Developing Durable, Env-Boosted CAR T Cells for HIV Cure

项目摘要/摘要 用嵌合抗原受体(CAR)分子修饰自体T细胞首次被提出近30年 几年前作为艾滋病毒携带者的治疗方法。从那时起，CAR-T细胞已经成为一种强大和高度 成功治疗液体肿瘤，而HIV特异性CAR-T细胞才刚刚开始在大范围内显示疗效 动物模型和临床试验。基于我们长期以来对CAR-T细胞疗法治疗HIV的兴趣，我们假设 限制这种方法治疗潜力的三个主要障碍。首先，艾滋病毒-1抗原水平较低 细胞表面(即环境蛋白)，特别是在抗逆转录病毒治疗(ART)期间，呈现潜伏感染的细胞 CAR-T细胞和其他病毒特异性免疫效应器几乎看不见。第二，知识的财富 关于病毒包膜蛋白的细胞运输，尚未在包膜病毒的背景下得到彻底的应用。 依赖艾滋病毒的治疗策略。第三，在完成之前，CAR-T细胞的持久性和功能会随着时间的推移而减弱 清除潜伏的艾滋病毒蓄水池。我们突破性的初步数据勾勒出了一条克服这些问题的途径 限制。我们最近报告了在四只感染了类似HIV病毒的恒河猴身上的发现， 被ART抑制，然后输注含有CD4胞外区的病毒特异性CAR-T细胞 (CD4CAR)。为了在体内扩大这些有效的抗病毒效应物，接下来用照射过的细胞增强动物。 稳定表达HIV-1env的菌株。在ART治疗中断(ATI)后，观察到2例病毒控制 4只动物，与CD4CAR-T细胞的旺盛和环境依赖的扩增一致。这样做的中心目标是 建议的目的是提高这一方法的效力和可行性。在目标1中，我们将转变我们的环境提升 从永生化细胞系到FDA批准的信使核糖核酸脂质纳米粒(mRNA-LNP)平台的战略， 类似于针对SARS-CoV-2的莫德纳和辉瑞/BioNtech疫苗。环境免疫原将得到优化 用于CD4CAR T细胞的相互作用，并发展为Env mRNA-LNP疫苗。在AIM 2中，我们将使用CRISPR- Cas9基因编辑以延长CD4CAR-T细胞的耐受性和功能。我们将比较一系列汽车 携带灭活的免疫检查点等位基因的产品，我们假设这些产品将支持更耐用 功能，并有效地清除持久性病毒库。在目标3中，我们将对Env mRNA-LNP和免疫进行基准测试 我们审查的非人类灵长类(NHP)HIV基因治疗模型中的检查点基因编辑策略。这些 实验将以强大的竞争性种群研究设计为特色，提供关于基本 在临床研究中无法收集到的汽车生物学。总之，这些目标建立在我们认为的 迄今报道的最有前景的抗HIV细胞和基因治疗方法。我们独一无二的、信息量大的 HIV持续存在和CAR-T细胞治疗的NHP模型将填补关于CAR-T细胞的关键知识空白 在有限的抗原环境中的安全性和功能性，并促进在已开发和 发展中国家。我们从这些研究中学到的教训将不仅适用于治疗 HIV-1，但用于一系列疾病，如实体肿瘤，在这些疾病中，CAR-T细胞治疗必须得到类似的加强。

PROJECT SUMMARY/ABSTRACT Modification of autologous T cells with chimeric antigen receptor (CAR) molecules was first proposed nearly 30 years ago as a therapy for people living with HIV. Since then, CAR-T cells have emerged as a potent and highly successful therapy for liquid tumors, while HIV-specific CAR-T cells have only begun to show efficacy in large animal models and clinical trials. Based on our longstanding interest in CAR-T cell therapies for HIV, we posit three primary barriers that limit the curative potential of this approach. First, low levels of HIV-1 antigen at the cell surface (namely Env protein), especially during antiretroviral therapy (ART), render latently infected cells nearly invisible to CAR-T cells and other virus-specific immune effectors. Second, the wealth of knowledge regarding cellular trafficking of the viral Env protein has yet to be thoroughly applied in the context of Env- dependent HIV cure strategies. Third, CAR-T cell persistence and function wane over time, prior to complete clearance of the latent HIV reservoir. Our groundbreaking preliminary data outlines a path to overcome these limitations. We recently reported findings in four rhesus macaques that were infected with an HIV-like virus, suppressed by ART and then infused with virus-specific CAR-T cells containing the CD4 extracellular domain (CD4CAR). To expand these potent antiviral effectors in vivo, animals were next boosted with an irradiated cell line stably expressing HIV-1 Env. Following ART treatment interruption (ATI), viral control was observed in 2 of 4 animals, consistent with robust and Env-dependent expansion of CD4CAR-T cells. The central goal of this proposal is to increase the potency and feasibility of this approach. In AIM 1, we will transition our Env boosting strategy from an immortalized cell line to an FDA-approved mRNA lipid nanoparticle (mRNA-LNP) platform, analogous to the Moderna and Pfizer/BioNTech vaccines for SARS-CoV-2. Env immunogens will be optimized for CD4CAR T cell interactions and developed as Env mRNA-LNP vaccines. In AIM 2, we will use CRISPR- Cas9 gene editing to extend the durability and function of CD4CAR-T cells. We will compare a series of CAR products that carry inactivated immune checkpoint alleles, which we hypothesize will support more durable function and efficiently clear persistent viral reservoirs. In AIM 3, we will benchmark Env mRNA-LNP and immune checkpoint gene editing strategies in our vetted nonhuman primate (NHP) model of HIV gene therapy. These experiments will feature a powerful competitive repopulation study design, providing critical information on basic CAR biology that cannot be gathered in clinical studies. Together, these aims build on what we believe to be the most promising anti-HIV cell and gene therapy approach reported to date. Our unique and highly informative NHP model of HIV persistence and CAR-T cell therapy will fill in critical gaps in knowledge regarding CAR-T cell safety and function in limited antigen environments, and facilitate clinical translation both in developed and developing nations. The lessons we learn from these studies will be applicable not only as a curative therapy for HIV-1, but for a range of diseases such as solid tumors where CAR-T cell therapies must be similarly augmented.