Synthetic immunostimulatory oligonucleotides in experimental and clinical practice

(1)

Minireview

Synthetic immunostimulatory oligonucleotides in experimental and clinical practice

Pawe³ Bodera, Wanda Stankiewicz, Janusz Kocik

Military Institute of Hygiene and Epidemiology, Kozielska 4, PL 01-163 Warszawa, Poland Correspondence: Pawe³ Bodera, e-mail: pbodera@gmail.com

Abstract:

Background: Oligonucleotides belong to a class of macromolecules with great potential for research and various therapeutic appli- cations. Their mechanisms of action are extremely diverse, although they are rather homogeneous in composition. Single-stranded oligodeoxynucleotides are not only inhibitors of gene expression, but their CpG sequence motifs may activate the innate immune response. Recent progress made in preclinical and clinical testing, as well as the case of the most recently discovered RNA interference technology, will help to overcome efficacy problems of the previous approaches of the ‘standard therapy’ of such diseases as tumors and various infections.

Methods: The aim of this article is to present various therapeutic aspects of oligonucleotides, and to review the most significant therapeutic applications of synthetic oligonucleotides. This paper presents a comprehensive review of current literature on various therapeutic properties of synthetic oligonucleotides.

Conclusions: The available results gathered from preclinical and clinical studies suggest that TLR9-targeted therapy of oligonu- cleotides can stimulate both innate and adaptive immunity. It also appears that CpG ODNs are generally safe, although moderate adverse effects, based on a backbone-related mechanism have been reported. The presented studies demonstrate that adjuvant CpG ODN can unify an immune response that leads to enhanced antigen-specific Ab formation. CpG ODN may therefore provide a unique approach to enhancing the efficacy of immunization, including the strengthening of antitumor immunity.

Key words:

immunostimulatory oligonucleotides, short interfering RNA, toll-like receptors, cytotoxic T-lymphocyte antigen 4, regulatory T cells

Abbreviations: Ab – antibody, B16 – mouse melanoma cell line, Bcl-2 – B-cell lymphoma 2, CTLA-4 – Cytotoxic T- Lymphocyte Antigen 4, CDR – complementarity determining region, CpG DNA – unmethylated CpG sequences in DNA, CpG ODN – oligodeoxynucleotide containing CpG motif, DTIC – dacarbazine, dsRNA – double-stranded RNA, HBsAg – surface antigen of the hepatitis-B virus, HBV – hepatitis B virus, HSV-2 – herpes simplex virus, IFN-a – interferon a, IFN-I – type I interferon, MC38 – murine colon carcinoma cell line, NHL – Non-Hodgkin’s Lymphoma, NK – natural killer cells, NSCLC – non-small-cell lung carcinoma, ODN – synthetic oligodeoxynucleotide, PDC – pyruvate dehydrogenase complex, PRR – pattern recognition receptor, RIG-I – retinoid-inducible gene 1, RNP – ribonucleoprotein enzyme,

siRNA – short interfering RNA, ssRNA – single-stranded RNA, STAT3 – signal transducer and activator of transcription 3, TH1 – T helper 1 cells, TLR7 – toll-like receptor 7

Introduction

A robust immunological defence system is vital to support the survival of the host organism. The adaptive immune system is based on lymphoid cell-surface

Pharmacological Reports 2012, 64, 10031010 ISSN 1734-1140

(2)

so-called pattern recognition receptors (PRRs) which recognize certain molecular structures that are present in pathogens. In recent years, significant progress in the understanding of innate immunity has been made through the identification of several families of pathogen sensors.

Oligonucleotides with the appropriate immunostimulatory sequences can be useful as vaccine adjuvants. The pharmaceutical compositions containing nucleic acids can be administered by any suitable route for administering medications.

The unmethylated CpG sequences in DNA (CpG DNA) have the ability to induce tumor regression by activating innate immunity, enhancing Ab dependent cellular cytotoxicity, and serving as potent vaccine adjuvants that elicit a specific, protective immune response. CpG oligonucleotides may also be good candidates for the treatment of asthma and allergy, various types of cancers and viral infectious diseases.

