358 and H520 models. Alimta was given i.p. at 100 mg/kg on days 11, 18, and 25 to the mice bearing H520 tumors.

Cell Survival
The percentage of cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (37, 38). A549 cells were grown in 96-well plates and exposed to the immunomodulatory oligonucleotide or control oligonucleotide (0, 1, 5, 10, 50, and 100 nmol/L) with or without Lipofectin (7 µg/mL; Life Technologies, Gaithersburg, MD) for 48 hours. The percentage of cell survival was calculated by dividing the mean absorbance of wells containing treated cells by that of vehicle-control wells.

Cell Proliferation
Proliferation was evaluated using a cell proliferation kit from Oncogene (La Jolla, CA; refs. 37, 38). Cells were seeded in 96-well plates and transfected with the immunomodulatory oligonucleotide (100 nmol/L) or the control oligonucleotide (100 nmol/L) for 24 hours. BrdUrd was added to the medium 10 hours before treatment termination. The levels of BrdUrd incorporated into cells were quantified by anti-BrdUrd antibody and absorbance was measured at dual wavelengths (450/540 nm).

Detection of Apoptosis
Treated and control cells in early and late stages of apoptosis were detected with an Annexin V-FITC apoptosis detection kit from BioVision (Mountain View, CA; refs. 37, 38). In brief, cells were transfected with the immunomodulatory oligonucleotide (100 nmol/L) or the control oligonucleotide (100 nmol/L) and incubated for 24 hours before analysis. Media and cells were collected and washed with serum-free medium. Cells that were positive for Annexin V-FITC alone (early apoptosis) and Annexin V-FITC and propidium iodide (late apoptosis) were counted and the apoptotic index was calculated.

Evaluation of TLR9 Expression
A549 cells were evaluated for TLR9 mRNA and protein expression. mRNA: Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) and was quantified and used to create cDNA. Amplification of TLR9 was accomplished using primers as published by Bauer et al. (28) and a portion of the PCR product was visualized using ethidium bromide on an agarose gel. ß-Actin was coamplified with TLR9 to verify the quality and expression level of the mRNA. Protein: TLR9 and ß-actin protein levels were evaluated by Western blot using an anti-TLR9 antibody from Calbiochem/EMD Biosciences following procedures previously described (37, 38). TLR9 mRNA expression in treated cells: A549 cells were treated with 100 nmol/L immunomodulatory oligonucleotide or control oligonucleotide, with or without the transfection agent Lipofectin for 24 hours. The mRNA was extracted, amplified, and evaluated as above.

Data and Statistical Analysis
The antitumor activity (as measured by differences in tumor mass) was expressed as mean and SD, and the significance of differences was analyzed by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo Administration of the Immunomodulatory Oligonucleotide Inhibits Tumor Growth
Several models of human lung cancer were used to show that the immunomodulatory oligonucleotide is effective against lung cancer with different genetic backgrounds (i.e., of p53). The immunomodulatory oligonucleotide inhibited tumor growth in the H520 model by >46% on day 27 (Fig. 1A ). The H358 model showed stronger results, with tumor growth inhibition by 67% on day 36 (Fig. 1B). In both of these models, the control oligonucleotide showed minimal effects on tumor growth. For two other models, a different dose of the immunomodulatory oligonucleotide was used to evaluate whether a lower dose could still inhibit tumor growth. In animals bearing A549 xenografts, tumor growth was inhibited by ~32% on day 35 when animals were given 0.5 mg/kg/d of the immunomodulatory oligonucleotide (compared with 1 mg/kg/d immunomodulatory oligonucleotide for the other two models; Fig. 1C). In the H1299 model, tumor growth was inhibited by ~78% on day 35 with the 0.5 mg/kg/d dose (Fig. 1D). There were no observable side effects in any of the four models. Splenomegaly was observed for animals treated with the immunomodulatory oligonucleotide, indicating its immunostimulatory effects.


Figure 1
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  		Figure 1. The immunomodulatory oligonucleotide inhibits the growth of xenograft tumors. Treatment with the immunomodulatory oligonucleotide results in potent antitumor effects in H520 (A), H358 (B), A549 (C), and H1299 (D) models of NSCLC.

