Development of immunotherapy for brain tumors targeting carbohydrate antigens of glycolipids

本项目的主要目标是开发一种新的胶质瘤免疫治疗方法，其中糖脂抗原进行系统分析和定量，并根据单个胶质瘤的抗原谱选择一组合适的抗体。我们首先分析了11个人神经胶质瘤细胞系，一个黑色素瘤细胞系和一个成纤维细胞系的鞘糖脂组成。胶质瘤细胞系的鞘糖脂组成与正常神经细胞不同。我们还发现，人胶质瘤细胞系U118 MG可以通过某些细胞色素调节鞘糖脂的表达。接下来，我们分析了10个人胶质瘤的糖脂抗原，发现主要由GM 3和GM 2组成。接下来检查针对这些神经节苷脂的单克隆抗体作为化学分离抗原或细胞表面抗原的反应性。在薄层层析板上制备的神经节苷脂与抗体的结合强度呈剂量依赖性，而在细胞表面的抗体反应较复杂，GM3有正、负结合阈，GM2在GM3几乎等量共存时反应较强。接下来我们分析了细胞周期相关的糖脂在某个胶质瘤中的表达。结果表明，总糖脂在有丝分裂过程中表达了近两次，nLc4Cer在该阶段显着增强。结果提示抗体对有丝分裂细胞有较好的反应。在另一个实验中，我们已经发现细胞密度极大地影响针对GM3的抗体结合水平。影响糖脂抗原抗体反应的因素很多。我们现在的重点是神经胶质瘤组织的手术标本的糖脂分析，以及参与对这些抗原的抗体反应性的因素。

The main objective of this project is to develop a novel immunotherapy for gliomas, in which glycolipid antigens are analyzed and quantified systematically and a set of appropriate antibodies would be selected according to the antigenic profile of an individual glioma. We first analyzed glycosphingolipid composition of 11 human glioma cell line, one melanoma cell line, and one fibroblast cell line. The glycosphingolipid composition of glioma cell line are different from that of normal nerve cell. We also found that a human glioma cell line U118MG could modulate glycosphingolipid expression via certain cytodines. We next analyzed the glycolipid antigens of 10 human gliomas, which were revealed to consist mainly of GM3 and GM2 . The reactivity of monoclonal antibodies against those gangliosides was examined next either as chemically isolated antigens or cell surface ones. The intensity of antibody binding was dose-dependent as the gangliosides were prepared on a thin-layr chromatographic plate, On the contrary, antibody reaction was more complex on the cell surface ; GM3 showed positive and negative threshould of antibody binding, and GM2 gave stronger reaction when GM3 co-existed nearly equally on the cell surface. We next analyzed cellcycle related glycolipids expression on a certain glioma. It was revealed that the total glycolipids were expressed nearly twice during mitosis, and nLc4Cer was significantly enhanced during that phase. The results may suggest better antibody react ions against the mitotic cells. In another experiment, we have found that the cell dendity greatly affects the level of antibody binding against GM3. There should be a variety of factors that influence the antibody reactions against glycolipid antigens. We are now focusing on the glycolipid analysis of surgical specimens of glioma tissue as well as on the factors participating in the antibody reactivity against those antigens.