The above mentioned synthetic oligonucleotides’

therapeutic applications are presented in this paper.

RNA oligonucleotides

Human telomerase is a ribonucleoprotein (RNP) enzyme, consisting of protein components and an RNA template which are able to catalyze telomere elonga- tion through the addition of TTAGGG repeats. Te- lomerase function has been attributed to aging and the immortalization of cancer cells. Human telomerase, a specialized reverse transcriptase, synthesizes telo- meric DNA and contributes to the maintenance of functional telomeres in immortal or highly prolifera- tive cells such as germline cells, hematopoietic pre- cursors and tumor cells [35].

Cross linking is followed by RNA digestion; two proteins in the extract, in competition with one an- other, compete for the same RNA site. Cross-linking experiments were performed with the labelled RNA and various relative amounts of the two recombinant proteins. The bound RNA regions should be protected against the action of the probes, and identified by comparison of the cleavages and modifications obtained under the same conditions in the two probes of

plexes formed upon incubation of in vitro transcribed RNA in a cellular or nuclear extract.

Short interfering RNA

Increased tumor cell survival and immune escape are two hallmarks of tumor development. Single-targeted treatments are often ineffective, bearing in mind genetic and epigenetic plasticity allows tumors to pro- liferate [18].

A new method of melanoma treatment was recently presented, utilizing short interfering RNA (siRNA) containing triphosphate groups at the 5‘ ends (3p- siRNA) [30].

The 3p-siRNA comprises two distinct and independ- ent functional activities in one molecule: silencing of anti-apoptotic bcl-2, and activation of the cytosolic helicase of retinoic acid-inducible gene I (RIG-I).

Considerable anti-tumor activity in a metastatic melanoma model was elicited with systemic treatment with bcl-2-specific 3p-siRNA elicited.

NK cells and type I interferon (INF-I) are associated with downregulation of bcl-2 in metastatic tumor cells in vivo on a single cell level, which is necessary to support the overall therapeutic activity of 3p- siRNA in vivo. 3p-siRNA represents a novel single molecule-based combinatorial approach in which the retinoid-inducible gene 1 (RIG-I) activation on both the immune and the tumor cell level corrects immune ignorance, and in which gene silencing is used to correct key molecular events that govern tumor cell survival.

Kortylewski and co-authors [23] recently presented results of their study, comprising a new strategy to alter the balance in the tumor microenvironment to re-awaken an anticancer immune response. When the toll-like receptor (TLR) agonist was coupled with small interfering RNA (siRNA), the double blow of TLR-mediated immune activation and targeted silencing of immunosuppressive genes suppressed tumor growth in mouse models.

siRNA, which targets the signal transducer and activator of transcription 3 (STAT3), an oncogenic tran-

(3)

scription factor, was coupled with TLR9-specific CpG oligonucleotides, which are currently in clinical trials for the treatment of cancer, including melanoma.

STAT3 controls the expression of immunosuppressive and angiogenic factors, and is associated with the tumor environment characterized by a lack of tumor- specific cytotoxic T cells, an inhibition of T helper 1 (TH1) cells and an excess of regulatory T cells, acting together with the infiltration by myeloid-derived sup- pressor cells. STAT3 is also constantly active in many cancer cells of different origin, which accelerates tumor growth and survival as well as resistance to anticancer drugs. In addition, STAT3 is known to sup- press TLR-mediated TH1-type antitumor response.

CpG alone or CpG-conjugated siRNA with random sequence of CpG-siRNA inhibited the growth of subcutaneous B16 and K1735 melanoma cells and cancer cells, preventing the CT26 colon carcinoma cells to a greater extent, and inducing tumor regression in the MC38 colon cancer model.

