Microsphere-based glypican-3 specific immunotherapy of hepatocellular carcinoma

免疫疗法是现有癌症疗法的一种很有前途的替代疗法，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极大关注。肝细胞癌具有免疫原性，是理想的免疫治疗靶点。虽然共抑制抗体的使用已经在个别患者中证明了临床效果，但由于全身免疫反应较低，生产癌症特异性疫苗的尝试只产生了很小的临床效果。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，以提高肿瘤特异性免疫反应的幅度。我们可以证明，肿瘤特异性免疫反应既可以被放大，也可以被加速，该免疫方案由抗原包裹的PLGA微球免疫、TLR3激动剂和随后的单核细胞增生性李斯特氏菌载体加强免疫组成。除了疫苗研究，还建立了适合于疫苗接种战争评估的本土肝癌模型。为此，使用了转座子侧翼的质粒，允许任何转基因稳定地整合到细胞的DNA中。利用成分活性的NRAS、AKT、抗P53的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的肿瘤完全消退，在总存活率方面优于传统的树突状细胞疫苗。在拟议的研究项目中，疫苗抗原的范围将从目前仅限于模型抗原卵白蛋白和黑色素瘤抗原Trp2扩展到肝癌特异性抗原Glypcan-3。为此，将克隆两个含有人或小鼠glypcan-3的李斯特菌载体。小鼠李斯特氏菌载体LM-mGPC3随后将在PLGA-LM Prime Boost疫苗的背景下进行测试，用于治疗C57BL/6小鼠中Glypcan-3阳性的肝细胞癌，为在人类进行相应的研究做准备。此外，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T细胞中确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，并使其能够产生有效和持久的癌症特异性免疫反应。

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.