Modulation of the tumor microenvironment by CAR-engineered NK cells

CAR 工程 NK 细胞调节肿瘤微环境

• 批准号: 413727587
• 负责人: Professor Dr. Winfried Wels
• 金额: --
• 依托单位: Georg-Speyer-Haus, Institut für Tumorbiologie und experimentelle Therapie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/413727587?language=en
• 关键词: Modulation; tumor; microenvironment; CAR; engineered

Natural killer (NK) cells play a critical role in antitumor immunity by directly eliminating malignant cells and by regulating tumor-specific adaptive immune responses. NK-cell-based cancer immunotherapies are typically based on adoptive transfer of donor-derived allogeneic NK cells. To enhance their antitumor activity, NK cells can be further engineered to express chimeric antigen receptors (CARs) that facilitate selective recognition and killing of tumor cells, with early stage clinical development of several such approaches ongoing. Nevertheless, in addition to pro-inflammatory cytokines such as interferon (IFN)-gamma, activated NK and CAR NK cells secrete high levels of immuno¬regulatory interleukin (IL)-10, which may counteract pro-inflammatory factors and dampen tumor-suppressive activities of bystander immune cells in the tumor microenvironment. With the proposed project, we aim to better understand the role of NK-cell-derived IL-10 in the interaction of CAR-engineered NK cells with host immune cells within solid tumors, and to enhance endogenous tumor-suppressive mechanisms by modulation of the CAR effector cells' cytokine profile. Using continuously expanding ErbB2 (HER2)-specific CAR NK cells derived from the human NK cell line NK-92 as a clinically relevant model, we will investigate the consequences of IL-10 depletion by antibody-mediated inhibition and CRISPR/Cas9-mediated gene knockout. Cell-intrinsic effects of IL-10 downregulation on growth, cytotoxicity and cytokine production of the CAR NK cells will be analyzed. In addition, in co-culture assays the influence of IL-10-depleted CAR NK cells on polarization of macrophages and maturation of dendritic cells will be studied, since these two cell types appear most crucial for overcoming an immunosuppressive tumor microenvironment and induction of adaptive antitumor immunity. Similarly, the potential enhancement of bystander immune cell activation by CAR NK cells through ectopically expressed pro-inflammatory cytokines such as IL-12 and IL-15 will be investigated. Subsequently, IL-10-depleted CAR NK cells and variants modified to express pro-inflammatory factors will be tested in immunocompetent mouse models with syngeneic, ErbB2-positive tumors in comparison to unmodified CAR NK cells. Similar experiments will be performed in tumor xenograft models in immunodeficient mice transplanted with human peripheral blood mononuclear cells to also address the influence of NK-derived factors on bystander immune cells of human origin in vivo. We expect that insights from this project will not only be relevant for improvement of adoptive cancer immunotherapy with CAR NK-92 cells, but may lead to a generally applicable approach to enhance endogenous antitumor immunity through the activity of genetically engineered primary NK and T cells.

自然杀伤（NK）细胞通过直接消除恶性细胞和调节肿瘤特异性适应性免疫反应在抗肿瘤免疫中发挥关键作用。基于NK细胞的癌症免疫疗法通常基于供体来源的同种异体NK细胞的过继转移。为了增强其抗肿瘤活性，NK细胞可以进一步工程化以表达促进选择性识别和杀死肿瘤细胞的嵌合抗原受体（汽车），其中几种此类方法的早期临床开发正在进行中。然而，除了促炎细胞因子如干扰素（IFN）-γ之外，活化的NK和CAR NK细胞分泌高水平的免疫调节性白细胞介素（IL）-10，其可以抵消促炎因子并抑制肿瘤微环境中旁观者免疫细胞的肿瘤抑制活性。通过拟议的项目，我们的目标是更好地了解NK细胞衍生的IL-10在实体瘤中CAR工程化NK细胞与宿主免疫细胞相互作用中的作用，并通过调节CAR效应细胞的细胞因子谱来增强内源性肿瘤抑制机制。使用来自人NK细胞系NK-92的连续扩增的ErbB 2（HER 2）特异性CAR NK细胞作为临床相关模型，我们将研究通过抗体介导的抑制和CRISPR/Cas9介导的基因敲除的IL-10消耗的后果。将分析IL-10下调对CAR NK细胞的生长、细胞毒性和细胞因子产生的细胞内在效应。此外，在共培养试验中，将研究IL-10耗尽的CAR NK细胞对巨噬细胞极化和树突细胞成熟的影响，因为这两种细胞类型对于克服免疫抑制性肿瘤微环境和诱导适应性抗肿瘤免疫似乎最关键。类似地，将研究CAR NK细胞通过异位表达的促炎细胞因子如IL-12和IL-15对旁观者免疫细胞活化的潜在增强。随后，与未修饰的CAR NK细胞相比，将在具有同基因ErbB 2阳性肿瘤的免疫活性小鼠模型中测试IL-10耗尽的CAR NK细胞和经修饰以表达促炎因子的变体。将在移植有人外周血单核细胞的免疫缺陷小鼠的肿瘤异种移植物模型中进行类似的实验，以也解决NK衍生因子对人源性旁观者免疫细胞的体内影响。我们预计，该项目的见解不仅与CAR NK-92细胞的过继性癌症免疫治疗的改善有关，而且可能导致通过基因工程原代NK和T细胞的活性增强内源性抗肿瘤免疫的普遍适用的方法。