G3139 and other CpG-containing immunostimulatory phosphorothioate oligodeoxynucleotides are potent suppressors of the growth of human tumor xenografts in nude mice

Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.     

Antitumor mechanisms of oligodeoxynucleotides with CpG and polyG motifs in murine prostate cancer cells: decrease of NF-kappaB and AP-1 binding activities and induction of apoptosis

Previous studies have shown that CpG oligodeoxynucleotides (ODNs) have substantial immunostimulatory effects with anticancer applications. The antitumor applications that have been described previously are mediated through the CpG-induced activation of the host immune system, not through direct antitumor effects. Using cytostasis and cell proliferation assays, we demonstrated that specific ODNs inhibit the proliferation of RM-1 cells, a murine prostate cancer cell line. Flow cytometry analysis using propidium iodide (PI) nuclear staining confirmed the direct proapoptotic effect of ODNs on prostate cancer cells. This effect was dose dependent. Further studies using Western blot analysis and electrophoresis mobility shift assay (EMSA) revealed that the treatment of prostate cancer cells with specific ODNs activated the caspase pathway(s) and decreased the binding activities of AP-1 and NF-kappaB in a time-dependent manner. Evaluation of a panel of ODNs containing different DNA motifs demonstrated that the optimal proapoptotic sequences required polyG sequences but that CpG motifs were not essential. Finally, in vivo antitumor studies showed that the proapoptotic polyG motifs significantly inhibited prostate tumor growth. PolyG motifs inhibited tumor growth, and the effects were enhanced by CpG immune activating sequences. ODN containing both polyG and CpG motifs may have enhanced efficacy in tumor therapy through multiple mechanisms of action, including direct antitumor activities and immune activation.     

CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents

We demonstrate a new design for immunomodulatory CpG DNA containing two sequences each with as few as five or six-nucleotides joined together via 3(')-3(') linkers. These do not require the -PuPu(Py)CGPyPy- hexameric motif generally found essential for CpG DNA immune stimulation. These novel, short-immunomers show potent immunostimulatory activity manifested by IL-12 and IL-6 secretion in murine spleen cell and PBMC cultures and splenomegaly in vivo. Short-immunomers show strong activation of NF-kappaB and stress-activated signaling pathways and induce cytokines in J774 cell cultures. The same sequences also induce cytokines in healthy human PBMC cultures whereas conventional CpG DNA requires different optimal sequences for murine and human immune cells. Additionally, short-immunomers inhibit IL-5 secretion and induce IFN-gamma secretion in conalbumin-sensitized mouse spleen cell cultures, suggesting reversal of established Th2 responses to Th1 type responses. Short-immunomer also inhibits growth of MCF-7 human tumor xenograft in nude mice. This is the first report of activity with such short DNA sequences and also of sequences lacking hexameric motifs proposed in earlier studies.     

In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response

A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9⁺ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.     

Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA)

Locked nucleic acid (LNA) is an RNA derivative that when introduced into oligodeoxynucleotides (ODN), mediates high efficacy and stability. CpG ODNs are potent immune stimulators and are recognized by toll-like receptor-9 (TLR9). Some phosphorothioate antisense ODNs bearing CpG dinucleotides have been shown to possess immune modulatory capacities. We investigated the effects of LNA substitutions on immune stimulation mediated by antisense ODN G3139 or CpG ODN 2006. LNA ODNs were tested for their ability to stimulate cytokine secretion from human immune cells or TLR9-dependent signaling. Phosphorothioate chimeric LNA/DNA antisense ODNs with phosphodiester-linked LNA nucleobases at both ends showed a marked decrease of immune modulation with an increasing number of 3' and 5' LNA bases. In addition, guanosine-LNA and cytosine-LNA or simply cytosine-LNA substitutions in the CpG dinucleotides of ODN 2006 led to strong decrease or near complete loss of immune modulation. TLR9-mediated signaling was similarly affected. These data indicate that increasing amounts of LNA residues in the flanks or substitutions of CpG nucleobases with LNA reduce or eliminate the immune stimulatory effects of CpG-containing phosphorothioate ODN.     

Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment

The components of the apoptotic program are targets for anticancer therapy. Bcl-2 protein inhibits apoptosis and confers resistance to treatment with traditional cytotoxic chemotherapy, radiotherapy, and monoclonal antibodies (mAb). Oblimersen sodium (G3139, Genasense, Genta Inc., Berkeley Heights, NJ) is an antisense oligonucleotide (AS-ON) compound designed to specifically bind to the first 6 codons of the human bcl-2 mRNA sequence, resulting in degradation of bcl-2 mRNA and subsequent decrease in Bcl-2 protein translation. Oblimersen is the first oligonucleotide to demonstrate proof of principle of an antisense effect in human tumors by the documented downregulation of the target Bcl-2 protein. A growing body of preclinical and clinical evidence suggests that oblimersen synergizes with many cytotoxic and biologic/immunotherapeutic agents against a variety of hematologic malignancies and solid tumors. Randomized clinical trials are currently underway to evaluate the efficacy and tolerability of oblimersen in combination with cytotoxic chemotherapy in chronic lymphocytic leukemia, multiple myeloma, malignant melanoma, and non-small cell lung cancer. In addition, nonrandomized trials are under way to evaluate oblimersen in non-Hodgkin's lymphoma, acute myeloid leukemia, and hormone-refractory prostate cancer. Preclinical data also support the clinical evaluation of oblimersen in additional tumor types, including chronic myelogenous leukemia and breast, small cell lung, gastric, colon, bladder, and Merkel cell cancers. Enhancement of the efficacy of anticancer treatments with oblimersen Bcl-2 antisense therapy represents a promising new apoptosis-modulating strategy, and ongoing clinical trials will test this therapeutic approach.     

CpG RNA: identification of novel single-stranded RNA that stimulates human CD14+CD11c+ monocytes

Synthetic immunostimulatory nucleic acids such as CpG DNA are being harnessed therapeutically as vaccine adjuvants, anticancer or antiallergic agents. Efforts to identify nucleic acid-based agents capable of more specifically modulating the immune system are being developed. The current study identifies a novel class of single-stranded oligoribonucleotides (ORN) containing unmethylated CpG motifs and a poly(G) run at the 3' end (CpG ORN) that directly stimulate human CD14+CD11c+ monocytes but not dendritic cells or B cells. CpG ORN activate NF-kappaB and p38 MAPK, resulting in IL-6 and IL-12 production and costimulatory molecule up-regulation but not IFNalpha. Methylation of cytosine at the 5' portion in core CpG motif abrogates such activation. TLR3, 7, 8, or 9 alone did not confer response to CpG ORN, in contrast to previously reported respective nucleic acid ligands. These data suggest that CpG ORN represent a novel class of synthetic immunostimulatory nucleic acids with distinct target cells, receptors, and functions from that of previously known immunomodulatory nucleic acids.     

Plasmacytoid dendritic cell-derived IFN-alpha induces TNF-related apoptosis-inducing ligand/Apo-2L-mediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation

Immunostimulatory oligodeoxynucleotides (ODN) containing the CpG motif are being tested as immune adjuvants in many disease settings. Of the human PBMC examined, plasmacytoid dendritic cells (pDC) are a major source of type I IFN upon stimulation with CpG ODN. IFNs have numerous immunostimulatory effects, including the induction of TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L on monocytes, NK cells, and T cells. Importantly, IFN has also been linked to antitumor responses. Thus, we tested whether CpG ODN stimulation of PBMC led to TRAIL/Apo-2L-induced tumor cell death. When PBMC were stimulated with CpG ODN, TRAIL/Apo-2L-dependent tumor cell death was observed. Further examination of CpG ODN-stimulated PBMC revealed that TRAIL/Apo-2L expression was limited to CD14(+) cells, which, when depleted, led to a loss of the TRAIL/Apo-2L-mediated tumor cell killing. Moreover, pDC depletion also abolished the TRAIL/Apo-2L-mediated killing of tumor cell targets. Analysis of the pDC showed IFN-alpha production after CpG ODN stimulation. Finally, inclusion of neutralizing IFN-alpha antiserum with the PBMC during CpG ODN stimulation abrogated TRAIL/Apo-2L-mediated tumor cell killing. These results define a mechanism by which CpG ODN induces TRAIL/Apo-2L-dependent killing of tumor cells by CD14(+) PBMC, in which CpG ODN-activated pDC produce IFN-alpha that stimulates CD14(+) PBMC to express functional TRAIL/Apo-2L.     

