抗人VEGFR2与抗人EGFR的双特异性重组蛋白治疗结直肠癌的体内外研究  

The generation of a VEGFR2/EGFR targeted bispecific singlechain diabody which against the colorectal carcinoma in vitro and vivo

作　　者：沈莹 高啸 谭晓燕 SHEN Ying;GAO Xiao;TAN Xiao-yan(Department of Geriatrics,Liyuan hospital,Tongji Medical College,Huazhong University of Science and Technology.Wuhan 430077,China)

机构地区：[1]华中科技大学同济医学院附属梨园医院老年病科,湖北武汉430077 [2]华中科技大学同济医学院附属梨园医院内分泌科,湖北武汉430077 [3]华中科技大学同济医学院附属梨园医院消化内科,湖北武汉430077

出　　处：《解剖学研究》2018年第4期248-252,共5页Anatomy Research

基　　金：湖北省自然科学基金(2014CFB434)

摘　　要：目的构建一种可以同时靶向抑制VEGFR2和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。方法通过Overlap PCR的方法通过柔性肽G4S将雷莫芦单抗的可变区与西妥昔单抗连接,构建入工程载体,并通过毕赤酵母表达体系进行表达纯化,获得同时靶向VEGFR2和EGFR的双特异性单链抗体(scDb),命名为VE-scDb;流式细胞术检测双特异性单链抗体对EGFR和VEGFR2过表达的结直肠癌细胞系HT-29的结合能力;将单链抗体与碱性荧光染料罗丹明B偶联,使用激光共聚焦检测单链抗体在体外对HT-29细胞的靶向能力;建立HT-29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。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VE-scDb,经Western blot鉴定验证了VE-scDb表达及装配正确。流式细胞术检测VE-scDb与结直肠癌细胞HT-29的结合率为50.1%,与亲本雷莫芦单抗(55.0%)与西妥昔单抗结合率(53.2%)相当。体外靶向性实验验证VE-scDb可以在体外很好的靶向结直肠癌细胞系HT-29,VE-scDb组的细胞平均荧光强度为97.24±6.72,明显优于阻断对照组(26.29±3.02)(P<0.01)。通过裸鼠移植瘤模型的体内抗肿瘤检验,VE-scDb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(73.70±6.16)%,明显优于亲本雷莫芦单抗(44.80±5.91)%和西妥昔单抗组(37.75±14.04)%(P<0.01)。结论本文采用毕赤酵母表达体系成功构建并表达了双特异性单链抗体VE-scDb。VE-scDb具有良好的体外肿瘤靶向性,并可能有效的抑制结直肠癌细胞HT-29的体内外增殖。该设计为抗肿瘤治疗提供了新的思路,有潜在的临床应用前景。Objective To improve the anti-tumor effect in vitro and vivo and reduces risks of drug resistance, a bispecific single-chain diabody（scDb） was constructed from the vairable domain genes of two antibodies directed against vascular endothelial growth factor receptor 2 （VEGFR2）and epidermal growth factor receptor （EGFR）. Methods The new bispecific single-chain diabody was obtained by overlapping PCR between variable region of Ramucirumab , G4S linker and variable region of Cetuximab. And Pichia pastoris was used to express VE-scDb. The binding activity of VE-scDb to colorectal carcinoma cell line HT-29 was detected by flow eytometry. The in-vitro targeting prosperities of VE-scDb to colorectal cancer cell line HT-29 was detected by laser confocal microscope through linking VE-scDb with alkaline dye Rhodamine B. Finally, mouse tumor model was generated by endermic injecting HT-29 cells, and we used this model to detect the antitumor activity of VE-scDb in vivo, Results The bispecific single-chain diabody （scDb） was purified from culture supernatants by nickel affinity chromatograph. And the Western blot result showed that VE-scDb has a correct molecular weight. What＇s more, VE-scDb maintained binding activity to HT-29 and the binding ratio was 50.1%, which similar to patented Ramucirumab （55.0%） and Cetuximab （53.2%）. From the in-vitro targeting experiment VE-scDb can target HT-29 in vitro. And the average fluorescence intensity of VE-seDb was 97.24±6.72, which better than that of blocking group （26.29±3.02） （P 〈 0.01 ）. In vivo assay of the HT-29 cell xenografts in nude mice showed that VE-scDb play a stronger antitumor effect in vivo, and the tumor inhibitory rate of high close group （73.70±6.16）% was better than patented Ramueirumab（44.80±5.91）% and Cetuximab （37.75±14.04）%（P 〈 0.05 or P 〈 0.01 ）. Conclusion We established and express the bispecific singlechain diabody, which maintained the binding activity of parent antibody. Comparing with single paren

关 键 词：血管内皮生长因子受体2 表皮生长因子受体 结直肠癌 双特异性单链抗体 

分 类 号：R735.34[医药卫生—肿瘤]