Dissecting the Differential Impacts of Toll-like Receptor 9 Agonism on the Capacity of Human Natural Killer Cells to Mediate Target Cell Killing

剖析 Toll 样受体 9 激动剂对人类自然杀伤细胞介导靶细胞杀伤能力的不同影响

• 批准号: 10730451
• 负责人: PAUL W. DENTON
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF NEBRASKA OMAHA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-08 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730451
• 关键词: Activated Natural Killer Cell; Affect; Agonist; Antibodies; Antibody-Dependent Enhancement; Arts; Biological Assay; Cell physiology; Cells; Clinical; Clinical Trials; Data; Disease; Effector Cell; Environment; Enzyme-Linked Immunosorbent Assay; Exhibits; FCGR3B gene; Faculty; Flow Cytometry; Funding; Future; Generations; Goals; Human; Immunobiology; Immunology; Immunotherapy; Infection; Intervention; Knowledge; Leukocytes; Literature; Malignant - descriptor; Malignant Neoplasms; Measures; Mediating; Methods; Monoclonal Antibodies; Mus; NK Cell Activation; Natural Killer Cells; Nebraska; Outcome; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Production; Public Health; Publishing; Reporting; Research; Research Personnel; Review Literature; Science; Series; Students; Surface; T-Lymphocyte; TLR9 gene; Testing; Therapeutic; Time; United States National Institutes of Health; Universities; Vaccine Adjuvant; Variant; Work; antibody-dependent cell cytotoxicity; cell killing; clinical development; college; cytokine; efficacy evaluation; experience; extracellular; granulocyte; hands on research; improved; in vivo; innovation; manufacture; monocyte; novel; pre-clinical; preclinical study; receptor; response; trial design; undergraduate student

PROJECT SUMMARY Toll-like receptor 9 (TLR9) agonist treatment increases the ability of human natural killer (NK) cells to directly kill target (e.g., malignant; infected) cells. The concept that TLR9 agonist treatment increases the capacity of human NK cells to mediate antibody dependent cellular cytotoxicity (ADCC) can be found in the literature, yet there are no published data directly demonstrating that this increase occurs. Nevertheless, several clinical trials registrations indicate that their trial design anticipates robust impacts of TLR9 agonist therapy on NK cell functions. Given the lack of a complete understanding of the impact(s) of TLR9 agonism on human NK cells, there is an urgent need to determine the effects of TLR9 agonism on the capacity of human NK cells to mediate ADCC. Thus, the overall objective of this application is to understand the impacts of TLR9 agonism on human NK cell-mediated ADCC while using direct killing as a positive control. Our central hypothesis, which was formulated based on our pilot data, is that TLR9 agonism causes a loss of CD16 from the surface of NK cells – thereby reducing the capacity of these cells to mediate ADCC. We will test this hypothesis by completing the following specific aims: Aim 1: Establish a time course for CD16 shedding from NK cells and for cytokine production when human PBMCs experience TLR9 agonism +/- ADAM17 inhibition. To do this, we will use flow cytometry, ELISA, and multiplex strategies to track CD16 levels and cytokines throughout our experimental window. Aim 2: Quantify the capacity of human NK cells to mediate killing when TLR9 agonism is combined with ADAM17 inhibition. To do this, we will use our novel NK-SADKA (Natural Killer cell – Simultaneous ADCC and Direct Killing Assay) flow cytometry-based approach. In addition to measuring the impact of TLR9 agonism on human NK cells, this strategy controls for potential human-to-human variations in the response to this drug. Beyond our new killing assay, our study is innovative because the TLR9 agonist utilized is manufactured in a way that eliminates confounding observations associated with stabilization methods used in the generation of other TLR9 agonists. At the conclusion of this work, we expect to have a full understanding of the impacts TLR9 agonism has on human NK cells’ ability to mediate ADCC. The data we generate will be highly relevant for interpreting outcomes from the dozens of completed, ongoing, and planned clinical trials evaluating the efficacy of TLR9 agonists as treatments for malignancies or infections. Finally, our preclinical data will inform the potential for combining TLR9 agonism with ADAM17 inhibition for improved ADCC-mediated clearance of malignant or infected cells in future studies.

项目摘要 Toll样受体9（TLR9）激动剂治疗增加了人类天然杀手（NK）的能力 细胞直接杀死靶标（例如，恶性；感染）细胞。 TLR9激动剂的概念 治疗增加了人NK细胞介导抗体依赖性细胞的能力 细胞毒性（ADCC）可以在文献中找到，但没有直接发布的数据 证明这增加了。然而，几项临床试验注册 表明他们的试验设计预测TLR9激动剂疗法对NK细胞的强大影响 功能。鉴于缺乏对TLR9激动剂对影响的影响 人类NK细胞，迫切需要确定TLR9激动剂对 人NK细胞介导ADCC的能力。这，此应用程序的总体目标是 了解TLR9激动剂对人NK细胞介导的ADCC的影响，同时使用直接 杀死作为积极的控制。我们的中心假设是根据我们的飞行员提出的 数据是，TLR9激动剂会导致NK细胞表面的CD16损失 - 从而导致 降低这些细胞介导ADCC的能力。我们将通过完成 以下具体目的：目标1：建立从NK细胞和 当人PBMC经历TLR9激动剂+/- ADAM17抑制时，细胞因子的产生。 为此，我们将使用流式细胞仪，ELISA和多种策略来跟踪CD16级别和 整个实验窗口中的细胞因子。目标2：量化人NK细胞的能力 当TLR9激动剂与ADAM17抑制结合时，媒体杀死媒体。为此，我们将 使用我们的小说NK-SADKA（天然杀手细胞 - 同时进行ADCC和直接杀戮测定法） 基于流式细胞仪的方法。除了测量TLR9激动剂对 人类NK细胞，该策略控制着反应中潜在的人对人类变化 使用这种药物。除了我们的新杀戮测定法之外，我们的研究具有创新性，因为TLR9激动剂 利用的制造方式是消除与 其他TLR9激动剂的生成中使用的稳定方法。在此结束时 工作，我们希望对TLR9激动剂对人类NK的影响有充分的了解 细胞介导ADCC的能力。我们生成的数据将与解释高度相关 评估了数十个完整，正在进行和计划的临床试验的结果 TLR9激动剂作为恶性或感染的疗法。最后，我们的临床前 数据将为将TLR9激动剂与ADAM17抑制以改进的潜力提供信息 在以后的研究中，ADCC介导的恶性细胞或感染细胞清除。