Induction of immune activation by a novel immunomodulatory oligonucleotide without thymocyte apoptosis

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG DNA) can potently stimulate innate immunity. While the actions of CpG DNA resemble those of LPS, these molecules stimulate distinct Toll-like receptors as well as cell types. In a previous study, we showed that a CpG ODN could induce cytokine production but, unlike LPS, did not induce thymocyte apoptosis. In this study, we have further investigated these differences using as a model a second-generation immunostimulatory oligonucleotide called HYB2048. Following administration to normal BALB/c mice, HYB2048-induced IL-12 but not IL-6 production. Under conditions in which LPS induced thymocyte apoptosis, HYB2048 did not cause significant cell death and, furthermore, did not block apoptosis induced by LPS. The levels of corticosterone induced by HYB2048 were also significantly lower than those induced by LPS. This pattern of activation could distinguish CpG DNA from LPS in its effects on the immune system.     

Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses

Intravenous (i.v.) administration of a lipopolyplex consisting of a ternary complex of DOTAP:cholesterol cationic liposomes, protamine sulfate, and noncoding plasmid DNA (LPD-pDNA) is capable of stimulating a potent Th-1 cytokine response and inhibiting the growth of established tumors in mice. Both activities are mainly elicited by unmethylated CpG motifs in the plasmid DNA (pDNA) component, which are bacterial in origin. Since oligodeoxynucleotides (ODN) that possess a consensus immunostimulatory CpG motif of RRCpGYY (R is purine and Y is pyrimidine) can mimic the immunostimulatory actions of bacterial DNA, we hypothesized that i.v. administration of LPD prepared with GpG-ODN would mimic the ability of LPD-pDNA to stimulate Th-1 cytokines and antitumor activity and provide an improved vector for probing the immune mechanisms underlying the observed antitumor effects. These hypotheses were tested for the treatment of established 24JK experimental pulmonary metastases that are syngeneic in C57BL/6 mice. Mice treated with LPD containing 25 microg of the prototypical phosphodiester (PO) CpG-ODN 1668 (tccatGACGTTcctgatgct, motif capitalized) demonstrated a dramatic reduction in lung tumor burden (>80% inhibition, P<0.01) compared to dextrose-treated controls. The antitumor effect was dependent on the CpG dinulceotide and correlated with the ability to stimulate serum Th-1 cytokines (TNF-alpha, IL-12, and IFN-gamma). Both activities required assembly of CpG-ODN in a cationic liposome/DNA complex (lipoplex) or the LPD lipopolyplex. LPD delivery of both PO-1668 and phosphorothioated (PS)-1668 stimulated a greater cytokine response compared to delivery of free ODN. Furthermore, within the LPD complex, both PO- and PS-1668 had similar ability to stimulate Th-1 cytokines with respect to potency and duration of response, thus eliminating the need for the PS modification. In tumor cell lysis assays, LPD-CpG DNA stimulated development of an acquired, tumor-specific CD8+ cytotoxic T-lymphocyte (CTL) activity that was dependent on CpG DNA. LPD was also capable of stimulating NK activity; however, this was not dependent on CpG DNA. Only formulations that concomitantly stimulated NK activity and CpG-specific, Th-1 cytokine were capable of stimulating the development of tumor-specific CTL activity and significant inhibition of tumor growth. Thus, we propose a model where CpG DNA in complex with cationic liposome-based lipoplexes or lipopolyplexes stimulates antitumor NK activity and CpG-stimulated Th-1 cytokine production. The combination of these two activities of the innate immune system subsequently direct the development of an acquired, tumor-specific CTL response that in total are effective for inhibiting the growth of established tumors in mice.     