 
The Immunomodulatory Oligonucleotide Sensitizes Tumors to Chemotherapeutic Agents
Gemcitabine, a chemotherapeutic agent in current use for lung cancer therapy, was tested in combination with the immunomodulatory oligonucleotide. In the H520 model, gemcitabine alone led to a 36% inhibition of tumor growth on day 27. Although combination with a control oligonucleotide did not increase the antitumor effects, combining the immunomodulatory oligonucleotide with gemcitabine increased the tumor inhibition to almost 71%. The immunomodulatory oligonucleotide alone was only able to inhibit tumor growth by 46% on day 27 (Fig. 2A ).


Figure 2
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  		Figure 2. Combining the immunomodulatory oligonucleotide with a first-line chemotherapeutic agent, gemcitabine, leads to enhanced antitumor effects in the H520 (A) and H358 (B) NSCLC xenograft models. C, growth inhibition of the H358 tumors.

 
In the H358 model, gemcitabine was very effective, decreasing tumor growth by 78% when administered alone. Addition of the control oligonucleotide had minimal effects but addition of the immunomodulatory oligonucleotide led to almost complete (>98%) inhibition of tumor growth (Fig. 2B). Tumors were removed from animals at the end of the study and the growth inhibitory effects of the immunomodulatory oligonucleotide, control immunomodulatory oligonucleotide, and gemcitabine can be seen in Fig. 2C.

A more recently approved chemotherapeutic drug was also tested in combination with the immunomodulatory oligonucleotide. Alimta (pemetrexed sodium) is an antifolate approved in late 2004 as a second-line agent for advanced lung cancer and as a first-line agent for unresectable malignant mesothelioma. Although the dose of Alimta used for the study was much lower than the maximum tolerated dose, it still inhibited tumor growth by 26% on day 30 in the H520 model. Combination with the control oligonucleotide increased the inhibition to 39%. However, combining the immunomodulatory oligonucleotide with the same dose of Alimta led to a 74% inhibition of tumor growth (Fig. 3 ). This suggests that combination therapy with this agent and the immunomodulatory oligonucleotide may be particularly useful.


Figure 3
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  		Figure 3. The immunomodulatory oligonucleotide sensitizes NSCLC H520 tumors to treatment with Alimta.

 
Animals receiving gemcitabine experienced a brief weight loss following administration of the drug but gained the weight back after a few days. The low dose of Alimta that was used did not cause any apparent side effects. No increases in chemotherapeutic drug or immunomodulatory oligonucleotide toxicity were observed following combination treatments.

Immunomodulatory Oligonucleotide Treatment Leads to Decreased Survival, Decreased Proliferation, and Increased Apoptosis in NSCLC Cells
Because the immunomodulatory oligonucleotide had potent antitumor effects in vivo, we decided to examine whether the immunomodulatory oligonucleotide could also have direct effects on the cancer cells. We first examined the effects of the immunomodulatory oligonucleotide on A549 lung cancer cell survival in vitro. A dose-dependent inhibitory effect on cell survival was observed in cells treated with the immunomodulatory oligonucleotide, but only when the immunomodulatory oligonucleotide was administered with the transfection reagent Lipofectin, and to a lesser extent in cells treated with the control oligonucleotide (Fig. 4A ).


Figure 4
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  		Figure 4. In vitro effects of immunomodulatory oligonucleotide treatment on A549 cells. A, percent survival following immunomodulatory oligonucleotide or control oligonucleotide treatment determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. B, proliferation index of cells treated with the immunomodulatory oligonucleotide or control oligonucleotide determined by BrdUrd assay. C, apoptotic index of cells treated with the immunomodulatory oligonucleotide or control oligonucleotide determined by Annexin V-FITC staining.

 
We next evaluated the immunomodulatory oligonucleotide for effects on cell proliferation in vitro. Treatment with the immunomodulatory oligonucleotide resulted in decreased proliferation of A549 cells when administered with Lipofectin. The control oligonucleotide had minimal effects on proliferation (Fig. 4B).

