Activated NK CAR Cells to Cure HIV

激活 NK CAR 细胞治愈 HIV

基本信息

批准号：
10584560
负责人：
Branden S Moriarity
金额：
$ 75.35万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-02 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10584560
关键词：
Adoptive Transfer Allogenic Antigens Autologous B-Lymphocytes BLR1 gene CD28 gene CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes CRISPR/Cas technology Cell physiology Cells Clinical Communicable Diseases Cytotoxic T-Lymphocytes DNA Data Development Disease remission Dose Engineering Genome engineering Goals HIV HIV Infections HIV-1 Homing Human Immune Immunotherapy In Vitro Intervention Knock-out Lymphoid Follicle Lymphoid Tissue Macaca Macaca mulatta Methods Modeling Natural Killer Cells Patients Process Protocols documentation Public Health RNA Reagent Receptor Cell Recombinant adeno-associated virus (rAAV)Regimen Research Risk SIV Safety Signal Transduction Site T-Cell Receptor T-Lymphocyte Test Result Testing Therapeutic Uses Transgenic Organisms Viral Viral Load result Viral Physiology Viral reservoir Viremia Virus Virus Replication cancer therapy cell type chemokine chemokine receptor chimeric antigen receptor chimeric antigen receptor T cells comparative efficacy cytokine effective therapy engineered NK cell experience graft vs host disease immunoengineering improved in vivo individual patient insight interest manufacture manufacturing cost manufacturing process migration nonhuman primate novel pre-clinical preconditioning programmed cell death protein 1 response trafficking treatment strategy tumor viral rebound

项目摘要

ABSTRACT Virus-specific CD8 T cells exert antiviral activity against HIV-1/SIV in vitro and in vivo. Yet, despite these responses in HIV-1-infected humans and SIV- infected macaques, they are unable to fully suppress virus replication. This is likely due to the majority viral replication occurring in CD4+ T cells within B-cell follicles in secondary lymphoid tissues; where virus-specific CD8 T cells are relatively few in number. In fact, we found that in vivo effector virus-specific CD8 T cell to target SIV RNA+ cell ratios (E:T) were over 40-fold lower inside compared to outside of B cell follicles in lymphoid tissues. These findings indicate that B cell follicles are an immune privileged site in which low levels of virus-specific CD8 T cells permit ongoing viral replication. Furthermore, we found that few virus-specific CD8 T cells express the follicular homing molecule CXCR5, likely explaining their low levels in B cell follicles. These data suggest that the inability of virus specific CD8 T cells to fully suppress virus replication may be due to a deficiency of these T cells in B-cell follicles. These findings have led us to our central hypothesis that targeting HIV-specific immunotherapy to B cell follicles will lead to durable remission of HIV infection. In support of this hypothesis we have shown that increased levels of virus-specific CD8 T cells in B cell follicles is associated with lower viral loads. Although many immunotherapies utilize patient T cells to generate CAR-T therapies, there are special consideration in the treatment of HIV. One major short comings of CAR-T approaches is the fact that T cells need to be autologous due to the risk of graft versus host disease (GvHD), requiring complicated/expensive manufacturing processes of patient cells. This is also challenging in the HIV setting as the patient T cells are already compromised and processing cells which may contain active virus is risky. Alternatively, Natural Killer (NK) cells are highly suited for allogeneic use as they do not cause GvHD and thus hold significant clinical potential as an off-the-shelf cellular product. Thuts, we propose to evaluate NK immunotherapy that targets virus-specific CAR NK cells (expressing CD4-MBL-CAR and CXCR5) to B cell follicles. Moreover, we will use CRISPR/Cas9 to knockout negative regulators of NK cell function, such as PD1, which we have previously shown to enhance NK cell function. Our long-term goal is to develop an intervention that will lead to durable remission of HIV infection using CAR NK cells. To test our hypotheses, we propose the following aims. 1) Develop reagents and methods to generate human and rhesus macaque CAR/CXCR5/PD1KO NK cells. 2) Determine the ability of CAR/CXCR5/PD1KO NK cells to migrate into B cell follicles of SIV-infected rhesus macaques and to induce and maintain viral suppression. Our proposed studies targeting CAR NK cells to follicles will have a broad impact on the field by providing insights into cell trafficking, persistence, and pre-conditioning regimens for NK immunotherapy. Moreover, our methods for engineering rhesus macaque NK cells will enable studies assessing the therapeutic use of NK cells in preclinical NHP models. Moreover, these studies could result in an effective strategy to induce long-term sustained remission of HIV.

