Combined treatment approach with GD2-CAR T cells and immune checkpoint inhibitors for high-risk neuroblastoma

神经母细胞瘤（NB）是儿童颅外最常见的实体瘤。尽管最近的治疗进展，高风险（HR）NB患者的结局仍然很差。新的免疫治疗方法，如嵌合抗原受体（CAR）治疗，在化学难治性白血病中显示出令人印象深刻的结果。CAR T细胞是遗传修饰的T细胞，其获得对所选细胞表面抗原的特异性。尽管在B细胞恶性肿瘤中取得了成功，但CAR疗法在实体瘤中仍然面临着一些挑战。由于免疫原性差的肿瘤实体和由NB开发的多种免疫逃逸机制，抗肿瘤免疫应答在高度免疫抑制的肿瘤微环境（TM）中受到阻碍。因此，存在开发设计用于克服HR NB的TM的CAR疗法的强烈医学需求。因此，该项目的目的是关注通过由先进的CAR设计介导的共刺激增强GD 2特异性汽车功能的机制，以及与NB异种移植小鼠模型中抑制信号抗体的联合治疗方法。工程共刺激对CAR T细胞的持久性和功能有着深远的影响。现在已经证明，具有使用信号传导结构域CD 28和CD 3 z以及共表达4-1BB配体（CD 1928 z-41 BBL）的构型的CD 19定向的CAR T细胞导致上级肿瘤根除。增强的抗肿瘤活性与4- 1BBL介导的转录因子IRF 7对T细胞I型干扰素（IFN-I）的反式共刺激和诱导有关。到目前为止，尚未研究这些发现是否可用于增强CAR T细胞对实体瘤的抗肿瘤活性。为了解决这个问题，我将评估共刺激信号结构域的结构构象对GD 2-CAR T细胞功能的影响。我将产生不同的GD 2-CAR配置，包括GD 22 z-41 BBL，以提供最佳的共刺激支持，并在体外和体内NB移植小鼠模型中分析功能活性。此外，我将研究GD 2-CAR T细胞与靶向T细胞抑制分子（称为检查点抑制剂）的单克隆抗体的联合治疗，以克服NB介导的抑制。最后，我将评估IRF 7/IFN γ信号转导的抗肿瘤活性改善的分子机制的功能相关性。该项目旨在开发一种有效的免疫治疗方法，以提高HR NB的治愈率。

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite recent therapeutic advances, the outcome in patients with high-risk (HR) NB remains poor. Novel immunotherapeutic approaches, such as chimeric antigen receptor (CAR) therapy, have shown impressive results in chemorefractory leukemia. CAR T cells are genetically modified T cells that acquire specificity for selected cell surface antigens. Despite success in B-cell malignancies, CAR therapy still faces several challenges in solid tumors. Due to the poorly immunogenic tumor entity and multiple immune escape mechanisms developed by NB, antitumor immune responses are hampered in the highly immunosuppressive tumor microenvironment (TM). Thus, there is a strong medical need to develop CAR therapies designed to overcome the TM of HR NB. Therefore, the aim of this project is to focus on mechanisms to enhance the function of GD2-specific CARs through costimulation mediated by advanced CAR design and combined treatment approaches with antibodies against inhibitory signals in xenograft mouse models of NB. Engineered co-stimulation has a profound impact on CAR T-cell persistence and function. It has now been demonstrated that CD19-directed CAR T cells with a configuration using the signaling domains CD28 and CD3z as well as co-expression of 4-1BB ligand (CD1928z-41BBL) lead to superior tumor eradication. Enhanced antitumor activity is associated with 4-1BBL-mediated trans-costimulation and induction of T-cell type I interferon (IFN-I) by the transcription factor IRF7. Until now, it has not been investigated if these findings can be used to enhance antitumor activity of CAR T cells against solid tumors. To address this question, I will evaluate the impact of structural conformations of co-stimulatory signaling domains on GD2-CAR T-cell function. I will generate different GD2-CAR configurations, including GD22z-41BBL, to provide optimal co-stimulatory support and analyze functional activity in vitro and in a NB engrafted mouse model in vivo. Furthermore, I will investigate combined treatment of GD2-CAR T cells with monoclonal antibodies targeting T-cell inhibitory molecules - referred to as checkpoint inhibitors - in order to overcome NB-mediated inhibition. Finally, I will evaluate functional relevance of molecular mechanisms underlying improved antitumor activity with regards to IRF7/IFNß-signaling. This project aims at the development of a potent immunotherapeutic treatment approach to increase cure rates in HR NB.