Finally, we determined whether the immunomodulatory oligonucleotide has an effect on cell apoptosis. A significant increase in apoptosis was seen in immunomodulatory oligonucleotide-treated cells (Fig. 4C). The control oligonucleotide had a minimal effect on apoptosis. As with the evaluation of survival and proliferation, Lipofectin was required for administration of the immunomodulatory oligonucleotide to have any effect on apoptosis.

TLR9 Is Expressed in a NSCLC Cell Line
A549 cells were responsive to treatment with the immunomodulatory oligonucleotide even in the absence of immune cells. Lipofectin was required for there to be an effect on the cancer cells in vitro, suggesting that internalization of the immunomodulatory oligonucleotide is required. TLR9 is an intracellular receptor localized within endosomes and the endoplasmic reticulum (39, 40). The fact that internalization was required for an effect and that A549 cells were directly affected by the immunomodulatory oligonucleotide in vitro suggests that the cancer cells themselves express TLR9. There have been a few reports indicating that TLR9 may be expressed in nonimmune cells (41–43). Based on these data, we decided to evaluate the A549 NSCLC cell line for expression of TLR9.

We observed that TLR9 mRNA and protein are expressed at basal levels in A549 cells (Fig. 5A and B ) and that treatment with the immunomodulatory oligonucleotide in the presence of Lipofectin (but not in the absence of Lipofectin) may result in a slight increase in TLR9 mRNA expression (Fig. 5C). These data indicate that the immunomodulatory oligonucleotide may be able to act directly on tumor cells.


Figure 5
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  		Figure 5. Expression of TLR9 in A549 cells. TLR9 mRNA (A) and protein (B) are expressed in vitro in cultured A549 cells. Treatment with the immunomodulatory oligonucleotide in combination with a transfection agent (Lipofectin) may change the expression of TLR9 mRNA (C).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Lung cancer is a disease that is both more resistant to therapy than many other cancers and highly metastatic. Only 15% of patients survive for ≥5 years following diagnosis of lung cancer (1). The current therapeutic approaches are not optimally effective. There is an urgent need to search for novel, effective therapy for lung cancer. After half a century of nonspecific cytotoxic drugs being the standard for cancer therapy, progress made in the identification, cloning, sequencing, and further investigation of tumor-specific targets has made the use of rationally designed drugs possible.

The immunomodulatory oligonucleotide represents a novel class of rationally designed immunotherapy. This type of therapy does not rely on time- and cost-intensive isolation of tumor antigens nor does it rely on culturing immune cells ex vivo. In addition, the immunomodulatory oligonucleotide is more effective, more stable, and more specific than other TLR9 agonists, including the previous generation of CpG-containing oligonucleotides (30–32). In the present study, we have shown at least four important points: (a) treatment with the immunomodulatory oligonucleotide as a single agent led to potent antitumor effects, inhibiting tumor growth in all four in vivo models, regardless of p53 status; (b) combining the immunomodulatory oligonucleotide with a chemotherapeutic agent (gemcitabine or Alimta) led to enhanced effects following treatment, providing a novel combination regimen for lung cancer; (c) the immunomodulatory oligonucleotide increased apoptosis, decreased proliferation, and decreased survival in A549 cells in vitro, indicating a previously unrecognized mechanism of action for immunomodulatory oligonucleotides: direct effects on cancer cells; and (d) both TLR9 mRNA and protein were expressed in NSCLC cells, providing a basis for further development of TLR9-based therapy for lung cancer.

TLR9 expression has been shown in several immune cell subsets and was previously considered to only be expressed in immune cells (44). However, there is now increasing evidence of TLR9 expression in nonimmune cells (41–43). In this article, we have shown both TLR9 mRNA and protein expression in a NSCLC cell line. This suggests that tumors themselves may be responsive to TLR9 agonists. In vitro, administration of the immunomodulatory oligonucleotide to cells causes apoptosis and decreases cell proliferation, independent of the involvement of any immune cells. It may be possible that the immunomodulatory oligonucleotide is directly cytotoxic to tumor cells. In this case, the immunomodulatory oligonucleotide can be considered to have two mechanisms of action: stimulation of an antitumor immune response and direct cytotoxicity to tumor cells.

