Development of cancer immunotherapy using antibody specific for tumor antigens recognized by tumor-infiltration B cells

抗体免疫治疗的最新进展为我们提供了识别靶抗原作为一种新的免疫治疗的希望。我们以前用改良的SEREX方法证明了肿瘤浸润性B细胞识别肿瘤抗原并产生抗体。在本研究中，我们鉴定了两例肺癌患者的肿瘤相关抗原，并分析了这些抗原在肿瘤标志物和免疫治疗中的作用。在患者1(G603)中，发现其中一种已鉴定的抗原是MAGE-B2。在对患者血清的免疫监测中，MAGE-B2抗体滴度在术前较高，原发灶切除后下降。肾上腺转移时又升高，切除后下降，提示抗MAGE-B2抗体可作为肿瘤标志物。在患者2中，分离出22种不同的抗原。在这些抗原中，RT-PCR检测到X蛋白在9个肺癌细胞系中有5个是高表达的。此外，与相应的正常肺组织相比，X蛋白在15例肺癌组织中有9例过表达。制备了针对蛋白X的多克隆抗体，并对其抗原定位和抗肿瘤活性进行了分析。流式细胞术显示蛋白X在肿瘤细胞表面表达，且该抗原高表达。免疫组织化学分析显示，X蛋白在28例肿瘤组织中有7例表达于肿瘤细胞膜。接种抗蛋白X抗体后，SCID小鼠移植瘤消退。体外分析表明，CDC机制介导抗肿瘤活性。提示TIB识别的这些抗原可作为肿瘤标志物用于临床诊断和抗体介导的免疫治疗的临床应用。

Recent progress of immunotherapy by antibody have provided us hope of identification of target antigen as a novel immunotherapy. We previously demonstrated that tumor-infiltrating B cells recognized tumor antigens and produced antibodies against them by a using modified SEREX method. In the present study, we have identified tumor-associated antigens in two lung cancer patients and analyzed the usefulness of antigens for tumor marker and immunotherapy. In patient 1 (G603), one of identified antigens was revealed to be MAGE-B2. In the immuno-monitoring of the patient's sera, high antibody titer against MAGE-B2 was observed before operation and the titer decreased after resection of the primary tumor. It was elevated again at the time of adrenal metastasis, but then decreased after resection, indicating that anti-MAGE-B2 antibody could be used as tumor markers for the patient. In patient2, 22 distinct antigens were isolated. Of these antigens, Protein X was highly expressed in 5 out of 9 lung cancer cell lines by RT-PCR. Moreover, Protein X was overexpressed in 9 out of 15 lung cancer tissues compared with corresponding normal lung tissues. Polyclonal antibody against Protein X was generated and was analyzed for localization of antigens and for anti-tumor activity. Flow cytometory showed that Protein X expressed in the cell surface of tumor cells with high expression of this antigen. Immunohistochemical analysis revealed that Protein X was expressed in cell membrane of tumor cells in 7 out of 28 tumor tissues. Tumor implanted in SCID mouse regressed by the inoculation of anti-Protein X antibody. In vitro analysis, anti-tumor acticity was mediated by CDC mechanism. These result indicated that these antigens recognized by TIB could be usefull for clinical diagnosis as a tumor marker and for clinical application of antibody-mediated immunotherapy.