靶向HER2/EGFR分子双特异性单链抗体的构建及其体内外抗乳腺癌的研究  

Study on construction of targeted HER2/EGFR bispecific single-chain antibody and its anti-breast carcinoma in vitro and vivo

作　　者：郭艳[1] 张长庆 GUO Yan;ZHANG Changqing(Department of Breast Oncology,Affiliated People′s Hospital of Inner Mongolia Medical University,Hohhot,Inner Mongolia 010020,China)

机构地区：[1]内蒙古医科大学附属人民医院乳腺肿瘤科,呼和浩特010020

出　　处：《检验医学与临床》2018年第19期2887-2891,共5页Laboratory Medicine and Clinic

摘　　要：目的构建一种可以同时靶向抑制人类表皮生长因子受体2(HER2)和表皮生长因子受体(EGFR)分子的双特异性单链抗体,从而降低单一通路抑制而造成的耐药性,增强抗肿瘤效果。方法采用Overlap PCR法通过柔性肽(G4S)将曲妥珠单抗的可变区与西妥昔单抗连接,构建工程载体,并通过大肠埃希菌系统进行表达纯化,获得同时靶向HER2和EGFR的双特异性单链抗体;流式细胞术检测双特异性单链抗体对EGFR和HER2过表达的乳腺癌细胞BT474的结合能力;噻唑蓝(MTT)法检测单链抗体对BT474乳腺癌细胞的增殖抑制;建立BT474荷瘤小鼠动物模型,检测双特异性单链抗体的体内抗肿瘤活性;使用免疫组织化学检测单链抗体是否可以抑制表皮生长因子受体(EGFR)的磷酸化,并对肿瘤细胞增殖指标Ki-67进行检测。结果通过基因工程手段和毕赤酵母表达及镍柱纯化,成功获得双特异性单链抗体,经蛋白印迹法鉴定验证了双特异性单链抗体表达及装配正确。流式细胞术检测双特异性单链抗体与乳腺癌细胞BT474的结合率为53.3%,与曲妥珠单抗(69.0%)、西妥昔单抗结合率(60.3%)相当。MTT法中,与磷酸盐缓冲液(PBS)组相比,曲妥珠单抗组、西妥昔单抗组与双特异性单链抗体组对乳腺癌细胞BT474均有抑制作用,且抑制作用呈浓度依赖性。在蛋白水平为400nmol/L时,双特异性单链抗体组抑制率[(65.19±4.21)%]强于曲妥珠单抗组[(40.67±1.78)%]与西妥昔单抗组[(32.20±2.94)%]。通过裸鼠移植瘤模型的体内抗肿瘤实验,双特异性单链抗体组在体内仍能发挥较强的抗肿瘤效果,高剂量组肿瘤抑制率可以达到(74.32±4.37)%,明显优于曲妥珠单抗组(44.83±6.02)%与西妥昔单抗组(39.44±8.75)%。通过免疫组织化学对给药后的肿瘤组织进行分析,与PBS组相比,双特异性单链抗组可以明显抑制BT474皮下移植瘤组织中p-EGFR、Ki-67的表达量。结论采用毕赤酵母表达体系Objective To construct a bispecific single-chain antibody with targeted inhibition on HER2/EGFR,thus to reduce the drug resistance caused by single channel inhibition and enhance the anti-tumor effect. Methods The overlap PCR was used to link the variable region of trastuzumab and cetuximab by flexible peptide （G4S） for constructing the engineering vector.Then the expression purification was performed by Escherichia coli system for obtaining simultaneously targeted HER2 and EGFR bispecific single-chain antibody.The binding activity of bispecific single-chain antibody to HER2 and EGFR overexpression breast carcinoma cell line BT474 was detected by flow cytometry.The methyl thiazol tetrazolium （MTT） assay was used to detect the inhibition of single-chain antibody to the proliferation of breast carcinoma BT474 cells.Finally,the tumor-bearing mouse animal model of BT474 cells was established.Then the in vivo anti-tumor activity of bispecific monochain antibody was detected;the immunohistochemical method was used to detect whether the monochin antibody can inhibit the phosphorylation of dermal growth factor receptor （EGFR） and tumor cellular proliferative activity index Ki-67 was detected. Results The bispecific single-chain antibody was successfully obtained by using the genetic engineering technique,pichia pastoris expression and Ni-sepharose purification.The western blot identification verified the correct expression and assembling of bispecific single-chain antibody.The binding rate of bispecific single-chain antibody to BT474 cells was 53.3% by flow cytometry,which was similar to trastuzumab （69.0%） and cetuximab （60.3%）.In the MTT assay,compared with phosphade butter solution （PBS） group,trastuzumab group,cetuximab group and bispecific single-chain antibody group all had the inhibiting effect on breast carcinoma BT474 cells,moreover their inhibiting effect showed the concentration dependence.And when the protein level was 400 nmol/L,the inhibitory rate of bispecific single-chain antibo

关 键 词：人类表皮生长因子受体2 表皮生长因子受体 乳腺癌 双特异性单链抗体 

分 类 号：R737.9[医药卫生—肿瘤]