CpG Oligodeoxynucleotides for Anticancer Monotherapy from Preclinical Stages to Clinical Trials

CpG oligodeoxynucleotides (CpG ODNs), the artificial versions of unmethylated CpG motifs that were originally discovered in bacterial DNA, are demonstrated not only as potent immunoadjuvants but also as anticancer agents by triggering toll-like receptor 9 (TLR9) activation in immune cells. TLR9 activation triggered by CpG ODN has been shown to activate plasmacytoid dendritic cells (pDCs) and cytotoxic T lymphocytes (CTLs), enhancing T cell-mediated antitumor immunity. However, the extent of antitumor immunity carried by TLR agonists has not been optimized individually or in combinations with cancer vaccines, resulting in a decreased preference for TLR agonists as adjuvants in clinical trials. Although various combination therapies involving CpG ODNs have been applied in clinical trials, none of the CpG ODN-based drugs have been approved by the FDA, owing to the short half-life of CpG ODNs in serum that leads to low activation of natural killer cells (NK cells) and CTLs, along with increases of pro-inflammatory cytokine productions. This review summarized the current innovation on CpG ODNs that are under clinical investigation and explored the future direction for CpG ODN-based nanomedicine as an anticancer monotherapy.

607 Regional delivery of a TLR9 agonist to boost checkpoint inhibitor responsiveness in liver metastases

BackgroundClass C TLR9 agonists, CpG oligodeoxynucleotides (ODNs) enhance responsiveness to anti-PD1 therapy in solid tumors through favorable modulation of the tumor microenvironment (TME) [1]. Recently, we reported that regional delivery of a TLR9 agonist eliminated myeloid derived suppressor cells (MDSC) and promoted pro-inflammatory/anti-tumorigenic M1 macrophage programming in the TME of liver metastases (LM) [2]. Further, we found enhanced TLR9 activation in LM following regional TLR9 agonist infusion compared to the systemic treatment. We hypothesize that regional delivery of a TLR9A into LM will enhance the responsiveness to systemically infused anti-PD1 therapy.MethodsIn this study, we treated mice with established MC38-CEA-Luc LM with ODN-2395 (30µg/mouse) regionally with or without anti PD-1 antibody (250µg/mouse) intraperitoneally.ResultsControl of LM growth (Figure 1) was significantly higher with combinatorial treatment as compared to anti-PD1 (p<0.01) or PBS treatments (p<0.05). To study the impact of TLR9 activation on human MDSC, we treated healthy donor PBMCs with ODN-2395 or SD101. We found that both reduced the hu-MDSC (CD11b+CD33+HLADR-) population in a dose-dependent manner with an increase in PD-L1 expression as determined by flow cytometry (FC) analysis (Figure 2). Moreover, by using Luminex, demonstrated that ODN-2395 and SD101 enhanced expression of IL 29, IFNα, and NFκB, along with downstream cytokines IL 6 and IL 10. To investigate the effect of SD101 in modulating the differentiation of huMDSC from huPBMC, we treated huPBMC with IL6+GM-CSF in the presence or absence of SD101. By FC analysis, we found that SD-101 blocked huMDSC development induced by IL6+GM-CSF, preferentially limited the more immunosuppressive monocytic MDSC subtype, and drove M1 macrophage polarization. Treatment of SD101 only once for 48hrs was sufficient to inhibit huMDSC differentiation for two weeks.Abstract 607 Figure 1Combinatorial treatment of CPI and ODN’s reduces tPV = portal vein; IP = intraperitoneal.Eight to twelve weeks old male C57/BL6 mice were challenged intra-splenic with 0.5e6 MC38-CEA-Luc cells for a week. Bioluminescence value was determined by IVIS on D0, and mice were randomized accordingly and treated with 30 µg/mouse ODN2395 via PV with or without 250 µg/mouse anti-PD1 antibody via IP on D0, D+3 and D+6. PBS served as the vehicle (Veh) control and administered via PV. Fold change of the tumor burden was calculated based on D0 baseline bioluminescence. Tumor progression was analyzed unpaired t test among groups. (*p <0.05).Abstract 607 Figure 2Human PBMC treated with ODN2395 and SD101 reducesCtrl = control; MDSC = ODN = oligodeoxynucleotide’ PBMC = peripheral blood monocytes.Human PBMC were isolated from the Leukoreduction system chamber. 1e6/ml PBMCs were treated with increasing concentrations (0.04–10 µM) SD101, ODN2395 along with ctrl ODN5328 (1µM) for 48 hours. Panels A and B: MDSC population and their corresponding PD–L1 expression were evaluated (n=12). Four donors with three replicates were used. Data represented as mean ± SEM.ConclusionsBoth the in vitro and in vivo findings suggest that regional TLR9 stimulation in a model of LM improves responsiveness to systemic anti-PD-1 therapy through elimination of MDSC, and the effect on huMDSC was confirmed in vitro. Increased PDL-1 expression in response to TLR9 stimulation among MDSC may further enhance the anti-PD-1 effect. Therefore, combing regional infusions of a TLR9 agonist with systemic anti-PD-1 agents may be a promising approach for liver tumor treatment.ReferencesWang, S., et al., Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A, 2016. 113(46): p. E7240-E7249.Ghosh CC, H.K., O’Connell K, Laporte J, Guha P, Cox B, Jaroch D, Katz SC, Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion, Abstract: AACR Annual Meeting. 2021.

