Modeling HIV CAR-T cell trafficking and persistence in Non-Human Primates

项目2-摘要 用嵌合抗原受体(CAR)分子修饰自体T细胞使 治疗多种白血病，旨在实现任何表面表达的即插即用靶向 人类疾病的标志。我们对优化持续HIV-1感染的CAR-T疗法感兴趣。 重要的是，尽管可以应用CAR-T的血液系统恶性肿瘤的名单正在迅速扩大， 有几个障碍阻止了对HIV+患者的应用。首先，CAR-T功能经常 在转移到组织时下调或丢失，例如限制对实体肿瘤的靶向。这也是 是靶向潜伏的HIV-1感染细胞的关键限制，这些细胞驻留在包括淋巴结在内的部位， 肠子和大脑。此外，越来越多的证据表明，为了让CAR-T识别他们的同源词 靶细胞表面可能需要一个抗原表达的阈值水平。的中心目标是 我们的U19联盟是为了了解CAR T细胞在体内的命运。在我们的项目中，我们将比较特定于病毒的 针对CD20+白血病的CARS到CARS，抗原丰富与抗原稀疏的作用机制 环境，以及CAR-T在迁移到次要环境后保持强大的、特定于目标的功能的能力 已知的含有潜伏病毒的组织部位。我们将在我们久负盛名的非人类组织中解决这些问题 灵长类(NHP)抑制HIV-1感染的模型，重点研究开发的优化的CD4CAR分子 我们将首先将CD4CAR-T与之前描述的NHP版本进行比较 在6只未感染的动物身上，成功地靶向了CD20 CAR分子(“NHP CD20 CAR-T”)。接下来，我们 将重点研究12只先前感染猴/人类免疫缺陷的动物的CD4CAR-T 病毒(SIV)和抗逆转录病毒治疗(ART)抑制。我们的研究中共有18例未感染或感染， 被抑制的动物将为促进植入的机制提供前所未有的见解， 持久性和体内功能，和/或导致沉默或抑制抗原依赖的扩张。我们有 选择NHP模型进行我们的研究，因为我们方法的一个关键方面是更好地理解CAR-T贩运 并在组织中发挥作用。使用成熟的方法来测量组织驻留记忆T细胞(TRM)， 用免疫组织化学和转录谱方法，我们将表征CD4CAR-T的功能。 低和高抗原环境(即ART释放之前和之后)，以及ii)对这些活动进行基准 对照我们的阳性对照，NHP CD20 CAR-T。我们的NHP研究目标将在体内应用黄金标准 抑制HIV感染的模型，并与项目中提出的实验高度互补 1/惠里，项目3/莱利，项目4/Coughlin和Tebas。

Project 2 - Abstract Modification of autologous T-cells with chimeric antigen receptor (CAR) molecules has revolutionized the treatment of many leukemias, and is designed to enable “plug and play” targeting of any surface-expressed marker of human disease. We are interested in optimizing CAR-T therapies for persistent HIV-1 infection. Importantly, although the list of hematological malignancies to which CAR-T can be applied is rapidly expanding, several barriers have prevented application to HIV+ individuals. First and foremost, CAR-T function is frequently downregulated or lost upon migration to tissues, for example limiting targeting of solid tumors. This also represents a key limitation for targeting of latently HIV-1 infected cells that reside at sites including lymph nodes, gut, and the brain. Furthermore, increasing evidence suggests that in order for CAR-T to recognize their cognate targets, a threshold level of antigen expression may be required at the target cell surface. The central goal of our U19 consortium is to understand the fate of a CAR T-cell in vivo. In our project, we will compare virus-specific CARs to CARs directed against CD20+ leukemias, mechanisms of action in antigen-rich vs. antigen-sparse environments, and the ability of CAR-T to maintain potent, target-specific function after migrating to secondary tissue sites known to harbor latent virus. We will address these questions in our well-established nonhuman primate (NHP) model of suppressed HIV-1 infection, focusing on the optimized CD4CAR molecule developed by Dr. Riley in Project 3. We will first compare CD4CAR-T to a previously-characterized NHP version of the successful, leukemia-targeting CD20 CAR molecule (“NHP CD20 CAR-T”), in 6 uninfected animals. Next, we will focus on CD4CAR-T in 12 animals that have been previously infected with simian/human immunodeficiency virus (SHIV) and suppressed by antiretroviral therapy (ART). Our studies in a total of 18 uninfected or infected, suppressed animals will provide unprecedented insights into the mechanisms that promote engraftment, persistence, and function in vivo, and/or lead to silencing or inhibition of antigen-dependent expansion. We have chosen the NHP model for our studies, as a key aspect of our approach is to better understand CAR-T trafficking and function in tissues. Using well-established assays to measure tissue resident memory T-cells (TRM), along with immunohistochemistry and transcriptional profiling approaches, we will characterize CD4CAR-T function in low and high antigen environments (i.e. before and after release of ART), and ii) benchmark these activities against our positive control, NHP CD20 CAR-T. Our NHP research aims will apply the gold standard for in vivo modeling of suppressed HIV infection, and are highly complementary to experiments proposed in Project 1/Wherry, Project 3/Riley, and Project 4/Coughlin and Tebas.