A Specific Study for Clinical Application of a New Tumor-associated Antigen, MK-1,

肿瘤相关的MK-1抗原，识别单克隆抗体FU-MK-1，广泛表达于几乎所有的癌症。最近的分子鉴定证实MK-1是一种分子量为40 kDa的跨膜糖蛋白，由GA 733 -2基因编码。本研究首先利用家蚕核型多角体病毒表达系统在家蚕中表达MK-1重组蛋白，并以重组蛋白为免疫原制备抗MK-1单克隆抗体。建立了MK-1的EIA检测体系。测定范围为2 - 1，000 ng/ml。用MK-1酶免疫分析法检测了236例恶性肿瘤患者和20例健康人的血清样品。MK-1对恶性疾病的敏感性为10.2%（24/236），并且对于所有健康个体，MK-1浓度均低于检测限。该结果表明，MK-1可能对其他已建立的肿瘤标志物无任何升高的病例有用。我们构建了细菌超抗原金黄色葡萄球菌肠毒素J（SEA）和FU-MK的单链可变区片段（scFv）的重组融合蛋白，1抗体。SEA是一种非常有效的T淋巴细胞激活剂，当提出的MHC II类分子。通过细菌表达系统产生所得到的融合蛋白（SEA/FUscFv），并表征其抗肿瘤活性。SEA/FUscFv融合蛋白可诱导T-LAK细胞对肿瘤细胞的特异性杀伤作用，从而抑制SCID小鼠移植瘤的生长。这一结果表明，SEA/FUscFv融合蛋白可能作为一个潜在的有用的免疫试剂的人MK-1表达的肿瘤。

The tumor-associated MK-1 antigen, recognized by MAb FU-MK-1, is widely expressed on almost all carcinomas. Recent molecular characterization confirmed that MK-1 is a transmembrane glycoprotein with molecular mass of 40 kDa and is encoded by the GA733-2 gene. In this study, we investigated if MK-1 could be a useful tumor marker and also could be a useful target antigen for cancer immunotherapy.First, we produced a recombinant MK-1 protein in silkworms by the Bombyx mori nuclear polyhedorosis virus expression system, and newly generated anti-MK-1 MAbs using the recombinant MK-1 protein as immunogen. Then, we established an EIA system for determination of MK-1. The assay range was 2 - 1,000 ng/ml. Serum samples of 236 patients with malignant disease as well as of 20 healthy individuals were tested with the MK-1 EIA. The sensitivity of MK-1 for malignant disease was 10.2% (24/236) and for all healthy individuals the MK-1 concentrations were less than the detection limit This result suggests that MK-1 might be useful for in cases without any elevation of other established tumor markers.Second, we constructed a recombinant fusion protein of the bacterial superantigen staphylococcal enterotoxin J (SEA) and the single-chain variable fragment (scFv) of the FU-MK-1 antibody. SEA is an extremely potent activator of T lymphocytes when presented on MHC class II molecules. The resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system and characterized for the antitumor activity. The SEA/FUscFv fusion protein introduced a specific cytotoxicity of T-LAK cells to the tumor cells and consequently suppressed the tumor growth in a SCID mouse xenograft model. This result indicates that the SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1 -expressing tumors.