Mechanism of action and therapeutic utility of stimulatory CpG oligonucleotides

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The unmethylated CpG motifs present in bacterial DNA interact with toll-like receptor 9 to trigger a pro-inflammatory immune response. CpG DNA also improves antigen presenting cell function, thereby facilitating the development of adaptive immunity. My laboratory established that synthetic oligonucleotides expressing immunostimulatory CpG motifs (CpG ODN) could be conjugated to apoptotic tumor cells to generate tumor vaccines that were rapidly internalized by professional APCs, promot DC maturation, and boost the induction of tumor-specific immunity. In multiple murine models we found that vaccination with CpG-conjugated apoptotic cell vaccines significantly reduced susceptibility to tumor challenge. This effect was observed both in mice vaccinated and then challenged and in animals immunized after tumor challenge. Unfortunately, the ability of these vaccines to eradicate tumors waned as cancer burden increased. We found this reflected the ability of large established tumors to generate an immunosuppressive microenvironment capable of inhibiting Ag-specific cellular responses that interferes with CpG-mediated immunotherapy. Myeloid-derived suppressor cells (MDSC) represent an important constituent of this immunosuppressive milieu. Large numbers of MDSC are present in and near established tumors and have been shown to inhibit the activity of antigen-specific T and NK cells. Our subsequent studies demonstrated that when CpG ODN were injected into the tumor itself, the immunosuppressive activity of monocytic MDSC (mMDSC) was significantly reduced. We hypothesize that the signal provided via TLR9 ligation was sufficient to overcome the inhibitory signals provided by the cells infiltrating the tumor. In mice, mMDSC express TLR9 and respond to CpG stimulation by i) losing their ability to suppress T cell function, ii) producing Th1 cytokines and iii) differentiating into M-1 like macrophages with tumoricidal capability. Similar activity was observed with TLR7 agonists, and the combination of TLR7 plus TLR9 agonists was significantly more effective. Indeed, intra-tumoral injection of this agonist combination induced the regression of even large established tumors (500 mm3 at the time of initial treatment). We extended these studies to evaluate the effect of TLR agonists on mMDSC purified from normal human donors and cancer patients. Results show that stimulation with TLR7/8 agonists induce human mMDSC to mature and lose their immuno-suppressive activity, reproducing on human MDSC the activity that TLR9 ligation has on mouse MDSC. We conclude that a combination of TLR ligands may be harnessed to achieve two independent but mutually supportive functions: boosting the efficacy of anti-tumor vaccines and reducing the activity of cells at the tumor site that would otherwise reduce the efficacy of this anti-tumor response. Our subsequent research sought to identify the optimal means and therapeutic window for the delivery of TLR ligands to tumor nodules. We've found that agonists adsorbed onto microparticles can be administered to mice with lung cancer, and that local DC and macrophages transport these particles to the tumor bed where they can effectively prevent tumor growth. We are exploring other formulations designed to optimize the delivery of TLR agonists to the lungs for use in cancer therapy. Efforts to optimize the therapeutic utility of CpG ODN require a detailed understanding of the cells they activate (both directly and indirectly), their duration of action, and the regulatory pathways involved in mediating these responses. To clarify these issues, we are using microarray technology to identify the genes and networks central to the immune stimulation elicited by CpG ODN. Such experiments are conducted in vitro on highly purified cell subpopulations (including human pDC and MDSC) and in vivo studies of mice to monitor gene expression under physiologic conditions. Earlier results showed that CpG ODN consistently activated a set of genes that was largely dependent on autocrine type I interferon (IFN) signaling. Current studies are evaluating combination therapy with TLR agonists plus chemotherapeutic agents (Cytoxan) or PD1 inhibitors on the progression of primary tumors and lung metastases in a murine orthotopic breast cancer model. We further demonstrated that this regulatory pathway was mediated via IRF5 acting through Nf-kB. Indeed, proximity ligation assays showed that IRF5 col-localized with Nf-kB to accomplish this task. This stimulatory activity is reversed by IRF8, such that IRF8 inhibits CpG induced IFN signaling. The cytokines responsible for the maturation of human mMDSC into M1 vs M2 like macrophages have also been identified. Highly purified human mMDSC are driven to differentiate into M1 by a combination of TNFa plus either IL-6 or IL-10. In contrast, M-CSF drives them to mature down the M2 pathway. Functional studies support the phenotypic identification of these unique cell types. Ongoing studies are directed towards identifying TLR agonists that induce human monocytes to differentiate into either M1- or M2-like macrophages. Results indicated that although most TLR agonists support the generation of pro-inflammatory M1-like macs, PAM3 (a TLR2/1 agonist) uniquely induces the generation of M2-like macrophage. The cytokines/factors elicited by treatment with PAM3 were identified and could substitute as stimulants. In addition, the regulatory pathways responsible for driving monocytes to differentiate into either M1- or M2-like macrophage were identified by a combination of microarray, RNA sequencing, and IPA analysis. Most recently, these findings have been applied to the use of TLR agonists to prevent/treat cancer and autoimmune diseases. A combination of TLR 7 and 9 agonists was identified that significantly reduced tumor burden and extended lifespan in mice with primary mammary cancer metastatic to the lungs. This required intra-tumoral and intra-pulmonary delivery of the combination TLR agonists. Improved results were achieved by the additional of anti-PD.L1 to the therapeutic cocktail when control of tumor growth by the agonists alone began to fail. Of concern, considerable toxicity was observed after repeated intra-pulmonary Rx of tumor metastases. We've also studied the ability of PAM3 to treat NZB/W mice with lupus. Therapy was initiated either early (at 7 wk of age) or late (at 18 wk of age after glomerulonephritis had developed). Weekly PAM3 injection significantly reduced anti-DNA autoAb titers and slowed the progression of renal disease, thereby prolonging life. As PAM3 has the same effect on human and mouse cells, this evidence of in vivo activity supports the initiation of clinical trials.