Expression of MAGE tumor rejection antigen on lung cancer and application for cancer vaccine

(1)用GST-MAGE-1或4融合蛋白刺激肺癌患者外周血单核细胞（PBMC）。然而，我们不能诱导特异性CTL对抗MAGE抗原。另一方面，通过体外刺激HLA-A2中受限制的MART-1肽，可以诱导特异性ctl (Sugita et al., Int.；Immunol。， 8(5), 1996)。(2) MAGE基因在许多不同的癌症中经常表达，但在除睾丸和胎盘外的正常细胞或正常组织中不表达。MAGE家族蛋白的生物学作用仍有待研究。用重组MAGE-1或MAGE-4蛋白抗体免疫组织化学方法研究了重组MAGE-1或MAGE-4蛋白在睾丸细胞中的表达。这两种蛋白均在精原细胞和原代精母细胞的细胞核和细胞质中发现，但在精母细胞和Sertoli细胞中未发现。因此，MAGE蛋白是一种正常组织抗原区室蛋白，在精子发生的早期阶段起着重要作用（Takahashi et al ., Cancer Res., 55,1995）。(3)采用重组MAGE-4b蛋白（Shichijo et al., J Immunol.）的单克隆抗体和多克隆抗体，建立酶联免疫吸附法（ELISA）检测人肿瘤细胞表达的MAGE-4蛋白。方法,186,1995)。(4)通过对原发性肺癌患者血清中MAGE-4蛋白水平的研究，了解MAGE蛋白的生物学作用。在MAGE-4^+肿瘤细胞系的上清液和患者血清中检测到MAGE-4蛋白为未降解蛋白。肺癌患者（n=100，平均=1.17ng/ml）血清MAGE-4蛋白水平显著高于良性肺疾病患者（n=80,033ng/ml）或健康供者（n=68,032ng/ml）。100名癌症患者中有34名高于临界值（1.15ng/ml），但其他组中没有人高于临界值（Shichijo et al.， JJCR,in press, 1997）。(5)对16例非小细胞肺癌患者il -2活化的肿瘤浸润淋巴细胞的HLA分型限制及细胞毒性的肿瘤特异性进行了研究。建立了6条l型限制性肿瘤特异性CTL系：(i) 3例腺癌患者的HLA- a2限制性和腺癌特异性ctl， （ii） HLA- a3101 /3302+腺癌患者的HLA- a3101和a3302限制性和腺癌特异性ctl，以及（iii） HLA- a3302限制性ctl和（iv） HLA- a2402限制性ctl分别在HLA- a3302 +腺癌患者和HLA- a2402鳞状细胞癌患者中识别不同组织学类型的肿瘤(Seki等，cell。Immunol。、175、1997)。这些结果表明，存在HLA- l类限制性和肿瘤特异性ctl识别腺癌或SCC肿瘤部位HLA- a等位基因上的肽抗原的证据

(1) Peripheral blood mononuclear cells (PBMC) from lung cancer patients had been stimulared with GST-MAGE-1 or-4 fusion protein. However, we could not induct specific CTL against MAGE antigens. In the other hand, it had been possible to induce specific CTLs against MART-1 peptide restricted in HLA-A2 by the stimulation with this peptide in vitro (Sugita et al., Int. Immunol., 8 (5), 1996). (2) The MAGE genes are frequently expressed in many different cancers, but are not expressed in normal cells or normal tissues other than testis and placenta. Biological roles of proteins of MAGE family remained to be investigated. The expression of MAGE-1 or MAGE-4 protein in the testicular cells was studied immunohistochemically with the antibodies to the recombinant MAGE-1 or MAGE-4 protein. Both proteins were identified in the nucleus and cytoplasm of spermatogonia and in primary spermatocytes but not in spermatids or Sertoli's cells. Therefore, MAGE proteins are normal tissue antigens compartmen … More talized in particular testicular cells playing an important role in the early phase of the spermatogenesis (Takahashi et al, Cancer Res., 55,1995). (3) An enzyme-linked immunosorbent assay (ELISA) was established for measuring cellular MAGE-4 protein expressed on human tumor cells using a monoclonal antibody and polyclonal Ab to recombinant MAGE-4b protein (Shichijo et al., J Immunol. Method, 186,1995). (4) We investigated the level of MAGE-4 protein in sera of patients with primary lung cancer to understand the biological roles of the MAGE proteins. MAGE-4 protein was detected as a non-degraded from in both the supernatant of a MAGE-4^+ tumor cell line and in a patient's serum. Surum level of the MAGE-4 protein in lung cancer patients (n=100, mean=1.17ng/ml) was significantly higher than that in either patients with benign pulmonary diseases (n=80,033ng/ml) or healthy donors (n=68,032ng/ml). It was higher than the cutoff level (1.15ng/ml) in 34 of 100 cancer patients, but not in anyone in the other groups (Shichijo et al., JJCR,in press, 1997). (5) HLA class lrestriction and tumor specificity of cytotoxicity in the IL-2-activated tumor-infiltrating lymphocytes from 16 patients with non-small cell lung cancer were investigated. Six HLA class l-restricted and tumor-specific CTL lines were established : (i) HLA A2-restricted and adenocarcinoma-specific CTLs in three patients with adenocarcinoma, (ii) HLA-A3101-and A3302-restricted and adenocarcinoma-specific CTLs in an HLA-A3101/3302+patients with adenocarcinoma, and (iii) HLA-A3302-restricted CTLs and (iv) HLA-A2402-restricted CTLs recognizing tumors with different types of histology in an HLA-A3302+patients with adenocarcinoma and HLA-A2402 patients with squamous cell carcinoma, respectively (Seki et al., Cell. Immunol., 175,1997). These results showed evidence of the existence of HLA class l-restricted and tumor-specific CTLs recognizing peptide antigens on HLA-A alleles of adenocarcinoma or SCC in tumor sites of a Less