In the B16 lung metastasis model, the systematic supply of small quantities of CpG-siRNA molecules (< 1 mg per kg) led to a significant reduction in the number of lung metastases, compared with controls [23]. In addition, the tumor microenvironment analysis showed the increased expression of chemokines and costimulatory molecules of dendritic cells in tumor-draining lymph nodes, as well as an increase in TH1-type cytokines, reducing the number of regulatory T cells. This analysis also showed the increased infiltration of cytotoxic T cells. B16 tumor sections in mice treated with CpG-siRNA presented extensive apoptosis of cancer cells, which was associated with increased tumor infiltration of neutrophils.

Providing a possibility to optimize the CpG-siRNA design, this study demonstrated an elegant way to simultaneously target tumor-associated immune cells in order to silence tumor promotion or immunosuppressive parti- cles, and stimulate TLR of immune activation.

Immunomodulation

The interesting results which were obtained during the study relate particularly to the immune system modulation, through the use of oligonucleotide- derived compounds [25].

The main aspect was to provide immunostimulatory agents that are less expensive to produce than

currently available oligonucleotides. The long oligonucleotides are usually expensive because they have specificity of flanking sequences limits, which is associated with the breadth of their function. A potential benefit of immunotherapy is to provide improved efficacy by enhancing the patient’s own immune response to tumors while minimizing deleterious effects to normal cells.

Bi-specific antibodies having one binding site for a cell surface antigen, such as human Cytotoxic T-Lym- phocyte Antigen 4 (CTLA-4), and a second binding site for an Fc receptor on the surface of an effector cell, have been demonstrated in other research [32].

It also relates to antibodies having the amino acid sequences of the complementarity determining regions (CDRs) of the heavy and light chains. CTLA-4 is a member of the immunoglobulin (Ig) superfamily of proteins that downregulate T-cell activation and main- tain immunologic homeostasis.

An internal dinucleotide, in general, shall mean any pair of adjacent nucleotides connected by an internu- cleotide linkage. Phosphate modification of the backbone located at a particular phosphodiester internu- cleotide bridge and/or at a particular 3-D-ribose unit of RNA molecule is parallel to the same sequence of natural DNA.

CpG ODN PF-3512676/anti-CTLA-4 may be used in a combination therapy to treat renal cell carcinoma; breast, colorectal, ovarian, non-small cell lung cancer; melanoma, cutaneous T-cell, and Non-Hodgkin’s Lymphoma (NHL), including its indolent and aggressive forms [32].

Antiviral effects

The pattern recognition receptors (PRRs) are engaged by the innate immune system to recognize non-self material based on factors such as nucleotide sequence specificity and cellular localization [1]. Expression of these receptors depends on various cell types and their activation states.

The innate immunity is induced by viral infections through replication intermediates such as double- stranded RNA (dsRNA) and single-stranded RNA (ssRNA) sequence motifs.

Classical H1N1 influenza virus genomes were modi- fied during virus evolution in humans by preferential elimination of CpG motifs in an A/U context [20].

Synthetic immunostimulatory oligonucleotides

Pawe³ Bodera et al.

(4)

on the signaling through TLR7.

The direct evidence that CpG RNA motifs in a U-rich context control PDC activation and IFN-a-driven activation of NK cells, likely through TLR7 was presented by the authors credited above.

An extremely low CpG content is observed in the coding regions of many human innate immunity genes, particularly IFN-Is.

mRNAs are heavily expressed during the acute phase of the innate response, and have a bias toward low CpG content [15].

The above-presented facts allow us to hypothesize that both ssRNA viruses and host genes involved in the innate immune response have evolved to have low CpG content in order to avoid a CpG RNA sensing receptor. Limitation of the spread of influenza virus infection depends on IFN-a [13].

The disease severity is connected with subtle differences among individuals in IFN responsiveness.

Thus, the decreasing of the stimulatory CpG motif content in humans, in order to avoid IFN-a secretion, may be a result of evolution of influenza virus.

The vaginal instillation of synthetic oligonucleotides containing CpG that activate TLRs have been experimentally used to induce an antiviral state.

An innate mucosal antiviral state which afforded protection against intravaginal contagion with herpes simplex virus (HSV)-2 was demonstrated after delivery of CpG oligodeoxynucleotide (ODN) to vaginal mucosa.

