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.     

CpG oligodeoxynucleotides for immune stimulation in cancer immunotherapy

Synthetic oligodeoxynucleotides containing CG motifs (CpG ODNs) have potent immunostimulatory properties, and have potential as immunotherapeutic agents in cancer. Animal models suggest that CpG ODNs may be useful as both single agents and to enhance the efficacy of monoclonal antibody therapy. CpG ODNs can also enhance the efficacy of tumor immunization through use as an immune adjuvant, to activate antigen presenting cells, or as a component of DNA vaccines. Clinical trials are now beginning to assess whether the promise of these agents in the laboratory will translate into CpG ODNs becoming a valuable component of clinical cancer immunotherapy.     

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.     

Evaluation of Generation 2 and 3 Poly(Propylenimine) Dendrimers for the Potential Cellular Delivery of Antisense Oligonucleotides Targeting the Epidermal Growth Factor Receptor

PURPOSE: To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells. METHODS: Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels. RESULTS: G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximately 30 microg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4 degrees C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery. CONCLUSIONS: G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture.     

Studies on uptake, sub-cellular trafficking and efflux of antisense oligodeoxynucleotides in glioma cells using self-assembling cationic lipoplexes as delivery systems

The cellular uptake of antisense oligodeoxynucleotides (ODNs) may be enhanced by the use of carriers such as cationic liposomes or lipoplexes, but little is known about the intracellular fate and subcellular trafficking of these systems in target cells. In this study, we report on the cellular uptake and biodistribution of ODNs in the presence and absence of optimised self-assembled cationic lipoplexes using the C6 glioma cell line as an in vitro model. Biotin or radiolabelled 15-mer phosphorothioate (PS) ODNs were synthesised and their cellular uptake and subcellular biodistribution characterised in the presence and absence of an optimised cationic lipoplex delivery system using studies ranging from cellular association, cellular efflux and transmission electron microscopy (TEM). Ultrastructural studies clearly showed PS ODNs in the absence of liposomal delivery to be sequestered within endosomal and lysosomal vesicular bodies indicative of endocytic uptake. ODNs were also visible, to a lesser extent, in the nucleus and cytoplasm. By employing DOSPA (2'-(1",2"-dioleoyloxypropyldimethyl-ammonium bromide)-N-ethyl-6-amidospermine tetra trifluoroacetic acid) and DOPE (dioleoylphosphatidylethanolamine) complex in a 3 : 1 ratio, as a delivery system for ODNs at a optimal lipid/DNA charge ratio of 1 : 1, the level of ODN cellular association was significantly increased by approximately 10-12 fold with a concomitant change in subcellular distribution of PS ODN. TEM studies indicated enhanced penetration of ODN within the cytosol and the cell nucleus with reduced presence in vesicular compartments. Efflux studies confirmed that cationic lipoplexes promoted entry of ODNs into 'deeper' cellular compartments, consistent with endosomal release. Optimised cationic lipoplexes improved cellular delivery of ODNs by enhancing cell association, uptake and by favourably modulating the intracellular trafficking and distribution of ODNs into non-vesicular compartments including the cytosol and nucleus.     

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.     

Liposome-mediated delivery of oligodeoxynucleotides in vivo

Oligodeoxynuclotides (ODNs) are deoxyribonucleic-acid-based sequences showing therapeutic potential against such diseases as cancer, arteriosclerosis, arthritis, viral infections, and inflammation. Administration of these chemical entities using carriers has certain advantages over administration of free nucleic acid strands. Liposomes, one class of carriers, have been proven to be a popular choice for ODN delivery in vivo because of their potential to increase resistance of ODNs against nucleases and enhance circulation half-life. This result increases ODN uptake in target cells. Several liposomal formulations with proven potential for ODN delivery in vivo are discussed in this article.     

Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists

Toll-like receptor 9 (TLR9) agonists have demonstrated substantial potential as vaccine adjuvants, and as mono- or combination therapies for the treatment of cancer and infectious and allergic diseases. Commonly referred to as CpG oligodeoxynucleotides (ODN), TLR9 agonists directly induce the activation and maturation of plasmacytoid dendritic cells and enhance differentiation of B cells into antibody-secreting plasma cells. Preclinical and early clinical data support the use of TLR9 agonists as vaccine adjuvants, where they can enhance both the humoral and cellular responses to diverse antigens. In mouse tumor models TLR9 agonists have shown activity not only as monotherapy, but also in combination with multiple other therapies including vaccines, antibodies, cellular therapies, other immunotherapies, antiangiogenic agents, radiotherapy, cryotherapy, and some chemotherapies. Phase I and II clinical trials have indicated that these agents have antitumor activity as single agents and enhance the development of antitumor T-cell responses when used as therapeutic vaccine adjuvants. CpG ODN have shown benefit in multiple rodent and primate models of asthma and other allergic diseases, with encouraging results in some early human clinical trials. Although their potential clinical contributions are enormous, the safety and efficacy of these TLR9 agonists in humans remain to be determined.     

