【作者】 郭晓芳；

【导师】 甄永苏； 李电东； 邵荣光；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2009， 博士

【摘要】 背景与目的:表皮生长因子受体EGFR/HER1和HER2/neu都是跨膜受体酪氨酸激酶家族（ErbB）的成员,ErbB受体家族与人类癌症的关系非常密切,EGFR和HER2在多种肿瘤细胞表面异常表达,且ErbB受体发生改变的肿瘤通常更具有侵袭性,病人也往往预后不良,这使得EGFR和HER2成为抗肿瘤靶向治疗的理想靶点。力达霉素（Lidamycin,LDM）是迄今报道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素,由辅基蛋白（LDP）和烯二炔结构的发色团（AE）两部分组成,发色团是力达霉素分子的活性部分。本研究的目的是构建一种靶向EGFR和HER2的双特异性强化融合蛋白（Ec-LDP-Hr-AE）,并对其体内外抗肿瘤活性进行检测。方法:双特异性融合蛋白Ec-LDP-Hr的构建主要采用PCR扩增和DNA克隆技术。pelB信号肽序列、寡肽配体Ec基因序列、寡肽配体Hr基因序列以及（GGGGS）₂连接肽基因序列均采用人工合成的方法得到,并作为PCR引物,经过5轮PCR扩增以及DNA克隆后,得到了融合蛋白基因Ec-ldp-Hr,将该基因经过双酶切后插入到pET30a（+）表达载体中,得到重组表达载体pET-Ec-ldp-Hr。为了进行比较,本研究同时构建了单靶点的重组表达载体pET-Ec-ldp和pET-ldp-Hr。将三种表达载体均转化到大肠杆菌BL21（DE3）star^TM中,加入IPTG进行诱导表达,SDS-PAGE分析目的蛋白的表达情况。融合蛋白的提取采用渗透压震惊法,使用Ni²⁺亲和层析法分离纯化融合蛋白,HPLC检测融合蛋白的纯度,Western Blot对纯化出的融合蛋白进行鉴定。融合蛋白与肿瘤细胞表面EGFR和HER2的亲和活性主要采用了ELISA、细胞免疫荧光化学法、基于流式细胞术的免疫荧光试验、竞争性免疫荧光试验以及免疫共沉淀技术进行分析。将LDM活性发色团分子（AE）与融合蛋白在体外进行分子组装,得到强化融合蛋白（Ec-LDP-Hr-AE）,采用MTT法测定强化融合蛋白对肿瘤细胞的体外杀伤活性。细胞免疫荧光染色和基于流式细胞术的荧光检测用于分析融合蛋白是否可内吞进入细胞以及检测内吞效率。双特异性强化融合蛋白Ec-LDP-Hr-AE的体内抗肿瘤活性采用人卵巢癌SK-OV-3裸鼠移植瘤模型进行评价。结果:经过5轮PCR扩增以及DNA克隆后,得到了融合蛋白基因Ec-ldp-Hr,将其插入到表达载体后成功实现了在大肠杆菌中的表达。Ec-LDP-Hr蛋白和Ec-LDP蛋白主要以可溶性的形式分泌到了大肠杆菌周质腔中,而LDP-Hr蛋白除存在于周质腔中外,还分泌到了培养液上清中。Ni²⁺亲和层析对周质腔中的蛋白进行纯化,得到的目的蛋白经HPLC检测纯度均在95%以上。Ec-LDP-Hr、Ec-LDP和LDP-Hr三种融合蛋白的产量分别为每升发酵液得到9 mg、18 mg和60 mg活性蛋白。将纯化的蛋白用Western blot方法进行鉴定,证实了纯化的蛋白为带有His-tag标签的目的蛋白。ELISA和基于流式细胞术的免疫荧光试验结果显示Ec-LDP-Hr蛋白对EGFR和HER2高表达的肿瘤细胞系,如A431、SK-BR-3、SK-OV-3、MCF-7均有很强的结合活性,而对不表达EGFR和HER2的NIH 3T3细胞则无亲和活性。细胞免疫荧光化学试验证实了Ec-LDP-Hr蛋白可以与A431和SK-BR-3细胞表面的受体结合,免疫共沉淀试验则进一步证明了Ec-LDP-Hr蛋白是与A431和SK-BR-3细胞表面的EGFR和HER2结合的;竞争性免疫荧光试验结果显示,Ec-LDP-Hr蛋白可部分抑制抗EGFR和抗HER2抗体与各自抗原的结合能力,表明该蛋白可竞争性的与EGFR和HER2结合。将AE分子与融合蛋白在体外进行分子组装后,经HPLC检测在350nm处出现了特定的吸收峰,表明AE分子与融合蛋白进行了成功组装,得到了强化融合蛋白Ec-LDP-Hr-AE。MTT试验结果显示强化融合蛋白Ec-LDP-Hr-AE对不同的肿瘤细胞系均表现出了强烈的杀伤作用,对A431、SK-BR-3、SK-OV-3、MCF-7和NIH 3T3细胞的IC₅₀值分别为:4.25×10^-13mol/L、4.04×10^-14mol/L、1.55×10^-13mol/L、4.7×10^-12mol/L和2.33×10^-11mol/L。对于高表达EGFR和HER2的肿瘤细胞系,双特异性强化融合蛋白Ec-LDP-Hr-AE表现出了比LDM和单靶点的强化融合蛋白Ec-LDP-AE和LDP-Hr-AE更强的杀伤活性,而对于EGFR和HER2不表达的NIH 3T3细胞,Ec-LDP-Hr-AE蛋白的IC₅₀值则高于LDM和Ec-LDP-AE、LDP-Hr-AE蛋白,表明双特异性的Ec-LDP-Hr-AE蛋白提高了对肿瘤细胞作用的特异性。细胞内吞试验结果表明,Ec-LDP-Hr和Ec-LDP-Hr-AE均可通过EGFR和HER2的介导进入细胞质中发挥作用,并且与单靶点的Ec-LDP和LDP-Hr蛋白相比,双特异性的Ec-LDP-Hr蛋白表现出更高的内吞效率。强化融合蛋白对人卵巢癌SK-OV-3裸鼠移植瘤的治疗结果显示,Ec-LDP-Hr-AE蛋白可显著的抑制移植瘤的生长,在0.3、0.4和0.5 mg/kg的剂量下,其抑瘤率分别为64.7%、79.5%和91.3%,其中0.5 mg/kg剂量组与LDM处理组（抑瘤率为65.6%）有显著性差异。在相同剂量下,双特异性Ec-LDP-Hr-AE蛋白同样表现出了比单靶点的Ec-LDP-AE、LDP-Hr-AE蛋白更强的抑制肿瘤生长的作用,三种强化融合蛋白的抑瘤率分别为:90.2%、83.9%和80.3%。结论:本研究成功构建并表达了一种含有EGFR和HER2寡肽配体以及力达霉素的双特异性强化融合蛋白Ec-LDP-Hr-AE,该融合蛋白可特异地与肿瘤细胞表面的EGFR和HER2受体结合,并对体外培养的不同肿瘤细胞系具有强烈杀伤作用,在体内对人卵巢癌裸鼠移植瘤有很强的生长抑制作用。Ec-LDP-Hr-AE蛋白具有双靶点、小型化和高效化的特点,具有发展成肿瘤靶向性治疗药物的潜能。更多还原

【Abstract】 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） star^TM 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.更多还原