It is interesting that treatment with synthetic CpG ODN evoked local inflammation and splenomegaly, which was not observed during mucosal delivery of ligands for Toll-like receptor (TLR)-3, used for induced protection against genital HSV-2 challenge [3, 17, 31].

Cancer treatment

Optional treatment options may be useful in cancer treatment prior to and following surgery, radiation or chemotherapy. Important research relates to the use of anti-CTLA-4 Ab in combination with CpG oligonucleotides for cancer treatment [33].

class of investigational synthetic agonists for Toll-like receptor 9 – TLR-9, has been demonstrated to overcome tolerance, e.g., reverse or prevent anergy or tolerance to tumor antigens [23].

Conversely, inhibitory effects demonstrated by the regulatory T cells (Treg) may limit the effectiveness of CpG immunotherapy; therefore blocking these effects with an anti-CTLA-4 Ab should improve the efficacy of the CpG ODN. The applied methods enhanced antitumor response, allowing use of lower and/or less fre- quent doses of anti-CTLA-4 Ab and/or CpG ODN PF3512676 to reduce the incidence of adverse effects caused by the administration of anti-CTLA-4 Ab and/or CpG ODN PF-3512676 alone [32].

Agents that directly target angiogenic factors of their receptors offer the prospect for greater activity in receptor-competent hematologic malignancies by in- terrupting autocrine receptor signaling.

The first dose of studied ODN was administered in the period of one to three, or one to four months post- transplantation. Furthermore, the treatment methods that employ ODNs combined with radiation therapy are likely to be developed in the future.

Doses of the studied compounds for parenteral delivery for the purpose of effective enhancement of the systemic immune response varies from 2 to 1,000 times higher than the effective mucosal dose, more specifically 2 to 100 times higher, and usually 5 to 50 times higher.

The latest clinical study provides a method for treating or preventing NSCLC using therapeutically effective amount of a CpG ODN in the therapeutic dose of 0.01 to 5.0 mg/kg [21]. It offers combination therapies and treatment regimens for treatment of cancers, using an immunostimulatory oligodeoxynucleotide. Immunostimulatory CpG oligonucleotides contain specific sequences found to elicit the immune response. An oligonucleotide containing at least one unmethylated CpG dinucleotide is a molecule containing a cytosine-guanine dinucleotide sequence (i.e., CpG DNA or DNA with 5’ cytosine linked by a phosphate bond to a 3’ guanine) and is able to activate the immune system.

Regarding the mechanism of action and the pre- liminary preclinical pharmacology data, the studied entity PF-3512676 demonstrates immunotherapeutic properties and can be administered alone or as a com-

(5)

bination anticancer therapy [27]. Among others, the mentioned agent can be useful for melanoma, multiple myeloma, acute myeloid and chronic lymphocytic leukemia, prostate and lung cancer treatment. It also possesses the ability to act synergistically with a variety of chemotherapeutic agents and therapeutic mo- dalities in the treatment of cancer, such as: chemotherapy, radiation therapy, monoclonal antibodies and immunotherapy. Additionally, the quoted study provides a method for treating or preventing regression of melanoma. Therapeutic regimen comprises administration of a therapeutically effective amount of a CpG ODN, simultaneously, separately or sequen- tially in combination with a chemotherapeutic agent such as dacarbazine (DTIC), vinblastine, vincristine, vindesine, temozolomide, interferon or interleukin.

Tremelimumab, which was used in the other cited study, presented its immune activation by blocking the CTLA-4 negative co-stimulatory receptor with the an- tagonistic Ab [33]. Tremelimumab (formerly ticilimu- mab, CP-675,206) is a fully human IgG2 monoclonal Ab. It binds to the protein CTLA-4, which is expressed on the surface of activated T lymphocytes. Its administration to patients with locally advanced and metastatic melanoma has resulted a durable objective tumor regression. Its IgG2 isotype minimizes the possibility of cytotoxic effects evoked by activated T lymphocytes and cytokine release syndrome.