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.     

Biphasic lipid vesicles as a subcutaneous delivery system for protein antigens and CpG oligonucleotides

One of the major drawbacks to the development of novel vaccines has been the lack of safe yet effective adjuvants. Biphasic lipid vesicles are formulations suitable for the delivery of proteins, peptides and oligo/polynucleotides. They constitute a new class of delivery system into which antigens and adjuvants can be incorporated. The purpose of these studies was to investigate the ability of biphasic lipid vesicles (Vaccine-Targeting Adjuvants--VTA) to induce immune responses to bacterial antigens and to enhance the adjuvant activity of CpG ODNs. Immunization of mice with bacterial antigen and CpG ODNs in saline was not as effective at inducing immune responses as formulation in VTA vesicles. Results showed that formulation of CpG ODN in VTA significantly enhanced its adjuvanticity.     

Cancer immunotherapeutic effects of novel CpG ODN in murine tumor model

While CpG oligodeoxynucleotides (ODN) are excellent candidates for cancer immunotherapeutics, the numbers of usable CpG ODNs are limited in current clinical settings. To resolve this, we investigated whether novel CpG ODN (KSK-CpG) would be an effective immunotherapeutic in a murine tumor model by affecting in vivo and in vitro parameters, such as survival span, the number of tumor nodules, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activity and interleukin (IL)-6 or IL-12 cytokine release in splenocytes. We found that KSK-CpG was effective in the murine cancer model by way of prolonging survival span, reducing the number of tumor nodules, augmenting NK cell and CTL cytotoxicity, as well as evoking IL-6 and IL-12 cytokine release in splenocytes. Collectively, these data demonstrate that KSK-CpG is active against the highly malignant B16BL6 and EL4 tumor mouse model via innate immune augmentation.     

Section Review: Biologicals & Immunologicals: Applications of antisense technology to both basic and clinical research

Antisense oligodeoxynucleotides (ODNs) are synthetic molecules typically designed to hybridise to the sense strand of the target gene messenger RNA (mRNA). The activity of these molecules reduces gene expression by disrupting the flow of information from gene to protein. The specificity of this interaction arises from the antisense ODN having a unique affinity for the primary sequence of the target gene due to nucleotide base complementarity. Specific inhibition of target gene products allows a rapidly increasing number of researchers to investigate gene function. Additionally, the reversible and specific nature of this interaction has initiated interest in antisense ODNs as a potentially novel class of therapeutics. Antisense molecules are already undergoing clinical trials as candidate therapeutics for several indications. Recent advances in our understanding of the activity of antisense ODNs, together with the advances in ODN chemistry, has made the notion of therapeutically active antisense molecules realistic. However, there remain many challenges in this field which must be overcome. Notably, the most commonly used chemical class of antisense ODNs, the phosphorothioates, has been associated with significant non-specific activities. This brief review aims to present in vivo applications of antisense in the domains of both basic and clinical research.     

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。     

Recent advances in the development of immunostimulatory oligonucleotides

Some immune cells recognize distinct molecular structures present in pathogens through specific pattern recognition receptors that are able to distinguish prokaryotic DNA from vertebrate DNA. The detection of invading microbial DNA is based on the recognition of unmethylated deoxycytidyl-deoxyguanosin dinucleotide (CpG) motifs. Synthetic oligonucleotides (ODNs) containing these CpG motifs are able to activate both innate and acquired immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9). Depending on the sequence, length, as well as number and positions of CpG motifs in an ODN, distinct immunostimulatory profiles can be observed. These immunostimulatory profiles can be further modified and fine-tuned by appropriate chemical modifications, leading to preclinical and clinical development of CpG ODNs in cancer, allergy, asthma and infectious diseases.     

CpG oligodeoxynucleotides with double stem-loops show strong immunostimulatory activity

Based on the current understanding of TLR9 recognition of CpG ODN, we have tried to design a series of CpG ODNs that display double stem-loops when being analyzed for their secondary structures using ‘mfold web server’. Proliferation of human PBMC and bioassay for IFN production were used as technical platforms in primary screening. Interestingly, two of them, designated as DSL01 and D-SL03, belonging to B class CpG ODN and C class CpG ODN respectively, showed vigorous immunostimulatory activity and were chosen for further tests. Flow cytometry analysis showed that both of them could activate human B cells, NK cells, mononuclear cells and T cells and up-regulate expression of CD80, CD86 and HLA-DR on the surface of subsets in human PBMCs. Furthermore, we demonstrated that those two ODNs potently stimulated proliferation of PBMC/splenocytes obtained from diverse vertebrate species. Noticeably, both of them displayed anti-breast cancer effect in mice when administered by peritumoral injection.     

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.     

