卵巢上皮性癌6B11抗独特型微抗体疫苗免疫方案的探讨

Study of immunization protocol of ovarian carcinoma associated 6B11 anti-idiotypical minibody

目的探讨卵巢上皮性癌6B11抗独特型微抗体(6B11微抗体)的最佳免疫剂量和免疫间隔时间,为用于临床治疗卵巢上皮性癌打下基础。方法(1)采用基因工程方法构建6B11微抗体的重组表达载体,并将其转化到大肠杆菌BL21中进行表达。直接酶联免疫吸附试验(ELISA)法测定其免疫学活性。(2)将BALB/c小鼠随机分为A、B、C、D、E、F组,于第1、14、28天时均分别给予6B11微抗体150、100、50μg及小鼠IgG100μg、人IgG100μg、磷酸盐缓冲液100μl,其中A、B、C组为实验组,D、E、F组为对照组。间接ELISA法测定各组小鼠血清中抗卵巢上皮性癌6B11抗独特型抗体(6B11抗体)的抗体(即Ab3)的水平,并观察其变化规律。(3)将小鼠脾淋巴细胞作为效应细胞,人卵巢上皮性癌细胞株SKOV3细胞作为靶细胞,设定效靶比为250∶1、125∶1、60∶1、30∶1,51Cr释放实验检测各组小鼠脾淋巴细胞产生的抗体依赖的细胞毒效应(ADCC);设定血清稀释度为1∶2、1∶25、1∶50,51Cr释放实验检测各组小鼠脾淋巴细胞产生的补体依赖的细胞毒效应(CDC)。分别于紫外光波长490nm时测定其吸光度(A)值。结果(1)大肠杆菌BL21中成功表达6B11微抗体,并证实其保留了6B11抗体的免疫学活性。(2)用上述不同剂量6B11微抗体免疫BALB/c小鼠,在第14天注射后1周,血清中可检测到较低水平Ab3;第28天注射后1周,Ab3水平达到最高值,A、B、C组平均A值分别为1·16、1·11、1·06;此Ab3水平持续6周,6周内A、B、C组平均A值分别为1·05、1·06、0·94,B组略高于A、C两组,但3组间比较,差异无统计学意义(P>0·05);第7周Ab3水平明显下降。此时,给予加强免疫,1周后Ab3水平很快升高,并持续至少2周。(3)在4个不同的效靶比条件下,实验组小鼠脾淋巴细胞诱导产生的ADCC明显高于对照组(P<0·05);效靶比为1∶125时,A、B、C组51Cr自然释放率分别为23%、17%、12%,3组间比较,差异有统计学意义(P<0·05)。血清稀释度1∶50时,A、B、C组51Cr自然释放率分别为47%、39%、26%,A、B组BALB/c小鼠血清诱导产生的CDC明显高于C组(P<0·05),而A和B组相比,差异则无统计学意义(P>0·05)。结论6B11微抗体可作为卵巢上皮性癌疫苗,其较佳的动物免疫方案为:免疫剂量为100μg;基础免疫分别于第1、14、28天进行,共3次;间隔6周给予加强免疫。

Objective To probe better immunization dose and interval of anti-idiotypical minibedy （6B11 minibody） in animals and to guide the clinical trials of ovarian epithelial cancer treatment. Methods Minibody was induced with isopropyhhiogalactoside （IPTG） in E. coli and analyzed with direct enzyme linked immunosorbent assay （ELISA）. Normal BALB/c mice were randomly divided into groups A, B, C, D, E, F,immunized with the murine monoclonal anti-idiotypic antibody 6B11 minibody 150 μg, 100 μg,50 μg, and mice IgG 100 μg, human IgG 100 μg, phosphate buffered saline （PBS） 100 μl at d1 ,d14, d28 respectively. Anti-anti-idiotypic antibody （Ab3） level was tested and antibody-dependent cellular cytotoxicity（ADCC） and complement-dependent cytotoxicity（CDC） effect of mouse spleen cells to human ovarian carcinoma SKOV3 cells were measured using ^51Cr-rclease assay. UV absorption values were tested at 490 nm. Results 6B11 minibody was successfully expressed on E. coli and could react with a routine monoclonal antibody COC166-9. Ab3 could not be detected until one week after d14＇s boost, its level gradually elevated and peaked one week after d2s＇s boost, and maintained at high level for about six weeks then declined dramatically seven weeks after d28＇s boost. When boosted again at seven weeks after d28＇s boost , the Ab3 level elevated quickly one week after, and maintained at this level at least for two weeks. The Ab3 level of group B was higher than groups A and C,mean A （absorption） value during six weeks after d28 ＇s boost was 1.05 in group A, 1.06 in group B, 0. 94 in group C, and there was no significant difference （P 〉0. 05）. At four different effector cell/target cell （E/T） ratios, the ADCC was highest in group A and lowest in group C; when the E/T ratio was 1： 125, ^51Cr releasing rate was 23% in group A, 17% in group B, and 12% in group C, the difference between groups A, B, C had statistical significance （P 〈0.05）. When serum was diluted at 1：50, the CDC effects were higher in group A and B than in group C （^51Cr releasing rate was 47%, 39%, 26% respectively; P 〈 0. 05 ）, but there was no significant difference between groups A and B （P 〉 0. 05）. Conclusion 6B11 anti-idiotypic minibody of ovarian carcinoma can be used as tumor vaccine, the suitable immunization protocol in animals may be ： prime at d1, d14 and d28 at a dose of 100μg; prime interval is six weeks.