| Background and Objective:The epidermal growth factor receptor EGFR/HER1 and HER2/neu are members of receptor tyrosine kinase family.Overexpression of EGFR and HER2 has been observed in a variety of human tumors,making these receptors promising targets for directed tumor therapy.Lidamycin(LDM) is an enediyne antibiotic with highly potent antitumor activity.This study was to construct a bispecific fusion protein consisting of EGFR and HER2 peptide ligands and lidamycin,and investigate its antitumor efficacy.Methods:Polymerase chain reaction and DNA cloning techniques were used to construct the gene of fusion protein.The DNA sequences of pelB signal peptide,peptide ligand Ec,(GGGGS)2-linker and peptide ligand Hr were all synthesized and used as PCR primers.After five rounds of PCR and DNA cloning,the gene coding for fusion protein Ec-LDP-Hr was successfully constructed and was inserted into the expression vector pET30a(+) under an isopropyl-β-thiogalactopyranoside inducible promoter.Genes coding for monospecific fusion protein Ec-LDP and LDP-Hr were created by the same way.Three recombinant plasmids were transformed into E.coli strain BL21(DE3) starTM and the fusion proteins were expressed after the IPTG induction. Proteins from periplasmic space of E.coli were prepared with osmotic shock method and the fusion proteins were purified by Ni2+ affinity chromatography.SDS-PAGE and HPLC were used to analyze the purity of fusion proteins and Western blot was used to verify the fusion proteins.The binding affinity of fusion proteins to EGFR and HER2 on the membrane of different cancer cell lines was analyzed by ELISA,immunofluorescent cytochemical staining,FCM-based immunofluorescence detection,competitive immunofluorescence assay and co-immunoprecipitation assay.The energized fusion proteins Ec-LDP-Hr-AE,Ec-LDP-AE and LDP-Hr-AE were prepared by integrating the active enediyne chromophore(AE) of lidamycin into the Ec-LDP-Hr,Fc-LDP and LDP-Hr protein,respectively.Immunoftuorescent staining and FCM-based immunofluorescence detection were used to analyze the internalization efficiency of fusion proteins.The cytotoxicity of energized fusion proteins was measured by MTT assay,and the in vivo efficacy of growth inhibition by energized fusion proteins was evaluated with human ovarian carcinoma SK-OV-3 xenografls model.Results:The genes coding for fusion proteins Ec-LDP-Hr,Ec-LDP,and LDP-Hr were successfully constructed and the encoded proteins were expressed in the periplasmic space of E.coli. Fusion proteins were purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 95%as determined by HPLC.The production of Ec-LDP-Hr, Ec-LDP and LDP-Hr was 9 mg,18 mg and 60 mg per liter fermentation broth, respectively.ELISA and FCM-based immunofluorescence assay revealed that Ec-LDP-Hr protein has strong binding activity to cancer cell lines highly expressing EGFR and HER2,such as A431,SK-BR-3,SK-OV-3 and MCF-7 cells.However, Ec-LDP-Hr has no binding activity to EGFR and HER2 negative NIH 3T3 cell.The results of imrnunofluorescent cytochemical staining showed that Ec-LDP-Hr protein bound to the receptors of A431 and SK-BR-3 cells,and the co-immunoprecipitation assay proved that Ec-LDP-Hr protein specifically binds to the EGF receptor and HER2. The inhibition on the binding of anti-EGFR and anti-HER2 mAbs with their antigens by Ec-LDP-Hr protein obtained by competitive immunofluorescence assay provided new evidence that Ec-LDP-Hr binds to EGFR and HER2 with high affinity.Internalization assay indicated that Ec-LDP-Hr was taken up by SK-OV-3 cells.Furthermore, Ec-LDP-Hr protein was more effectively taken up by SK-OV-3 and SK-BR-3 cells than the monospecific fusion proteins.The bispecific energized fusion protein Ec-LDP-Hr-AE showed potent cytotoxicity to A431,SK-BR-3,SK-OV-3,MCF-7,and NIH 3T3 cells with IC50 value of 4.25×10-13mol/L,4.04×10-14mol/L,1.55×10-13mol/L,4.7×10-12mol/L, and 2.33×10-11mol/L,respectively.Ec-LDP-Hr-AE shows more potent cytotoxicity to EGFR and HER2 overexpressing cells(e.g.A431,SK-BR-3,SK-OV-3,MCF-7 cells), however,it is less cytotoxic to EGFR and HER2 negative NIH 3T3 cells compared with LDM and monospecific energized fusion proteins.Results of in vivo efficacy study have shown that the growth of established tumors was significantly inhibited when treated with Ec-LDP-Hr-AE.On day 30,Ec-LDP-Hr-AE at the dose of 0.3mg/kg,0.4mg/kg and 0.5mg/kg inhibited the growth of ovarian carcinoma SK-OV-3 xenografts by 64.7%, 79.5%and 91.3%,respectively.The group treated with 0.5 mg/kg Ec-LDP-Hr-AE showed significant differences with group treated with 0.05 mg/kg LDM which inhibited the xenografts by 65.6%.The antitumor activity of bispecific Ec-LDP-Hr-AE is more remarkable than that of monospecific energized fusion proteins Ec-LDP-AE and LDP-Hr-AE at the same dose,and these three energized fusion proteins inhibited the xenografts growth by 90.2%,83.9%,and 80.3%,respectively.Conclusion:A bispecific energized fusion protein Ec-LDP-Hr-AE was successfully constructed and expressed in E.coli.Ec-LDP-Hr-AE protein binds to EGF receptor and HER2 specifically and shows more potent cytotoxicity to different kinds of tumor cell lines overexpressing EGFR and HER2,as compared with corresponding monospecific energized fusion proteins.In addition,Ec-LDP-Hr-AE has remarkable antitumor activity in human ovarian carcinoma xenografts nude mice model.These properties,together with its two-receptor targeting and much smaller size,suggested that Ec-LDP-Hr-AE would be a promising candidate for cancer-targeting therapy.
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