抗EGFR/VEGF双特异性单链抗体的构建及其抗卵巢癌SKOV-3的研究

Construction of bispecific single-chain diabody of anti-EGFR/VEGF and against ovarian carcinoma SKOV-3

目的将表皮生长因子受体(EGFR)和血管内皮生长因子(VEGF)同时进行靶向治疗,可协同抑制作用,增强抗肿瘤效果。方法使用重叠聚合酶链式反应(PCR)手段,西妥昔单抗的可变区和贝伐珠单抗的可变区通过G4S柔性肽连接在一起,获得同时靶向EGFR和VEGF的双特异性单链抗体(scDb);通过酶联免疫吸附测定(ELISA)检测scDb与EGFR和VEGF的结合活性,并计算亲和力常数;流式细胞术检测scDb对受体过表达的卵巢癌细胞系SKOV-3的结合能力;MTT法检测scDb对卵巢癌细胞系SKOV-3的增殖抑制;Western Blot研究与单独给药相比scDb对SKOV-3相关信号通路的影响;建立卵巢癌SKOV-3细胞荷瘤小鼠动物模型,检测scDb的体内抗肿瘤活性。结果通过重叠PCR手段和毕赤酵母X-33表达,成功获得scDb,经Western blot鉴定验证了scDb的分子量为55kD,证明表达及装配正确。ELISA检测scDb与EGFR和VEGF均有结合活性,通过EC50计算,得到scDb与EGFR的亲和力常数为1.27nM,与VEGF的亲和力常数为0.75nM。流式细胞术检测scDb与SKOV-3的结合率为72.00%。MTT实验,亲本抗体组与双特异性单链抗体组对SKOV-3均有抑制作用。通过Western blot实验表明,scDb能同时阻断EGFR和激酶结构域受体(KDR)的磷酸化,最终导致丝氨酸/苏氨酸激酶等信号通路被强烈抑制。通过SKOV-3细胞裸鼠移植瘤模型的体内抗肿瘤实验,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(74.53±9.63)%。结论该文成功构建并表达了scDb。scDb具有良好的体内外抗肿瘤活性,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 build a bispecific single-chain diabody（scDb）targeting epidermal growth factor receptor（EGFR）and vascular endothelial growth factor（VEGF）to enhance anti-tumor effect by synergistic inhibition.Methods The scDb targeting EGFR and VEGF was obtained by linking variable region of Cetuximab and Bevacizumab with G4 Sby means of PCR.ELISA was used to analyze the affinity of scDb to EGFR and VEGF,and affinity constant was calculated.The binding activity of scDb to SKOV-3 was detected by flow cytometry,and MTT assay was used to detect the inhibition of cell proliferation.Western Blot was used to research the effects of scDb on SKOV-3 signal transduction pathway.Finally,mouse tumor model was generated by endermic injecting SKOV-3 cells to detect the antitumor activity of scDb in vivo.Results The scDb was purified with PCR and X-33 expression of pichia pastoris,and the Western blot results showed that scDb had a correct molecular weight of 55 kD,proofing correct expression and assembly.ELISA revealed affinity of scDb to EGFR and VEGF,and the affinity constant was 1.27 nM to EGFR and 0.75 nM to VEGF.Binding ratio of scDb to SKOV-3 was 72.00% detected by flow cytometry.MTT assay showed that both parental antibody group and scDb group had inhibitory effect on SKOV-3.Western Blot analysis revealed that scDb could block phosphorylation of EGFR and kinase domain region receptor（KDR）and thus signal transduction pathway of serine and threonine kinase was strongly inhibited.In vivo assay of the SKOV-3 cell xenografts in nude mice showed that scDb played a stronger antitumor effect in vivo.Tumor inhibition rate of group with high dose reached 74.53±9.63%.Conclusion In this study scDb is successfully established and expressed,which maintains good antitumor effect in vivo and vitro.A new thought is proposed with potential clinical application prospect.