Analysis on the expression of gene encoding human melanoma-associated antigen immunogenic in patients and application for active specific immunotherapy

本研究旨在鉴定和表征患者中的人黑素瘤相关抗原的免疫原性。所克隆的编码34 Kd肽抗原的cDNA与已报道的哺乳动物基因的核酸序列无明显同源性。克隆D-1cDNA与培养的人黑色素瘤、神经母细胞瘤、红白血病和肾癌细胞的mRNA杂交，而与正常成纤维细胞、淋巴细胞和肾癌细胞的mRNA不杂交。利用D-1 cRNA探针通过原位杂交分析了D-1对应的mRNA的组织分布，并显示其在体内黑色素瘤细胞上表达。在主要的正常器官和其他皮肤癌组织中未检测到明显的表达。用重组D-1肽免疫Balb/c小鼠，产生小鼠抗D-1多克隆抗体，并以几乎与原位杂交相同的方式显示D-1抗原在黑色素瘤细胞表面膜上的体内定位。但在某些黑色素瘤组织中，D-1抗原在胞浆中的强表达表明，D-1抗原需要某些亚类的HLA分子在黑色素瘤表面表达。与对照细胞K562共培养28天后，D-1重组肽体外活化的淋巴细胞对自体黑色素瘤细胞具有明显的细胞毒活性。D-1重组肽疫苗的主动特异性免疫治疗正在进行中，用于IV期黑色素瘤患者。对接种疫苗的患者血清进行血清学观察，发现有高滴度和低滴度两组。计划对这些组之间的HLA I类分型进行比较分析。

This study aimed at the identification and characterization of human melanoma-associated antigens immunogenic in patients. The cloned cDNA encoding 34 Kd peptide antigen showed no significant homology of nucleic acid sequence with previously reported mammalian genes. The clone D-1 cDNA hybridized with mRNA from cultured human melanomas, neuroblastoma, erythroleukemia and lympoma cells but not with mRNA from normal fibroblasts, lymphocytes and renal carcinoma cells. The tissue distribution of mRNA corresponding to D-1 was analyzed by in-situ hybridization utilizing D-1 cRNA prove, and showed to be expressed on melanoma cells in vivo. No significant expression on major normal organs and other skin cancers was detected. Murine polyclonal anti-D-1 antibodies were raised against Balb/c mice with immunization of recombinant D-1 peptide and showed the invivo localization of D-1 antigen on the surface membrane of melanoma cells in an almost identical fashion to in-situ hybridization. But the strong expression of D-1 antigen in the cytoplasm of some melanoma tissues indicated the need of HLA molecules with certain subclass to express D-1 antigen on the surface. The activated lymphocytes in vitro with D-1 recombinant peptide showed substantial cytotoxic activities to autologous melanoma cells comparing with the control cells K562 in co-culture for 28 days. Active, specific immunotherapy with D-1 recombinant peptide vaccine is in process on patients with StageIV melanoma. Serological observation of anti-D-1 antisera in patients received vaccination revealed two groups with high and low titer. The comparative analysis of HLAclass I type between these groups is planned.