Evaluation of lead FANA oligos targeting FOXP3, with and without anti-PD-1 mAb, as an approach for cancer immunotherapy

项目摘要/摘要 肺癌占男性和女性癌症相关死亡的25%，是最大的 癌症相关死亡的来源。尽管手术干预、放射治疗、化疗都取得了进步， 而免疫疗法，所有类型肺癌的5年存活率为19%。免疫的到来 检查点抑制疗法对部分肺癌患者疗效显著，但 &gt；75%的患者没有取得有意义的临床反应。目前，T调节(Treg)T细胞是 被认为是肿瘤免疫治疗临床成功应用的主要障碍之一。特雷格斯 有助于肿瘤的早期建立和发展，通过多种途径抑制效应细胞的反应 机制，并促进和支持亲肿瘤髓系来源的抑制细胞的发展和 功能。因此，Treg是肿瘤微环境免疫抑制状态的关键， 阻止有效的抗肿瘤免疫反应。临床前研究显示Tregs的靶向，通过他们的 耗竭或功能调整，单独或联合应用可产生显著的治疗效果 其他免疫调节方法。我们和其他人描述了大量积累的 肺癌患者的瘤内Treg，其瘤内数目的增加与不良相关 多种类型癌症的预后。 Treg由转录因子FOXP3的表达来定义，FOXP3控制Treg的发育， 稳定性和功能。Foxp3是多个Treg相关分子复合体的关键元件 调节Treg特异的基因网络和Treg介导的免疫调节机制。Foxp3是 位于细胞内，以前使用CD25表面标记靶向Treg的努力已经 无意中共靶向CD25+激活的T效应细胞，导致缺乏强大的抗肿瘤作用 临床前和临床研究的效率，以及严重的不良反应和毒性。另一方面 Hand，Foxp3特异性耗尽Treg的荷瘤DEREG转基因小鼠，联合 抗肿瘤疫苗治疗，导致强大的抗肿瘤免疫和缺乏显性自身免疫。 因此，存在对能够选择性靶向Treg细胞的疗法的迫切和未得到满足的需求，从而允许 身体对癌症建立正常的防御，而不会对其余的免疫系统产生负面影响 系统。 我们已经开发出第三代Fana反义寡核苷酸(Fana ASOS)，可以选择性地 通过下调FOXP3的表达来抑制Tregs。下一代ASO不同于他们的 通过化学修饰使它们能够高效地(体操)自我传递，甚至是休息 细胞，并使它们抵抗内源核酸酶的降解。在初步研究中，抗Foxp3 Fana Asos降低了小鼠Tregs的Foxp3表达和抑制功能，在50%的 同基因荷瘤小鼠，每天注射Fana Asos可完全破坏 与乱七八糟的对照相比，肿瘤没有明显的毒性。我们现在建议验证新的 开发了人FOXP3 FANAS，单独或与抗PD-1单抗联合治疗。 目的1将评估它们在人源化小鼠中敲除FOXP3并促进抗肿瘤免疫的能力 在活体内。目的2将评估它们对实际的人类肺癌相关Treg细胞的活性。

PROJECT SUMMARY/ABSTRACT Lung cancer accounts for >25% of cancer-related deaths in both men and women, making it the largest source of cancer-related mortality. Despite advancements in surgical intervention, radiation, chemotherapy, and immunotherapy, the 5-year survival rate for all types of lung cancer is 19%. The advent of immune checkpoint inhibitor therapy is associated with remarkable efficacy is some patients with lung cancers, but >75% of patients do not achieve meaningful clinical responses. Currently, T-regulatory (Treg) T cells are regarded as one of the major obstacles to the successful clinical application of tumor immunotherapy. Tregs contribute to the early establishment and progression of tumors, dampen effector cell responses by various mechanisms, and promote and support pro-tumoral myeloid-derived suppressor cell development and function. Tregs are thereby key to the immunosuppressive state of the tumor microenvironment that prevents effective anti-tumor immune responses. Pre-clinical studies show targeting of Tregs, by their depletion or functional modulation, can have significant therapeutic benefit, alone or in combination with other immunomodulatory approaches. We and others have described the significant accumulation of intratumoral Treg in lung cancer patients, and their increased intratumoral numbers correlate with poor prognosis in many types of cancers. Tregs are defined by their expression of the transcription factor FOXP3, which controls Treg development, stability, and function. FOXP3 is the key element of multiple Treg-associated molecular complexes that regulate the Treg-specific gene network and mechanisms of Treg-mediated immune regulation. FOXP3 is located intracellularly, and previous efforts to target Tregs using their CD25 surface marker have unintentionally co-targeted CD25+ activated T effector cells, causing a lack of robust anti-tumoral efficiency in pre-clinical and clinical studies, along with severe adverse effects and toxicity. On the other hand, Foxp3-specific depletion of Treg using tumor bearing DEREG transgenic mice, in combination with anti-tumor vaccine therapy, resulted in robust antitumor immunity and an absence of overt autoimmunity. Thus, there is an urgent and unmet need for therapies that can selectively target Treg cells, allowing the body to mount its normal defenses against the cancer, without negative influences on the rest of the immune system. We have developed third generation FANA antisense oligonucleotides (FANA ASOs) that selectively inhibit Tregs by knockdown of FOXP3 expression. This next generation of ASO differs from their predecessors by chemical modifications that allow their efficient (gymnotic) self-delivery, even to resting cells, and make them resistant to degradation by endogenous nucleases. In preliminary studies, anti-Foxp3 FANA ASOs reduced Foxp3 expression and suppressive functions of murine Tregs, and in 50% of syngeneic mice bearing lung tumors, daily injection of FANA ASOs showed complete destruction of the tumors as compared to scrambled controls and without overt toxicity. We now propose validation of newly developed human FOXP3 FANAs, alone or in conjunction with anti-PD-1 monoclonal antibody therapy. Aim 1 will assess their ability to knockdown FOXP3 and promote anti-tumor immunity in humanized mice in vivo. Aim 2 will evaluate their activity against actual human lung cancer associated Treg cells.