A CpG oligodeoxynucleotide potentiates the anti-tumor effect of HSP65-Her2 fusion protein against Her2 positive B16 melanoma in mice

Although being promising tumor vaccine candidates in animal models, heat shock protein (HSP)-based tumor vaccines have not yet succeeded in the clinical trials, implying the necessity to be formulated with appropriate adjutants to enhance their immunogenicity. In this study, we investigated whether a B-class CpG ODN (BW006), a TLR9 agonist, could facilitate HSP65-Her2, a recombinant protein between mycobacterial HSP65 and Her2-derived peptide, to induce vigorous anti-tumor activity against Her2 positive tumors in mice both prophylactically and therapeutically. It was found that BW006 could enhance prophylactic and therapeutic effect of HSP65-Her2 with improved survival of the mice bearing Her2(+) B16 melanoma and HSP65-Her2 specific Th1 response.     

Prophylaxis with cationic liposome-DNA complexes protects hamsters from phleboviral disease: importance of liposomal delivery and CpG motifs

Cationic liposome-DNA complexes (CLDC) are cationic/neutral lipid carriers complexed with plasmid DNA that when administered systemically results in a robust T(H)1 cytokine response. CLDC have been shown to be effective in prophylaxis and therapeutic treatment of animal models of viral disease. To determine the contribution of liposomal delivery and CpG content of the plasmid DNA to the efficacy of CLDC; plasmid, CpG-free plasmid DNA, or CpG-containing oligodeoxynucleotides (ODN) with and without liposomes, as well as poly(I:C(12)U), were evaluated for their ability to elicit protection against lethal Punta Toro virus (PTV, Bunyaviridae, phlebovirus) challenge in hamsters. CLDC-containing plasmid significantly improved survival, decreased systemic and liver viral loads, and reduced liver damage due to progression of viral infection. Mouse-reactive ODNs complexed with liposomes failed to protect hamsters, whereas ODNs known to cross-react with human and mouse (CpG 2006) or non-liposomal poly(I:C(12)U) showed survival benefit but did not limit liver injury. Liposomes complexed with a non-CpG motif-containing plasmid reduced liver viral load and tissue damage, but did not protect hamsters from death. To evaluate the mechanisms of the enhanced activity of CLDC, microarray experiments examined differences in the gene expression profile. The results suggest a broad T(H)1 response elicited by liposomal delivery of a diverse sequence containing CpG and non-CpG elements may be a more effective antiviral treatment than other nucleic acid based immunotherapeutics.     

Nanoparticulate systems for the delivery of antisense oligonucleotides

Antisense oligonucleotides are molecules that are able to inhibit gene expression being therefore potentially active for the treatment of viral infections or cancer. However, because of their poor stability in biological medium and their weak intracellular penetration, colloidal drugs carriers such as nanoparticles were developed for the delivery of oligonucleotides (ODN). ODN associated to nanoparticles were shown to be protected against degradation and to penetrate more easily into different types of cells. As a consequence, nanoparticles were shown to improve the efficiency of ODNs for the inhibition of the proliferation of cells expressing the point mutated Ha-ras gene. In vivo, polyalkylcyanoacrylate (PACA) nanoparticles were able to efficiently distribute the ODNs to the liver whereas the alginate nanosponges could concentrate the ODNs in the lungs. Finally, ODN loaded to PACA nanoparticles were able to improve in mice, the treatment of RAS cells expressing the point mutated Ha-ras gene.     

Site-specific administration of antisense oligonucleotides using biodegradable polymer microspheres provides sustained delivery and improved subcellular biodistribution in the neostriatum of the rat brain

Antisense oligonucleotides (ODNs) are being increasingly used in the central nervous system as biological tools, as drug-target validation agents and as potential therapeutic agents. Although the local delivery of naked ODNs to the brain can result in the desired biological effects, the duration of efficacy is relatively short lived due to the combined effects of rapid ODN degradation and elimination half-lives in vivo. In this study, we have examined the use of biodegradable polymer microspheres as a site-specific delivery system for targeting ODNs to the neostriatum of the rat brain. Model phosphorothioate backbone-modified ODNs were entrapped within poly(D,L-lactide-co-glycolide) (PLAGA) microspheres using a double emulsion-deposition method and the formulations characterised in terms of particle size, surface morphology, percent encapsulation efficiency, ODN loading and in vitro release profiles. For in vivo evaluation, PLAGA microspheres containing fluorescently-labelled ODNs were stereo-taxically administered to the neostriatum of the rat brain and biodistribution of ODNs monitored after 48 h. Administration of free fluorescently-labelled ODNs to the neostriatum resulted in a punctate cellular distribution of ODNs after 24 h with little or no ODN remaining in the neostriatum after 48 h. In comparison, fluorescently-labelled ODNs delivered using polymer microspheres were intensely visible in cells after 48 h post-administration and the fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Dual-label immunohistochemical analyses suggested that ODNs were mainly distributed to neuronal cells. These data indicate that site-specific administration of ODNs using biodegradable polymer microspheres will not only provide sustained delivery of nucleic acids but can also improve the cellular distribution of ODNs to brain cells. Sustained or controlled-release biodegradable polymer formulations, therefore, represent an attractive strategy for improved local delivery of ODNs to the CNS.