一种全人源IgG样抗EGFR/抗KDR双特异性抗体的构建与表达

Construction and Expression of a Human IgG-Like Anti-EGFR/AntiVEGFR2 Bispecific Antibody

为增强抗EGFR或抗VEGFR2(KDR)单克隆抗体的抗肿瘤作用,作者将抗人EGFR单克隆抗体(Cetuximab)可变区基因和抗人KDR单克隆抗体(m Ab-04)可变区基因融合并与人Ig G1的Fc段基因通过柔性肽连接在一起,得到全人源Ig G样抗EGFR/抗KDR双特异性抗体(Bi-Ab)的基因。将Bi-Ab基因插入载体p PICZα,转化毕赤酵母X-33并诱导表达。发酵液经硫酸铵沉淀法初步提取后采用Protein A亲和层析柱进一步分离纯化,得到电泳纯的Bi-Ab蛋白,其产量约为2.3 mg/L发酵液。表面等离子共振法(SPR)分析显示,Bi-Ab与EGFR或KDR的结合亲和力与其亲本Cetuximab或m Ab-04相近,表明该双特异性抗体Bi-Ab能与EGFR和KDR特异性结合,可用于进一步的抗肿瘤活性研究。

Recent studies have shown that both Epidermal growth factor receptor（ EGFR） and Vascular endothelial growth factor receptor 2（ VEGFR2,KDR） play critical roles in tumorigenesis of a variety of cancers,therefore anti-EGFR and anti-KDR antibodies are used to treat cancers in the current study. To improve the anti-tumor effect of anti-EGFR and anti-VEGFR2 antibodies,a recombinant human Ig G-like bispecific antibody（ Bi-Ab） co-targeting EGFR and KDR was developed. The gene of Bi-Ab was constructed from variable regions of two different antibodies,cetuximab targeting EGFR and m Ab-04（ a fully human anti-KDR antibody developed in our lab） targeting KDR,whereas the Fc fragment was obtained from human Ig G1. After fusing with a linker using overlap PCR,the gene of interest（ Bi-Ab） was inserted into p PICZα plasmid. The amplified recombinant plasmid was firstly cloned into Escherichia coli DH5α to obtain enough concentration to transform the ultimate host Pichia pastoris X-33 host. The expressed of target protein was induced by methanol and directed into the medium. After initial separation by ammonium sulfate precipitation,Bi-Ab was purified by Protein A affinity purification. Bi-Ab protein was characterized using Western blotting. Surface plasmon resonance spectroscopy（ SPR） was subsequently used to analyze the interactions and binding kinetics between the Bi-Ab and EGFR or KDR. The experimental results indicated that the Bi-Ab was expressed in the medium with 0. 5% methanol induction,and that it could be purified by ammonium sulfate precipitation and Protein A affinity purification. The coomassie brilliant blue kit was used to detect the quantitation of target protein,and the yield was about 2. 3 mg per liter medium. The SPR experimental results indicated that the equilibrium dissociation rate constant（ KD） of Bi-Ab to EGFR or KDR was 1. 45 × 10- 9mol / L and 2. 37 × 10- 9mol / L respectively. The affinity of Bi-Ab to EGFR / KDR was similar to m Ab-04 and cetuximab. In conclusion,Bi-Ab could strongly bind to both EGFR and KDR,and it could be used for further anti-tumor research.