Construction of an Energized Fusion Protein LDP-Hr-AE Targeting Human Epidermal Growth Factor Receptor 2 and Its Antitumor Activity

GUO Xiao-Fang, ZHU Xiao-Fei, ZHONG Gen-Shen, ZHEN Yong-Su

Chinese Pharmaceutical Journal ›› 2012, Vol. 47 ›› Issue (12) : 941-947.

Chinese Pharmaceutical Journal ›› 2012, Vol. 47 ›› Issue (12) : 941-947.

Construction of an Energized Fusion Protein LDP-Hr-AE Targeting Human Epidermal Growth Factor Receptor 2 and Its Antitumor Activity

  • GUO Xiao-fang 1， ZHU Xiao-fei 1， ZHONG Gen-shen 2， ZHEN Yong-su3*
Author information +
History +

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.

Key words

human epidermal growth factor receptor 2 / fusion protein / lidamycin / cell apoptosis / antitumor

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations
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

References

[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.

59

Accesses

0

Citation

Detail
Sections
Recommended

/

〈 〉