CpG oligodeoxynucleotide-based therapy of lymphoid malignancies

Preclinical and early clinical trials indicate synthetic oligodeoxynucleotides containing unmethylated CG dinucleotides (CpG ODN) have potent immunostimulatory effects. CpG ODN are being explored as immune adjuvants in vaccination strategies and as potential treatments for a wide variety of disorders including cancer and asthma. Therapeutic approaches designed to take advantage of this potent class of agents are based largely on the ability of CpG ODN to activate professional antigen presenting cells (APCs) that express the target receptor — Toll-Like Receptor 9 (TLR9). B-cell malignancies are unique in that the malignant cells themselves express TLR9. CpG ODN can have a direct effect on the malignant B cells and lead to activation induced cell death. CpG ODN also alter the phenotype of target malignant B cells as indicated by upregulation of MHC, immunostimulatory molecules, and antigens that serve as targets for other approaches to lymphoma immunotherapy such as CD20. B cell malignancies are also relatively sensitive to the cytokines that are produced by dendritic cells in response to CpG ODN. Thus, B cell malignancies appear to be uniquely sensitive to CpG ODN because of both the direct and indirect effects the CpG ODN on target cells and the sensitivity of B cell malignancies to an immune response. Preclinical studies support further exploration of the potential of CpG ODN as a component of therapy for lymphoid malignancies. Ongoing clinical trials are exploring the potential of CpG ODN, both alone and in combination with other agents.     

Approaches to enhancing immune responses stimulated by CpG oligodeoxynucleotides

CpG oligodeoxynucleotides (ODN) activate the immune system and are promising immunotherapeutic agents against infectious diseases, allergy/asthma and cancer. It has become apparent that while CpG ODN are potent immune activators in mice, their immune stimulatory effects are often less dramatic in humans and large animals. This disparity between rodents and mammals has been attributed to the differences in TLR9 expression in different species. This along with the sometimes transient activity of ODN may limit its potential immunotherapeutic applications. Several approaches to enhance the activity of CpG ODN have been explored including formulation of ODN in depot-forming adjuvants, and more recently, coadministration with polyphosphazenes, inhibitors of cytokines that downregulate TLR9 activation, and simultaneous activation with multiple TLR agonists. We will discuss these approaches and the mechanisms involved, with emphasis on what we have learned from large animal models.     

含非甲基化胞苷-磷酸盐-鸟苷寡脱氧核苷酸在非小细胞肺癌治疗中的应用进展

非甲基化胞苷-磷酸盐-鸟苷(CpG)寡脱氧核苷酸(ODN)属于Toll样受体9(TLR9)激动剂,其功能主要通过TLR9信号系统来体现。根据构效关系,共鉴别出K型、D型、C型和P型4个类别的CpG ODN,分别对B细胞和浆细胞样树状突细胞产生不同刺激作用。无论单独应用CpG ODN,抑或联合化疗、放疗,还是联合靶向药物或疫苗,对非小细胞肺癌的治疗均有一定的增效作用。该文也简述了CpG ODN的安全性。     

Toll-like receptors involved in the response to microbial pathogens: development of agonists for toll-like receptor 9

Upon microbial infection, a host has to mount a multiplicity of immune responses that target the invading pathogen. This is achieved in part by the use of particular receptors expressed on mammalian innate immune cells, the toll-like receptors (TLRs). Recognition of a given microbial molecular structure, such as bacterial or viral DNA, leads to the activation of signaling pathways that result in distinct sets of immune responses. The pathogen structures that are recognized by TLR9 in bacterial or viral DNA are deoxycytidyl-deoxyguanosine dinucleotides (CpGs) in specific sequence contexts (CpG motifs). The stimulatory activity of pathogen DNA can be mimicked by synthetic oligodeoxynucleotides (ODNs) containing such motifs (CpG ODNs). The TLR9-mediated stimulation of the vertebrate innate immune system, and subsequently of the adaptive immune system, allows the use of TLR9 agonists as highly effective vaccine adjuvants for infectious diseases, and as stand-alone therapies or in combination with other therapies in cancer.     

