EGFR靶向抗体融合蛋白激活免疫细胞杀伤肝癌细胞Huh7的研究

Generation of an EGFR Fusion Antibody Stimulating Immune Cells to Kill Huh7 Hepatoma Carcinoma Cell Line

目的探究靶向表皮生长因子受体(EGFR)的抗体西妥昔单抗(Cetuximab)与MHC-I(major histocompatibility complex-I)相关抗原分子MICB(MHC class I-related chain molecules B)的融合蛋白对肝癌细胞Huh7的杀伤效果。方法通过重叠PCR(polymerase chain reaction)的方法通过柔性肽G4S将西妥昔单抗重链C末端及人MICB胞外区基因连接,构建成工程载体,并通过毕赤酵母进行表达;流式检测融合蛋白对Huh7细胞的结合能力;噻唑蓝(MTT)法检测Huh7细胞的增殖抑制;通过抗体依赖的细胞介导的细胞毒作用(ADCC)实验验证融合蛋白是否能激活NK细胞杀伤肿瘤细胞。结果通过基因工程手段和毕赤酵母表达,成功获得融合抗体蛋白,经Western blot鉴定结果说明融合蛋白Cetuximab-MICB表达及装配正确。流式细胞术检测Cetuximab-MICB与肝癌细胞Huh7的结合率为72.8%,与母体西妥昔单抗结合率(75.5%)相当。细胞增殖抑制实验,与PBS组对照相比,西妥昔单抗、融合蛋白组对肝癌细胞Huh7均有抑制作用,且抑制作用呈浓度依赖性。在蛋白浓度为400 nmol/L时,Cetuximab-MICB融合蛋白组抑制率(66.09%)强于西妥昔单抗组(56.54%)。将免疫细胞PBMC(peripheral blood mononuclear cell)/NK92和肿瘤细胞Huh7共孵育,通过检测LDH(lactate dehydrogenase)的释放,发现融合蛋白比西妥昔单抗更有效地介导NK细胞对肿瘤细胞的特异性杀伤,以PBMC为效应细胞,效靶比为100:1时Huh7细胞裂解率可以达到43.58%,优于西妥昔单抗组(37.77%)。以NK92细胞为效应细胞,效靶比为10:1时Huh7细胞裂解率可以达到61.29%,明显优于西妥昔单抗组(40.54%)。结论采用毕赤酵母表达体系成功构建并表达了Cetuximab-MICB融合蛋白。Cetuximab-MICB融合蛋白具有良好的体外抗肿瘤活性,有效的抑制肿瘤细胞Huh7的增值,并能进一步通过NKG2D途径激活NK细胞加强免疫系统对肿瘤的免疫监视,为抗肿瘤治疗提供了新的思路,有潜在的临床应用前景。

Objective Understanding of the therapeutic effect of an EGFR fusion antibody Cetuximab-M ICB on Huh7 hepatoma carcinoma cell line. Methods A newfusion protein,Cetuximab-M ICB was obtained by overlapping PCR method containing the heavy chain of patented Cetuximab,G4 S linker and the major histocompatibility complex-I（ M ICB）. Pichia pastoris was used to express the fusion protein. The binding activity of Cetuximab-M ICB or Cetuximab to Huh7 was detected by flowcytometry. M ethyl thiazol tetrazolium（ M TT） assay was used to detect the inhibition of cell growth. Furthermore,antibody dependent cellular cytotoxicity（ ADCC） assay was performed to measure the killing effect of PBM Cs or NK92 on Huh7 tumor cells. Results The fusion protein,Cetuximab-M ICB was purified from culture supernatants by protein a affinity chromatograph,followed by Western blot analysis. Cetuximab-M ICB showed high binding activity on Huh7 cells and the binding rate was 72. 8%,similar to that of Cetuximab（ 75. 5%）. From the M ethyl thiazol tetrazolium（ M TT） assay,both Cetuximab and Cetuximab-M ICB groups restrained cell growth.When the concentration of antibodies was 400 n M,the inhibitory rate of Cetuximab-M ICB group（ 66. 09%）was higher than Cetuximab group（ 54. 54%）. Based on release of the lactate dehydrogenease（ LDH）,Cetuximab-M ICB also enhanced the killing effect of PBM C or NK92 on Huh7 tumor cells. At 100： 1 ratio of effective cells,PBM C to target cells,the cell lysis percent of Cetuximab-M ICB on Huh7 was 43. 58%,higher than that of Cetuximab group（ 37. 77%）. At 10： 1 ratio of PBM C to Hub7,the cell lysis percent ofCetuximab-M ICB on Huh7 became 61. 9%,higher than that of cetuximab（ 40. 54%）. Conclusion The fusion protein Cetuximab-M ICB show ed an enhanced anti-tumor activity in vitro on Huh7 hepatoma carcinoma cell line compared w ith the parent antibody Cetuximab,w hich could provide a rationale for drug development strategy forhepatoma carcinoma.