Nude mice bearing xenograft tumors are one of the most frequently used models to study cancer therapeutic agents. The establishment of tumors in these animals is facilitated by their immunocompromised state. It could be suggested that these animals represent a less appropriate model for studying an agent such as the immunomodulatory oligonucleotide, which has shown effects on the immune system. Whereas there is some validity to this claim, nude mice do, in fact, have immune system function. They still possess a normal number of B cells, natural killer cells, and macrophages, all of which have been shown to play a role in mediating the effects of TLR9 agonists (10, 16, 17). In addition, nude mice are often "leaky" and still have some functional T cells, although in lower numbers than normal mice. However, the immunocompromised condition of the nude mice and the results observed from these studies do suggest that full immune system function is not required for the TLR9-mediated antitumor effects. It may be that the direct effects on tumor cells via TLR9 are at least partially responsible for the potent effects against tumors. This is particularly promising for cancer patients who receive chemotherapy or radiation therapy or who have compromised immune function for other reasons because the immunomodulatory oligonucleotide represents a rationally targeted therapy with low toxicity, which can be used in combination with or following administration of other therapeutic strategies.

Moreover, our results indicate that the immunomodulatory oligonucleotide has similar therapeutic effects in tumors with or without functional p53 expression. This is an important property of the immunomodulatory oligonucleotide because it is believed that DNA damaging treatments, such as chemotherapeutic agents and radiation therapy, often exert their therapeutic effects via p53 activation, resulting in apoptosis and cell cycle arrest. The immunomodulatory oligonucleotide showed significant antitumor efficacy in various cancer models, regardless of the p53 status, indicating that the immunomodulatory oligonucleotide would have broad-spectrum utility against cancers with different genetic backgrounds. Combining traditional therapeutic agents with new agents such as the immunomodulatory oligonucleotide could also prove invaluable. Tumors are heterogeneous and cancer cells are often multidrug resistant, making combination therapy necessary to ensure that all cells within the tumor are destroyed. Using a combination of drugs targeting different pathways and receptors increases the likelihood of eradicating the cancer.

Immunomodulatory oligonucleotide or other TLR9 agonists might enhance the efficacy of traditional therapies in several ways. Beyond the simple increase due to targeting of different pathways, there are numerous other immune system-dependent and independent ways by which the immunomodulatory oligonucleotide may act additively or synergistically with other agents. First, the immunomodulatory oligonucleotide may protect immune cell subsets from cytotoxic agents or radiation, allowing for increased immunosurveillance and an increased likelihood of tumor rejection (45). Second, the immunomodulatory oligonucleotide may alter signal transduction, perhaps leading to an increase in apoptotic signaling such as has been shown following TLR2 agonism (46). It may also lead to other changes such as in epidermal growth factor receptor signaling and angiogenesis (47). Finally, immunomodulatory oligonucleotide treatment may lead to changes in the pharmacokinetics of other agents, such as has been seen with antisense oligonucleotides (48). Combining the immunomodulatory oligonucleotide with other therapeutic approaches, such as monoclonal antibodies, would also be likely to lead to enhanced therapeutic efficacy.

In conclusion, the immunomodulatory oligonucleotide was shown to be effective against NSCLC, both alone and in combination with traditional chemotherapeutic agents. The immunomodulatory oligonucleotide is currently in phase II trials for clear cell renal carcinoma. The results generated in this study suggest that the immunomodulatory oligonucleotide should be evaluated in the clinic for NSCLC, administered alone and/or in combination with conventional chemotherapeutic agents.


   Acknowledgments
 
We thank Dr. Gautam Prasad for excellent technical assistance and Drs. Robert B. Diasio and Donald L. Hill for helpful discussions.


   Footnotes
 
Grant support: University of Alabama at Birmingham, Idera Pharmaceuticals, Inc., University of Alabama at Birmingham Comprehensive Cancer Center for Cancer Pharmacology Laboratory, and Predoctoral Traineeship Award from the U.S. Department of Defense Prostate Cancer Research Program, grant no. W81XWH-06-1-0063 (E.R. Rayburn).The apoptosis analyses were done by the Flow Cytometry Core of the Arthritis and Musculoskeletal Center, which is supported in part by NIH grant P60 AR20614.

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.

Received 2/20/06; revised 3/15/06; accepted 4/13/06.


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 Introduction
 Materials and Methods
 Results
 Discussion
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