Microsphere-based glypican-3 specific immunotherapy of hepatocellular carcinoma

免疫疗法代表了一种有前途的替代已建立的癌症疗法，由于在实体癌治疗中有前途的临床结果，免疫疗法最近吸引了很多关注。肝细胞癌由于其免疫原性特征而成为免疫治疗的理想靶点。虽然共抑制抗体的使用已经在个体患者中证明了临床疗效，但是由于全身免疫应答低，产生癌症特异性疫苗的尝试仅导致较小的临床效果。在SFB TRR 77 "肝癌"的框架内进行的实验中，我们因此测试了各种疫苗接种方案以增加肿瘤特异性免疫应答的幅度。我们可以证明，肿瘤特异性免疫反应可以被放大和加速使用prime-boost疫苗接种方案，包括免疫抗原包被的PLGA微球，TLR3激动剂和随后的加强免疫接种与李斯特菌vector.In除了疫苗接种研究，一个本土肝癌模型适合于评估疫苗战争的开发。为此目的，使用转座子侧翼质粒，其允许任何转基因稳定整合到细胞的DNA中。利用组成型激活的NRAS、AKT、p53的shRNA和疫苗抗原，可以在一周内建立原位肝癌。在该肿瘤模型中，PLGA/TLR3-李斯特菌联合疫苗诱导了完全的肿瘤消退，并且在总体存活率方面优于传统的树突状细胞疫苗接种。在拟议的研究项目中，迄今为止仅限于模型抗原卵清蛋白和黑色素瘤抗原TRP 2的疫苗抗原谱将扩展到HCC特异性抗原磷脂酰肌醇蛋白聚糖-3。为此，将克隆两种含有人或鼠磷脂酰肌醇蛋白聚糖-3的李斯特菌载体。然后将在PLGA-LM初免加强疫苗的背景下测试鼠李斯特菌载体LM-mGPC3，用于治疗C57 B1/6小鼠中的磷脂酰肌醇蛋白聚糖-3阳性肝细胞癌，为人类中的相应研究做准备。此外，在接种后两周，将从荷瘤小鼠中分离癌症特异性耗尽的CD8 T细胞，并进行全基因组微阵列分析以产生T细胞耗尽的转录特征。在随后的实验中，将评估T细胞中鉴定的候选基因的功能相关性，以鉴定与PLGA/LM疫苗接种的潜在协同作用，并能够产生有效和持久的癌症特异性免疫应答。

Immunotherapy represents a promising alternative to established cancer therapies thas has recently attracted a lot of attention due to promising clinical results in the therapy of solid cancers. Hepatocellular carcinoma represents an ideal target for immuntherapy due to its immunogenic features. While the use of co-inhibitory antibodies has already demonstrated clinical efficacy in individual patients, the attempts to generate cancer-specific vaccines only resulted in minor clinical effects due to low systemic immune responses.In experiments performed within the framwork of the SFB TRR77 "Liver cancer" we therfore tested various vaccination regimens to increase the magnitude of the tumor-specific immune response. We could show that the tumor-specific immune response could be both amplified and accelerated using a prime-boost vaccination regimen consisting of an immunization with antigen-coated PLGA microspheres, a TLR3 agonist and a subsequent booster vaccination with a Listeria monocytogenes vector.In addition to the vaccination studies, an autochthonous liver cancer model suitable for the evaluation of the vaccinations war developed. For this purpose, transposon-flanked plasmids were used that allow for the stable integration of any transgene into the cell's DNA. Using constitutively active NRAS, AKT, shRNA against p53 and the vaccination antigen, orthotopic liver cancers could be established wihin one week. In this tumor model, the combined PLGA/TLR3-Listeria vaccine induced comlete tumor regressions and was superior to conventional dendritic cell vaccination with regard to overall survival.In the proposed research project the spectrum of the vaccination antigens which has so far been restricted to the model antigen ovalbumin and the melanoma antigen TRP2 will be extended to the HCC-specific antigen Glypican-3. To this extent two Listeria vectors containing either human or murine Glypican-3 will be cloned. The murine Listeria vector LM-mGPC3 will then be tested in the context of the PLGA-LM prime boost vaccine for the therapy of Glypican-3 positive hepatocellular carcinoma in C57Bl/6 mice, in preparations for corresponding studies in humans. Additionally, cancer-specific, exhausted CD8 T cells will be isolated from tumor-bearing mice two weeks after the vaccination and subjected to whole genome microarray analysis to yield a transcriptional signature of T cell exhaustion. In subsequent experiments the functional relevance of the candidate genes identified in the T cells will be assessed to identify potential synergies with the PLGA/LM vaccination and to enable the generation of both potent and long-lasting cancer-specific immune responses.