Electronic Cigarettes Induce Mitochondrial DNA Damage and Trigger TLR 9 (Toll-Like Receptor 9)-Mediated Atherosclerosis

Objective: Electronic cigarette (e-cig) use has recently been implicated in promoting atherosclerosis. In this study, we aimed to investigate the mechanism of e-cig exposure accelerated atherosclerotic lesion development. Approach and Results: Eight-week-old ApoE − /− mice fed normal laboratory diet were exposed to e-cig vapor (ECV) for 2 hours/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining and greatly upregulated TLR9 (toll-like receptor 9) expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by enhanced TLR9 expression in human femoral artery atherosclerotic plaques from e-cig smokers. Intriguingly, we found a significant increase of oxidative mitochondria DNA lesion in the plasma of ECV-exposed mice. Administration of TLR9 antagonist before ECV exposure not only alleviated atherosclerosis and the upregulation of TLR9 in plaques but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the plaque accumulation of lipid and macrophages, and decreased the frequency of blood CCR2 + classical monocytes. Surprisingly, we found that cytoplasmic mitochondrial DNA isolated from ECV extract-treated macrophages can enhance TLR9 activation in reporter cells and the induction of inflammatory cytokine could be suppressed by TLR9 inhibitor in macrophages. Conclusions: E-cig increases level of damaged mitochondrial DNA in circulating blood and induces the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacological target of ECV-related inflammation and cardiovascular diseases.

Helicobacter pylori-induced TLR9 Activation and Injury are Associated with the Virulence-Associated Adhesin HopQ

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma. The H. pylori cancer-associated cag pathogenicity island (cag-PAI) encodes a type IV secretion system (T4SS) which translocates microbial DNA and activates TLR9; however, most cag-PAI + infected persons do not develop cancer and cag-PAI-independent regulators of pathogenesis, including strain-specific adhesins, remain understudied. We defined the relationships between H. pylori HopQ adhesin allelic type, gastric injury, and TLR9 activation. Type I hopQ alleles were significantly associated with magnitude of injury, cag-T4SS function, and TLR9 activation. Genetic deletion of hopQ significantly decreased H. pylori-induced TLR9 activation, implicating this adhesin in H. pylori-mediated disease.

Electronic cigarettes induce mitochondrial DNA damage and trigger toll-like receptor 9-mediated atherosclerosis

ObjectiveBoth electronic cigarette (e-cig) use and toll-like receptor 9 (TLR9) activation have been implicated in promoting atherosclerosis. In this study we aimed to investigate the causative relationship of e-cig exposure on TLR9 activation and atherosclerosis development.Approach and ResultsEight-week-old ApoE-/- mice fed normal chow diet were exposed to e-cig vapor (ECV) for 2 h/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining of aortic root and greatly upregulated TLR9 expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by upregulated TLR9 expression in human femoral artery atherosclerotic plaques in e-cig smokers. Intriguingly, we found a significant increase of damaged mitochondria DNA level in the circulating blood of ECV exposed mice. Furthermore, administration of TLR9 antagonist prior to ECV exposure not only alleviated atherosclerotic lesion and the upregulation of TLR9 in plaques, but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the accumulation of lipid and macrophages, and decreased the frequency of blood CCR2+ classical monocytes. Surprisingly, we found that the cytoplasmic mtDNA isolated from ECV extract-treated cells can greatly enhance the expression of TLR9 in reporter cells.ConclusionE-cig induces mtDNA damage and the mtDNA in circulating blood stimulates the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacologic target of ECV-related inflammation and cardiovascular diseases.

TLR1/2 and 5 induce elevated cytokine levels from rheumatoid arthritis monocytes independent of ACPA or RF autoantibody status

Objective RA is an autoimmune inflammatory joint disease. Both RF and ACPA are associated with more progressive disease and higher levels of systemic inflammation. Monocyte activation of toll-like receptors (TLRs) by endogenous ligands is a potential source of increased production of systemic cytokines. RA monocytes have elevated TLRs, some of which are associated with the disease activity score using 28 joints (DAS28). The aim of this study was to measure TLR-induced cytokine production from monocytes, stratified by autoantibody status, to assess if their capacity to induce cytokines is related to autoantibody status or DAS28. Methods Peripheral blood monocytes isolated from RA patients and healthy controls were stimulated with TLR1/2, TLR2/6, TLR4, TLR5, TLR7, TLR8 and TLR9 ligands for 18 h before measuring IL-6, TNFα and IL-10. Serum was used to confirm the autoantibody status. Cytokine levels were compared with RF, ACPA and DAS28. Results RA monocytes demonstrated significantly increased IL-6 and TNFα upon TLR1/2 stimulation and IL-6 and IL-10 upon TLR5 activation. TLR7 and TLR9 activation did not induce cytokines and no significant differences were observed between RA and healthy control monocytes upon TLR2/6, TLR4 or TLR8 activation. When stratified by ACPA or RF status there were no correlations between autoantibody status and elevated cytokine levels. However, TLR1/2-induced IL-6 did correlate with DAS28. Conclusions Elevated TLR-induced cytokines in RA monocytes were not related to ACPA or RF status. However, TLR1/2-induced IL-6 was associated with disease activity.