In vivo role of p38 mitogen-activated protein kinase in mediating the anti-inflammatory effects of CpG oligodeoxynucleotide in murine asthma

DNA containing unmethylated CpG motifs is intrinsically immunostimulatory, inducing the production of a variety of cytokines and chemokines by immune cells. The strong Th1 response triggered by CpG oligodeoxynucleotide (ODN) inhibits the development of Th2-mediated allergic asthma in mice. This work documents that CpG ODN-induced IL-12 production plays a critical role in this process, because intrapulmonary CpG ODN inhibits allergic inflammation in wild-type but not IL-12(-/-) mice. CpG ODN rapidly localized to alveolar macrophages (AM), thereby triggering the phosphorylation of p38 mitogen-activated protein kinase (MAP kinase). AM cultured with CpG but not control ODN up-regulated IL-12 p40 expression and release, and these effects were blocked by the highly specific p38 MAP kinase inhibitor SB202190. Intrapulmonary administration of this inhibitor blocked the ability of CpG ODN to produce IL-12 in the lungs and reversed the anti-inflammatory effects of CpG ODN on allergic lung inflammation. These findings indicate that IL-12 production by AM is stimulated by intrapulmonary CpG ODN administration through a p38 MAP kinase-dependent process, and IL-12 is a key cytokine that mediates CpG ODN-induced protection against allergic lung inflammation.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.     

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K_i between 0.2 and 0.5 µM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 µM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 µM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability. respiration; voltage-dependent anion-selective channel; apoptosis; cell death     

Stimulation of thymocyte proliferation by phosphorothioate DNA oligonucleotides

DNA is a complex macromolecule the immunological properties of which depend on short sequence motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences are mitogenic for B cells and can stimulate macrophage cytokine production. While these sequences do not directly activate T cells, they can augment effects of stimulation via the TCR. Furthermore, ISS can affect T cells because of macrophage production of IL-12 and IFN-alpha/beta. In these studies, we further evaluated the immune effects of DNA on T cells, testing the possibility that certain T cell populations can respond directly to this stimulus. We therefore tested the in vitro responses of thymocytes to a series of phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) varying in sequence. In in vitro cultures, phosphorothioate ODNs (sODNs) containing CpG motifs induced significant proliferation of murine thymocytes, although phosphodiester compounds lacked activity. The magnitude of stimulation varied with sequences flanking the CpG motifs, as both dA and dT sequences enhanced the stimulatory capacity of the CpG motif. Furthermore, CpG sODNs were strong costimulators of anti-CD3-mediated thymocyte activation, increasing proliferation compared to anti-CD3 in the absence of DNA. This activation was only partially inhibited by cyclosporine A and was not dependent on a calcium influx. Together, these results indicate that phosphorothioate oligonucleotides containing CpG motifs can directly induce thymocyte proliferation as well as augment TCR activation. These observations thus extend the range of actions of CpG DNA and suggest additional mechanisms for its function as an immunomodulatory agent or adjuvant.     

Specific lipoplex-mediated antisense against Bcl-2 in breast cancer cells: a comparison between different formulations

G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3ss[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms--either large unilamellar vesicles (approximately 100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.     

Specific Lipoplex-Mediated Antisense Against Bcl-2 in Breast Cancer Cells: A Comparison between Different Formulations

G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3β[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms — either large unilamellar vesicles (～100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.     

