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.

项目总结/摘要 用抗HIV嵌合抗原受体（CAR）工程化T细胞和自然杀伤（NK）细胞已经出现， 这是一个很有希望的根除艾滋病毒感染细胞的策略。目前的抗HIV汽车主要是基于以下设计的： 识别HIV包膜糖蛋白gp 160的单个广泛中和抗体（bNAb）。然而，在这方面， 它们受到靶向单一表位的限制，这不能对抗HIV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种通用的CAR-NK细胞平台， 不同HIV-1克隆的各种包膜表位。我们不是直接针对HIV-1 gp 160，而是建议 设计识别二硝基苯基（DNP）的CAR，一种小分子标签。我们的核心假设是抗DNP CAR-NK细胞可以通过使用DNP修饰的bNAb作为衔接分子重定向到靶向HIV-1 gp 160。在 在初步研究中，我们用基于CD 28/CD 3 β的抗DNP CAR工程化人NK细胞系NK-92。我们表明 这种CAR-NK细胞可以识别并杀死表达gp 160的细胞，我们还发现， 影响了DNP修饰的bNAb重定向抗DNP CAR-NK细胞对抗gp 160+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测试我们的假设：1）开发和验证一个 增强的通用CAR-NK平台在体外靶向HIV感染的细胞;和2）确定抗HIV功效 在人源化小鼠模型中的通用CAR-NK细胞。在第一个目标中，我们将开发新的抗DNP汽车 使用NK细胞特异性信号传导结构域，如NKG 2D、2B 4、DAP 10和CD 16，我们将鉴定一种 最佳CAR-NK构建体以及一组具有最高效力的DNP修饰的bNAb，以激活CAR-NK。 NK细胞在体外对抗HIV感染细胞。在第二个目标中，我们将检查通用CAR的能力- NK细胞与基于bNAb的衔接分子的混合物组合，以抑制HIV复制， 减少感染HIV的人源化小鼠中的病毒逃逸。我们还将确定通用CAR-NK 细胞可以与潜伏逆转试剂偶联以减少或消除潜伏的HIV库。拟议 在我们看来，研究是创新的，因为模块化方法非常灵活，可以一起使用 基本上都是抗艾滋病病毒的bNAbs这项拟议的研究意义重大，因为它的成功完成将 导致具有显著扩大的表位覆盖的通用CAR-NK细胞原型的开发， 使我们有可能克服HIV-1的多样性。这种方法，如果证明是成功的，也可以广泛地 申请工程改造NK细胞、T细胞和造血干细胞以针对艾滋病毒和其他传染病 以及癌症。