Mechanism of action and therapeutic utility of stimulatory CpG oligonucleotides

存在于细菌DNA中的未甲基化的CpG基序与toll样受体9相互作用以触发促炎免疫应答。CpG DNA还改善抗原呈递细胞功能，从而促进获得性免疫的发展。我的实验室建立了表达免疫刺激性CpG基序的合成寡核苷酸（CpG ODN）可以缀合到凋亡的肿瘤细胞，以产生肿瘤疫苗，该疫苗被专业APC迅速内化，促进DC成熟，并增强肿瘤特异性免疫的诱导。在多种鼠模型中，我们发现用CpG缀合的凋亡细胞疫苗接种显著降低了对肿瘤攻击的易感性。在接种疫苗然后攻击的小鼠中以及在攻击后免疫长达5周的动物中均观察到这种效应。添加已知可增强NK和T细胞活化的药物（例如4-1BB MAb）可协同增强CpG ODN的抗肿瘤作用。通过测试6种不同的肿瘤类型并在每种情况下显示活性来建立这种方法的一般效用。不幸的是，这些疫苗根除肿瘤的能力随着肿瘤负荷的增加而减弱。我们认为这反映了大型肿瘤产生免疫抑制微环境的能力，该免疫抑制微环境能够抑制Ag特异性细胞应答，从而干扰CpG介导的免疫治疗。骨髓源性抑制细胞（MDSC）是这种免疫抑制环境的重要组成部分。大量的MDSC存在于已建立的癌症中和附近，并且已显示出抑制抗原特异性T和NK细胞的活性。我们的研究表明，当将CpG ODN注射到肿瘤中时，它们对单核细胞（CD 11b+，Ly 6 G neg，Ly 6C high）MDSC的免疫抑制活性显著降低。我们假设通过TLR 9连接提供的信号足以克服肿瘤细胞提供的抑制信号。单核MDSC表达TLR 9并通过i）失去其抑制T细胞功能的能力，ii）产生Th 1细胞因子和iii）分化成具有杀肿瘤能力的M-1样巨噬细胞来响应CpG刺激。这些发现提供了一种新的机制，CpG ODN有助于肿瘤消退，并支持肿瘤内注射作为其交付的最佳途径。我们已经将这些研究扩展到包括来自正常人类供体和癌症患者的mMDSC。结果显示，用适当的TLR激动剂刺激诱导人mMDSC成熟并失去其免疫抑制活性。令人感兴趣的是，CpG ODN对人MDSC相对无活性，而TLR 7/8激动剂对人MDSC再现了TLR 9连接对小鼠MDSC的活性。因此，我们得出结论，TLR配体的组合可以被利用来实现两个独立但相互支持的功能：提高抗肿瘤疫苗的功效和降低肿瘤部位细胞的活性，否则会降低这种抗肿瘤反应的功效。我们正在进行的研究旨在确定用于递送CpG ODN和其他TLR配体的最佳治疗窗口，并检查它们引发的保护性免疫应答是否可以通过将它们与其他免疫调节剂（例如额外的TLR配体和小分子激动性免疫增强剂）组合来加速和/或放大。优化CpG ODN治疗效用的努力需要详细了解它们激活的细胞（直接和间接），它们的作用持续时间以及介导这些反应的调控途径。为了澄清这些问题，我们正在使用微阵列技术来识别CpG ODN引起的免疫刺激的基因和网络。这些实验在体外对高度纯化的细胞亚群（包括人pDC和MDSC）进行，并在小鼠体内进行研究以监测生理条件下的基因表达。最近的研究结果表明，一个子集的基因的特点是共享的抗病毒活性一致上调的ODN，否则介导离散功能。这组基因在很大程度上依赖于自分泌I型干扰素（IFN）信号传导，因为它们的诱导被靶向I型IFN受体的中和抗体阻断。将这些实验与其他已发表作品的荟萃分析相结合，鉴定出一组32个功能保守的基因，这些基因在不同物种和细胞类型中被不同类型的CpG DNA可重复激活。在功能上，这些核心基因支持I型IFN对病毒感染的应答，并且不同于仅由单一类型的CpG ODN上调的基因。这些发现有助于确定通过TLR 9触发的基因激活的保守和序列特异性模式，并提高我们对CpG ODN引起的免疫调节作用的理解。对从正常人供体和患有各种类型癌症的患者分离的MDSC的分析表明，暴露于TLR 7/8激动剂均匀地诱导这些细胞成熟为杀肿瘤巨噬细胞，而没有抑制活性。这些发现增加了TLR激动剂联合治疗在人类中应用的可能性。

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, promoted DC maturation, and boosted 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 up to 5 weeks post challenge. The addition of agents known to boost NK and T cell activation, such as 4-1BB MAb, synergistically enhanced the anti-tumor effect of CpG ODN. The general utility of this approach was established by testing 6 different tumor types and showing activity in each case. Unfortunately, the ability of these vaccines to eradicate tumors waned as tumor burden increased. We believe this reflects 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 cancers and have been shown to inhibit the activity of antigen-specific T and NK cells. Our studies demonstrate that when CpG ODN are injected into the tumor, they immunosuppressive activity of monocytic (CD11b+, Ly6G neg, Ly6C high) MDSC is significantly reduced. We hypothesize that the signal provided via TLR9 ligation is sufficient to overcome the inhibitory signals provided by the tumors cells. Monocytic MDSC 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. These findings provide insight into a novel mechanism by which CpG ODN contribute to tumor regression, and support intra-tumoral injection as the optimal route for their delivery. We have extended these studies to include mMDSC from normal human donors and cancer patients. Results show that stimulation with the appropriate TLR agonist induces human mMDSC to mature and lose their immunosuppressive activity. Of interest, CpG ODN are relatively inactive on human MDSC whereas TLR 7/8 agonists reproduce on human MDSC the activity that TLR9 ligation has on mouse MDSC. Thus, we conclude that a combination of TLR ligands can be being 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 ongoing research aims to identify the optimal therapeutic window for the delivery of CpG ODN and other TLR ligands and examine whether the protective immune responses they elicit can be accelerated and/or magnified by combining them with other immunomodulatory agents (such as additional TLR ligands and small molecule agonistic immune potentiators). 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. Recent results show that a subset of genes characterized by shared anti-viral activity was consistently up-regulated by ODNs that otherwise mediate discrete functions. This group of genes was largely dependent on autocrine type I interferon (IFN) signaling, as their induction was blocked by neutralizing antibody targeting the type I IFN receptor. Coupling these experiments with a meta-analysis of other published works led to the identification of a set of 32 functionally conserved genes that was reproducibly activated by different types of CpG DNA in different species and cell types. Functionally, these core genes support a type I IFN response to viral infection, and differ from genes up-regulated by only a single type of CpG ODN. These findings help define the conserved and sequence-specific patterns of gene activation triggered via TLR9 and improve our understanding of the immunomodulatory effects elicited by CpG ODN. Analysis of MDSC isolated from normal human donors and patients with various types of cancer demonstrate that exposure to TLR 7/8 agonists uniformly induce such cells to mature into tumoricidal macrophages without suppressive activity. These findings increase the likelihood that combination TLR agonist therapy will find utility in humans.