Mechanism of action and therapeutic utility of immunostimulatory CpG oligonucleo

免疫刺激性 CpG 寡核的作用机制和治疗用途

• 批准号: 8552864
• 负责人: Dennis Klinman
• 金额: $ 112.47万
• 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/8552864
• 关键词: Agonist; Animals; Antigen-Presenting Cells; Antigens; Apoptotic; Bacterial DNA; Cancer Patient; Cancer Vaccines; Cell physiology; Cells; Conserved Sequence; Coupling; DNA; Dendrimers; Development; Drug Formulations; Gene Activation; Gene Expression; Gene Expression Profiling; Generations; Genes; Goals; Human; ITGAM gene; Immune; Immune response; Immunity; Immunosuppressive Agents; Immunotherapy; In Vitro; Individual; Inflammatory; Injection of therapeutic agent; Interferon Type I; Laboratories; Ligands; Malignant Neoplasms; Mediating; Meta-Analysis; Microarray Analysis; Modeling; Mus; Myelogenous; Natural Killer Cells; Oligonucleotides; Pattern; Physiological; Predisposition; Production; Publishing; Regulation; Regulator Genes; Regulatory Pathway; Research; Route; Signal Transduction; Site; Speed; Suppressor-Effector T-Lymphocytes; Systems Biology; T-Cell Activation; T-Lymphocyte; TLR9 gene; Testing; Therapeutic; Time; Toll-like receptors; Tumor Burden; Vaccinated; Vaccination; Vaccine Adjuvant; Vaccines; Viral Physiology; Virus Diseases; Work; Wound Healing; adaptive immunity; autocrine; base; cell killing; cell type; chemokine; cytokine; immunogenicity; improved; in vivo; insight; interest; killings; macrophage; neoplastic cell; neutralizing antibody; novel; research study; response; small molecule; therapeutic target; tumor; type I interferon receptor

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. Since joining the NCI, 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 killed cell vaccines significantly reduced susceptibility to tumor challenge. This effect was observed both in mice pre-vaccinated and then challenged and in animals immunized with our CpG-adjuvanted apoptotic tumor vaccine 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.However, the ability of this approach to eradicate tumors waned as tumor burden increased. To some extent, we believe this reflects the induction of an immunosuppressive microenvironment surrounding the large established cancers that inhibits Ag-specific cellular responses and thus 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 tumor sites where they inhibit the activity of antigen-specific T and NK cells. Our ongoing studies indicate that when CpG ODN are injected directly into a tumor, they reduce the immunosuppressive activity of monocytic (CD11b+, Ly6G neg, Ly6C high) MDSC. 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 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've now 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. Thus, we envision TLR activation as being harnessed for two independent but mutually supportive functions: boosting the efficacy of anti-tumor vaccines and reducing the activity of cells at the tumor site that 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 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.

细菌DNA中未甲基化的CpG基序与toll样受体9相互作用，引发促炎免疫反应。CpG DNA还可以改善抗原提呈细胞的功能，从而促进适应性免疫的发展。自加入NCI以来，我的实验室确定了表达免疫刺激CpG基序（CpG ODN）的合成寡核苷酸可以偶联到凋亡的肿瘤细胞上，产生肿瘤疫苗，这些疫苗被专业apc迅速内化，促进DC成熟，促进肿瘤特异性免疫的诱导。在多个小鼠模型中，我们发现接种CpG结合的死细胞疫苗可显著降低对肿瘤攻击的易感性。这种效果在预先接种并随后攻毒的小鼠以及在攻毒后5周接种cpg佐剂凋亡肿瘤疫苗的动物中都观察到了。加入已知的促进NK和T细胞活化的药物，如4-1BB MAb，协同增强了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出活性，确立了该方法的一般效用。然而，这种方法根除肿瘤的能力随着肿瘤负荷的增加而减弱。在某种程度上，我们认为这反映了在大型肿瘤周围的免疫抑制微环境的诱导，这种微环境抑制ag特异性细胞反应，从而干扰cpg介导的免疫治疗。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存在于肿瘤部位及其附近，它们抑制抗原特异性T细胞和NK细胞的活性。我们正在进行的研究表明，当CpG ODN直接注射到肿瘤中时，它们会降低单核细胞（CD11b+， Ly6G阴性，Ly6C高）MDSC的免疫抑制活性。单核细胞MDSC表达TLR9，对CpG刺激的反应是：1)失去抑制T细胞功能的能力，2)产生Th1细胞因子，3)分化为具有杀瘤能力的巨噬细胞。这些发现为CpG ODN促进肿瘤消退的新机制提供了见解，并支持肿瘤内注射作为其递送的最佳途径。我们现在已经将这些研究扩展到包括来自正常人类捐赠者和癌症患者的mMDSC。结果表明，适当的TLR激动剂刺激可诱导人mMDSC成熟并丧失其免疫抑制活性。因此，我们设想TLR激活可用于两种独立但相互支持的功能：提高抗肿瘤疫苗的效力，降低降低抗肿瘤反应效力的肿瘤部位细胞的活性。我们正在进行的研究旨在确定CpG ODN和其他TLR配体递送的最佳治疗窗口，并检查它们引发的保护性免疫反应是否可以通过与其他免疫调节剂（如额外的TLR配体和小分子激动性免疫增强剂）联合来加速和/或放大。优化CpG ODN的治疗效用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节途径。为了澄清这些问题，我们正在使用微阵列技术来识别CpG ODN引发的免疫刺激的核心基因和网络。这些实验是在体外高度纯化的细胞亚群（包括人pDC）和小鼠体内研究中进行的，以监测生理条件下的基因表达。最近的研究结果表明，具有共同抗病毒活性的基因子集在odn中持续上调，否则odn会介导离散功能。这组基因在很大程度上依赖于自分泌I型干扰素（IFN）信号，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对病毒感染的反应，并且与仅被单一类型的CpG ODN上调的基因不同。这些发现有助于定义通过TLR9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引发的免疫调节作用的理解。