The CpG ODNs are readily soluble in aqueous buffers and can be administered by most routes (e.g., intravenously, subcutaneously, intraperitoneally, in- trapulmonarily and intranasally). Subcutaneous application of CpG ODNs turned out to be particularly ad- vantageous, as the ODN is detected at relatively high levels in the lymph nodes, resulting in excellent cytokine induction. As a result of the activation of both innate and adaptive immunity, CpG ODNs can be applied in the therapy of cancer, infectious diseases, and asthma or allergy, but also as highly effective adjuvants in vaccination [34].

Asthma and allergy

Combination of Engerix-B vaccine and CpG ODN significantly improved the effect of the HBV vaccine alone. HBsAG-specific Ab responses appeared sooner, and levels were significantly higher in volunteers receiving CpG ODN as adjuvant [41].

CpG ODNs are very effective agents for inducing a Th1-like immune response and therefore, they might stand for a good basis for the development of therapeutics which interfere with the core cause of the allergic disease rather than curing the symptoms only.

CpG ODNs were able to prevent allergen-induced airway inflammation in a previously sensitized mouse, suggesting that exposure to CpG ODN may protect against asthma. It was also shown that a single dose of CpG ODN inhibited airway eosinophilia at least as efficiently as daily injections of corticosteroids, the standard treatment for allergic airway disease [1]. Th1 and Th2 cells interact in a counter-regulatory fashion, since the Th2-type cytokines Il-4 and IL-10 promote Th2 development and inhibit Th1 cell and cytokine production. The Th2-type cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 play a central role in the pathogene- sis of allergic diseases, including asthma. While the Th2-type cytokines are secreted by activated CD41 T-cells, the Th1-type cytokines IL-2 and IFN-g are produced by Th1 cells.

Co-administration of CpG ODNs with an antigen efficiently prevented airway eosinophilia, Th2 cytokine induction, IgE production and bronchial hyper- reactivity in a murine model of asthma [2, 4].

Discussion

Chemical modification of CpG ODNs can be used to enhance stability against nucleases, to improve cellular uptake and compartmentalization, and to modulate binding to TLR-9 [19]. However, some of the reported structure-activity relationship (QSAR) data in the literature are conflicting, which may be explained by the fact that different sequences were used to study the influence of chemical modifications on immune stimulation [5, 29].

Many CpG ODNs contain multiple CpG motifs within a sequence and the effect of the chemical modification can be position dependent. The species- specific differences offer the possibility to use either the human (50-GTCGTT) or mouse (50-GACGTT) consensus hexanucleotide motif. Sometimes, ODNs that contain the human consensus motif have been as- sayed in murine cells and vice versa. The first genera- tion of CpG ODN drug candidates were fully

(6)

being examined for cancer immunotherapy [37].

Although initial reports by Carpentier and col- leagues demonstrated an 88% cure rate and long-term immunity in rats with i.c. gliomas [6, 9], others have shown variable CpG antiglioma responses ranging from no efficacy [40], through partial response [11] to complete tumor eradication [14]. These contrasting reports may be explained by differences in animal models and various CpG structures. Nevertheless, results from early-stage human clinical trials on patients with recurrent malignant gliomas, utilizing conven- tional CpG therapy have been disappointing, with only modest antitumor response in a few patients [7, 8].

TLR9 which was located in the endosomal compartment, such as those available in bacterial DNA, was shown to specifically respond to unmethylated CpG motifs (Fig. 1 and 2).

Synthetic CpGs contain repeat unmethylated CpG motifs, which were designed to enhance TLR9 en- gagement and phosphorothioate linkages in order to prevent therapy breakdown [38].

Other work suggests that DNA recognition by TLR9 depends on the 20 deoxyribose phosphate backbone. Therefore, neither phosphorothioate linkage nor specific sequences (i.e., CpG motifs) are necessary to induce the immune response [16].

The tenet that unmethylated CpG motifs constitute the ‘foreign signature’ that triggers TLR9 was con- tested by these findings. Therefore, phosphorothioate linkages and CpG motifs appear to increase CpG stability, aggregation, and uptake into cells instead.

