A Universal CAR-NK Cell Targeting Various Epitopes of HIV-1

一种针对 HIV-1 多种表位的通用 CAR-NK 细胞

• 批准号: 10215523
• 负责人: Jianming Xie
• 金额: $ 14.2万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-13 至 2023-01-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10215523
• 关键词: 2,4-Dinitrophenol; Affect; Antibodies; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; Cell Line; Cell surface; Cells; Communicable Diseases; Complex; Coupled; Cytotoxic T-Lymphocytes; Development; Engineering; Epitopes; Escape Mutant; FCGR3B gene; Generations; Genetic Engineering; Goals; HIV; HIV Envelope Protein gp120; HIV Envelope Protein gp160; HIV envelope protein; HIV-1; Hematopoietic stem cells; Human; Immobilization; Immune; In Vitro; Individual; Interruption; Lead; Location; Malignant Neoplasms; Molecular; Mutate; Natural Killer Cells; Reagent; Research; Signal Transduction; Structure; T-Lymphocyte; Testing; Variant; Viral; Viral reservoir; Virus; Virus Latency; antiretroviral therapy; base; chimeric antigen receptor; chimeric antigen receptor T cells; design; dinitrophenyl; engineered NK cell; engineered T cells; flexibility; gp160; humanized mouse; immunoengineering; innovation; interest; latent HIV reservoir; mouse model; neutralizing antibody; pressure; prototype; receptor; viral rebound

PROJECT SUMMARY/ABSTRACT Engineering T cells and natural killer (NK) cells with anti-HIV chimeric antigen receptors (CAR) has emerged as a promising strategy to eradicate HIV-infected cells. Current anti-HIV CARs are mostly designed based on individual broadly neutralizing antibodies (bNAb) recognizing the HIV envelope glycoprotein gp160. However, they are limited by targeting a single epitope, which cannot counter the enormous diversity and mutability of HIV. The overall objective of our proposed research is to develop a universal CAR-NK cell platform that can target various envelope epitopes of different HIV-1 clones. Instead of targeting HIV-1 gp160 directly, we propose to design a CAR recognizing dinitrophenyl (DNP), a small molecular tag. Our central hypothesis is that anti-DNP CAR-NK cells can be redirected to target HIV-1 gp160 by using DNP-modified bNAbs as adaptor molecules. In preliminary studies, we engineer a human NK cell line NK-92 with a CD28/CD3-based anti-DNP CAR. We show that this CAR-NK cell can recognize and kill gp160-expressing cells, and we also find that the epitope location on gp160 affects the ability of DNP-modified bNAb to redirect anti-DNP CAR-NK cells against gp160+ cells. Here, we propose to further test our hypothesis by pursuing two specific aims: 1) Develop and validate an enhanced universal CAR-NK platform to target HIV-infected cells in vitro; and 2) Determine the anti-HIV efficacy of universal CAR-NK cells in humanized mouse models. In the first aim, we will develop new anti-DNP CARs using NK cell-specific signaling domains, such as NKG2D, 2B4, DAP10, and CD16, and we will identify an optimal CAR-NK construct as well as a panel of DNP-modified bNAbs with the highest potency to activate CAR- NK cells against HIV-infected cells in vitro. In the second aim, we will examine the ability of the universal CAR- NK cells, in combination with a cocktail of bNAb-based adaptor molecules, to suppress HIV replication and reduce viral escape in humanized mice infected with HIV. We will also determine whether the universal CAR-NK cells can be coupled with latency reversal reagents to reduce or eliminate latent HIV reservoirs. The proposed research is innovative, in our opinion, because the modular approach is highly flexible and can be used together with essentially any anti-HIV bNAbs. The proposed research is significant because its successful completion will lead to the development of a universal CAR-NK cell prototype with significantly expanded epitope coverage, making it possible to overcome the diversity of HIV-1. This approach, if proved successful, can also be broadly applied for engineering NK cells, T cells, and hematopoietic stem cells to target HIV and other infectious diseases as well as cancer.

项目概要/摘要 具有抗 HIV 嵌合抗原受体 (CAR) 的工程化 T 细胞和自然杀伤 (NK) 细胞已成为 根除艾滋病毒感染细胞的一个有前途的策略。目前的抗HIV CAR大多是基于 识别 HIV 包膜糖蛋白 gp160 的个体广泛中和抗体 (bNAb)。然而， 它们受到针对单一表位的限制，无法对抗艾滋病毒的巨大多样性和可变性。 我们提出的研究的总体目标是开发一个通用的 CAR-NK 细胞平台，可以靶向 不同HIV-1克隆的不同包膜表位。我们建议不直接针对 HIV-1 gp160 设计一个识别二硝基苯基 (DNP)（一种小分子标签）的 CAR。我们的中心假设是抗 DNP 通过使用 DNP 修饰的 bNAb 作为衔接分子，可以将 CAR-NK 细胞重定向至靶向 HIV-1 gp160。在 初步研究中，我们用基于 CD28/CD3 的抗 DNP CAR 设计了人类 NK 细胞系 NK-92。我们展示 该CAR-NK细胞可以识别并杀死表达gp160的细胞，并且我们还发现表位位置 gp160 上的 gp160 影响 DNP 修饰的 bNAb 将抗 DNP CAR-NK 细胞重定向到 gp160+ 细胞的能力。 在这里，我们建议通过追求两个具体目标来进一步检验我们的假设：1）开发并验证 增强的通用 CAR-NK 平台可在体外靶向 HIV 感染细胞； 2) 确定抗HIV功效 通用 CAR-NK 细胞在人源化小鼠模型中的应用。第一个目标，我们将开发新的抗DNP CAR 使用 NK 细胞特异性信号传导域，例如 NKG2D、2B4、DAP10 和 CD16，我们将鉴定一个 最佳的 CAR-NK 构建体以及一组 DNP 修饰的 bNAb，具有最高的激活 CAR-的效力 NK 细胞在体外对抗 HIV 感染细胞。第二个目标，我们将检验通用CAR的能力 NK 细胞与基于 bNAb 的衔接分子混合物相结合，可抑制 HIV 复制并 减少感染艾滋病毒的人源化小鼠的病毒逃逸。我们还将确定是否通用CAR-NK 细胞可以与潜伏逆转试剂结合，以减少或消除潜伏的 HIV 储存库。拟议的 我们认为，研究具有创新性，因为模块化方法非常灵活并且可以一起使用 基本上与任何抗 HIV bNAb 一起使用。拟议的研究意义重大，因为其成功完成将 导致通用 CAR-NK 细胞原型的开发，其表位覆盖范围显着扩大， 使得克服 HIV-1 的多样性成为可能。这种方法如果被证明是成功的，也可以广泛应用 应用于工程 NK 细胞、T 细胞和造血干细胞以针对 HIV 和其他传染病 还有癌症。