Activated NK CAR Cells to Cure HIV

激活 NK CAR 细胞治愈 HIV

• 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
• 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

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.

抽象病毒特异性CD8 T细胞在体外和体内对HIV-1/SIV发挥抗病毒活性。但是，尽管如此 这些反应在HIV-1感染的人和SIV感染的猕猴中，它们无法完全抑制病毒 复制。这可能是由于B细胞卵泡中的CD4+ T细胞中发生的多数病毒复制引起的 继发性淋巴组织；病毒特异性CD8 T细胞的数量相对较少。实际上，我们发现 体内效应病毒特异性CD8 T细胞至靶标SIV RNA+细胞比率（E：T）的内部低于40倍以上 与淋巴组织中B细胞卵泡外部相比。这些发现表明B细胞卵泡是 免疫特权部位，低水平的病毒特异性CD8 T细胞允许持续的病毒复制。 此外，我们发现很少有病毒特异性的CD8 T细胞表达卵泡分子CXCR5，可能 解释了它们在B细胞卵泡中的低水平。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这些T细胞在B细胞卵泡中的缺乏造成的。这些发现有 导致我们提出了核心假设，即针对HIV特异性免疫疗法对B细胞卵泡将导致持久 艾滋病毒感染的缓解。为了支持这一假设，我们表明病毒特异性水平升高 B细胞卵泡中的CD8 T细胞与较低的病毒负荷有关。尽管许多免疫疗法利用患者 T细胞生成CAR-T疗法，在HIV治疗中有特殊的考虑。一个主要的缩写 CAR-T方法的启动是，由于移植物与宿主的风险，T细胞需要自体 疾病（GVHD），需要患者细胞的复杂/昂贵的制造过程。这也是 由于患者的T细胞已经被妥协，并且可能会在HIV环境中挑战 包含活性病毒是有风险的。另外，天然杀手（NK）细胞非常适合同种异体使用 不会引起GVHD，因此具有巨大的临床潜力作为现成的细胞产品。 thuts，我们提出 评估针对病毒特异性CAR NK细胞的NK免疫疗法（表达CD4-MBL-CAR和CXCR5） 到B细胞卵泡。此外，我们将使用CRISPR/CAS9来敲除NK细胞功能的负面调节剂，例如 作为PD1，我们以前已证明它可以增强NK细胞功能。我们的长期目标是发展 干预措施将导致使用CAR NK细胞对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）开发产生人类和恒河猕猴的试剂和方法 CAR/CXCR5/PD1KO NK细胞。 2）确定CAR/CXCR5/PD1KO NK细胞迁移到B细胞的能力 SIV感染的恒河猕猴的卵泡并诱导和维持病毒抑制。我们提出的研究 将CAR NK细胞靶向卵泡将通过提供对细胞贩运的见解，对现场产生广泛的影响， NK免疫疗法的持久性和预先调节方案。而且，我们的工程方法 恒河猕猴NK细胞将启用评估临床前NHP模型中NK细胞的治疗用途的研究。 此外，这些研究可能导致有效的策略诱导长期持续的艾滋病毒缓解。