Combination Of CpG Oligodeoxynucleotides Research Articles

Combination of CpG Oligodeoxynucleotide and Anti-4-1BB Antibody in the Treatment of Multiple Hepatocellular Carcinoma in Mice.

BackgroundTo investigate the effect of topical application of CpG oligodeoxynucleotide (CpG-ODN) combined with anti-4-1BB antibodies on mouse HCC multiple tumor-bearing models and the degree of improvement of anti-tumor immune response in mice.Materials and MethodsWe inoculated each BALB/c male mouse subcutaneously with one tumor in the axillae of the four limbs and divided them into four groups. We only selected the tumor-bearing part of the left lower limb for drug treatment. We measured the tumor-bearing volume of mice in each group. Then, we tested the organ coefficients of mice, the concentrations of IL-12 and IFN-γ in peripheral blood, the ratio of spleen Tregs and CD8+T cells, the spleen CTL killing activity, and the survival time of mice.ResultsWe found that the tumor-bearing volume decreased significantly after the combination of CpG-ODN and anti-4-1BB antibody (P<0.001). The organ coefficients of treated mice were not significantly different from normal mice (P>0.05). The concentration of IL-12 and IFN-in serum and the ratio of CD8+T cells in spleen were increased, while the ratio of spleen Tregs was decreased. CTL activity of spleen was increased. The survival time of mice was significantly prolonged (P<0.001).ConclusionThe treatment programme combining CpG-ODN with an anti-4-1BB antibody can significantly reduce tumor growth at the treatment site, slow the growth rate of metastases and improve host prognosis.

Recombinant plasmids containing CpG with porcine host defense peptides (PR-39/pBD-1) modulates the innate and adaptive intestinal immune responses (including maternal-derived) in piglets

CpG oligodeoxynucleotides (CpG-ODN) is an immunoenhancer, which is composed of unmethylated cytosine and guanine. Host Defense Peptides (HDPs) are small molecule polypeptides with various immunological activities that have been shown to induce a stronger innate immune response in piglets with synthetic CpG-ODN. Therefore, combination of CpG-ODN and HDPs was expected to be a novel immunoadjuvant with high efficiency, low toxicity and great potential. However, cost of synthetic HDPs or CpG-ODN is too high to be advantageous for animal farming. In this study, in order to improve the immune function of vaccine and reduce cost, a series of recombinant plasmids (containing HDPs gene (PR-39/pBD-1) and different numbers of CpG motifs) were constructed. In vitro, porcine lymphocytes were stimulated by recombinant plasmids to verify the immunostimulatory function of recombinant plasmids. In vivo, recombinant plasmids were used to immunize piglets with Enterotoxigenic Escherichia coli (ETEC) vaccine to analyze effects of recombinant plasmids on the mucosal immune responses. In addition, dosage screening and capability of maternal antibody responses were also investigated. Our results showed that recombinant plasmids had strong adjuvant effects especially the plasmid pVAX49-PR-39 and pVAX49-pBD-1. Moreover, there was no diarrhea in piglets using pVAX49-PR-39 or pVAX49-pBD-1 as adjuvants. These findings suggested that recombinant plasmids (containing PR-39/pBD-1 and CpG) as adjuvants of vaccines could enhance immune stimulation better than HDPs or CpG alone. It has a good protective effect on maintaining health of newborn piglets. Among them, both plasmids pVAX49-PR-39 and pVAX49-pBD-1 could be used as effective vaccine adjuvants for piglets.

Drug Combinations Co-Targeting Myeloid Cell Leukemia-1 (Mcl-1) Protein Can Overcome Microenvironmentally-Induced Multi-Drug Tolerance in Non-Hodgkin Lymphomas

Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (Btk) and venetoclax (VEN), an inhibitor of Bcl-2 have been used in Chronic Lymphocytic Leukemia (CLL), but single agent responses to these drugs are often incomplete and not durable. We previously reported synergistic cytotoxicity for the IBR+VEN combination and have initiated a trial to test that combination (NCT02419560). However, we noted extensive variability in response to IBR+VEN between patient samples ex vivo, suggesting intrinsic tolerance even to this combination. CLL cells with an “activation” phenotype (CD5+/19+/69+) that occurs from interactions with the microenvironment were less sensitive to IBR+VEN, implicating the cancer microenvironment as an inducer of drug tolerance. This was supported by our finding that CLL cells display decreased sensitivity to IBR+VEN in co-culture with agonists and cells that emulate the cancer cell microenvironment. The combination of CpG-oligodeoxynucleotides (ODN), sCD40L, and IL10 (“agonist mix”) induced near complete loss of sensitivity to IBR+VEN and upregulated Mcl-1 and Bcl-xL expression in CLL cells. However, Mcl-1 or Bcl-xL inhibitors alone were weakly effective in these cells, suggesting that exploitation of these targets requires drug combinations.Here we report a combination drug screen with an Mcl-1 inhibitor in a CLL microenvironment model ex vivo and novel drug combinations that overcome multi-drug tolerance. CLL patient PBMCs were cultured in the microenvironment model consisting of HK follicular dendritic cells, HUVEC, Jurkat T cells expressing CD40L, and CpG-ODN. Markers characteristic of activation and drug tolerance in vivo were assessed in CLL (CD5+/19+) cells using flow cytometry. We found increased expression of Ki67, CD69, and anti-apoptotic proteins Mcl-1 and Bcl-xL and decreased CXCR4 expression, as expected upon microenvironmental stimuli in vivo.Also, CLL cells from 2 IBR-treated patients were still responsive to agonist-induced drug tolerance, suggesting Btk inhibition may be less effective in blocking these microenvironmental stimuli in vivo. By comparing the ratio of anti-apoptotic proteins with cognate pro-apoptotic proteins, we noted targetable dependence of CLL cells for diverse pro-survival proteins (Mcl-1 and Bcl-xL) in our model.Flow cytometry analysis demonstrated that inhibitors of Mcl-1 (S63845), Bcl-xL (A1155643), or Bcl-2 (VEN) when used as single agents failed to induce significant apoptosis in CLL cells cultured in our model, suggesting induction of multi-drug tolerance. Drug combinations inhibiting Mcl-1 with Bcl-xL, Bcl-2, or Bcl-xL and Bcl-2 have effectively overcome multi-drug tolerance. As direct and simultaneous inhibition of multiple pro-survival proteins could be fatal to healthy cells, we carried out a Mcl-1-anchored combinatorial drug screen with inhibitors of other survival pathways in 3 CLL patient PBMCs cultured in our ex vivo model to identify drug combination(s) that induce selective toxicity in multi-drug tolerant CLL cells. Cytotoxicity was determined by analyzing cleaved PARP and dead cell staining in CLL and healthy T (CD3+/5+) cells by flow cytometry. Results showed that inhibitors of apoptotic proteins (A1155643, VEN, ABT737), IRAK4 (CA-4948, compound 26, and AS2444697), intracellular TLRs (chloroquine), Hsp90 (ganetespib), CDK (ribociclib), proteasome (bortezomib), Btk (IBR), AKT (MK2206), and HDAC (SAHA and panobinostat) or activator of PP2A (DBK1532 and NZ8061) were synergistically toxic with Mcl-1 inhibitor (S63845) in CLL cells. By comparing toxicity in CLL and healthy T cells, drug combinations targeting Mcl-1 with proteasome, IRAK4, TLRs, Btk, Hsp90, and CDK showed selective toxicity in CLL cells, indicating a potential therapeutic window for these combinations. Drug combinations targeting Mcl-1 with other apoptotic proteins were highly toxic to healthy T cells.In conclusion, CLL cells in our ex vivo model display characteristics of microenvironmentally-induced multi-drug tolerance. A similar phenotype was noted using post IBR therapy samples, suggesting IBR alone may not be effective in overcoming this multi-drug tolerance in vivo. Drug combinations targeting Mcl-1 and proteasome, IRAK4, TLRs, Btk, Hsp90, or CDK selectively overcame multi-drug tolerance in CLL cells. Thus, these combinations may be effective in patients showing intrinsic tolerance to multiple drugs. DisclosuresPortell:TG therapeutics: Research Funding; AbbVie: Research Funding; Infinity: Research Funding; Genentech/Roche: Consultancy, Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Kite: Research Funding; Amgen: Consultancy. Narla:University of Michigan: Patents & Royalties: Small molecule PP2A activators. Williams:Takeda: Research Funding; TG Therapeutics: Consultancy; Seattle Genetics: Consultancy; Verastem: Consultancy; Juno: Consultancy; Sandoz: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy; Abbvie: Consultancy; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Kite: Consultancy; Gilead: Consultancy, Research Funding.

