Development of new multiepitope DNA vaccines for active immunotherapy against neuroblastoma combined with immune checkpoint blockade

神经母细胞瘤(NB)是一种具有挑战性的儿童期癌症，预后差。最近，GD2导向的免疫疗法将高危NB患者的存活率提高了15%。与大多数被动免疫疗法一样，这种策略不能诱导持久的免疫反应，因此重复应用抗体是必要的。这一障碍可以通过DNA疫苗来避免。在本项目中，我们的目标是开发一种新的针对NB的多表位DNA疫苗。我们之前成功地制备了一种基于IL-15的双顺反子DNA疫苗，针对在NB中过度表达的肿瘤相关抗原(TAA)酪氨酸羟基酶(TH)。在我们的同基因小鼠模型中对该疫苗的评估清楚地表明，与不含IL-15的对照TH疫苗相比，IL-15增强了抗肿瘤效果。我们在DNA疫苗领域的专业知识和新知识将被用于产生更有效的新一代针对NB的多表位DNA疫苗。为了提高免疫原性，进一步的针对NB的TAA(ALK、MYCN、Survivin)将在TH的基础上整合到疫苗中。这使得即使在一些TAA被肿瘤细胞丢失后也能发挥抗肿瘤作用。为了产生持久的效果，将进行额外的T辅助细胞激活以刺激抗原提呈细胞(APC)，从而激活肿瘤特异性细胞毒性T细胞(CTL)。为此，编码通用T辅助细胞表位的DNA序列将被整合到疫苗中。此外，为了有效地将TAA和T辅助表位分别装载到MHC-I和MHC-II分子上，溶酶体和蛋白酶体抗原递呈途径都将由DNA疫苗依赖的突变泛素的合成(多泛素化)来诱导。疫苗介导的IL-15合成将实现对APC的额外刺激。在产生和鉴定之后，新的DNA疫苗的抗肿瘤效果将在我们的临床前NB模型中进行评估。为此，将使用一种新的基于非病毒脂质体的纳米颗粒疫苗接种策略。为了克服肿瘤对免疫反应的抑制，将对两个已被证明在癌症中发挥重要作用的免疫检查点(PD-L1+TIGIT)进行额外的阻断。综上所述，我们的目标是产生一种针对不同NB特异性TAA的新一代基于细胞因子的DNA疫苗，该疫苗将与免疫检查点阻断一起进行测试，以诱导对NB的长期免疫反应。

Neuroblastoma (NB) is a still challenging cancer of childhood with poor prognosis. Recently, GD2-directed immunotherapies improved survival of high-risk NB patients by 15%. As with most passive immunotherapies, such a strategy does not induce a long-lasting immune response and repeated Ab applications are necessary. This obstacle can be avoided by DNA vaccination.In the present project, we aim to generate a new mutiepitope DNA vaccine against NB. We previously successfully generated a bicistronic IL-15-based DNA vaccine against the tumor-associated antigen (TAA) tyrosine hydroxylase (TH) overexpressed in NB. Evaluation of this vaccine in our syngeneic mouse model clearly showed increased antitumor efficacy by IL-15 compared to the IL-15-free control TH vaccine. Our expertise and new knowledge in the field of DNA vaccination will be used for the generation of a more effective new generation multiepitope DNA vaccine against NB. To increase immunogenicity, further NB-specific TAAs (ALK, MYCN, survivin) will be integrated into the vaccine additionally to TH. This allows antitumor effects even after loss of some TAAs by tumor cells. To induce long-lasting effects, additional activation of T helper cells stimulating antigen presenting cells (APCs) and thereby activating tumor-specific cytotoxic T cells (CTLs) will be performed. For that, DNA sequences encoding universal T helper cell epitopes will be integrated into the vaccine. Furthermore, for effective loading of TAA- and T helper epitopes onto MHC-I and –II molecules, respectively, both lysosomal and proteasomal antigen presentation pathways will be induced by the DNA vaccine-dependent synthesis of the mutated ubiquitin with increased stability (polyubiquitination). An additional stimulation of APCs will be achieved by the vaccine-mediated synthesis of IL-15. After generation and characterization, antitumor efficacy of the new DNA vaccine will be evaluated in our preclinical NB model. For that, a new non-viral liposome-based nanoparticle vaccination strategy will be utilized. To overcome a tumor-mediated inhibition of immune response, an additional blockade of two immune checkpoints (PD-L1 + TIGIT) that have been shown to play an important role in cancer will be performed.In summary, we aim to generate a new generation cytokine-based DNA vaccine against different NB-specific TAAs that will be tested in combination with immune checkpoint blockade for induction of a long-lasting immune response against NB.