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.

项目摘要/摘要 具有抗HIV嵌合抗原受体（CAR）的工程T细胞和天然杀手（NK）细胞已出现 放射性HIV感染细胞的有希望的策略。当前的反HIV汽车主要是基于 识别HIV包膜糖蛋白GP160的个体中和抗体（BNAB）。然而， 它们的限制是针对单个发作的，该发作无法应对艾滋病毒的巨大多样性和可变性。 我们拟议的研究的总体目标是开发一个通用的汽车-NK单元平台，可以针对 不同HIV-1克隆的各种信封表位。我们建议，而不是直接靶向HIV-1 GP160 设计识别二硝基苯基（DNP）的汽车，这是一个小分子标签。我们的中心假设是抗DNP 可以将CAR-NK细胞重定向到靶向HIV-1 GP160，通过使用DNP修饰的BNAB作为衔接子分子。在 初步研究，我们使用基于CD28/CD3的抗DNP汽车来设计人类NK细胞系NK-92。我们显示 该汽车-NK单元可以识别并杀死表达GP160的细胞，我们还发现表位置位置 在GP160上，会影响DNP修饰的BNAB对GP160+细胞重定向抗DNP CAR-NK细胞的能力。 在这里，我们建议通过追求两个具体目的进一步检验我们的假设：1）开发和验证 增强了通用的CAR-NK平台，以便在体外靶向HIV感染的细胞； 2）确定抗HIV效率 人源化小鼠模型中的通用汽车-NK细胞。在第一个目标中，我们将开发新的反DNP汽车 使用NK细胞特异性信号域，例如NKG2D，2B4，DAP10和CD16，我们将确定一个 最佳CAR-NK构建体以及DNP修饰的BNAB面板，具有最高效力以激活CAR- NK细胞在体外针对HIV感染的细胞。在第二个目标中，我们将研究普遍汽车的能力 - NK细胞与基于BNAB的衔接子分子的鸡尾酒结合使用，以抑制HIV复制和 减少感染HIV的人源性小鼠的病毒逃生。我们还将确定是否通用汽车-NK是否 细胞可以与潜伏期反向试剂结合起来，以减少或消除潜在的HIV储层。提议 在我们看来，研究具有创新性，因为模块化方法非常灵活，可以一起使用 基本上有任何抗HIV BNAB。拟议的研究很重要，因为它的成功完成将 导致具有明显扩大表位覆盖范围的通用汽车-NK细胞原型的发展， 可以克服HIV-1的多样性。这种方法，如果被证明是成功的，也可以广泛 申请工程NK细胞，T细胞和造血干细胞靶向HIV和其他传染病 以及癌症。