Thus, stabilization and improvement of CpG uptake into endosomal compartments may enhance its stimulatory functions [23].

It is worth emphasizing that some nanoparticles have been employed to augment CpG uptake and their function [26]. CpG uptake and immunopotency may be improved by lipid nanoparticles. Lipid nanoparticles, for example, can not only improve CpG uptake and immunopotency [39], as recently demonstrated, they are also critical in TLR9 migration from endo- plasmic reticulum to the endosomal compartment, where it can interact with CpG [10].

Since antisense ODNs of the first generation belong to the same class of chemical substances, the ADME (adsorption, distribution, metabolism, elimination)

properties have been well investigated and the phosphorothioate class effects are pretty much understood, at least for the B-class CpG ODNs which do not form stable secondary structures in solution. Metabolism of C-class CpG ODNs differs from that of B-class ODNs

Fig. 1. Structure of the CpG dinucleotide in the unmethylated form where R is H, which activates TLR9. The methylated form where R is CH₃, is poorly recognized by TLR9

Fig. 2. The structural basis for selective binding of non-methylated CpG islands. CpG islands contain a high density of CpG content and embrace the promoters of most genes in human genome

(7)

in that the usually observed degradation by 30- exonucleases is slowed down because of duplex formation of the 30-palindromic sequence [28].

B-class CpG ODNs have been studied in various cancer indications, including non-Hodgkin’s lymphoma, cutaneous T cell lymphoma, basal cell carcinoma, breast cancer, renal cell cancer, melanoma and non-small-cell lung carcinoma (NSCLC) [42]. It seems that TLR-9 agonists could be promising drug candidates which are capable of triggering T helper cell 1 (Th1)-type immune responses, e.g., secretion of Th1-promoting chemokines and cytokines [22].

Th-1 cytokine response, which has been detected among the patients treated with CpG ODNs, provided evidence for the immune stimulatory potency of such compounds [24].

CpG ODNs could also be good candidates for the therapy of infectious diseases.

Preclinical experiments have demonstrated that CpG ODNs are able to protect mice against lethal challenges with a variety of pathogens, such as Lis- teria monocytogenes, malaria, anthrax and Ebola vi- rus. B-class CpG ODNs have also been studied for their adjuvant effects as a combination with commer- cial vaccines, such as the hepatitis B virus (HBV) vaccine Engerix-B [12].

References:

1. Akira S, Uematsu S, Takeuchi O: Pathogen recognition and innate immunity. Cell, 2006, 124, 783–801.

2. Ashino S, Wakita D, Zhang Y, Chamoto K, Kitamura H, Nishimura T: CpG-ODN inhibits airway inflammation at effector phase through down-regulation of antigen- specific Th2-cell migration into lung. Int Immunol, 2008, 20, 259–266.

3. Ashkar AA, Yao XD, Gill N, Sajic D, Patrick AJ, Rosen- thal KL: Toll-like receptor (TLR)-3, but not TLR4, agonist protects against genital herpes infection in the ab- sence of inflammation seen with CpG DNA. J Infect Dis, 2004, 190, 1841–1849.

4. Banerjee B, Kelly KJ, Fink JN, Henderson JD, Bansal NK, Kurup VP: Modulation of airway inflammation by immunostimulatory CpG oligodeoxynucleotides in a murine model of allergic aspergillosis. Infect Immun, 2004, 72, 6087–6094.

5. Barøy T, Sørensen K, Lindeberg MM, Frengen E:

shRNA expression constructs designed directly from siRNA oligonucleotide sequences, Mol Biotechnol, 2010, 45, 116–120.

6. Carpentier AF, Chen L, Maltonti F, Delattre JY: Oligode- oxynucleotides containing CpG motifs can induce rejec-

tion of a neuroblastoma in mice. Cancer Res, 1999, 59, 5429–5432.

7. Carpentier A, Laigle-Donadey F, Zohar S, Capelle L, Behin A, Tibi A, Martin-Duverneuil N et al.: Phase 1 trial of a CpG oligodeoxynucleotide for patients with recurrent glioblastoma. Neuro Oncol 2006, 8, 60–66.

