Effects of entinostat and neoantigen vaccination on bladder cancer

项目摘要/摘要 膀胱癌是一种流行和致命的癌症，每年有超过8万新病例和1.7万人死亡。 美国。晚期膀胱癌的5年生存率只有15%。最有效的方法之一 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，但大多数反应不是持久的。一种有希望的新疗法被确定 我们小组对改善膀胱癌的ICB反应是选择性的1类组蛋白去乙酰化酶抑制剂 内抑素。在小鼠模型中，内抑素联合抗PD-1(αPD-1)ICB诱导了完整的、持久的反应 67%的老鼠。恩替比妥减少了肿瘤内M-MDSC和Treg细胞的数量，减少了肿瘤的单个- 体内SNV新抗原负荷，体外某些SNV新抗原表达增加， 并提高了体内对这些新抗原的T细胞特异性。然而，响应背后的大部分机制 Eninostat联合αPD-1的作用尚不清楚，特别是Eninostat如何减少免疫抑制种群和 影响新抗原景观的表达。了解这一机制对于预测 患者会有反应，并有可能通过抗原导向治疗改善反应。我们假设 内抑素诱导的ICB反应是由M-MDSC分化增加，M-MDSC减少 迁移和表达受抑制的免疫原性新抗原，增强对 新抗原疫苗接种。我将研究两种推动内源性降钙素+αPD-1应答的成分：M-MDSCs 和新的抗原。计算和湿实验室免疫学、肿瘤生物学和遗传学、原位培训 小鼠肿瘤模型，以及临床试验的转化研究，将帮助我成为一个独立的 受资助的内科科学家，领导癌症免疫学研究实验室，照顾膀胱癌患者。 在我们专注于M-MDSC的目标1中，我将使用M-MDSC进行流式细胞术和跨井迁移分析 原位膀胱癌模型肿瘤对M-MDSC分化和迁移的影响 通过内抑素治疗。我将对来自LCCC1827的人类肿瘤进行免疫荧光染色 内抑素窗口试验(NCT03978624)，以评估在ICB治疗中加入内抑素是否降低M- 人类体内的MDSCs。文森特实验室已经开发了LENS，这是一个从 多个基因组来源。在我们专注于新抗原表达的Aim 2中，我将使用镜头来识别所有 金实验室培育的3株小鼠膀胱癌新抗原检测T细胞新抗原的特异性 高通量ELISPOT，并使用统计建模来预测新抗原的免疫原性。我也会 从LCCC1827验证Eninostat诱导的免疫编辑是否发生在人类肿瘤中。在我们的新抗原中 疫苗接种目标3，我将测试接种新抗原疫苗是否能改善肿瘤对内抑素的反应 外加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用于测量未耗尽的新抗原特异性CD8+T细胞的丰度。

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.