鼻咽癌人源抗独特型单链抗体的制备及筛选

Construction and Screening of Human Anti-Idiotypic Single Chain Antibodies of Nasopharyngeal Carcinoma

背景与目的:抗独特型抗体作为肿瘤抗原替代物可用于肿瘤治疗,这已在临床试验中得到证实。但由于目前所使用的抗独特型抗体多为鼠源性,用于人体可产生人抗鼠抗体反应,从而影响疗效。本实验拟构建噬菌体人源抗独特型抗体库,并从中筛选出能模拟鼻咽癌相关抗原的β型抗独特型单链抗体scFv(Ab2βscFv),以解决鼠源性抗独特型抗体用于临床所产生的人抗鼠抗体反应。方法:体外致敏并用EB病毒(Epstein-Barrvirus,EBV)转化鼻咽癌患者的外周血单个核细胞(peripheralbloodmononuclearcell,PBMC),用RT-PCR分别扩增VH和VL基因并连接成scFv基因,将scFv基因与载体fUSE5连接后,转化大肠杆菌MC1061,构建噬菌体呈现型scFv库。在用单抗FC2对文库进行4轮筛选后,用SandwichELISA和结合抑制法从中筛选出β型Ab2scFv。结果:用单抗FC2体外致敏并经EBV转化的10例鼻咽癌患者的PBMC中,8例有鼻咽癌抗独特型抗体产生。经PCR分别扩增出5种VH(γ、μ)和7种VL(κ、λ)基因,经连接组成14种scFv基因。在与载体连接后,导入大肠杆菌MC1061,得到库容为1.5×108的初级噬菌体抗独特型抗体库。经富集筛选后,从中随机挑取270个克隆进行ELISA筛选,得到91个Ab2scFv单克隆,阳性率为33.7%。再用结合抑制法从中初步筛选出5个可能为β型的Ab2scFv。

BACKGROUND &OBJECTIVE: The potential of anti idiotypic antibody as a surrogate of tumor antigen for cancer therapy has been demonstrated in clinical investigations. But at present, many anti idiotypic antibodies are mouse original antibodies, which can cause human anti mouse antibody (HAMA) response and decrease the curative effect. The objective of this study was to construct phage human anti idiotypic antibody library and select βtype anti idiotypic single chain antibodies bearing the internal image of the nasopharyngeal carcinoma (NPC) associated antigen to overcome human anti mouse antibody response caused by application of mouse original anti idiotypic antibody. METHODS: Peripheral blood mononuclear cells (PBMCs) of patients with NPC were immunized in vitro by anti NPC monoclonal antibody FC2 and transformed by Epstein Barr virus (EBV). VH and VL genes were amplified by RT PCR and combined to single chain fragments of variable region (scFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into E.coli MC1061 to construct the scFv displaying phage library. After four rounds of panning with monoclonal antibody (mAb) FC2,the βtype Ab2 scFv were selected by Sandwich ELISA and binding inhibition test. RESULTS: Of 10 NPC patients, 8 patients showed their B cells immunized by FC2 and transformed by EBV produced anti idiotypic antibodies to NPC. Five types of VH genes and 7 types of VL genes were obtained by RT PCR amplification and then connected to form 14 scFv genes. ScFv genes were transducted into E.coli MC1061. The library capacity was 1.5×108 clones. After panning, 270 phage clones were selected randomly and 91 FC2 positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. Five clones,which might display βtype Ab2 scFv, were selected by binding inhibition test. CONCLUSION: The strategy for preparing phage anti idiotypic antibody library and selecting βtype Ab2 scFv by immunization in vitro, EBV transformation, and phage display technique is feasible, which provide a way for preparing cancer vaccine using βtype Ab2 scFv.