人源化单链抗体与白细胞介素2融合蛋白靶向治疗肿瘤的实验研究

Suppression of established melanoma in mice by a humanized single-chain Fv antibody/human interleukin-2 fusion protein therapy

目的评价人源化抗Her2/neu单链抗体及人白细胞介素2(hIL-2)双功能融合蛋白H520C9scFv-hIL-2治疗Her2/neu阳性肿瘤的效果。方法将构建的表达载体转染293细胞,G418筛选阳性克隆建立稳定表达细胞系。免疫印迹法(Western blotting)、酶联免疫吸附试验(ELISA)及[3H]脱氧胸苷(3H-TdR)渗入法检测融合蛋白浓度和生物学活性。建立荷瘤小鼠肿瘤模型,静脉给药,观测肿瘤体积变化判断治疗效果。结果细胞培养上清中融合蛋白浓度达(1.2±0.23)mmol/L,5μl上清即可在46000处见到清晰蛋白条带。纯化后的融合蛋白两个功能单位活性良好,在荷瘤小鼠肿瘤模型治疗实验中显示了明确的治疗效果。结论哺乳动物细胞可高效表达生物活性良好的融合蛋白H520C9scFv-hIL-2,该蛋白在体内实验中有明显的抑瘤作用,显示了较好的应用前景。

Objective To evaluate the potent antitumor responses of a humanized single-chain Fv antibody/human interleukin-2 fusion protein （H520CgscFv-hIL-2） in tumor-bearing mice by targeting cytokine activity to the tumor microenvironment. Methods The constructed expression vectors were transfected into 293 cells. The positive clone was obtained to make stable cell line with G418 selection. Western blot, ELISA and 3 H-TdR were used to measure its concentration and bioactivities. The antitumor effect was evaluated in an animal tumor model by measurement of tumor volume after i. v injection of the fusion protein. Results The concentration of fusion protein was （ 1.2 ± 2.4） mmol/L in supernatant, and only 5 μl of the supernatant was able to be shown to migrate as single band of 47 000 by Western blot analysis. The fusion protein was determined to retain the bioactivities of hIL-2（P 〉 0.05 vs standard rhIL-2 ） and the antigen-binding activity （P 〉 0. 05 vs H520CgscFv）. It was also shown to reduce markedly tumor burden in tumor-bearing mice. Conclusions Taken together, the efficient expression of the fusion protein is performed in 293 cells. The protein retains both hIL-2 bioactivities and antigen-binding specificity. It is observed to inhibit the growth of established tumor, and indicates that the therapy with tumor targeted IL-2 provides an approach for the rational design novel cancer immunotherapy modalities.