Microsphere-based glypican-3 specific immunotherapy of hepatocellular carcinoma

免疫疗法是一种有希望的替代癌症治疗方法，最近由于在治疗实体癌方面有希望的临床结果而引起了人们的广泛关注。肝细胞癌由于其免疫原性特点，是免疫治疗的理想靶点。虽然共抑制抗体的使用已经在个别患者中证明了临床疗效，但由于全身免疫反应较低，产生癌症特异性疫苗的尝试只产生了轻微的临床效果。因此，在SFB TRR77“肝癌”框架内进行的实验中，我们测试了各种疫苗接种方案，以增加肿瘤特异性免疫反应的强度。我们可以证明，使用由抗原包被PLGA微球、TLR3激动剂和随后的单核细胞增生李斯特菌载体加强疫苗接种组成的初级增强疫苗方案，肿瘤特异性免疫反应可以被放大和加速。在疫苗接种研究的基础上，建立了适合于疫苗接种战争评价的肝癌模型。为此，使用转座子侧的质粒，允许任何转基因稳定地整合到细胞的DNA中。利用组成型活性NRAS、AKT、抗p53 shRNA和疫苗抗原，可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肿瘤完全消退，并且在总生存率方面优于常规树突状细胞疫苗。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TRP2的疫苗接种抗原的谱将扩展到hcc特异性抗原Glypican-3。在此范围内，将克隆两个李斯特菌载体，其中包含人类或小鼠Glypican-3。小鼠李斯特菌载体LM-mGPC3将在PLGA-LM初级增强疫苗的背景下进行测试，用于治疗C57Bl/6小鼠Glypican-3阳性肝细胞癌，为相应的人类研究做准备。此外，在接种疫苗两周后，将从荷瘤小鼠中分离出癌症特异性的、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，将评估在T细胞中鉴定的候选基因的功能相关性，以确定与PLGA/LM疫苗接种的潜在协同作用，并使产生有效和持久的癌症特异性免疫反应成为可能。

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.