DNA- and protein vaccination in combination with immune checkpoint blockade for active immunotherapy against neuroblsatoma

该更新提案描述了两种治疗策略（DNA和蛋白质疫苗接种）用于主动免疫治疗神经母细胞瘤（NB），这是儿童最常见的实体肿瘤。由于大多数病例在诊断时发现转移，因此针对这种侵袭性恶性肿瘤的新治疗方法的发展非常重要。我们的第一个治疗策略是基于接种双链DNA质粒，编码肿瘤相关抗原（TAA）和免疫刺激细胞因子。与TAA相关的泛素序列确保其蛋白酶体降解并增强对细胞毒性T淋巴细胞（ctl）的呈递，从而增加细胞抗nb免疫应答。在抗原呈递的微环境中，一种生物活性细胞因子的分泌刺激了ctl，而在全身给药时不会引起众所周知的细胞因子依赖的副作用。第二种治疗策略实现了基于交叉呈递原理的基于igg的蛋白质疫苗激活CTL的概念。为此，我们的目的是研究用与人免疫球蛋白G1恒定重链连接的TAA表位组成的融合蛋白接种疫苗的抗肿瘤作用。在这里，IgG结构促进抗原呈递细胞对融合蛋白的摄取，从而增加TAA表位的呈递。第一个双电子DNA疫苗已经产生和表征。该疫苗编码在NB中过表达的TAA酪氨酸羟化酶（TH）和CTL刺激细胞因子白细胞介素15 （IL-15）。本项目由DFG资助（项目启动时间：01.07.2015，资助时间：2年，预计总工期：4年）。目前，我们在临床前模型中评估该疫苗与免疫检查点阻断（PD-1， CTLA-4和调节性T细胞（Treg））联合使用。与基于TH+ il -15的疫苗接种策略类似，将产生针对mycn阳性肿瘤的第二种双子DNA疫苗，这些肿瘤对标准治疗无反应且与生存率低相关。为了提高疫苗接种的效力，一个额外的DNA序列编码细胞因子IL-21，刺激CTL和抑制Treg，将被整合到疫苗中。此外，将研究由MYCN表位和IgG1重链组成的基于igg的MYCN融合蛋白的新概念。最后，基于mycn的疫苗将与免疫检查点阻断（PD-1, CTLA-4, Treg）联合进行评估。这些多模式策略很重要，因为它们将taa特异性抗肿瘤反应与免疫刺激剂（细胞因子和/或免疫检查点抑制）结合起来，从而提供了附加或协同抗肿瘤功效的潜力。

This renewal proposal describes two therapeutic strategies (DNA- and protein vaccination) for active immunotherapy against neuroblastoma (NB), the most common solid tumor in childhood. Since metastases are found in a majority of cases at diagnosis, the development of new therapeutic approaches against this aggressive malignancy is of great importance.The first therapeutic strategy we aim to use is based on vaccination with a bicistronic DNA plasmid encoding for both a tumor-associated antigen (TAA) and an immune stimulating cytokine. An ubiquitin sequence linked to the TAA ensures its proteasomal degradation and enhances presentation to cytotoxic T lymphocytes (CTLs), thus increasing a cellular anti-NB immune response. The secretion of a bioactive cytokine in microenvironment of the antigen presentation stimulates CTLs without induction of well-known cytokine-dependent side effects when administered systemically. The second therapeutic strategy implements a poor investigated concept of CTL activation by IgG-based protein vaccines based on the principle of cross-presentation. For this, we aim to investigate anti-tumor effects of vaccination with a fusion protein consisting of TAA epitopes linked to the constant heavy chain of human immunoglobulin G1. Here, the IgG structure facilitates the uptake of the fusion protein by antigen presenting cells resulting in increased presentation of TAA epitopes.The first bicistronic DNA vaccine is already generated and characterized. This vaccine encodes for the TAA tyrosine hydroxylase (TH), which is over expressed in NB, and the CTL stimulating cytokine interleukin 15 (IL-15). This project was funded by the DFG (project start: 01.07.2015; funding: 2 years; presumable overall duration: 4 years). Currently, we evaluate this vaccine in a pre-clinical model in combination with an immune checkpoint blockade (PD-1, CTLA-4 and regulatory T cells (Treg)).By analogy with the TH+IL-15-based vaccination strategy, a second bicistronic DNA vaccine against MYCN-positive tumors, that do not respond to standard therapy and are associated with poor survival, will be generated. To increase the efficacy of vaccination, an additional DNA sequence encoding for the cytokine IL-21, which stimulates CTL and inhibits Treg, will be integrated into the vaccine. Moreover, the new concept of an IgG-based MYCN fusion protein consisting of MYCN epitopes and IgG1 heavy chain will be investigated. Finally, the MYCN-based vaccines will be evaluated in combination with immuncheckpoint blockade (PD-1, CTLA-4, Treg).These multimodal strategies are important in that they combine TAA-specific anti-tumor response with immune stimulating agents (cytokines and/or inhibition of immune checkpoints), thus offering the potential for additive or synergistic anti-tumor efficacy.