Mechanism of action and therapeutic utility of immunostimulatory CpG oligonucleo

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。在过去的几年里，我的实验室确定，表达免疫刺激性CpG基序（CpG ODN）的合成寡核苷酸降低了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，并在治疗癌症中发挥作用。最近，我们证明了它们也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可用于治疗已确定的疾病和加速创伤或（肿瘤）手术后的伤口愈合。部分基于这些临床前数据，一些I期至III期临床试验探索了CpG ODN的安全性和有效性，这些临床试验已经由获得我们技术许可的团体发起。我实验室正在进行的临床前研究旨在确定CpG ODN递送的最佳治疗窗口，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）组合来加速和/或放大。 我的实验室用于检测CpG ODN佐剂活性的模型系统之一涉及AVA，即许可的炭疽疫苗。我们最近的研究结果表明，加入CpG ODN的AVA显着延长保护性免疫的持续时间通过两个不同的机制。首先，含CpG佐剂的AVA可产生更快和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超过一年（明显长于单独的AVA）。第二，CpG佐剂化的AVA诱导长期持续的高亲和力记忆B细胞群的产生。这些B细胞对炭疽感染的反应如此迅速，以至于即使在血清抗体水平下降后，它们也能对宿主产生抵抗力。这代表了一种提供长期保护免受生物恐怖病原体侵害的新机制。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进行，并在体内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在ODN施用的15分钟内是可检测的，并且持续至少9天。TNFa、IL-1b和IFNg被确定为在CpG介导的基因激活的初始上调中起关键作用（其最终扩展到涉及近700个基因的表达）。个别基因上调的幅度受主要在CpG ODN刺激的24小时内激活的另外的共调节因子的影响。出乎意料的是，我们观察到两个峰的基因激活后，CpG ODN交付到小鼠或人细胞。在3小时的初始峰主要涉及与免疫调节/活化相关的基因，而在第二峰（在第5天）中的基因主要与细胞分裂相关。这第二个峰有助于解释CpG ODN在体内延长的活性。在CpG ODN给药后3天，上调基因的数量减少了85%以上。这种效应主要由一组下调因子（包括MYC、FOS和SOCS）介导，这些下调因子积极抑制CpG诱导的基因表达。这些抑制子靶向上述关键的上调基因，从而关闭整个刺激网络。 该项目的另一个主要治疗目标是提高肿瘤特异性免疫的诱导。我们最近证明了将CpG ODN缀合至杀死的肿瘤细胞，提高了专业APC对癌症疫苗的摄取，并促进了体液和细胞介导的免疫应答的诱导。在多种鼠模型中，我们发现用CpG缀合的杀伤细胞疫苗接种显著降低了对肿瘤攻击的易感性。这种效果不仅在预先接种然后攻击的小鼠中观察到，而且在用我们的CpG杀死的肿瘤疫苗免疫的动物中观察到攻击后长达5周。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。

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.