腺病毒介导的全抗体基因治疗卵巢癌的实验研究

目的　探讨应用基因治疗方法表达完全抗体的可行性及其对肿瘤的治疗作用。方法构建携带曲妥珠单抗 Trastuzumab ,商品名 :赫赛汀 (Herceptin) ]全抗体基因的腺病毒载体 ,利用重组技术获得增殖缺陷型腺病毒Ad SG Her ,用Ad SG Her转染人正常肝细胞株L 0 2 ,判断体外肝细胞是否可以表达Herceptin抗体 ;将卵巢癌SK OV 3细胞接种于裸鼠皮下 ,3d后把 4 0只成瘤裸鼠随机分为3个治疗组和 1个对照组 ,每组各 10只。治疗组裸鼠尾静脉分别注射不同滴度的Ad SG Her :治疗A组注射 2× 10 9空斑形成单位 ( pfu) /只 ,B组注射 1× 10 9pfu/只 ,C组注射 5× 10 8pfu/只。用酶联免疫吸附试验 (ELISA)分别在体外转染Ad SG Her后的第 3天和第 7天 ,以及裸鼠接受Ad SG Her治疗后的第 3、7、10、14、2 1、2 8、35天检测Herceptin的表达量 ;通过间接免疫荧光法 (IFA)和Western印迹法分别检测血清中的抗体与Her2过表达卵巢癌细胞株SK OV 3结合的特异性以及所表达全长抗体的完整性 ;同时观察不同滴度的Ad SG Her对荷SK OV 3实体瘤裸鼠的治疗作用。结果　构建的带有全长Herceptin基因的重组腺病毒Ad SG Her ,在体外及体内均能高效表达人源化抗体Herceptin。Western印迹显示 :该人源化抗体与商品化Herceptin具有相同大小的轻链与

Gene therapy for ovarian cancers by adenovirus-mediated complete antibody gene

OBJECTIVE: To study the feasibility of adenoviral transduction of Herceptin complete antibody gene and its effect on Her2 over-expressing cancer. METHODS: The genes of VH and VL from the monoclonal antibody Herceptin were cloned into the genome of replication-defective adenovirus by viral recombination technology to produce the recombinant adenovirus Ad-SG-Her. Normal human liver cells of the line L-02 were transfected with Ad-SG-Her and ELISA was used to detect the expression of Herceptin antibody 3 and 7 days after. Forty BALB/c nude mice were inoculated with Her2 high-expressing oophoroma cells of SK-OV-3 line and were randomized into 4 equal groups: group A injected with Ad-SG-Her at the dosage of: 2 x 10(9) plaque forming unit (pfu) through the caudal vein, group B injected with Ad-SG-Her at the dosage of 1 x 10(9) pfu, group C injected with Ad-SG-Her at the dosage of 5 x 10(8) pfu, and control group. On the days 3, 7, 10, 14, 21, 28, and 35 after the injection of virus, the antibody expression in the serum was measured by ELISA and the size of tumor was measured vernier caliper. Western blot and IFA was used to detect the specificity for Her2-overexpressing ovarian cancer cell lines SK-OV-3 and the integrity of complete antibody. Anti-tumor effects were also observed in nude mice bearing SK-OV-3 tumors. RESULTS: The constructed recombinant adenovirus Ad-SG-Her could express Herceptin efficiently both in vitro and in vivo. The biological activity of the expressed antibody was similar to that of the commercial Herceptin as shown by Western blotting, IFA, and ELISA. Herceptin expression of Ad-SG-Her was detected since day 3 after treatment in the groups A, B, and C and reached the peak on days 7 - 10. The expression lasted for four weeks or so. The expression level was significantly different between group A and the groups B and C (all P < 0.05), however, without a significant difference between the group B and group C. The antibody expression of group A might increase to 103.5 micro g/ml, high enough to inhibit tumor growth and induce tumor cell apoptosis. The antibody expression of the group B was below 40 micro g/ml, and that of the group C was below 30 micro g/ml. Furthermore the expressed antibody doses were statistically significantly different at different time points. Almost no tumor growth was seen in the group A during the observation period in comparison with the groups B and C and the control group (all P < 0.05). The tumor growth was almost not influenced in the group B and C and the control group. CONCLUSION: Ad-SG-Her efficiently expresses humanized complete Herceptin with effective bioactivity and induces long-term stable expression both in vitro and in vivo. The system may serve as a new antitumoral gene therapy strategy in antibody field.