Zamboni Silvia, Mallano Alessandra, Flego Michela, Ascione Alessandro, Dupuis Maria Luisa, Gellini Mara, Barca Stefano, Cianfriglia Maurizio
Section of Pharmacogenetics, Drug Resistance and Experimental Therapeutics, Department of Drug Research and Evaluation, Istituto Superiore di Sanità, I-00161 Rome, Italy.
Int J Oncol. 2008 Jun;32(6):1245-51. doi: 10.3892/ijo_32_6_1245.
We report the genetic construction and expression of a fusion protein between a single chain fragment variable (scFv) human antibody (E8) specific for CEA cell surface antigen and yeast cytosine deaminase (yCD). Sequences encoding for the scFvE8 human monoclonal antibody recognizing an epitope shared by CEACAM1, CEACAM3 and CEACAM5 isoforms were assembled with a monomer of yCD. The construct was placed under the transcriptional regulation of the lac promoter, and in frame with 6xHis tag for protein purification. After transformation and induction of E. coli, the protein was recovered from cell lysates and processed for purification. The scFvE8:yCD fusion protein possessed the binding specificity for melanoma (Mel P5) and colon carcinoma (LoVo) cell lines similar to its cognate human scFv antibody. The scFv8:yCD system showed the ability to render tumor cells susceptible to the far less toxic substrate 5-fluorocytosine (5-FC) by its enzymatic conversion into 5-fluorouracil (5-FU). In vitro pre-treatment of Mel P5 and LoVo cell lines with scFvE8:yCD followed by cell washing and incubation with 5-FC, resulted in significant cell killing supporting the utility of this fusion protein as an agent for tumor-selective prodrug activation. This study shows the feasibility of constructing fusion proteins in a prokaryotic cell based system consisting of a human scFv antibody and yCD to convert the antifungal agent 5-FC to 5-FU, one of the widely used anticancer agent.
我们报告了一种融合蛋白的基因构建和表达，该融合蛋白由对癌胚抗原（CEA）细胞表面抗原具有特异性的单链可变片段（scFv）人抗体（E8）和酵母胞嘧啶脱氨酶（yCD）组成。编码识别CEACAM1、CEACAM3和CEACAM5同种型共有的一个表位的scFvE8人单克隆抗体的序列与yCD的一个单体组装在一起。该构建体置于乳糖启动子的转录调控之下，并与用于蛋白质纯化的6xHis标签读框一致。在大肠杆菌转化和诱导后，从细胞裂解物中回收该蛋白并进行纯化处理。scFvE8:yCD融合蛋白对黑色素瘤（Mel P5）和结肠癌细胞系（LoVo）具有与同源人scFv抗体相似的结合特异性。scFv8:yCD系统显示出通过将毒性小得多的底物5-氟胞嘧啶（5-FC）酶促转化为5-氟尿嘧啶（5-FU），使肿瘤细胞对其敏感的能力。用scFvE8:yCD对Mel P5和LoVo细胞系进行体外预处理，然后细胞洗涤并与5-FC孵育，导致显著的细胞杀伤，支持了这种融合蛋白作为肿瘤选择性前药激活剂的效用。这项研究表明在由人scFv抗体和yCD组成的原核细胞系统中构建融合蛋白以将抗真菌剂5-FC转化为5-FU（一种广泛使用的抗癌剂之一）的可行性。
