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: 7965763
• 负责人: Dennis Klinman
• 金额: $ 120.92万
• 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/7965763
• 关键词: Adjuvant; Affinity; Allergic; Animals; Anthrax Vaccines; Anthrax disease; Antigen-Presenting Cells; B-Lymphocytes; Bacterial DNA; Biological Models; Cancer Vaccines; Cell division; Cell physiology; Cells; Clinical Data; Clinical Trials; Communicable Diseases; DNA; Development; Disease; Gene Activation; Gene Expression; Gene Expression Profiling; Generations; Genes; Goals; Human; Immune; Immune response; Immunity; In Vitro; Individual; Infection; Inflammation; Inflammatory; Laboratories; Licensing; Ligands; Malignant Neoplasms; Mediating; Memory B-Lymphocyte; Microarray Analysis; Mus; Oligonucleotides; Phase; Physiological; Play; Population; Predisposition; Process; Regulation; Regulator Genes; Regulatory Pathway; Resistance; Role; Safety; Serum; Speed; Surgical Oncology; TLR9 gene; Technology; Therapeutic; Trauma; Tumor Cell Line; Up-Regulation; Vaccinated; Vaccination; Vaccine Adjuvant; Wound Healing; base; cancer therapy; design; expectation; improved; in vivo; killings; neoplastic cell; nonhuman primate; novel; pathogen; pre-clinical; pre-clinical research; preclinical study; research study; response; small molecule; therapeutic target; tumor

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. Over the past several years, my laboratory established that synthetic oligonucleotides expressing immunostimulatory CpG motifs (CpG ODN) could reduce host susceptibility to infection and allergic inflammation, were effective as vaccine adjuvants, and had a role in the treatment of cancer. Most recently, we demonstrated that they can also accelerate wound repair in mice and non-human primates. These pre-clinical studies suggest that CpG ODN may be used to both treat established diseases and speed wound healing after trauma or (oncologic) surgery. Based in part on this pre-clinical data, a number of phase I through III clinical trials exploring the safety and efficacy of CpG ODN have been initiated by groups who have licensed our technology. Ongoing pre-clinical research in my lab is designed to identify the optimal therapeutic window for CpG ODN delivery, 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). One of the model systems used by my lab to examine the adjuvant activity of CpG ODN involves AVA, the licensed anthrax vaccine. Our recent results show that adding CpG ODN to AVA significantly prolongs the duration of protective immunity via two distinct mechanisms. First, CpG-adjuvanted AVA elicits a faster and stronger initial Ab response, with anti-anthrax Ab levels persisting in the protective range for more than one year after a single vaccination (significantly longer than AVA alone). Second, CpG-adjuvanted AVA induces the generation of a high affinity memory B cell population that persists long-term. These B cells respond to anthrax infection so rapidly that they confer resistance to the host even after serum Ab levels have waned. This represents a novel mechanism for providing long-term protection against bioterror pathogens. 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 and in vivo to monitor gene expression under physiologic conditions. Results indicate that significant changes in gene expression are detectable within 15 minutes of ODN administration and persist for at least 9 days. TNFa, IL-1b, and IFNg were identified as playing key roles in the initial up-regulation of CpG mediated gene activation (which eventually spreads to involve the expression of nearly 700 genes). The magnitude with which individual genes are up-regulated is influenced by additional co-regulators that are predominantly activated within 24 hr of CpG ODN stimulation. Unexpectedly, we observed two peaks of gene activation following CpG ODN delivery to mouse or human cells. An initial peak at 3 hr primarily involves genes associated with immune regulation/activation while genese in the second peak (at day 5) a largely associated with cell division. This second peaks help explain the prolonged activity of CpG ODN in vivo. By three days post CpG ODN administration, the number of up-regulated genes had decreased by >85%. This effect is largely mediated by a group of down-regulators (including MYC, FOS, and SOCS) that actively suppress CpG-induced gene expression. These suppressors target the critical up-regulatory genes described above, thereby shutting down entire stimulatory networks. The other major therapeutic goal of this project is to improve the induction of tumor-specific immunity. We recently demonstrated by by conjugating our CpG ODN to killed tumor cells, we can vaccinate normal mice and significantly reduce their susceptibility to tumor challenge. This effect is not only observed in mice pre-vaccinated and then challenged, but in animals immunized with our CpG-killed tumor vaccine up to 5 weeks post challenge with the slow-growing TRAMP tumor cell line.