8. Carpentier A, Metellus P, Ursu R, Zohar S, Lafitte F, Barrié M, Meng Y et al.: Intracerebral administration of CpG oligonucleotide for patients with recurrent glioblastoma: a phase II study. Neuro Oncol, 2010, 12, 401–408.

9. Carpentier AF, Xie J, Mokhtari K, Delattre JY: Success- ful treatment of intracranial gliomas in rat by oligodeoxynucleotides containing CpG motifs. Clin Cancer Res, 2000, 6, 2469–2473.

10. de Jong SD, Basha G, Wilson KD, Kazem M, Cullis P, Jefferies W, Tam Y: The immunostimulatory activity of unmethylated and methylated CpG oligodeoxynucleotide is dependent on their ability to colocalize with TLR9 in late endosomes. J Immunol, 2010, 184, 6092–6102.

11. El Andaloussi A, Sonabend AM, Han Y, Lesniak MS:

Stimulation of TLR9 with CpG ODN enhances apoptosis of glioma and prolongs the survival of mice with experimental brain tumors. Glia, 2006, 54, 526–535.

12. Flynn PM, Cunningham CK, Rudy B, Wilson CM, Kapogiannis B, Worrell C, Bethel J et al.: Hepatitis B vaccination in HIV-infected youth: a randomized trial of three regimens. J Acquir Immune Defic Syndr, 2011, 56, 325–332.

13. García-Sastre A, Durbin RK, Zheng H, Palese P, Gertner R, Levy DE, Durbin JE: The role of interferon in influenza virus tissue tropism. J Virol, 1998, 72, 8550–8558.

14. Grauer OM, Molling JW, Bennink E, Toonen LW, Sut- muller RP, Nierkens S, Adema GJ: TLR ligands in the local treatment of established intracerebral murine gliomas. J Immunol, 2008, 181, 6720–6729.

15. Greenbaum BD, Rabadan R, Levine AJ: Patterns of oligonucleotides sequences in viral and host cell RNA iden- tify mediators of the host innate immune system. PLoS One, 2009, 4, 5969.

16. Haas T, Metzger J, Schmitz F, Heit A, Müller T, Latz E, Wagner H: The DNA sugar backbone 2’ deoxyribose de- termines toll-like receptor 9 activation. Immunity, 2008, 28, 315–323.

17. Harandi AM, Eriksson K, Holmgren J: A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection. J Virol, 2003, 77, 953–962.

18. Hartmann G: Silencing and RIG-1 activation by dual function oligonucleotides, WIPO Patent Application, 2010, WO/2010/002851.

19. Hofmann MA, Kors C, Audring H, Walden P, Sterry W, Trefzer U: Phase 1 evaluation of intralesionally injected TLR9-agonist PF-3512676 in patients with basal cell carcinoma or metastatic melanoma. J Immunother, 2008, 31, 520–527.

20. Jimenez-Baranda S, Greenbaum B, Manches O, Handler J, Rabadán R, Levine A, Bhardwaj N: Oligonucleotide motifs that disappear during the evolution of influenza virus in humans increase alpha interferon secretion by plas- macytoid dendritic cells. J Virol, 2011, 85, 3893–3904.

(8)

activation of signal transducer and activator of transcription 3 constrains its agonist-based immunotherapy.

Cancer Res, 2009, 69, 2497–2505.

23. Kortylewski M, Swiderski P, Herrmann A, Wang L, Kowolik C, Kujawski M, Lee H et al.: In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses. Nature Biotech, 2009, 27, 933–940.

24. Krieg AM, Efler SM, Wittpoth M, Al Adhami MJ, Davis HL: Induction of systemic TH1-like innate immunity in normal volunteers. J Immunother, 2004, 27, 460–471.

25. Marion J, Vollmer J, Uhlmann E: Oligoribonucleotides and uses thereof. WIPO Patent application, 2008, WO/2008/139262.