摘要病毒特异性 CD8 T 细胞在体外和体内发挥针对 HIV-1/SIV 的抗病毒活性。然而，尽管感染 HIV-1 的人类和感染 SIV 的猕猴的这些反应，它们无法完全抑制病毒复制。这可能是由于大多数病毒复制发生在 B 细胞滤泡内的 CD4+ T 细胞中。次级淋巴组织；其中病毒特异性 CD8 T 细胞的数量相对较少。事实上，我们发现体内效应病毒特异性 CD8 T 细胞与目标 SIV RNA+ 细胞的比率 (E:T) 比体内低 40 倍以上与淋巴组织中 B 细胞滤泡外部相比。这些发现表明 B 细胞滤泡是免疫特权位点，其中低水平的病毒特异性 CD8 T 细胞允许病毒持续复制。此外，我们发现很少有病毒特异性 CD8 T 细胞表达滤泡归巢分子 CXCR5，可能解释了它们在 B 细胞滤泡中的低水平。这些数据表明病毒特异性 CD8 T 细胞无法完全抑制病毒复制可能是由于 B 细胞滤泡中这些 T 细胞的缺陷。这些发现有引导我们得出中心假设，即针对 B 细胞滤泡的 HIV 特异性免疫疗法将带来持久的治疗效果。 HIV感染的缓解。为了支持这一假设，我们已经证明病毒特异性水平的增加 B 细胞滤泡中的 CD8 T 细胞与较低的病毒载量相关。尽管许多免疫疗法利用患者 T细胞生成CAR-T疗法，在治疗HIV方面有特殊考虑。一大短篇 CAR-T 方法的一个特点是，由于存在移植物抗宿主的风险，T 细胞需要是自体的疾病（GvHD），需要复杂/昂贵的患者细胞制造过程。这也是在 HIV 环境中具有挑战性，因为患者 T 细胞已经受到损害，并且处理细胞可能会含有活性病毒是有风险的。另外，自然杀伤 (NK) 细胞非常适合同种异体使用，因为它们确实不会引起 GvHD，因此作为现成的细胞产品具有显着的临床潜力。因此，我们建议评估针对病毒特异性 CAR NK 细胞（表达 CD4-MBL-CAR 和 CXCR5）的 NK 免疫疗法到 B 细胞滤泡。此外，我们将使用CRISPR/Cas9来敲除NK细胞功能的负调节因子，例如例如 PD1，我们之前已经证明它可以增强 NK 细胞功能。我们的长期目标是发展使用 CAR NK 细胞进行干预，将导致 HIV 感染的持久缓解。为了检验我们的假设，我们提出以下目标。 1）开发人类和恒河猴生成试剂和方法 CAR/CXCR5/PD1KO NK 细胞。 2) 测定CAR/CXCR5/PD1KO NK细胞迁移至B细胞的能力感染 SIV 的恒河猴的毛囊，诱导和维持病毒抑制。我们提出的研究将 CAR NK 细胞靶向毛囊将通过提供对细胞运输的见解对该领域产生广泛的影响， NK 免疫治疗的持续性和预处理方案。此外，我们的工程方法恒河猴 NK 细胞将使研究能够评估 NK 细胞在临床前 NHP 模型中的治疗用途。此外，这些研究可能会产生一种有效的策略来诱导艾滋病毒的长期持续缓解。