细菌DNA中存在的非甲基化CpG基序与Toll样受体9相互作用 引发促炎免疫反应CpG DNA也可改善抗原呈递细胞 功能，从而促进适应性免疫的发展。过去几 多年来，我的实验室建立了表达免疫刺激因子的合成寡核苷酸， CpG基序（CpG ODN）可以降低宿主对感染和过敏性炎症的易感性， 作为疫苗佐剂是有效的，并且在治疗癌症中有作用。最近， 我们证明了它们也可以加速小鼠和非人灵长类动物的伤口修复。 这些临床前研究表明，CpG ODN可用于治疗既有疾病， 加速创伤或（肿瘤）手术后的伤口愈合。部分基于此 临床前数据，一些I期到III期临床试验探索了安全性， CpG ODN的功效已经由已经许可我们的技术的团体发起。正在进行 我实验室的临床前研究旨在确定CpG的最佳治疗窗口 ODN递送，并检查它们引起的保护性免疫应答是否可以被 通过将它们与其它免疫调节剂（例如 另外的TLR配体和小分子激动性免疫增强剂）。模型之一 我的实验室用于检测CpG ODN佐剂活性的系统涉及AVA， 炭疽疫苗我们最近的研究结果表明，在AVA中加入CpG ODN可以显著降低AVA的表达， 通过两种不同的机制获得保护性免疫力。首先，CpG佐剂化的AVA洗脱液 更快和更强的初始抗体反应，抗炭疽抗体水平持续在 单次接种后超过一年的保护范围（明显长于 AVA单独）。第二，CpG佐剂化的AVA诱导高亲和力记忆B细胞的产生 人口长期存在。这些B细胞对炭疽感染的反应如此迅速， 它们甚至在血清Ab水平下降后也赋予宿主抗性。这表示 新的机制，提供长期保护，防止生物恐怖病原体。努力 优化CpG ODN的治疗效用需要详细了解它们 激活（直接和间接）、其作用持续时间和调节途径 参与调解这些反应。为了澄清这些问题，我们正在使用微阵列 技术，以确定基因和网络的核心免疫刺激引起的 CpG ODN。这些实验在体外对高度纯化的细胞亚群进行，并且在体外进行。 以监测生理条件下的基因表达。结果表明 在ODN给药15分钟内可检测到基因表达的显著变化 并持续至少9天。TNF α、IL-1b和IFNg被确定为在 CpG介导的基因激活的初始上调（最终扩散到涉及 近700个基因的表达）。单个基因的大小 上调受额外的共调节因子的影响，这些共调节因子主要在 CpG ODN刺激24小时。出乎意料的是，我们观察到两个基因激活峰， 将CpG ODN递送至小鼠或人细胞。3小时的初始峰值主要涉及基因 与免疫调节/激活相关，而基因在第二个高峰（第5天）a 主要与细胞分裂有关。第二个峰值有助于解释 CpG ODN的表达。在CpG ODN给药后3天，上调的细胞数量增加。 基因减少了85%。这种效应很大程度上是由一组 下调因子（包括MYC、FOS和SOCS），其主动抑制CpG诱导的基因 表情这些抑制子靶向上述关键上调基因，从而 关闭整个刺激网络。该项目的另一个主要治疗目标是 以改善肿瘤特异性免疫的诱导。我们最近通过 将我们的CpG ODN结合到杀死的肿瘤细胞上，我们可以接种正常小鼠， 降低他们对肿瘤攻击的敏感性。这种效应不仅在小鼠身上观察到 但在用我们的CpG杀死的肿瘤疫苗免疫的动物中， 用缓慢生长的TRAMP肿瘤细胞系攻击后长达5周。