CpG-ODNs and Budesonide Act Synergistically to Improve Allergic Responses in Combined Allergic Rhinitis and Asthma Syndrome Induced by Chronic Exposure to Ovalbumin by Modulating the TSLP-DC-OX40L Axis.

The experimental model of combined allergic rhinitis and asthma syndrome (CARAS) has shown that CpG oligodeoxynucleotides (CpG-ODNs) are potential inhibitors of type 2 helper cell-driven inflammatory responses. Currently available CpG-ODNs modestly inhibit allergic responses in CARAS, while a combination strategy for upper airway treatment by co-administration of CpG-ODNs and glucocorticoids may show good efficacy. This study aimed to assess the therapeutic effects of CpG-ODNs combined with budesonide (BUD) on upper and lower-airway inflammation and remodeling in mice with CARAS induced by chronic exposure to ovalbumin (OVA), exploring the possible underlying molecular mechanisms. A BALB/c mouse model of chronic CARAS was established by systemic sensitization and repeated challenge with OVA. Treatment with CpG-ODNs or BUD by intranasal administration was started 1h after OVA challenge. Then, nasal mucosa and lung tissues were fixed and stained for pathologic analysis. The resulting immunologic variables and TSLP-DC-OX40L axis parameters were evaluated. Both CpG-ODNs and BUD intranasal administration are effective on reducing Th2-type airway inflammation and tissue remodeling. Co-administration of CpG-ODNs and BUD was more effective than each monotherapy in attenuating upper and lower-airway inflammation as well as airway remodeling in chronic CARAS. Notably, combination of CpG-ODNs with BUD modulated the TSLP-DC-OX40L axis, as demonstrated by decreased TSLP production in the nose and lung, alongside decreased TSLPR and OX40L in DC. Intranasal co-administration of CpG-ODNs and BUD synergistically alleviates airway inflammation and tissue remodeling in experimental chronic CARAS, through shared cellular pathways, as a potent antagonist of the TSLP-DC-OX40L axis.