Effect of dendritic cells treated with CpG ODN on atopic dermatitis of Nc/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease and the pathogenesis of AD is associated with the release of various cytokines/chemokines due to activated Th(2) immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide in the context of particular base sequence (CpG motifs) are known to have the immunostimulatory activities in mice and to convert from Th(2) to Th(1) immune responses in AD. We aimed to investigate that CpG ODN, especially phosphodiester form, can stimulate the protective immunity in NC/Nga mice with AD. We isolated BMDCs from NC/Nga mice and then, cultured with GM-CSF and IL-4 for 6 days, and treated for 2 days by either phosphorothioate ODN or phosphodiester ODN. CpG ODN-treated DCs resulted in more production of IL-12. When CpG ODN-treated DCs were intravenously injected into the NC/Nga mice, the NC/Nga mice with CpG ODN-treated DCs showed significant improvement of AD symptoms and decrease of IgE level. Histopathologically, the NC/Nga mice skin with CpG ODN-treated DCs showed the decreased IL-4 and TARC expression comparing with non-injected mice. These results may suggest that phosphodiester CpG ODN-treated DCs might function as a potent adjuvant for AD in a mouse model.     

Encapsulation in liposomal nanoparticles enhances the immunostimulatory,adjuvant and anti-tumor activity of subcutaneously administered CpG ODN

Immunostimulatory oligodeoxynucleotides (ODN) containing cytosine-guanine (CpG) motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through triggering of the Toll-like receptor 9. We have previously shown that encapsulation in liposomal nanoparticles (LN) enhances the immunostimulatory activity of CpG ODN (LN-CpG ODN) (Mui et al. in J Pharmacol Exp Ther 298:1185, 2001). In this work we investigate the effect of encapsulation on the immunopotency of subcutaneously (s.c.) administered CpG ODN with regard to activation of innate immune cells as well as its ability to act as a vaccine adjuvant with tumor-associated antigens (TAAs) to induce antigen (Ag)-specific, adaptive responses and anti-tumor activity in murine models. It is shown that encapsulation specifically targets CpG ODN for uptake by immune cells. This may provide the basis, at least in part, for the significantly enhanced immunostimulatory activity of LN-CpG ODN, inducing potent innate (as judged by immune cell activation and plasma cytokine/chemokine levels) and adaptive, Ag-specific (as judged by MHC tetramer positive T lymphocytes, IFN-γ secretion and cytotoxicity) immune responses. Finally, in efficacy studies, it is shown that liposomal encapsulation enhances the ability of CpG ODN to adjuvanate adaptive immune responses against co-administered TAAs after s.c. immunization, inducing effective anti-tumor activity against both model and syngeneic tumor Ags in murine tumor models of thymoma and melanoma.     

A functionally improved locked nucleic acid antisense oligonucleotide inhibits Bcl-2 and Bcl-xL expression and facilitates tumor cell apoptosis

We previously reported the Bcl-2/Bcl-xL-bispecific activity of the 2'-O-(2-methoxy)ethyl (2'-MOE)-modified gapmer antisense oligonucleotide 4625. This oligonucleotide has 100% complementarity to Bcl-2 and three mismatches to Bcl-xL. In the present study, the isosequential locked nucleic acid (LNA)-modified oligonucleotide 5005 was generated, and its ability to further improve the downregulation of the two antiapoptotic targets in tumor cells was examined. We demonstrate that compared with 4625, 5005 more effectively decreased the expression of the mismatching Bcl-xL target gene in MDA-MB-231 breast and H125 lung cancer cells. In both cell lines, antisense activity caused decreased cell viability by induction of apoptosis. Moreover, in combination with various anticancer agents, 5005 reduced tumor cell viability more effectively than 4625. We describe for the first time the functional comparison of isosequential Bcl-2/Bcl-xL-bispecific 2'-MOE and LNA-modified antisense oligonucleotides and report that the LNA analog more effectively downregulated the two apoptosis inhibitors overexpressed in human tumors. Our data underscore the ability of LNA modifications to enhance the efficacy and favorably modulate the target specificity of antisense oligonucleotides.