Early development of the Toll-like receptor 9 agonist,PF-3512676, for the treatment of patients with advanced cancers

Background: Unmethylated oligodeoxynucleotides (ODNs) with cytosine-phosphate-guanine (CpG) motifs can potently activate the immune system through Toll-like receptor (TLR) 9. PF-3512676 is a synthetic CpG ODN that induces strong Th1-type immune responses through TLR9 and is now in clinical development. Objective: To review discovery and development of synthetic CpG ODNs and their effects on immune cells and to relate preclinical and early clinical development of PF-3512676. Methods: A literature search was performed on databases available through the National Library of Medicine (PubMed), the European Society of Medical Oncology and the American Society of Clinical Oncology. Results/conclusions: Unmethylated CpG motifs were identified as the element of bacillus Calmette-Guérin responsible for immunostimulatory activity. Preclinical studies identified the mechanism of action (i.e., TLR9) and an optimal human sequence for antitumor activity. On the basis of preclinical studies, PF-3512676, a B-class CpG ODN, was selected for further clinical development. Phase I/II clinical trials have shown PF-3512676 to be well tolerated and to have antitumor activity as a single agent in patients with several types of advanced cancer, and to show promise as a vaccine adjuvant.     

Lipid-based delivery of CpG oligodeoxynucleotides for cancer immunotherapy

The anti-tumor activity of CpG-containing oligodeoxynucleotides (ODNs) has been well established in numerous animal models and confirmed in a number of early clinical trials. While the use of chemical modifications has effectively reduced the sensitivity of ODNs to nuclease degradation and a number of human trials have yielded promising results, the clinical utility of free CpG ODN still faces several significant challenges that must be addressed to achieve optimal potency and therapeutic activity. These include unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and poor intracellular uptake. To overcome these challenges, lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution characteristics, enhance immune cell targeting and facilitate intracellular uptake. In preclinical cancer models, lipid-mediated delivery has demonstrated the capacity to increase the immunopotency of CpG ODNs and dramatically enhance their anti-tumor efficacy as monotherapies, vaccine adjuvants and combination therapies with monoclonal antibodies or chemotherapy. This review will focus on investigating CpG ODNs as a cancer immunotherapeutic and the promising enhancement in efficacy that can be achieved through the use of lipid nanoparticles as delivery vehicles.     

Toll-like receptor 9 agonists as cancer therapeutics

INTRODUCTION: Toll-like receptor 9 (TLR9) agonists, commonly referred to as CpG oligodeoxynucleotides (ODN), have been added to the arsenal of anti-cancer drugs as monotherapy or in combination with chemotherapy, radiotherapy and other immunotherapeutic approaches as they increase antigen presentation and boost anti-tumor T- and B-cell responses. Several synthetic TLR9 agonists have been developed for clinical grade use and displayed substantial efficacy in the preclinical and clinical models. AREAS COVERED: This review summarizes TLR9 signaling and the impact of TLR9 agonists on the immune response. The most recent experimental and clinical data are analyzed as well as the development of new TLR9 agonists in current clinical trials. EXPERT OPINION: Application of TLR9 agonists, in particular, combination strategies with chemo- or radiotherapy seem a promising and efficient immunotherapeutic approach in cancer patients even with refractory disease. Simultaneous application of TLR9 agonists aims at supporting the patient's immune response and overcoming specific immunosuppressant strategies developed by tumors. Combinatory approaches of the future might also seek for synergism of TLR9 agonists with other immunomodulatory strategies such as B-cell activation using the CD40-CD40L system.     

Lipid-based delivery of CpG oligonucleotides enhances immunotherapeutic efficacy

There has been significant interest in the potential of cytosine-guanine (CpG) containing oligodeoxynucleotides (ODN) as an immunotherapy for malignant, infectious and allergic diseases. While human trials have yielded promising results, clinical use of free CpG ODN still faces several challenges which limit their effectiveness. These include suboptimal in vivo stability, toxicity, unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and the requirement for intracellular uptake. To overcome these challenges, optimized lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution so as to enhance immune cell targeting and facilitate intracellular uptake. Ultimately, lipid-mediated delivery has the capacity to increase the immunopotency of CpG ODN and enhance their prophylactic or therapeutic efficacy in a range of diseases. Lipid-encapsulation provides a feasible strategy to optimize the immunostimulatory activity and immunotherapeutic efficacy of CpG ODN, thereby allowing their full clinical potential to be realized.     

