摘要
目的 制备一种以人表皮生长因子受体2（human epidermal growth factor receptor 2， HER2）为靶点的由抗体重链超变区多肽与力达霉素组成的强化融合蛋白LDP-Hr-AE，并初步探讨其抗肿瘤活性。方法 通过聚合酶链式反应扩增出抗人表皮生长因子受体2抗体C6.5重链CDR3区的20个氨基酸残基基因，将其与力达霉素辅基蛋白基因连接构建出融合蛋白基因ldp-Hr，转化至大肠杆菌中进行诱导表达。融合蛋白LDP-Hr采用HisTrap Ni2+亲和色谱柱进行分离纯化，高效液相色谱法检测其纯度。细胞免疫荧光法和基于流式细胞术的亲和实验分析融合蛋白与肿瘤细胞的结合活性。纯化的LDP-Hr蛋白与力达霉素发色团在体外进行组装，构建出强化融合蛋白LDP-Hr-AE。采用MTT法检测强化融合蛋白对肿瘤细胞的杀伤活性，Annexin V-FITC/PI双染结合流式细胞术的方法检测LDP-Hr-AE蛋白对细胞凋亡的影响。结果 成功构建并表达了融合蛋白LDP-Hr，目的蛋白以可溶形式分泌至大肠杆菌培养液和周质腔中，产量可达每升发酵液40 mg活性蛋白，纯化后的蛋白经高效液相色谱法检测纯度为97.4%。细胞免疫荧光实验和基于流式细胞术的亲和实验检测结果均显示LDP-Hr蛋白与HER2高表达的肿瘤细胞系（如SK-BR-3和SK-OV-3细胞）都有很强的结合活性。LDP-Hr蛋白与力达霉素发色团在体外组装后经高效液相色谱法检测350 nm处出现特定吸收峰，表明强化融合蛋白LDP-Hr-AE构建成功。采用MTT法检测了强化融合蛋白的体外杀伤活性，结果显示，LDP-Hr-AE对肿瘤细胞有强烈的杀伤作用，且其杀伤活性要高于力达霉素。细胞凋亡检测结果也表明LDP-Hr-AE在极低的浓度下（如0.1 nmol·L-1）即可强烈的诱导细胞发生凋亡，且凋亡的比率随着蛋白浓度的升高而增加。结论 本实验制备的强化融合蛋白LDP-Hr-AE可以特异的与人表皮生长因子受体2结合，对肿瘤细胞具有强烈的杀伤活性和诱导凋亡能力，具有发展为抗肿瘤靶向药物的潜能。
Abstract
OBJECTIVE To construct a novel fusion protein consisting of oligopeptides specific for human epidermal growth factor receptor 2 (HER2) and lidamycin (LDM)， and investigate its antitumor activity. METHODS Coding sequences of oligopeptides from complementarity determining region 3 (CDR3) of anti-HER2 antibody C6.5 heavy chain was fused to apoprotein of lidamycin to obtain the fusion gene ldp-Hr. Fusion protein LDP-Hr was expressed in E.coli and purified by affinity chromatography. The purity of LDP-Hr was analyzed by high HPLC. Immunofluorescence assay and flow cytometry-based binding assay were used to investigate the binding activity of LDP-Hr to HER2 overexpressed cancer cells. The energized fusion protein LDP-Hr-AE was prepared by integrating the active enediyne chromophore (AE) of lidamycin into the LDP-Hr protein. MTT assay was used to measure the in vitro cytotoxicity of LDP-Hr-AE and Annexin V-FITC/PI staining assay was used to analyze its apoptosis-inducing efficacy. RESULTS Fusion protein LDP-Hr was constructed correctly and expressed in E.coli in a secretory manner. The production of LDP-Hr was 40 mg per liter fermentation broth， and the purity of fusion protein was 97.4% as analyzed by HPLC. LDP-Hr showed strong binding activity to cancer cells highly expressing HER2， such as SK-BR-3 and SK-OV-3 cells. The energized fusion protein LDP-Hr-AE exhibited more potent cytotoxicity to SK-BR-3 and SK-OV-3 cells than LDM as measured by MTT assay. The results from Annexin V-FITC/PI staining assay also revealed that LDP-Hr-AE significantly induced cell apoptosis even at very low concentrations. CONCLUSION The novel energized fusion protein LDP-Hr-AE bounds to HER2 specifically， and shows potent cytotoxicity and apoptosis-inducing activity to cancer cells， which suggests that it would be a promising candidate for targeted cancer therapy.
关键词
人表皮生长因子受体2 /
融合蛋白 /
力达霉素 /
细胞凋亡 /
抗肿瘤
{{custom_keyword}} /
Key words
human epidermal growth factor receptor 2 /
fusion protein /
lidamycin /
cell apoptosis /
antitumor
{{custom_keyword}} /
郭晓芳, 朱小飞, 钟根深, 甄永苏.
以人表皮生长因子受体2为靶点的强化融合蛋白LDP-Hr-AE的构建及抗肿瘤活性研究[J]. 中国药学杂志, 2012, 47(12): 941-947
GUO Xiao-Fang, ZHU Xiao-Fei, ZHONG Gen-Shen, ZHEN Yong-Su.
