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免疫治疗胶质瘤的方法，系统地分析和量化糖脂抗原，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个人类胶质瘤细胞系、一个黑色素瘤细胞系和一个成纤维细胞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U118MG可以通过某些细胞素调节鞘糖脂的表达。我们进一步分析了10个人类胶质瘤的糖脂抗原，发现这些糖脂抗原主要由GM3和GM2组成。接下来，作为化学分离抗原或细胞表面抗原，检测了针对这些神经节苷脂的单克隆抗体的反应性。在薄层层析板上制备神经节苷脂时，抗体结合强度呈剂量依赖性，相反，在细胞表面的抗体反应更为复杂；GM3对抗体的结合表现出正、负三价，当GM3在细胞表面几乎均匀共存时，GM2的反应更强。接下来，我们分析了细胞周期相关的糖脂在某种胶质瘤中的表达。结果表明，总糖脂在有丝分裂期间表达了近2倍，nLc4Cer在有丝分裂期间显著增强。结果可能提示抗体反应离子对有丝分裂细胞有更好的作用。在另一项实验中，我们发现细胞依赖性极大地影响了针对GM3的抗体结合水平。应该有多种因素影响针对糖脂抗原的抗体反应。我们现在专注于胶质瘤组织手术标本的糖脂分析，以及参与抗体对这些抗原反应性的因素。

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.