Synthetic oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODNs) and modified analogs as novel anticancer therapeutics

Oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODNs) activate the host immune system, leading to innate and acquired immune responses. The immune stimulatory effects of CpG ODNs are being exploited as a novel therapeutic approach to treatment of human diseases, and some CpG ODNs are being evaluated in clinical trials. The cellular recognition of CpG motifs requires the presence of the Toll-like receptor (TLR) 9, which triggers cell signaling and immune responses. There are three main types of first-generation CpG ODNs, which mimic the immunostimulatory activity of bacterial DNA and are recognized by TLR9, A-, B- and C-Class ODNs. Although all three CpG ODN classes stimulate TLR9-dependent signaling, there are striking differences in the cell types they activate and their dose-dependent immunostimulatory efficacy. Second-generation CpG ODNs, with advanced nucleic acid chemistry and unique modifications to their sequences and structures are being developed. Medicinal chemistry studies suggest that the immunomodulatory activity of CpG ODNs can be altered by site-specific incorporation of modifications in order to develop disease-specific drugs. Both first- and second-generation CpG ODNs have potential for treatment of various human diseases, such as infections, immunodeficiencies, and cancers. This article will focus on the recent advances in developing CpG ODNs as novel anti-cancer therapeutic agents.     

CpG oligodinucleotides induce strong humoral and cellular responses to swine streptococcic septicemia vaccine in piglets in vivo

Oligodinucleotides containing CpG motifs (CpG ODN) are strong adjuvants for immune responses, particularly in mice, but data on humoral and cellular immune responses in piglets are scarce. In this report, using the swine streptococcus as model bacteria, CpG ODN was used as immunoadjuvants to enhance the immune responses of the piglets to swine streptococcic septicemia killed vaccine (SSSK vaccine). The titre of specific antibodies to SSSK vaccine, the proliferation of lymphocytes, SSSK-specific interferon-gamma(IFN-gamma) and IL-6, the expression of major histocompatibility complex class II (MHC-II) and CD14 of peripheral blood mononuclear cells (PBMCs) were examined to identify the immune responses of the piglets. The results were found that the above-mentioned immune responses of the piglets with CpG ODN were significantly stronger than standard immunization protocols. All these data suggested that immunostimulatory CpG ODN was promising immune enhancers for vaccination against SSSK vaccine.     

TLR agonists are highly effective at eliciting functional memory CTLs of effector memory phenotype in peptide immunization

Given the importance of memory cytotoxic T lymphocytes (CTLs) in eliminating altered self-cells, including virus-infected and tumor cells, devising effective vaccination strategies for generating memory CTLs is a priority in the field of immunology. Herein, we elaborate upon a novel boosting approach that utilizes synthetic peptides and Toll-like receptor (TLR) agonists as adjuvants to generate sufficient numbers of memory CTLs to protect against infection in mice. Peptide boosting with lipopolysaccharide (LPS), a TLR4-ligand, has been shown to progressively enhance memory CTLs. Whether this result is strictly dependent on activation of TLR4 or can be similarly achieved by signaling through other TLRs is of practical interest in vaccine development but is yet unknown. In this report, we present evidence that intravenous peptide boosting together with TLR3 and TLR9 agonists (Poly IC and CpG, respectively) is highly effective and induces large quantities of memory CTLs of effector memory phenotype after three boosts. Compared to LPS, CpG and Poly IC generate more robust immune responses after the first and second boosts, indicating that a protective level of CTLs might be achieved with fewer boosts when CpG or Poly IC is used. Lastly, the resultant memory CTLs from boosting with different TLR agonists as adjuvant are equally protective against pathogen challenge and are not immune senescent. Therefore, TLR agonists are effective adjuvants in intravenous peptide boosting for the generation of functional memory CTLs.     

TLR based therapeutics

Toll-like receptors (TLRs) play a crucial role in innate immune responses to infection. Binding of agonists to TLRs promotes maturation of antigen presenting cells, such as dendritic cells, which in turn directs the induction of adaptive immune responses. For this reason TLR agonists are being exploited as vaccine adjuvants for infectious disease or cancer and as therapeutics against tumors. However TLR agonists also promote inflammatory cytokine production and have a pathogenic role in many diseases with an inflammatory basis, including autoimmune diseases. Consequently, antibodies to TLRs and inhibitors of TLR signalling pathways have considerable potential as therapeutics for inflammatory disorders. Some have shown to be efficacious in pre-clinical models, and have now entered clinical trials.     