26. Malyala P, O’Hagan DT, Singh M: Enhancing the therapeutic efficacy of CpG oligonucleotides using biodegradable microparticles. Adv Drug Deliv Rev, 2009, 61, 218–225.

27. Murad YM, Clay TM, Lyerly HK, Morse MA:

CPG-7909 (PF-3512676, ProMune): toll-like receptor-9 agonist in cancer therapy, Expert Opin Biol Ther, 2007, 7, 1257–1266.

28. Noll BO, McCluskie MJ, Sniatala T, Lohner A, Yuill S, Krieg AM, Schetter C et al.: Biodistribution and metabolism of immunostimulatory oligodeoxynucleotide CPG 7909 in mouse and rat tissues following subcutaneous administration. Biochem Pharmacol, 2005, 69, 981–991.

29. Paliakov E, Henary M, Say M, Patterson SE, Parker A, Manzel L, Macfarlane DE et al.: Fujita-Ban QSAR analysis and CoMFA study of quinoline antagonists of immunostimulatory CpG-oligodeoxynucleotides. Bioorg Med Chem, 2007, 15, 324–332.

30. Poeck H, Besch R, Maihoefer C, Renn M, Tormo D, Morskaya SS, Kirschnek S et al.: 5’-Triphosphate- siRNA: turning gene silencing and Rig-I activation against melanoma. Nat Med, 2008 14, 1256–1263.

31. Pyles RB, Higgins D, Chalk C, Zalar A, Eiden J, Brown C, Van Nest G, Stanberry LR: Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection.

J Virol, 2002, 76, 11387–11396.

32. Readett DR, Jungnelius JU, Gomez-Novarro J, Hanson DC, Krieg AM: Anti-CTLA-4 Ab and CPG-motif- containing synthetic oligodeoxynucleotide combination

gen 4 blocking monoclonal AbAb in clinical development for patients with cancer. Oncologist, 2007, 12, 873–883.

34. Senti G, Johansen P, Haug S, Bull C, Gottschaller C, Müller P, Pfister T et al.: Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trial. Clin Exp Allergy, 2009, 39, 562–570.

35. Tuaillon E, Tabaa YA, Petitjean G, Huguet MF, Pajeaux G, Fondere JM, Ponseille B et al.: Detection of memory B lymphocytes specific to hepatitis B virus (HBV) surface antigen (HBsAg) from HBsAg-vaccinated or HBV- immunized subjects by ELISPOT assay. J Immunol Methods, 2006, 315, 144–152.

36. Uhlmann E: Immune Stimulatory Oligonucleotides. In:

Therapeutic Oligonucleotides. Ed. Kurreck J, Royal So- ciety of Chemistry, London, 2008, 142–160.

37. Vollmer J, Krieg AM: Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Adv Drug Deliv Rev, 2009, 61, 195–204.

38. Wagner H: The sweetness of the DNA backbone drives Toll-like receptor 9. Curr Opin Immunol, 2008, 20, 396–400.

39. Wilson KD, de Jong SD, Tam YK: Lipid-based delivery of CpG oligonucleotides enhances immunotherapeutic efficacy. Adv Drug Deliv Rev. 2009, 61, 233–242.

40. Wu A, Oh S, Gharagozlou S, Vedi RN, Ericson K, Low WC, Chen W, Ohlfest JR: In vivo vaccination with tumor cell lysate plus CpG oligodeoxynucleotides eradicates murine glioblastoma. J Immunother, 2007, 30, 789–797.

41. Yasuko K, Seiji K: Telomerase RNA inhibition using antisense oligonucleotide against human telomerase RNA linked to a 2’,5’-oligoadenylate. Methods Mol Biol, 2007, 405, 97–112.

42. Zha L, Qiao T, Yuan S, Lei L: Enhancement of radiosen- sitivity by CpG-oligodeoxyribonucleotide-7909 in human non-small cell lung cancer A549 cells. Cancer Bio- ther Radiopharm, 2010, 25, 165–70.

Received: March 8, 2011; in the revised form: April 30, 2012;

accepted: May 22, 2012.