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

肿瘤相关的MK-1抗原，被单克隆抗体FU-MK-1识别，在几乎所有的癌症中广泛表达。最近的分子鉴定证实MK-1是一个分子质量为40 kDa的跨膜糖蛋白，由GA733-2基因编码。在这项研究中，我们探讨了MK-1是否可以作为一种有用的肿瘤标志物，也可以作为一种有用的肿瘤免疫治疗的靶抗原。首先，我们利用家蚕核型多角体病毒表达系统在家蚕体内制备了重组MK-1蛋白，并以重组MK-1蛋白为免疫原制备了新的抗MK-1单克隆抗体。然后，我们建立了一个EIA系统来测定MK-1。测定范围为2 ~ 1000 ng/ml。对236例恶性肿瘤患者和20例健康人的血清样本进行了MK-1 EIA检测。MK-1对恶性疾病的敏感性为10.2%(24/236)，所有健康个体的MK-1浓度均低于检测限。这一结果表明，MK-1可能对没有其他肿瘤标志物升高的病例有用。其次，构建了细菌超抗原葡萄球菌肠毒素J （SEA）与FU-MK-1抗体单链可变片段（scFv）的重组融合蛋白。SEA是一种非常有效的T淋巴细胞激活剂，当呈递给MHC II类分子时。该融合蛋白（SEA/FUscFv）通过细菌表达系统产生，具有抗肿瘤活性。SEA/FUscFv融合蛋白引入了T-LAK细胞对肿瘤细胞的特异性细胞毒性，从而抑制了SCID小鼠异种移植模型中的肿瘤生长。这一结果表明SEA/FUscFv融合蛋白可能作为一种潜在的有用的免疫治疗试剂用于人表达MK-1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.