TLR7/9 antagonists as therapeutics for immune-mediated inflammatory disorders

There is an increasing interest in ligands of nucleic acid-sensing Toll-like receptors (TLR), especially TLR7 and TLR9, for pharmacological intervention in various diseases. The TLR7 agonist imiquimod is currently used as a topical treatment for genital warts caused by human papillomavirus (HPV), actinic keratosis (AK) and superficial basal cell carcinoma. Oligodeoxynucleotides (ODN) TLR9 agonists are currently in clinical trials for use in lung cancer, as anti-viral therapy, as adjuvants and as immune modulators in asthma and allergies. TLR7/9 antagonists, such as the anti-malaria drugs chloroquine, hydroxychloroquine and quinacrine, have been used since the 1950s to treat immune-mediated inflammatory disorders (IMID) such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sj?gren's syndrome. However, the use of these anti-malarials in IMID is limited due to the side effects or suboptimal efficacy. Pre-clinical animal models as well as genetic linkage studies have indicated that TLR7/9 play a pivotal role in the aforementioned as well as other IMID such as multiple sclerosis (MS), inflammatory bowl disease (IBD)/colitis and psoriasis. Recent evidence has suggested that selective, specific antagonists for TLR7 and/or 9 might be more beneficial in certain diseases, such as SLE. Thus, the use of suppressive ODN or novel small molecule TLR7/9 inhibitors with a larger safety window and differentiated selectivity may potentially have significant clinical utility in these IMID. Herein, we review efforts to develop novel TLR7/9 antagonists and the rationale for the use of such therapeutics in a variety of IMID.     

Modifying toll-like receptor 9 signaling for therapeutic use

Toll-like receptor (TLR) 9 recognizes synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG) motifs and mimics the immunostimulatory activity of bacterial DNA. Both innate and adaptive immune systems are activated through TLR9 signaling and thus its synthetic agonists or inhibitors have potential significance as a target for therapeutic use in immunological disorders. Interestingly, TLR9 found in the dendritic cells and B cells produce differential outcome in response to structurally distinct CpG-ODNs. While one class of CpG-ODN activates B cells and produce immunoglobulin, other can either redirect plasmacytoid dendritic (pDC) cells to secrete high level of IFNalpha or myeloid dendritic cells (mDC) to produce Th1-like cytokines and chemokines necessary for asthma control. This review focuses on potential use of various synthetic CpG to modify TLR9 signaling for therapeutic treatment of multiple diseases including cancer, asthma, allergy and systemic lupus erythematosus (SLE).     

A comparative study of the antigen-specific immune response induced by co-delivery of CpG ODN and antigen using fusion molecules or biodegradable microparticles

CpG ODN are toll-like receptor 9 (TLR9) agonists that can enhance antigen presentation by antigen presenting cells (APCs) such as dendritic cells (DCs). The most potent antigen-specific responses are seen when CpG ODN and the antigen are co-localized in the same APC. CpG ODN-antigen fusion molecules and biodegradable microparticles entrapping CpG ODN and antigen can ensure both components are delivered to the same APC. In this study, we compared the efficacy of the CpG-ODN fusion molecules against biodegradable microparticles entrapping antigen and CpG ODN. Microparticles were prepared using a double emulsion solvent evaporation methodology. CpG ODN-OVA fusion molecules were prepared by mixing maleimide-activated protein with thiolated CpG ODN. Both CpG ODN-OVA fusion molecules and microparticles co-entrapping CpG ODN and OVA generated stronger IgG2a and interferon-gamma (IFN-gamma) responses than delivery of soluble CpG ODN and OVA. The microparticles generated stronger IgG2a and IFN-gamma immune responses than did CpG ODN-antigen fusion molecules.     

Innovative strategies for co-delivering antigens and CpG oligonucleotides

Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODN) is a recent class of immunostimulatory adjuvants that includes unmethylated CpG dinucleotide sequences similar to those commonly found in bacterial DNA. CpG ODN specifically triggers toll like receptor 9 (TLR9), which is found within phagoendosomes of antigen presenting cells (APCs) such as dendritic cells (DCs). CpG ODN triggers activation and maturation of DCs and helps to increase expression of antigens. CpG ODN can be used to induce polarized Th1 type immune responses. Several studies have shown that antigens and CpG ODN must be co-localized in the same APC to generate the most potent therapeutic antigen-specific immune responses. Delivery vehicles can be utilized to ensure co-delivery of antigens and CpG ODN to the same APCs and to significantly increase uptake by APCs. These strategies can result in antigen-specific immune responses that are 5 to 500-fold greater than administration of antigen alone. In this review, we discuss several recent and innovative strategies to co-delivering antigens and CpG ODN adjuvants to APCs. These approaches include the utilization of conjugate molecules, multi-component nanorods, liposomes, biodegradable microparticles, pulsatile release chips and cell-microparticle hybrids.     

CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs act as immune adjuvants, accelerating and boosting antigen-specific immune responses. CpG motifs promote the induction of Th1 and pro-inflammatory cytokines and support the maturation/activation of professional antigen presenting cells (particularly plasmacytoid dendritic cells). These effects are optimized by maintaining close physical contact between the CpG ODN and the immunogen. Co-administering CpG ODN with a variety of vaccines has improved the resultant humoral and/or cellular immune responses, culminating in enhanced protective immunity in rodent and primate challenge models. Ongoing clinical studies indicate that CpG ODN are safe and well-tolerated when administered as adjuvants to humans, and that they can support increased vaccine-specific immune responses.     

Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4, TLR9 mRNA expressions and transcription factor NF-kappa B activation

Bacterial DNA (bDNA) and lipopolysaccharide (LPS) are potent activators of immune cells such as monocytes and macrophages, which contribute to systemic inflammatory response syndrome (SIRS) and sepsis. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and currently there is only one adjuvant therapy in clinical use, e.g. activated protein C. Artesunate (AS), a water-soluble derivative of dihydroartemisinin, has recently been demonstrated to protect against LPS-induced human umbilical vein endothelial cell (HUVEC) activation and injury by inhibiting tumor necrosis factor-alpha (TNF-alpha) mRNA expression. In the present study, heat-killed Escherichia coli was used to induce sepsis in the animal models. We observed that AS could protect mice against a lethal challenge with heat-killed E. coli in a dose-dependent manner. This protection was associated with reductions in serum TNF-alpha and measurable endotoxin levels. In addition, the treatment of murine peritoneal macrophage cells with AS strongly inhibited the release of TNF-alpha and IL-6 induced by CpG oligodeoxynucleotide (CpG ODN), LPS, or heat-killed E. coli in a dose-dependent manner. Experiments using affinity sensor technology revealed that AS could not directly bind to CpG ODN or LPS. Moreover, AS could not neutralize LPS in vitro. Further, flow cytometry revealed that AS could not alter the binding of CpG ODN to cell surfaces but could promote CpG ODN accumulation within RAW264.7 cells. Furthermore, AS reduced the expressions of TLR4 and TLR9 mRNA that were stimulated by LPS, CpG ODN, or heat-killed E. coli and inhibited heat killed E. coli-induced NF-kappaB activation. In conclusion, our results demonstrated that AS-mediated protection against a lethal heat-killed E. coli challenge was associated with a reduction in proinflammatory cytokine release and endotoxin levels via a mechanism involving a decrease in TLR4, TLR9 mRNA expression and NF-kappaB activation.     

Decreased intracellular TLR9 confers hyporesponsiveness of RAW264.7 cells to subsequent CpG ODN challenge

Low-dose CpG ODN pretreatment is known to induce effective protective immunity against acute infectious diseases. In the present study, using primary murine peritoneal macrophages and the macrophage-like cell line, RAW264.7, we investigated whether low-dose CpG ODN pretreatment would induce hyporesponsiveness in response to a subsequent high-dose CpG ODN challenge and further investigated the molecular mechanisms underlying this event. Our results showed that pretreatment with a low dose of CpG ODN inhibits TNF-alpha production stimulated by later high-dose CpG ODN stimulation in a dose- and time-dependent manner. Interestingly, anti-mouse TLR9 blocking antibody added prior to CpG ODN pretreatment did not affect TNF-alpha release, but antibody added after CpG ODN pretreatment augmented the pretreatment effect of CpG ODN. This difference suggests that cell-surface TLR9 is indeed functional on activated cells. Flow cytometry revealed that low-dose CpG ODN pretreatment decreased cell-surface binding and internalization of a subsequent high-dose stimulation, suggesting that decreased internalization of succeeding CpG ODN is associated with reduced TNF-alpha release. Although both intracellular and cell-surface TLR9 expression are observed, low dose of CpG ODN pretreatment increased only cell-surface TLR9 levels. Importantly, low-dose CpG ODN pretreatment also significantly inhibited the activation of NF-kappaB, an important downstream regulator of various proinflammatory cytokines. In summary, our results demonstrate that suppression of TNF-alpha production by low dose of CpG ODN pretreatment correlates with decreased binding and internalization of subsequent CpG ODN, decreased intracellular content of TLR9, and inhibition of NF-kappaB activation.     

