Myeloid cell-derived Granzyme B as an inducible enhancer of cancer immunotherapy

项目摘要 我们广泛、长期的目标是了解Fcγ受体（FcγR）功能的分子细节， 其目的是改善癌症的单克隆抗体治疗。抗体治疗的抗肿瘤作用 主要由FcγR介导，Fc γ R在与 抗体的单核细胞和巨噬细胞是抗体治疗有效所必需的，FcγR 这些细胞中的活化可导致多效性反应。这些包括吞噬作用，抗体依赖性 细胞毒性（ADCC）、炎性细胞因子的产生或这些的组合。在这些 尽管单核细胞/巨噬细胞具有ADCC活性，但其进行ADCC的机制仍然是最不了解的。 我们最近发现FcγR聚集可以诱导人单核细胞产生颗粒酶B， Toll样受体（TLR）4和8的配体可以显著增强这种反应，以及诱导 颗粒酶B本身。颗粒酶B是一种主要由自然杀伤（NK）细胞表达的丝氨酸蛋白酶 和CD 8 + T细胞，并且是它们的细胞毒性功能所必需的。鉴于粒酶B在NK细胞中的核心作用- 介导的ADCC，我们发现单核细胞也可以产生颗粒酶B，这是值得注意的，因为它提供了一个作为 单克隆抗体尚未发现的抗肿瘤功能。最值得注意的是，我们还发现护士- 从CLL患者血液中产生的类细胞（NLC）在FcγR活化后也产生颗粒酶B， TLR 8配体治疗，并且它们可以参与CLL细胞的颗粒酶依赖性ADCC。后一 观察结果特别令人感兴趣，因为NLC通常促进CLL细胞存活和增殖。因此我们 假设单核细胞和NLC产生颗粒酶B代表了效应子 对抗体包被的靶细胞的反应。我们建议具体探讨这些发现的意义 本研究的主要目的是：1）阐明颗粒酶B的作用机制 FcγR在单核细胞/巨噬细胞和NLC中的诱导作用，2）确定增强 免疫调节剂诱导的Fcγ R诱导的颗粒酶B产生和3）分析免疫调节剂诱导的颗粒酶B的表达和功能。 CLL患者样本和小鼠模型中单核细胞和单核细胞系细胞的颗粒酶B。 在完成这项研究后，我们将充分探索一种全新的抗体介导的机制。 单核细胞/巨噬细胞和NLC对肿瘤的破坏。这些机制研究将大大 增强我们对这些细胞介导的ADCC的理解，并可能提供信息， 抗体治疗的进一步增强。

PROJECT SUMMARY Our broad, long term objective is to understand the molecular details of Fcγ receptor (FcγR) function, with the goal of improving monoclonal antibody therapy for cancer. The antitumor effects of antibody therapy are largely mediated by FcγR, which become clustered and activated upon binding to the Fc portion of antibodies. Monocytes and macrophages are essential for antibody therapy to be effective, and FcγR activation in these cells can lead to pleiotropic responses. These include phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), production of inflammatory cytokines, or a combination of these. Amongst these activities, the mechanisms by which monocytes / macrophages carry out ADCC remain the least understood. We have recently found that FcγR clustering can induce Granzyme B production by human monocytes and that ligands for Toll-like receptors (TLR) 4 and 8 can significantly enhance this response, as well as induce Granzyme B themselves. Granzyme B is a serine protease expressed predominantly by natural killer (NK) cells and CD8+ T cells, and is required for their cytotoxic functions. Given the central role of Granzyme B in NK cell- mediated ADCC, our finding that monocytes can also produce Granzyme B is remarkable as it provides an as- yet undiscovered anti-tumor function of monoclonal antibodies. Most notably we have also found that nurse- like cells (NLCs) generated from CLL patient blood also produce Granzyme B following FcγR activation and TLR8 ligand treatment, and they can engage in Granzyme-dependent ADCC of CLL cells. This latter observation is of particular interest, as NLCs typically promote CLL-cell survival and proliferation. We therefore hypothesize that Granzyme B production by monocytes and NLCs represents a critical aspect of the effector response to antibody-coated target cells. We propose to explore the significance of these findings specifically related to CLL immunotherapy in the following 3 specific aims: 1) Elucidate the mechanisms of Granzyme B induction by FcγR in monocytes/macrophages and NLCs, 2) Determine the mechanism of augmentation of FcγR-induced Granzyme B production by immune modulators and 3) Analyze the expression and function of Granzyme B by monocytes and monocyte-lineage cells in patient samples and mouse models of CLL. Upon completion of this study we will have fully explored an entirely new mechanism of antibody-mediated destruction of tumors by monocytes/macrophages and NLCs. These mechanistic studies will significantly enhance our understanding of ADCC mediated by these cells, and will likely provide information that can lead to the further enhancement of antibody therapy.