Development of First-in-Class RIPK1 Degraders to Improve Cancer Immunotherapies

摘要 癌症免疫疗法取得了前所未有的临床反应，并正在给癌症带来革命性的变化 治疗。尽管癌症免疫疗法取得了巨大的成功，但仍不清楚为什么只有一种亚群 对治疗有反应的个体，以及如何使无反应者变得有反应。受体相互作用 蛋白激酶1(RIPK1)调节多种天然免疫下游的细胞命运和致炎信号 途径，包括由肿瘤坏死因子-α(肿瘤坏死因子-α)、Toll样受体配体和干扰素启动的途径。 在肿瘤坏死因子-α信号转导中，RIPK1的激酶活性是诱导细胞凋亡和坏死下垂所必需的。RIPK1还 作为一种不依赖于激酶的支架蛋白来招募NF-κB激活复合体，从而导致激活 核因子-κB途径与细胞存活的关系。有趣的是，带有激酶死亡Ripk1突变的小鼠(模仿 抑制剂)和Ripk1基因敲除(模拟降解物)表现出完全不同的表型。最近， 我们的工作以及另外两项独立研究表明，在癌细胞中敲除RIPK1 显著地使肿瘤对抗PD1增敏，导致肿瘤微环境的有利变化。 人类对纯合子功能丧失RIPK1突变的耐受性很好。完全丧失的患者 RIPK1蛋白仅表现出仅限于免疫系统的症状，并伴有原发免疫缺陷和/或 肠道炎症，表明药物RIPK1的降解可能是安全和可耐受的， 尤其是在临床环境中进行一过性干预。利用蛋白水解靶向嵌合体(PROTAC) 技术，我们开发了一流的RIPK1降解器LD4172。在我们的初步研究中，我们表明 LD4172能有效地降解一组人类癌细胞中的RIPK1蛋白，并抑制NF-κB的活性。这个 蛋白质组学分析证实了LD4172的降解特异性。小鼠黑色素瘤 免疫活性模型中，LD4172与抗PD1抗体有显著的协同作用。在这个项目中，我们将继续我们的 药物化学活动，开发一种口服RIPK1降解剂，并在一组 具有不同免疫亚型的临床前模型。这个项目的成功完成将导致 RIPK1相关生物化学探针的研究进展 推进癌症免疫治疗的治疗剂。我们努力使用不同的临床前模型来测试 RIPK1降解剂用于识别生物标志物，这对未来临床识别有反应的患者非常重要 翻译。

Abstract Cancer immunotherapies have achieved unprecedented clinical responses and are revolutionizing cancer treatments. Despite of the tremendous success of cancer immunotherapies, it remains unclear why only a subset of individuals responds to treatment and how to turn non-responders to become responsive. Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by tumor necrosis factor-α (TNF-α), toll-like receptor ligands, and interferons. In TNF-α signaling, the kinase activity of RIPK1 is required for inducing apoptosis and necroptosis. RIPK1 also serves as a kinase-independent scaffolding protein to recruit the NF-κB activation complex, leading to activation of the NF-κB pathway and cell survival. Interestingly, mice with a kinase-dead Ripk1 mutation (mimicking inhibitors) and with Ripk1 knockout (mimicking degraders) showed completely different phenotypes. Recently, our work, along with two other independent studies, showed that knockout of RIPK1 in cancer cells significantly sensitized tumors to anti-PD1, leading to favorable changes in the tumor microenvironment. Homozygous loss-of-function RIPK1 mutations are well tolerated in humans. Patients with complete loss of RIPK1 protein only showed symptoms confined to the immune system with primary immunodeficiency and/or intestinal inflammation, suggesting that pharmacological RIPK1 degradation can be potentially safe and tolerable, especially with transient intervention in clinical settings. Leveraging the Proteolysis targeting chimera (PROTAC) technology, we developed a first-in-class RIPK1 degrader LD4172. In our preliminary study, we showed that LD4172 potently degrades RIPK1 protein in a panel of human cancer cell lines and inhibits NF-κB activities. The degradation specificity of LD4172 was confirmed with proteomics profiling. In a mouse melanoma immunocompetent model, LD4172 significantly synergized with anti-PD1. In this project, we will continue our medicinal chemistry campaign to develop an orally available RIPK1 degrader and test its efficacy in a panel of preclinical models with different immune subtypes. Successful completion of this project will lead to the development of not only a chemical probe to interrogate RIPK1 related biology but also a highly promising therapeutic agent to advance cancer immunotherapies. We strive to use diverse preclinical models to test the RIPK1 degrader to identify biomarkers, which is very important to identify responsive patients in future clinical translations.