鼻咽癌抗独特型疫苗的制备及临床研究

Production and application of anti-idiotypic vaccine for patients of nasopharyngeal carcinoma

目的 :制备具有内影像特征的抗独特型抗体 ,用于鼻咽癌病人的主动免疫治疗以探讨其抗肿瘤效应。方法 :用杂交瘤技术制备针对Ab1可变区的抗独特型单抗 (Ab2 ) ,免疫同系动物诱导产生免疫应答 ,并用氢氧化铝凝胶沉淀法制成抗独特型疫苗 ,对 19例晚期鼻咽癌放疗病人作主动免疫治疗 ,9例放疗加生理盐水注射为对照组 ,用ELISA检测治疗前后病人血清抗体和细胞因子水平。用原位Northern杂交检测外周血单个核细胞 (PBMC)IL 2mRNA的表达。结果 :用抗鼻咽癌单抗FC2、HNL5 (Ab1)为免疫原 ,获得了两株抗独特型单抗 2H4和 5D3(Ab2 )。用Ab2免疫小鼠诱导产生了能与相应Ab1竞争结合靶抗原的抗抗独特型抗体 (Ab3)。并能诱发特异性迟发型超敏反应 (DTH)和细胞增殖反应。用Ab2治疗的病人无一例有过敏或其他毒副反应 ,血清中Ab3、抗肿瘤抗体 (Ab1’)水平均有不同程度的增高 ,但也产生了人抗鼠抗体 (HAMA)。血清细胞因子TNF α、IFN γ和IL 2水平在大多数治疗组病人中升高 ,而对照组Ab1’、IFN γ、TNF α和IL 2血清水平均未升高。病人血清的IL 2水平含量与PBMCIL 2mRNA的表达呈正相关关系 (r=0 882 9)。结论 :(1) 2H4、5D3具有鼻咽癌相关抗原的内影像 ,能模拟鼻咽癌相关抗原在同系鼠诱导体液和细胞免疫反应。 (2 )

Purpose:To generate anti idiotypic monoclonal antibodies (Ab2) bearing the internal image of Nasopharyngeal Carcinoma (NPC) associated antigen and investigate active immunotherapy with Ab2 for patients with NPC. Methods:Using NPC monoclonal antibody (Ab1)as immunogen, the hybridoma cell clone were obtained by fusion of SP2/0 myeloma cell with immunized murine spleen cell. Positive clone were screened by Sandwich ELISA and binding inhibition test. To determine whether Ab2 posses the internal image of the original antigen or not, mice immunized with Ab2. Ab2 in anti sera were tested by ELISA and the competitive inhibition assay. Cell mediated immunity to tumors induced by Ab2 was investigated by delayed type hypersensitivity (DTH) response and mouse T cell proliferation assays. Vaccined Ab2 were used in active immunotherapy to NPC patients receiving radiotherapy. Using ELISA, antibodies and cytokine levels in patients' sera were determined before and after active immunotherapy. IL 2 mRNA expression in peripheral blood mononuclear cell (PBMC) were measured as well by in situ hypridization. Results:We have generated Ab2 against FC2 and HNL5 that recognize NPC associated antigen, these Ab2, which designated 2H4 and 5D3, could inhibit the binding of FC2 or HNL5 to NPC cell lines. The anti sera from the immunized mice, which contain anti anti idiotype antibodies (Ab3),could compete with FC2 or HNL5 for binding with NPC cell by a competitive inhibition assay. Mice immunized with 2H4 or 5D3 couple with KLH, occurred positive and specific DTH reaction after stimulated by NPC cell. The results of mice T cell proliferative assay indicated that there was a significantly higher proliferative response of the splenocytes in experimental groups than that in control groups. Nineteen patients with NPC at stage Ⅳ were treated with Alum precipitated 2H4 or 5D3. Neither hypersensitivity nor adverse side effects was observed. Ab3 were elevated, the levels of anti NPC antibodies (Ab1') were increased and human anti mouse antibodies (HAMA) were occurred in nineteen patients of the experimental group, where as the levels of Ab1' didn't rise in control group. Serum IL 2, IFN γ and TNF α levels were increased in most patients in experimental group. While in the control group,there were no difference of Ab1' and cytokine level between pro therapy and post therapy. In addition, IL 2 mRNA expression in PBMC of NPC patients was closely related to serum IL 2(r=+0.8829) by in situ hybridization. Conclusions:(1) 2H4 and 5D3 are the Ab2 beta bearing the internal image of the NPC associated antigen, that either 2H4 or 5D3 expressing three dimensional shapes which resemble the structure of natural antigens could induce immune response. (2) Vaccined Ab2 was safe for clinic active immunotherapy and might enhance humoral and/or cellular immunity of NPC patients receiving radiotherapy.