Modulation of the tumor microenvironment by CAR-engineered NK cells

自然杀伤(NK)细胞通过直接清除肿瘤细胞和调节肿瘤特异性获得性免疫反应，在抗肿瘤免疫中发挥重要作用。以NK细胞为基础的癌症免疫疗法通常基于供者来源的同种异体NK细胞的过继转移。为了增强NK细胞的抗肿瘤活性，可以进一步改造NK细胞表达嵌合抗原受体(CARS)，以促进对肿瘤细胞的选择性识别和杀伤，几种此类方法的早期临床开发正在进行中。然而，除了干扰素-γ等促炎细胞因子外，激活的NK细胞和CAR-NK细胞还分泌高水平的免疫调节性白介素10，这可能会对抗促炎因子，并抑制肿瘤微环境中旁观者免疫细胞的抑瘤活性。通过这个项目，我们的目标是更好地了解NK细胞衍生的IL-10在实体瘤内CAR工程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来增强内源性肿瘤抑制机制。以人类NK细胞系NK-92来源的持续扩增的ErbB2(HER2)特异性CAR NK细胞为临床相关模型，我们将通过抗体介导的抑制和CRISPR/Cas9介导的基因敲除来研究IL-10缺失的后果。将分析IL-10下调对CAR NK细胞生长、细胞毒性和细胞因子产生的内在影响。此外，在共培养试验中，将研究IL-10缺失的CAR NK细胞对巨噬细胞极化和树突状细胞成熟的影响，因为这两种细胞似乎对克服免疫抑制的肿瘤微环境和诱导适应性抗肿瘤免疫最为关键。同样，CAR NK细胞通过异位表达的促炎细胞因子，如IL-12和IL-15，对旁观者免疫细胞激活的潜在促进作用也将被研究。随后，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同基因ErbB2阳性肿瘤的免疫活性小鼠模型中进行测试，并与未经修饰的CAR NK细胞进行比较。类似的实验将在免疫缺陷小鼠移植人外周血单个核细胞的肿瘤异种模型中进行，以研究NK衍生因子对体内人源性旁观者免疫细胞的影响。我们预计，该项目的见解不仅将与改进CAR NK-92细胞过继癌症免疫治疗相关，而且可能导致一种普遍适用的方法，通过基因工程的初级NK和T细胞的活性来增强内源性抗肿瘤免疫。

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.