整合NGR肽抗EGFR单链抗体制备及其抗肿瘤活性研究  

Preparation of NGR motif-integrated anti-EGFR single-chain Fv and its antitumor activity

作　　者：盛唯瑾[1] 商悦[1] 甄永苏[1] 

机构地区：[1]中国医学科学院.北京协和医学院医药生物技术研究所,北京100050

出　　处：《中华肿瘤防治杂志》2014年第13期969-973,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(31000416)

摘　　要：目的:利用大肠埃希菌表达基于抗表皮生长因子受体(epidermal growth factor receptor,EGFR)单链抗体和靶向CD13环状NGR肽的双靶点重组融合蛋白ER(Fv)-NGR,研究其对肿瘤细胞的亲和能力和体内外抑制作用。方法:用基因工程的方法构建重组质粒pET-scfv-ngr,转入大肠埃希菌BL21,IPTG诱导表达带有His-tag标签肽的重组蛋白ER(Fv)-NGR,用Ni离子亲和柱进行纯化。运用细胞免疫荧光检测ER(Fv)-NGR与肿瘤细胞的结合;流式细胞术分析重组蛋白与肿瘤细胞的亲和力;克隆形成实验测定重组蛋白对肿瘤细胞体外增殖的抑制作用;利用人乳腺癌MCF-7裸鼠移植瘤模型对ER(Fv)-NGR的体内抗肿瘤活性进行研究。结果:重组蛋白ER(Fv)-NGR由抗EGFR单链抗体和3个连续的环状NGR肽构成,相对分子质量约为27×104,以包涵体形式在大肠埃希菌内表达,表达量约为5mg/L。细胞免疫荧光结果表明,重组蛋白与EGFR和CD13皆高表达MCF-7细胞有较强的结合能力,而与正常HEK293细胞结合较弱。ER(Fv)-NGR与MCF-7细胞结合的解离常数为8.4×10-7 nmol/L。ER(Fv)-NGR在体外对肿瘤细胞的增殖有抑制作用,作用于MCF-7和A549细胞的IC50值分别为1.2×10-6和4.7×10-6 mol/L。在体内对人乳腺癌MCF-7裸鼠移植瘤有生长抑制作用,在10mg/kg药物剂量下28d对照组与实验组瘤体积分别为(1.06±0.17)和(0.63±0.11)cm3,F=18.7,P=0.003;瘤质量分别为(0.95±0.12)和(0.52±0.07)g,F=42.7,P<0.001;抑瘤率为45.4%。结论:成功制备了基于单链抗体和靶向肽的双靶点融合蛋白ER(Fv)-NGR,其对肿瘤细胞具有良好的亲和力和体外增殖抑制作用,能够有效抑制裸鼠移植瘤的生长,为研制新型双靶点抗肿瘤药物提供依据。OBJECTIVE： To express a recombinant fusion protein ER（Fv）-NGR based on anti-EGFR single-chain variable fragment （scFv） and cyclic NGR peptide against CD13 with Escherichia coli,and study its antitumor activity. METHODS： The recombinant plasmids pET-scfv-ngr was constructed and transformed into competent E. coli BL21. Recombinant protein was produced after IPTG induction,and then purified with Ni ion affinity chromatography. Immunofluorescence was used for measuring binding efficiency of ER（Fv）-NGR to tumor cells, and flow cytometry was applied to analyze binding activity. The cytotoxicity against tumor cells in vitro was assessed by colony formation assay, and inhibitory effects in vivo were observed in nude mice bearing human breast cancer MCF-7 xenografts. RESULTS：Recombinant fusion protein,consisting of an anti-EGFR single-chain variable fragment （scFv） and tri-CNGRC （Cys-Asn-Gly-Arg-Cys） peptide about 2.7 x 10^4 ,labled with His-tag was produced in the form of inclusion bodies,and was purified with Ni ion affinity chromatography. Finally about 5 mg of ER（Fv）-NGR was obtained from 1 L of culture medium. Recombinant protein bound specially to the membrane of tumor cells,and possessed powerful affinity to MCF-7 cells with the dissociation constant （Kd） of 8.4 X 10^-7 mol/L. ER（Fv）-NGR showed eytotoxicity against tumor cells in vitro, with the IC50 value of1.2X10.6 mol/L for MCF-7 and 4. 7 X 10^-6 mot/L for A549 cells. In animal experiment, ER（Fv）-NGR at dose of 10 mg/kg moderately inhibited the growth of＇ MCF-7 xenografts with the inhibitory rate of 45.4%. The tumor volume were（1.06±0.17） cm3 for ER（Fv）-NGR and（0.63±0.11） cm3 for control （F=lS. 7,P=0. 003） ,and the tumor weigh were （0.95±0.12） g for ER（Fv）-NGR and（0.52±0.07） g for control （F=42.7 ,P〈0. 001）. CONCLUSION： The prep- aration of scFv/peptide-based bispecific fusion protein with binding affinity to tumor ceils and antitumor activity provides the basis for the developme

关 键 词：肿瘤 单链抗体 靶向肽 表皮生长因子受体 CD13 流式细胞术 

分 类 号：R73-36[医药卫生—肿瘤]