Mechanism of action and therapeutic utility of stimulatory CpG oligonucleotides

刺激性 CpG 寡核苷酸的作用机制和治疗用途

• 批准号: 9343728
• 负责人: Dennis Klinman
• 金额: $ 112.43万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9343728
• 关键词: Agonist; Animals; Antigen-Presenting Cells; Antigens; Apoptotic; Automobile Driving; Bacterial DNA; Beds; Biological Assay; Cancer Burden; Cancer Patient; Cancer Vaccines; Cell physiology; Cells; DNA; Development; Formulation; Gene Expression Profiling; Generations; Genes; Goals; Human; Immune; Immune response; Immunity; Immunosuppressive Agents; Immunotherapy; In Vitro; Inflammatory; Injection of therapeutic agent; Interferon Type I; Interferons; Interleukin-10; Interleukin-6; Laboratories; Ligands; Ligation; Lung; Macrophage Colony-Stimulating Factor; Malignant neoplasm of lung; Mediating; Microarray Analysis; Molecular; Mus; Myelogenous; Natural Killer Cells; Nodule; Oligonucleotides; Pathway interactions; Pattern; Physiological; Predisposition; Production; Regulatory Pathway; Research; Signal Transduction; Site; Speed; Suppressor-Effector T-Lymphocytes; T-Lymphocyte; TLR7 gene; TLR9 gene; Testing; Therapeutic; Time; Toll-like receptors; Vaccinated; Vaccination; Vaccine Adjuvant; Vaccines; Work; adaptive immunity; autocrine; base; cancer cell; cancer therapy; cell type; chemokine; cytokine; design; immune clearance; immunogenicity; improved; in vivo; interest; killings; macrophage; mouse model; nanoparticle; neoplastic cell; novel; particle; prevent; research study; response; targeted delivery; tumor; tumor growth

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 ongoing research aims 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 aset of genes that was largely dependent on autocrine type I interferon (IFN) signaling. Recent work demonstrates that this regulatory pathway is 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.

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。本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）可以与凋亡的肿瘤细胞结合，产生肿瘤疫苗，这些疫苗可以被专业的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肿瘤攻击后免疫的动物中均观察到这种效应。不幸的是，这些疫苗根除肿瘤的能力随着癌症负担的增加而减弱。我们发现，这反映了大型肿瘤产生免疫抑制微环境的能力，这种免疫抑制微环境能够抑制Ag特异性细胞反应，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存在于已建立的肿瘤中和附近，并且已显示出抑制抗原特异性T和NK细胞的活性。我们随后的研究表明，当将CpG ODN注射到肿瘤本身时，单核细胞MDSC（mMDSC）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由浸润肿瘤的细胞提供的抑制信号。在小鼠中，mMDSC表达TLR 9并通过i）失去其抑制T细胞功能的能力，ii）产生Thl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。用TLR 7激动剂观察到类似的活性，并且TLR 7加TLR 9激动剂的组合显著更有效。实际上，肿瘤内注射该激动剂组合诱导甚至大的已建立肿瘤（在初始治疗时500 mm 3）的消退。我们扩展了这些研究，以评估TLR激动剂对从正常人供体和癌症患者纯化的mMDSC的作用。结果显示，用TLR 7/8激动剂刺激诱导人mMDSC成熟并失去其免疫抑制活性，在人MDSC上再现TLR 9连接对小鼠MDSC具有的活性。我们的结论是，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将TLR配体递送至肿瘤结节的最佳方法和治疗窗口。我们已经发现，吸附在微粒上的激动剂可以施用于患有肺癌的小鼠，并且局部DC和巨噬细胞将这些颗粒转运到肿瘤床，在那里它们可以有效地防止肿瘤生长。我们正在探索其他制剂，旨在优化TLR激动剂向肺部的递送，用于癌症治疗。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。早期的结果表明，CpG ODN持续激活一组基因，这些基因在很大程度上依赖于自分泌I型干扰素（IFN）信号。最近的工作表明，这一调节途径是通过IRF 5介导的，IRF 5通过Nf-kB起作用。事实上，邻近连接测定显示IRF 5与Nf-kB共定位以完成该任务。这种刺激活性被IRF 8逆转，使得IRF 8抑制CpG诱导的IFN信号传导。还鉴定了负责人mMDSC成熟为M1与M2样巨噬细胞的细胞因子。高度纯化的人mMDSC通过TNF α加IL-6或IL-10的组合被驱动分化成M1。相反，M-CSF驱动它们沿着M2途径成熟。功能研究支持这些独特细胞类型的表型鉴定。