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

近年来抗体免疫治疗的研究进展为靶抗原的筛选提供了新的希望。我们以前证明，肿瘤浸润B细胞识别肿瘤抗原，并产生针对他们的抗体，通过使用修改SEREX方法。在本研究中，我们鉴定了两名肺癌患者的肿瘤相关抗原，并分析了抗原在肿瘤标志物和免疫治疗中的有用性。在患者1（G603）中，发现鉴定的抗原之一是MAGE-B2。在患者血清的免疫监测中，在手术前观察到抗MAGE-B2的高抗体滴度，并且在原发肿瘤切除后滴度降低。在肾上腺转移时再次升高，但在切除术后下降，表明抗MAGE-B2抗体可作为患者的肿瘤标志物。在患者2中，分离出22种不同的抗原。RT-PCR结果显示，9株肺癌细胞系中有5株高表达X蛋白。此外，与相应的正常肺组织相比，蛋白X在15例肺癌组织中的9例中过表达。产生了针对蛋白X的多克隆抗体，并分析了抗原定位和抗肿瘤活性。流式细胞术显示，蛋白X在高表达该抗原的肿瘤细胞的细胞表面表达。免疫组化结果显示，28例肿瘤组织中有7例的肿瘤细胞膜表达X蛋白。通过接种抗蛋白X抗体，植入SCID小鼠的肿瘤消退。体外抗肿瘤活性分析表明，其抗肿瘤活性是由CDC机制介导的。这些结果表明，TIB识别的这些抗原可作为肿瘤的临床诊断标志物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.