Effects of entinostat and neoantigen vaccination on bladder cancer

项目总结/摘要 膀胱癌是一种流行且致命的癌症，在美国，每年有超过80，000例新发病例和17，000例死亡。 美国的晚期膀胱癌的5年生存率只有15%。一个最有效的 晚期膀胱癌的治疗是免疫检查点阻断（ICB），但只有20-30%的患者 晚期膀胱癌有反应，大多数反应不是持久的。一种有前途的新疗法被发现 由我们组提高膀胱癌ICB反应的选择性1类组蛋白去乙酰化酶抑制剂 恩替司他。在小鼠模型中，恩替司他加抗PD-1（αPD-1）ICB诱导了完全、持久的反应， 67%的老鼠恩替司他减少了肿瘤内M-MDSC和Treg群体，减少了肿瘤单个核细胞， 体内核苷酸变体（SNV）新抗原负荷，体外一些SNV新抗原的表达增加， 并增加体内T细胞对这些新抗原的特异性。然而，反应背后的机制 恩替司他加αPD-1的效果尚不清楚，特别是恩替司他如何减少免疫抑制人群， 影响新抗原景观的表达。了解这种机制对于预测哪种机制很重要 患者将响应，并通过抗原导向治疗潜在地改善响应。我们假设 恩替司他诱导的ICB反应是由增加的M-MDSC分化、减少的M-MDSC 免疫原性新抗原的表达增加，增强对 新抗原疫苗接种。我将研究驱动恩替司他加αPD-1反应的两种组分：M-MDSC 和新抗原。在计算和湿实验室免疫学，肿瘤生物学和遗传学，原位 小鼠肿瘤模型和临床试验的转化研究，将帮助我成为一个独立的 他是一位受资助的医生兼科学家，领导一个癌症免疫学研究实验室，并照顾膀胱癌患者。 在我们以M-MDSC为重点的目标1中，我将用来自以下的M-MDSC进行流式细胞术和transwell迁移测定： 原位膀胱癌模型肿瘤，以评估M-MDSC分化和迁移是否受到影响 恩替司他治疗我将对来自LCCC 1827的人类肿瘤进行免疫荧光染色 恩替司他窗口试验（NCT 03978624），以评估在ICB治疗中添加恩替司他是否会降低M- 人类的MDSC文森特实验室开发了透镜，这是一种软件平台，可以识别来自 多种基因来源。在我们关注新抗原表达的目标2中，我将使用透镜来识别所有的 Kim Lab开发的3种鼠膀胱癌细胞系中的新抗原，通过以下方法测试T细胞新抗原特异性： 高通量ELISPOT，并使用统计建模来预测新抗原免疫原性。我也会 验证恩替司他诱导的免疫编辑是否发生在来自LCCC 1827的人类肿瘤中。在我们的新抗原中 以疫苗接种为重点的目标3，我将测试新抗原疫苗接种是否改善肿瘤对恩替司他的反应 加上αPD-1。我将治疗原位肿瘤，测量它们的生长，并进行流式细胞术和单细胞 RNAseq测量未耗尽的新抗原特异性CD 8 + T细胞丰度。

Project Summary/Abstract Bladder cancer is a prevalent and deadly cancer, with over 80,000 new cases and 17,000 deaths annually in the United States. Advanced bladder cancer has only a 15% 5-year survival rate. One of the most effective treatments for advanced bladder cancer is immune checkpoint blockade (ICB), but only 20-30% of patients with advanced bladder cancer respond and most responses are not enduring. A promising new treatment identified by our group to improve bladder cancer ICB response is the selective class 1 histone deacetylase inhibitor entinostat. In a mouse model, entinostat plus anti-PD-1 (αPD-1) ICB induced complete, enduring responses in 67% of mice. Entinostat decreased intratumoral M-MDSC and Treg populations, decreased tumor single- nucleotide variant (SNV) neoantigen burden in vivo, increased expression of some SNV neoantigens in vitro, and increased T cell specificity for these neoantigens in vivo. However, much of the mechanism behind response to entinostat plus αPD-1 is unknown, particularly how entinostat decreases immunosuppressive populations and affects expression of the neoantigen landscape. Understanding this mechanism is important to predict which patients will respond and to potentially improve responses through antigen-directed therapy. We hypothesize that entinostat-induced ICB response is driven by increased M-MDSC differentiation, decreased M-MDSC migration, and increased expression of suppressed immunogenic neoantigens, augmenting response to neoantigen vaccination. I will investigate two components driving response to entinostat plus αPD-1: M-MDSCs and neoantigens. The training in computational and wet lab immunology, tumor biology and genetics, orthotopic murine tumor models, and translational research with a clinical trial, will assist me in becoming an independently funded physician-scientist leading a cancer immunology research lab and caring for bladder cancer patients. In our M-MDSC-focused Aim 1, I will perform flow cytometry and transwell migration assays with M-MDSCs from orthotopic bladder cancer model tumors to assess whether M-MDSC differentiation and migration are affected by entinostat treatment. I will conduct immunofluorescence staining of human tumors from the LCCC1827 entinostat window trial (NCT03978624) to assess whether adding entinostat to ICB treatment decreases M- MDSCs in humans. The Vincent Lab has developed LENS, a software platform to identify neoantigens from multiple genomic sources. In our neoantigen expression-focused Aim 2, I will use LENS to identify all the neoantigens in 3 murine bladder cancer lines developed by the Kim Lab, test T cell neoantigen specificity by high-throughput ELISPOT, and use statistical modeling to predict neoantigens immunogenicity. I will also validate whether entinostat-induced immunoediting occurs in human tumors from LCCC1827. In our neoantigen vaccination-focused Aim 3, I will test whether neoantigen vaccination improves tumor response to entinostat plus αPD-1. I will treat orthotopic tumors, measure their growth, and perform flow cytometry and single cell RNAseq to measure non-exhausted neoantigen-specific CD8+ T cell abundance.