CpG 7909: PF 3512676, PF-3512676

CpG 7909 [PF-3512676] is an immunomodulating synthetic oligonucleotide designed to specifically agonise the Toll-like receptor 9 (TLR9). It is being developed for the treatment of cancer [ProMune] as a monotherapy and in combination with chemotherapeutic agents, and it is also under development as an adjuvant [VaxImmune] for vaccines against cancer and infectious diseases. CpG 7909, acting through the TLR9 receptor present in B cells and plasmacytoid dendritic cells, stimulates human B-cell proliferation, enhances antigen-specific antibody production and induces interferon-alpha production, interleukin-10 secretion and natural killer cell activity. Coley Pharmaceutical Group originally developed CpG 7909 using its CpG DNA technology. In March 2005, Coley granted Pfizer an exclusive global license to develop and commercialise CPG 7909 [ProMune] for the treatment, control and prevention of multiple cancer indications. Coley licensed CpG 7909 [VaxImmune] to Chiron Corporation for adjuvant use with Chiron's prophylactic vaccine candidates against infectious diseases in December 2003. Chiron was acquired by and merged into Novartis in April 2006. In 2002, GlaxoSmithKline (GSK) was granted a worldwide, non-exclusive licence to Coley's CpG immunostimulatory oligonucleotides, including CpG 7909 [VaxImmune], for their use as adjuvants for cancer vaccines. In 2000, Coley entered into a co-exclusive licensing agreement with GSK for the development of therapeutic and prophylactic vaccines against infectious diseases. This licensing agreement included CpG 7909 [VaxImmune] and other CpG-based immunostimulatory oligonucleotides. In September 2004, Coley Pharmaceuticals was awarded a 16.9 million US dollars, 5-year contract from the National Institute of Allergy and Infectious Diseases (NIAID), one of the National Institutes of Health (NIH), to support the development of novel immune-activating drugs for defense against bioterror agents. This contract will be used to expand Coley's proprietary line of TLR Therapeutic products. Together with prior awards, the new contract brings the total committed biodefense funding for Coley to 35 million US dollars. During the first quater of 2006, Pfizer disclosed its intention to develop CpG 7909 for breast cancer. A phase I/II trial in patients with NHL has also been conducted in 24 patients with relapsed or refractory disease at the University of Iowa. Pfizer initiated two international phase III trials under the special protocol assessment (SPA) procedure of the US FDA. These trials are evaluating CpG 7909 in combination with chemotherapy versus chemotherapy alone as a first-line treatment for patients with advanced (stage IIIb or IV) non-small-cell lung cancer (NSCLC). Approximately 800 patients will be enrolled in each trial. The primary endpoint is overall survival time. In 2005, Coley completed a multinational phase IIb trial of CpG 7909 in combination with chemotherapy in 112 patients with NSCLC. The goal of the study was to improve the outcome of standard first-line chemotherapeutic regimens (taxane and platinum) for NSCLC by adding CpG 7909. Coley has been granted 11 US patents, covering key aspects of the company's CpG TLR9 antagonist technologies. Three US patents relating to CpG 7909 and the use of certain oligonucleotides for treating cancer are due to expire in 2014. Coley also has pending US patent applications covering the specific sequence of CpG 7909 and its use to treat cancer, which, if issued, would be expected to expire between 2014 and 2017. In April 2004, Coley received US Patent No. 6,727,230 covering the use of oligonucleotides containing at least one phosphorothioate linkage to stimulate cellular immune responses. In November 2003, Coley received US Patent No. 6,653,292, which protects the use of TLR9-containing oligonucleotides to treat or prevent cancer or to enhance multimodal cancer treatment regimens. In July 2002, Coley received US Patent No. 6,406,705 covering its CpG oligonucleotide immunostimulants for use in combination with conventional adjuvants. In April 2001, Coley was issued with US Patent No. 6,207,646 covering the composition and use of its immune stimulants including CpG 7909. In September 2001, Coley was granted US Patent No. 6,214,806, which expands the coverage on the use of CpG oligonucleotides in the treatment of certain respiratory disorders.