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

项目总结/摘要 用嵌合抗原受体（CAR）分子修饰自体T细胞首次提出近30年。 作为艾滋病病毒感染者的一种治疗方法。从那时起，CAR-T细胞已经成为一种有效的、高度 成功治疗液体肿瘤，而HIV特异性CAR-T细胞才开始在大规模 动物模型和临床试验。基于我们对CAR-T细胞治疗HIV的长期兴趣，我们 三个主要障碍限制了这种方法的治疗潜力。第一，艾滋病毒-1抗原水平低， 细胞表面（即Env蛋白），特别是在抗逆转录病毒治疗（ART）期间， CAR-T细胞和其他病毒特异性免疫效应子几乎看不到。第二，知识的丰富 关于病毒Env蛋白的细胞运输，尚未在Env的背景下彻底应用。 依赖艾滋病毒治疗策略。第三，CAR-T细胞的持久性和功能随着时间的推移而减弱，在完成之前， 清除潜伏的HIV病毒库。我们突破性的初步数据概述了克服这些问题的途径 局限性。我们最近报道了在四只感染了艾滋病毒样病毒的恒河猴身上的发现， 通过ART抑制，然后输注含有CD 4胞外结构域的病毒特异性CAR-T细胞 （CD4CAR）。为了在体内扩大这些有效的抗病毒效应物，动物接下来用辐照过的细胞加强免疫 稳定表达HIV-1 Env.在ART治疗中断（ATI）后，在2例受试者中观察到病毒控制。 4只动物，与CD 4CAR-T细胞的稳健和Env依赖性扩增一致。这个项目的中心目标是 我们的建议是增加这种方法的效力和可行性。在AIM 1中，我们将把我们的Env提升 从永生化细胞系到FDA批准的mRNA脂质纳米颗粒（mRNA-LNP）平台的策略， 类似于用于SARS-CoV-2的Moderna和Pfizer/BioNTech疫苗。将优化Env免疫原 用于CD 4CAR T细胞相互作用，并开发为Env mRNA-LNP疫苗。在AIM 2中，我们将使用CRISPR- Cas9基因编辑延长CD 4CAR-T细胞的耐久性和功能我们将比较一系列的汽车 携带灭活免疫检查点等位基因的产品，我们假设这将支持更持久的 功能和有效地清除持久性病毒水库。在AIM 3中，我们将对Env mRNA-LNP和免疫 在我们审查的HIV基因治疗的非人灵长类动物（NHP）模型中使用检查点基因编辑策略。这些 实验将采用强大的竞争性再种群研究设计，提供有关基本的关键信息， CAR生物学不能在临床研究中收集。总之，这些目标建立在我们认为是 最有前途的抗HIV细胞和基因治疗方法的日期报告。我们独特的和高度信息化的 HIV持续性和CAR-T细胞治疗的NHP模型将填补有关CAR-T细胞的知识的关键空白 在有限的抗原环境中的安全性和功能，并促进发达国家和 发展中国家。我们从这些研究中学到的教训不仅适用于治疗 HIV-1，但对于一系列疾病，如实体瘤，CAR-T细胞疗法必须类似地增强。

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.