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

神经母细胞瘤(NB)是儿童最常见的颅外实体瘤。尽管最近在治疗上取得了进展，但高危(HR)NB患者的预后仍然很差。新的免疫治疗方法，如嵌合抗原受体(CAR)治疗，在化疗难治性白血病中显示出令人印象深刻的结果。CAR T细胞是对选定的细胞表面抗原具有特异性的转基因T细胞。尽管CAR疗法在治疗B细胞恶性肿瘤方面取得了成功，但在实体肿瘤方面仍面临着几个挑战。在高度免疫抑制的肿瘤微环境(TM)中，由于NB免疫原性差和多种免疫逃逸机制，抗肿瘤免疫反应受阻。因此，有一种强烈的医学需求来开发旨在克服HR NB的TM的CAR疗法。因此，本项目的目的是通过先进的CAR设计和联合抗抑制信号抗体的治疗方法，在Nb异种移植小鼠模型中，通过共刺激增强GD2特异性CARS的功能。工程共刺激对CAR T细胞的持久性和功能有深远的影响。目前已证明，CD19导向的CAR T细胞具有使用CD28和CD3z信号域的配置以及4-1BB配体(CD1928z-41BBL)的共表达，从而导致更好的肿瘤根除。增强的抗肿瘤活性与4-1BBL介导的反式共刺激和转录因子IRF7诱导的T细胞I型干扰素(IFN-I)有关。到目前为止，还没有研究这些发现是否可以用来增强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域的结构构象对GD2-CAR T细胞功能的影响。我将产生不同的GD2-CAR配置，包括GD22z-41BBL，以提供最佳的共刺激支持，并在体外和体内移植NB的小鼠模型中分析功能活性。此外，我将研究针对T细胞抑制分子的单抗--称为检查点抑制物--联合治疗GD2-CAR T细胞，以克服NB介导的抑制。最后，我将评估与IRF7/干扰素？信号有关的抗肿瘤活性增强背后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.