Dual targeting of tumoral microenvironment and tumoral cells by blocking the IL-33/ST2 pathway

项目摘要/摘要：我们对免疫学肿瘤的认识存在着根本的差距 急性髓系白血病(AML)和其他致命儿童发病过程中的微环境 癌症。弥合这一差距可能会带来新的免疫疗法。我们的长期目标是开发新的 白血病和儿童实体肿瘤的免疫疗法。我们在此应用程序中的目标是调查 表达IL-33受体刺激-2(ST2)的Foxp3+调节性T细胞(Tregs)在慢性粒细胞白血病中的作用 微环境以及ST2对肿瘤细胞的影响。我们的中心假设是我们可以针对肿瘤 中和双特异性阻断IL-33/ST2通路的微环境与肿瘤细胞 我们将产生的抗体。这一假设是基于我们未发表的初步数据而形成的。 结果表明：首先，T-bet基因缺陷的非恶性小鼠骨髓浸润性ST2+Tregs数量增加 与野生型(WT)小鼠(分别为45%和25%)相比，表明ST2/IL33和T- BET/干扰素-γ(干扰素-γ)在来自BM利基的Treg中。其次，我们观察到10倍以上的Tregs表达 携带AML的小鼠骨髓中的激活标记物显著多于非恶性的小鼠。 第三，在接受供者同基因ST2基因敲除的小鼠中，MLL-AF9 AML细胞的增殖显著降低 (ST2-/-)T细胞多于接受供者同基因WT T细胞的小鼠(分别为2%和17%)。ST2封锁 也降低了Treg的激活(即KLRG1)，增加了CD8+T细胞中的1型信号以及 减少了它们的消耗，表明ST2-/-小鼠的抗肿瘤反应恢复了。其基本原理是 这项研究是，一旦我们能够了解免疫微环境的生物学在 恶性的小生境，我们可以提出个体化的治疗方案，阻断耐受的通路。同样， 了解ST2/IL-33在肿瘤细胞上过度表达的生物学机制将有助于靶向 这条路。这一假设将以三个特定的目的进行检验：1)探索ST2和IL-33在 人类和小鼠的液体和固体儿童癌症的肿瘤细胞和肿瘤微环境； 2)检测ST2/IL-33阻断对恶性骨肉瘤瘤巢和实体瘤免疫功能的影响 微环境作为一种新的抗肿瘤免疫疗法的原理验证；以及3)优化抗ST2 针对小鼠和人类靶标的翻译中和抗体。这种方法是创新的。 因为，据我们所知，ST2+Tregs在恶性微环境中的功能几乎仍然存在 未被开发的。它还将提供对ST2+Tregs和ST2+肿瘤细胞本质的生物学见解。 并确定其阻断是否能恢复抗肿瘤活性和减少肿瘤 通过双重机制进行扩散。这项拟议的研究具有重要意义，因为新型抗肿瘤药物 比经典的化疗/放射疗法更有针对性和毒性更低的免疫疗法是 需要的。

PROJECT SUMMARY/ABSTRACT: A fundamental gap exists in our knowledge of the immunologic tumoral microenvironment during the development of acute myeloid leukemia (AML) and other deadly childhood cancers. Bridging of this gap could lead to novel immunotherapies. Our long-term goals are to develop novel immunotherapies for leukemia and pediatric solid tumors. Our objective in this application is to investigate the role of Foxp3+ regulatory T cells (Tregs) expressing Stimulation-2 (ST2), the IL-33 receptor in the microenvironment as well as ST2 on tumoral cells. Our central hypothesis is that we can target the tumoral microenvironment and tumoral cells by blocking the IL-33/ST2 pathway with neutralizing and bispecific antibodies that we will develop. This hypothesis was formed based on our unpublished preliminary data showing that: First, nonmalignant mice deficient in T-bet have elevated numbers of BM-infiltrating ST2+ Tregs compared to wild-type (WT) mice (45% vs 25%, respectively), suggesting reciprocal roles of ST2/IL33 and T- bet/interferon-γ (IFN-γ) in Tregs from the BM niche. Second, we observed 10-fold more Tregs expressing significantly more activation markers in the BM niche of AML-bearing mice than in the nonmalignant niche. Third, MLL-AF9 AML cell proliferation was significantly lower in mice receiving donor syngeneic ST2 knock-out (ST2-/-) T cells than in mice receiving donor syngeneic WT T cells (2% vs 17%, respectively). ST2 blockade also decreased Treg activation (i.e., KLRG1) and increased type 1 signaling in CD8+ T cells as well as decreased their exhaustion, suggesting restoration of an antitumoral response in ST2-/- mice. The rationale for this study is that once we are able to understand the biology of the immunological microenvironment in the malignant niche, we can propose personalized treatment plans to block the tolerogenic pathways. Similarly, understanding the biology responsible for the overexpression of ST2/IL-33 on the tumoral cell, will help target this pathway. This hypothesis will be tested with three specific aims: 1) Explore ST2 and IL-33 expression on tumoral cells and in the tumoral microenvironment from liquid and solid childhood cancers in human and mice; 2) Examine whether ST2/IL-33 blockade can impact tumor immunity in the malignant BM niche and solid tumor microenvironment as a proof-of-principle of a novel antitumoral immunotherapy; and 3) Optimizing anti-ST2 neutralizing antibodies against murine and human targets for translational purpose. This approach is innovative because, to our knowledge, the function of ST2+ Tregs in the malignant microenvironment, remains virtually unexplored. It will also provide biological insights into the nature of ST2+ Tregs and ST2+ tumoral cells in the microenvironment and determine whether their blockade can restore antitumoral activity and decrease tumoral proliferation via a dual mechanism. The proposed research is significant because novel antitumoral immunotherapies that are more targeted and less toxic than classical chemotherapy/irradiation regimens are needed.