Abstract 08: Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies

Abstract The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is FDA approved for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). However, IBR responses are incomplete in majority of cases, and not durable in MCL and high-risk CLL. We hypothesize that de novo resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be cotargeted with BTK to achieve deeper and durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generates synergistic cytotoxicity with IBR in MCL lines (Axelrod et al. Leukemia 2014;28:407-10). Recently, we extended these findings to circulating leukemic B cells in CLL, MCL (Jayappa et al. ASH 2016), and follicular lymphoma (FL) patient PBMCs treated ex vivo. Based on these results we initiated a phase I/Ib trial for IBR+VEN combination in MCL. Our ex vivo cytotoxicity studies showed a surprisingly high frequency of de novo resistance to IBR+VEN. Resistance did not correlate with previous therapy or clinical characteristics of patients. However, FACS analysis of primary CLL cells showed that cells displaying an “activation” phenotype (CD5+/CD19+/CD69+) were less sensitive to cytotoxicity of IBR+VEN, suggesting that cellular activation by extrinsic factors could generate drug resistance. To test this, we cocultured or preincubated CLL patient PBMCs with various stromal cells, cytokines, and other agonists and found that soluble CD40L, IL10, and CpG oligodeoxynucleotides (ODN) generated variable resistance to IBR+VEN in CLL samples, with CpG ODN, a specific TLR9 agonist, being the most effective. The combination of CpG ODN, sCD40L, and IL10 (“agonist mix”) generated near-complete resistance to IBR+VEN in most CLL(N=22)/MCL(N=7)/FL(N=4) patient PBMCs. The agonist mix treatment significantly upregulated nuclear localization of RelA and RelB proteins in CLL/MCL/FL cells analyzed by ImageStream, suggesting activation of NF-kB signaling. Agonist mix treatment also induced the NF-kB dependent overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, and inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame this drug resistance (Jayappa et al. ASH 2016). To further explore the role of TLR9 and other innate immune receptors in resistance, we performed IBR+VEN cytotoxicity analysis in CLL/MCL patient PBMCs cocultured with agonists of various TLRs and NOD1/2 receptors. Preliminary results showed that TLR9 agonist CpG ODN generated significant resistance to IBR+VEN in most CLL/MCL samples, and agonists of TLR1/2, TLR7, and NOD1/2 induced a moderate level of resistance in a MCL sample. The CpG ODN treatment induced a significant increase in NF-kB signaling measured by increased nuclear localization of RelB in CLL patient samples. To determine if soluble agonists in the extranodal macroenvironment of patients could generate NF-kB dependent resistance to IBR+VEN, we analyzed drug cytotoxicity in CLL(N=6) and MCL(N=1) patient PBMCs cultured with autologous plasma. We found that preincubation with autologous plasma induced variable levels of resistance to IBR+VEN in CLL/MCL samples, but did not induce resistance to a NF-kB inhibitor BMS-345541. In conclusion, soluble factors in the patient nodal/extranodal macroenvironment, particularly a TLR9 agonist, generate resistance to IBR+VEN by activating NF-kB signaling and overexpression of multiple anti-apoptotic proteins. The same resistance mechanism, based on innate immune signaling, occurs in three mature B-cell malignancies, CLL, MCL and FL, that differ in etiology and clinical course. The signaling pathway responsible for resistance contains new targets for therapeutic intervention with potential to prevent or reverse resistance to IBR and VEN in these and perhaps other cancers as well. Citation Format: Kallesh D. Jayappa, Craig A. Portell, Vicki L. Gordon, Timothy P. Bender, Michael E. Williams, Michael J. Weber. Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 08.

CXCL14 Acts as a Specific Carrier of CpG DNA into Dendritic Cells and Activates Toll-like Receptor 9-mediated Adaptive Immunity

CXCL14 is a primordial chemokine that plays multiple roles in tumor suppression, autoimmune arthritis, and obesity-associated insulin resistance. However, the underlying molecular mechanisms are unclear. Here, we show that CXCL14 transports various types of CpG oligodeoxynucleotide (ODN) into the endosomes and lysosomes of bone marrow-derived dendritic cells (DCs), thereby activating Toll-like receptor 9 (TLR9). A combination of CpG ODN (ODN2395) plus CXCL14 induced robust production of IL-12 p40 by wild-type, but not Tlr9-knockout, DCs. Consistent with this, ODN2395-mediated activation of DCs was significantly attenuated in Cxcl14-knockout mice. CXCL14 bound CpG ODN with high affinity at pH7.5, but not at pH6.0, thereby enabling efficient delivery of CpG ODN to TLR9 in the endosome/lysosome. Furthermore, the CXCL14-CpG ODN complex specifically bound to high affinity CXCL14 receptors on DCs. Thus, CXCL14 serves as a specific carrier of CpG DNA to sensitize TLR9-mediated immunosurveillance.

Modulatory effect of CpG oligodeoxynucleotide on a DNA vaccine against nervous necrosis virus in orange-spotted grouper (Epinephelus coioides).

We report the development of a DNA vaccine pcMGNNV2 against nervous necrosis virus (NNV), a leading cause of mass mortality in grouper larvae. In addition, the modulatory effect of CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 agonist, on the DNA vaccine was evaluated. The DNA vaccine alone elicited the production of NNV-specific antibodies, indicating that the vaccine was capable of triggering adaptive humoral response. Furthermore, significant induction of TLR9, Mx and IL-1β was observed in the spleen on day 7 post-vaccination, supporting that the vaccine could trigger TLR9 signaling. The incorporation of CpG ODN at high dose did not significantly affect the level of NNV-specific antibodies, but was able to moderately enhance the expression of Mx and IL-1β on day 7, indicating its ability in modulating innate response. After challenge with NNV, the vaccine alone enhanced the survival rate in infected larvae at both 1 and 2 weeks post-vaccination. The combination of CpG ODN further increased the survival rate at week 1 but not week 2. Interestingly, at week 2 the ODN appeared to induce a Th1-like response, as indicated by upregulation of T-bet (a Th1 marker) and downregulation of GATA-3 (a Th2 marker). Thus, the results suggest that the boosted Th1 response by CpG ODN does not augment the protection efficacy of pcMGNNV2 vaccine. To our best knowledge, this is the first report of a successful DNA vaccine against NNV in grouper.

TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN.

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type-1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K-type) induces IFNs only weakly and STING ligands rather induce type-2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type-1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING-ligand cyclic GMP–AMP (cGAMP), culminating in NK cell IFN-γ (type-II IFN) production, is due to the concurrent effects of IL-12 and type-I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type-1 adjuvant, capable of inducing strong Th1-type responses, as demonstrated by enhanced antigen-specific IgG2c and IFN-γ production, as well as cytotoxic CD8+ T-cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen-free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type-II IFN inducer.

Abstract 2572: CpG oligodeoxynucleotide enhances the efficacy of anticancer monoclonal antibody in an in vivo xenograft model using human endometrial cancer cell

Abstract Background: CpG oligodeoxynucleotides (ODN) has been shown to exhibit a potent immunostimulatory activity. Therefore, CpG ODN has been considered to be a promising material for enhancing the therapeutic effect of anticancer monoclonal antibody. We have previously identified bone marrow stromal antigen 2 (BST2) as a therapeutic target of endometrial cancer cells and also demonstrated that an anti-BST2 antibody showed a potent anti-tumor effect by antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against BST2 positive cells. The aim of this study was to evaluate the synergistic anti-tumor effect by combination of anti-BST-2 antibody and CpG ODN, and to clarify the mechanisms of its anti-tumor effect, using xenograft model of anti-BST2 positive endometrial cancer cells. Material and Method: We implanted 7-8 week-old female SCID mice with 5×106 HEC-88 nu endometrial cancer cells subcutaneously. Mice with tumor were treated by intra-peritoneal injection of an anti-BST2 antibody alone (0 - 200 μg/mouse, twice a week for four weeks), intra-tumoral injection of CpG ODN alone (0 - 30 μg/mouse, twice a week for four weeks) or combination therapy (anti-BST2 antibody and CpG ODN). Anti-tumor effect was evaluated by monitoring the changes of tumor volume and also pathological analysis. In addition, activation of NK cells was evaluated. Results: Intraperitoneal treatment of anti-BST2 antibody showed a significant anti-tumor effect, dose dependently. Similarly, intratumoral injection of CpG ODN showed a significant anti-tumor effect, in a dose dependent manner. Interestingly, the combination therapy was more effective than the single-agent therapy. In addition, the combination therapy also resulted in a high activity of NK cells. Conclusion: We showed that CpG ODN increases the efficacy of anti-tumor effect of anti-BST2 antibody induced ADCC activity via potentiating NK cells mediated cancer cell injury. Combination of CpG ODN and anti-tumor monoclonal antibody is useful cancer therapy. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Kouji Kobiyama, Akiko Morimoto, Toshihiro Kimura, Kiyoshi Yoshino, Minoru Fujimoto, Ken J. Ishii, Masami Fujita, Tetsuji Naka. CpG oligodeoxynucleotide enhances the efficacy of anticancer monoclonal antibody in an in vivo xenograft model using human endometrial cancer cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2572. doi:10.1158/1538-7445.AM2014-2572

Combination of CpG-oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective immunity against Toxoplasma gondii infection

