Mechanism of action and therapeutic utility of immunostimulatory CpG oligonucleo

细菌DNA中存在的未甲基化的CpG基序与Toll样受体9相互作用，触发促炎免疫反应。CpG DNA还可改善抗原提呈细胞功能，从而促进获得性免疫的发展。在过去的几年里，我的实验室证实，表达免疫刺激CpG基序的合成寡核苷酸(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，并在癌症治疗中发挥作用。最近，我们证明了它们还加速了小鼠和非人类灵长类动物的伤口修复。这些临床前研究表明，CpG ODN既可用于治疗已有的疾病，又可加速创伤或(肿瘤)手术后的伤口愈合。部分基于这些临床前数据，获得我们技术许可的团体已经启动了一些探索CpG ODN安全性和有效性的I-III期临床试验。我的实验室正在进行的临床前研究旨在确定CpG ODN传递的最佳治疗窗口，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配体和小分子激动型免疫增强剂)相结合来加速和/或放大。我的实验室用来检查CpG ODN佐剂活性的一个模型系统涉及获得许可的炭疽疫苗AVA。我们最近的研究结果表明，在AVA中加入CpG ODN可以通过两种不同的机制显著延长保护性免疫的持续时间。首先，CpG佐剂的AVA可诱导更快、更强的初始抗体应答，单次接种后，抗炭疽抗体水平持续在保护范围内一年以上(明显长于单独接种AVA)。其次，CpG佐剂的AVA诱导产生高亲和力的记忆B细胞群体，并长期存在。这些B细胞对炭疽感染的反应如此之快，以至于即使在血清抗体水平下降后，它们也会对宿主产生抵抗力。这代表了一种针对生物恐怖病原体提供长期保护的新机制。为了优化CpG ODN的治疗效果，需要详细了解它们(直接和间接)激活的细胞、它们的作用持续时间以及参与调节这些反应的调节通路。为了阐明这些问题，我们正在使用微阵列技术来识别CpG ODN诱导的免疫刺激的核心基因和网络。这些实验在体外对高纯度的细胞亚群进行，并在体内进行，以监测生理条件下的基因表达。结果表明，基因表达的显著变化可以在给药后15分钟内检测到，并持续至少9天。TnFa、IL-1b和IFNG被认为在CpG介导的基因激活(最终扩散到涉及近700个基因的表达)的初始上调中发挥关键作用。单个基因上调的幅度受其他辅助调节因子的影响，这些协同调节因子主要在CpG ODN刺激后24小时内被激活。出乎意料的是，我们观察到在将CpG ODN传递到小鼠或人类细胞后，基因激活的两个高峰。最初的高峰在3小时，主要涉及与免疫调节/激活相关的基因，而第二个高峰(在第5天)的基因主要与细胞分裂有关。这第二个峰值有助于解释CpG ODN在体内活性延长的原因。在CpG ODN给药后3天，上调基因的数量减少了85%以上。这种作用在很大程度上是由一组下调调控因子(包括MYC、FOS和SOCS)介导的，它们积极抑制CpG诱导的基因表达。这些抑制物针对上述关键的上调基因，从而关闭了整个刺激网络。该项目的另一个主要治疗目标是提高肿瘤特异性免疫的诱导。我们最近证明，将CpG ODN连接到被杀死的肿瘤细胞可以改善专业APC对癌症疫苗的摄取，并增强体液和细胞介导的免疫反应的诱导。在多个小鼠模型中，我们发现接种CpG结合的灭活细胞疫苗显著降低了对肿瘤攻击的敏感性。这种影响不仅在预先接种疫苗然后挑战的小鼠中观察到，而且在攻击后5周内用我们的CpG灭活肿瘤疫苗免疫的动物中也观察到。已知的促进NK和T细胞活化的药物，如4-1BB单抗的加入，协同增强了CpG ODN的抗肿瘤作用。

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) reduced 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 also accelerated 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 greater than 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 conjugating CpG ODN to killed tumor cells improved the uptake of the cancer vaccine by professional APCs, and boosted the induction of humoral and cell mediated immune responses. In multiple murine models, we found that vaccination with CpG conjugated killed cell vaccines significantly reduced 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. 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.