靶向表皮生长因子受体Ⅲ型突变体的第三代嵌合抗原受体的构建与表达

Construction and expression of a third generation chimeric antigen receptor target epidermal growth factor receptor variant Ⅲ-specific

目的:构建第三代靶向表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)的嵌合抗原受体表达载体,为肿瘤过继免疫治疗提供实验基础。方法:运用分子克隆方法将嵌合抗原受体的胞外抗原结合区(EGFRvⅢ单克隆抗体的轻链和重链可变区,EGFRvⅢsc Fv,726 bp)、铰链区/穿膜区(213 bp)、共刺激分子(CD28和CD137的胞内信号区,123 bp和126 bp)和免疫受体酪氨酸活化基序(CD3ζ链,336 bp)连接成EGFRvⅢsc Fv-CD28-CD137-CD3ζ(EGFRvⅢ/3CAR),克隆入真核表达载体p CDHCMV-MCS-EF1-cop GFP的EcoRⅠ和Bam HⅠ位点,转染293T细胞48 h后提取蛋白,采用SDS-PAGE和Western blotting检测EGFRvⅢ/3CAR的表达。结果:EGFRvⅢsc Fv和基因合成的CD28-CD137-CD3ζ表达框通过重叠延伸PCR拼接成EGFRvⅢ/3CAR,限制性内切酶片段分析和DNA测序均验证了重组载体序列完全正确,Western blotting应用抗人-CD3ζ抗体检测到EGFRvⅢ/3CAR在293T细胞中的完整表达(相对分子质量约58 k D)。结论:成功构建嵌合抗原受体EGFRvⅢ/3CAR表达载体,为嵌合抗原受体修饰T细胞靶向治疗肿瘤提供研究基础。

Objective: To design and construct a vector expressing a human epidermal growth factor receptor variant Ⅲ( EGFRvⅢ)-specific third generation chimeric antigen receptor( CAR) for potential use in adoptive immunotherapy of cancer. Methods: An anti-EGFRvⅢ CAR,referred to as EGFRvⅢ /3CAR,comprising the variable fragment( EGFRvⅢsc Fv),hinge region /transmembrane region,CD28 & CD137 intracellular signal region,and CD3ζ chain of a monoclonal antibody against human EGFvⅢ was generated and inserted in frame into p CDH-CMV-MCS-EF-cop GFP EcoR1 and Bam H1 sites. The integrity of the resultant recombinant vector was confirmed by restriction enzyme mapping and sequence analysis. For further functional integrity verification,the recombinant vector was transfected into 293 T cells and the fusion protein expression was assessed by Western blotting. Results: DNA sequencing and restriction enzyme mapping demonstrated the sequence of EGFRvⅢ /3CAR was corret. Western blotting detected EGFRvⅢ /3CAR protein at approximately58 k D in 293 T cells transfected with the EGFRv Ⅲ /3CAR construct. Conclusion: We have successfully designed and constructed a recombinant vector expressing a human EGFRvⅢ-specific third generation CAR,which may prove usefull in further studies on CAR-based cancer immunotherapy.