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

项目摘要/摘要 具有抗HIV嵌合抗原受体(CAR)的工程T细胞和自然杀伤(NK)细胞已经出现 根除艾滋病毒感染细胞的有希望的战略。目前的抗艾滋病毒汽车大多是基于 识别HIV包膜糖蛋白gp160的个体广谱中和抗体(BNAb)。然而， 它们受到针对单一表位的限制，这不能对抗艾滋病毒的巨大多样性和变异性。 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，该平台可以针对 不同HIV-1克隆的不同包膜表位。我们不是直接针对HIV-1 gp160，而是建议 设计了一辆能识别小分子标签二硝基苯基(DNP)的汽车。我们的中心假设是反DNP 通过使用DNP修饰的bNAbs作为接头分子，CAR-NK细胞可以被重定向到靶向HIV-1gp160。在……里面 初步研究，我们用CD28/CD3为基础的抗DnP汽车构建了人NK-92细胞系。我们展示了 该CAR-NK细胞能够识别和杀伤表达gp160的细胞，我们还发现该细胞的表位位置 On gp160影响DNP修饰的bNAb将抗DNP CAR-NK细胞重定向至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验证我们的假设：1)开发和验证一个 增强型通用CAR-NK平台体外靶向HIV感染细胞；2)测定抗HIV疗效 人源化小鼠模型中通用CAR-NK细胞的研究。在第一个目标中，我们将开发新型反DNP汽车 使用NK细胞特异性信号域，如NKG2D、2B4、DAP10和CD16，我们将识别 优化的CAR-NK构建以及一组DNP修饰的bNAbs具有最高的激活CAR-NK的效力 NK细胞体外抗HIV感染细胞。在第二个目标中，我们将检查通用汽车的能力- NK细胞，结合基于bNAb的接头分子的鸡尾酒，抑制HIV复制和 减少感染艾滋病毒的人源化小鼠的病毒逃逸。我们还将确定万能汽车NK是否 细胞可以与潜伏期逆转试剂相结合，以减少或消除潜在的艾滋病毒宿主。建议数 在我们看来，研究是创新的，因为模块化方法高度灵活，可以一起使用 基本上任何抗HIV的bNAb。拟议的研究具有重要意义，因为它的成功完成将 导致了具有显著扩大表位覆盖率的通用CAR-NK细胞原型的开发， 使克服艾滋病毒-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。 应用于设计NK细胞、T细胞和造血干细胞，以对抗HIV和其他传染病 以及癌症。

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.