Construction of an Energized Fusion Protein LDP-Hr-AE Targeting Human Epidermal Growth Factor Receptor 2 and Its Antitumor Activity[J]. Chinese Pharmaceutical Journal, 2012, 47(12): 941-947
中图分类号：
R730.3
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] OLAYIOYE M A， NEVE B M， LANE H A， et al. The ErbB signaling network： receptor heterodimerization in development and cancer [J]. EMBO J， 2000， 19(13)： 3159-3167.[2] BASELGA J， ARTEAGA C L. Critical update and emerging trends in epidermal growth factor receptor targeting in cancer [J]. J Clin Oncol， 2005， 23(11)： 2445-2459.[3] REID A， VIDAL L， SHAW H， et al. Dual inhibition of ErbB1 (EGFR/HER1) and ErbB2 (HER2/neu) [J]. Euro J Cancer， 2007， 43（4）： 481-489.[4] HYNES N E， LANE H A. ERBB receptors and cancer： the complexity of targeted inhibitors [J]. Nat Rev Cancer， 2005， 5(5)：341-354.[5] ROWINSKY E K. The erbB family： targets for therapeutic development against cancer and therapeutic strategies using monoclonal antibody and tyrosine kinase inhibitors [J]. Annu Rev Med， 2004， 55（3）： 433-457.[6] MARIANI G， FASOLO A， DE BENEDICTIS E， et al. Trastuzumab as adjuvant systemic therapy for HER2-positive breast cancer [J]. Nat Clin Pract Oncol， 2009， 6(2)： 93-104.[7] RUSNAK D W， AFFLECK K， COCKERILL S G， et al. The characterization of novel， dual ErbB-2/EGFR， tyrosine kinase inhibitors： potential therapy for cancer [J]. Cancer Res， 2001， 61(19)： 7196-7203.[8] PASTAN I， HASSAN R， FITZGERALD D J， et al. Immunotoxin treatment of cancer [J]. Annu Rev Med， 2007， 58（3）： 221-237.[9] ZHEN Y S. Anticancer Drug Research and Development (抗肿瘤药物研究与开发) [M]. Vol. 1. Beijing： Chemical Industry Press， 2004： 570-585.[10] ACCARDI L， DI BONITO P. Antibodies in single-chain format against tumor-associated antigens： present and future applications [J]. Curr Med Chem， 2010， 17(17)： 1730-1755.[11] MIAO Q F， LIU X Y， SHANG B Y， et al. An enediyne-energized single-domain antibody-containing fusion protein shows potent antitumor activity [J]. Anticancer Drugs， 2007， 18(2)： 127-137.[12] SCHIER R， MARKS J D， WOLF E J， et al. In vitro an in vivo characterization of picomolar affinity anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library [J]. Immunotechnology， 1995， 1(1)： 73-81.[13] SCHIER R， MCCALL A， ADAMS G P， et al. Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site [J]. J Mol Biol， 1996， 263(4)： 551-567.[14] SHIRAI H， KIDERA A， NAKAMURA H. H3-rules： identification of CDR-H3 structures in antibodies [J]. FEBS Lett， 1999， 455(1-2)： 188-197.[15] KURODA D， SHIRAI H， KOBORI M， et al. Structural classification of CDR-H3 revisited： a lesson in antibody modeling [J]. Proteins， 2008， 73(3)： 608-620.[16] QIU X Q， WANG H， CAI B， et al. Small antibody mimetics comprising two complementarity-determining regions and a framework region for tumor targeting [J]. Nat Biotechnol， 2007， 25(8)： 921-929.[17] GOVINDAN S V， GOLDENBERG D M. New antibody conjugates in cancer therapy [J]. Scientific World J， 2010， 10（10）： 2070-2089.[18] TRAIL P A， KING H D， DUBOWCHIK G M. Monoclonal antibody drug immunoconjugates for targeted treatment of cancer [J]. Cancer Immunol Immunother， 2003， 52(5)： 328-337.[19] SHAO R G， ZHEN Y S. Enediyne anticancer antibiotic lidamycin： chemistry， biology and pharmacology [J]. Anticancer Agents Med Chem， 2008， 8(2)： 123-131.[20] RATHORE D， NAYAK S K， BATRA J K， et al. Expression of ribonucleolytic toxin restriction in Escherichia coli： purification and characterization [J]. FEBS Lett， 1996， 392(3)： 259-262.[21] FRANKEL A E， WOO J H. Bispecific immunotoxins [J]. Leuk Res， 2009， 33(9)： 1173-1174.[22] BAGSHAWE K D. Antibody-directed enzyme prodrug therapy (ADEPT) for cancer [J]. Expert Rev Anticancer Ther， 2006， 6(10)： 1421-1431.[23] VALLERA D A， TODHUNTER D A， KUROKI D W， et al. A bispecific recombinant immunotoxin， DT2219， targeting human CD19 and CD22 receptors in a mouse xenograft model of B cell leukemia/lymphoma [J]. Clin Cancer Res， 2005， 11(10)： 3879-3888.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