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) have been characterized as Th1-promoting immunopotentiators, an adjuvant activity desirable for vaccination against intracellular parasites like Toxoplasma gondii. In an attempt to find new antigen–adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the extent of protection in mice immunized with ROP2 and GRA4 recombinant proteins when co-administered with CpG-ODN. Both GRA4+CpG-ODN and ROP2+CpG-ODN formulations were shown to induce a strong humoral Th1-biased response characterized by a high IgG2a to IgG1 antibody ratio. Both vaccination regimens led to increased secretion of IFN-γ and IL-10, and negligible amounts of IL-4, upon specific re-stimulation of spleen cells from these groups of mice. After a non-lethal challenge with tissue cysts of a moderately virulent strain, only the brains from mice vaccinated with ROP2 or GRA4 in combination with CpG-ODN showed a significant reduction (63% and 62%, respectively) in their parasite load compared to the controls. The rate of protection obtained with GRA4+ROP2+CpG-ODN resulted equivalent (66%) to those achieved with the single antigens plus CpG-ODN. Taken together, these results indicate that CpG-ODN is an important candidate adjuvant for use in potential multicomponent anti-T. gondii vaccines for animals and humans.

The novel adjuvant combination of CpG ODN, indolicidin and polyphosphazene induces potent antibody- and cell-mediated immune responses in mice

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-α, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-γ secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.

Treatment with LL-37 Peptide Enhances Antitumor Effects Induced by CpG Oligodeoxynucleotides Against Ovarian Cancer

There is an urgent need for innovative therapies against ovarian cancer, one of the leading causes of death from gynecological cancers in the United States. Immunotherapy employing Toll-like receptor (TLR) ligands, such as CpG oligodeoxynucleotides (CpG-ODN), may serve as a potentially promising approach in the control of ovarian tumors. The CpG-ODN requires intracellular delivery into the endosomal compartment, where it can bind to TLR9 in order to activate the immune system. In the current study, we aim to investigate whether the antimicrobial polypeptide from the cathelicidin family, LL-37, could enhance the immunostimulatory effects of CpG-ODN by increasing the uptake of CpG-ODN into the immune cells, thus enhancing the antitumor effects against ovarian cancer. We found that treatment with the combination of CpG-ODN and LL-37 generated significantly better therapeutic antitumor effects and enhanced survival in murine ovarian tumor-bearing mice compared with treatment with CpG-ODN or LL-37 alone. We also observed that treatment with the combination of CpG-ODN and LL-37 enhanced proliferation and activation of natural killer (NK) cells, but not CD4(+) or CD8(+) T cells, in the peritoneal cavity. Furthermore, in vivo antibody depletion experiments indicated that peritoneal NK cells played a critical role in the observed antitumor effects. Thus, our data suggest that the combination of CpG-ODN with LL-37 peptide may lead to the control of ovarian tumors through the activation of innate immunity.

CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors

Background and purpose Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess anti-tumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074–7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361–9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC). Materials and methods CpG ODN 1826 (100 μg) was given sc three times: when leg tumors were 6 mm, when they grew to 8 mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10 Gy) were given when tumors were 8 mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter. Results Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. Conclusion Overall results show that CpG ODN 1826 can markedly improve tumor response to radiation and chemotherapy (DOC), suggesting that CpG ODNs have potential to be beneficial when used singly or in combination with other standard treatment modalities such as taxane chemotherapy, radiotherapy or both.

Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia

AbstractIn chronic lymphocytic leukemia (CLL), chromosomes usually evade detailed cytogenetic analyses because cells poorly respond to the traditionally used set of mitogens. We applied novel technologies, such as stimulation of CLL cells either with CD40 ligand or with a combination of CpG-oligodeoxynucleotides and IL-2, to increase the freequncy of metaphase spreads for detailed chromosome analysis in 96 patients with CLL. This approach revealed that translocations occurred in 33 of 96 (34%) of our patients with CLL. The presence of translocations defined a new prognostic subgroup because these patients have significantly shorter median treatment-free survival (24 months vs 106 months; P < .001) and significantly inferior overall survival (OS; median, 94 months) than patients without translocations (346 months; P < .001). In multivariate analysis—including Binet stage, complex karyotype, CD38 expression, and 17p deletions—translocation proved to be the prognostic marker with the highest impact for an unfavorable clinical outcome (P < .001). In summary, we identified a new subgroup of patients with CLL defined by chromosomal trans-locations and poor prognosis. Our data may facilitate the identification of molecular events crucial for transforming activity in this disease and should have implications for risk-adapted clinical management of patients with CLL. (Blood. 2006;107:742-751)