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

这份更新提案描述了两种治疗策略(DNA疫苗和蛋白质疫苗)，用于针对神经母细胞瘤(NB)进行主动免疫治疗，NB是儿童最常见的实体肿瘤。由于大多数病例在确诊时发现转移，因此针对这种侵袭性恶性肿瘤开发新的治疗方法是非常重要的。我们目标使用的第一种治疗策略是基于接种编码肿瘤相关抗原(TAA)和免疫刺激细胞因子的双顺反子DNA质粒。与TAA相连的泛素序列确保了其蛋白酶体的降解，并增强了对细胞毒性T淋巴细胞(CTL)的呈递，从而增强了细胞抗NB免疫反应。在抗原呈递的微环境中分泌生物活性的细胞因子刺激CTL，而不会在全身给药时引起众所周知的细胞因子依赖的副作用。第二种治疗策略基于交叉呈递原理实现了基于免疫球蛋白的蛋白疫苗激活CTL的概念，这一概念尚未得到充分研究。为此，我们的目标是研究用TAA表位与人免疫球蛋白G1的恒定重链相连的融合蛋白接种的抗肿瘤效果。在这里，免疫球蛋白的结构有助于融合蛋白被抗原提呈细胞摄取，从而增加TAA表位的呈递。第一个双顺反子DNA疫苗已经研制成功并进行了鉴定。该疫苗编码在NB中过度表达的TAA酪氨酸羟化酶(TH)，以及CTL刺激细胞因子白介素15(IL-15)。该项目由德国发展集团资助(项目启动时间：2015年7月1日；资助时间：2年；预计总期限：4年)。目前，我们在临床前模型中结合免疫检查点阻断(PD-1、CTLA-4和调节性T细胞(Treg))对该疫苗进行评估。通过与基于IL-15的疫苗接种策略类似，将产生针对MYCN阳性肿瘤的第二个双顺反子DNA疫苗，这些肿瘤对标准治疗无效，且存活率较低。为了提高疫苗接种的效果，疫苗中将整合另外一个编码细胞因子IL-21的DNA序列，这种细胞因子可以刺激CTL并抑制Treg。此外，还将研究基于免疫球蛋白的MYCN融合蛋白的新概念，该融合蛋白由MYCN表位和IgG1重链组成。最后，基于MYCN的疫苗将与免疫检查点阻断(PD-1、CTLA-4、Treg)相结合进行评估。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)相结合，从而提供了相加或协同抗肿瘤效果的潜力。

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.