免疫毒素2E8去甲斑蝥素的研制及其体外靶向抑制效应研究

Preparation of the immunotoxin 2ES-norcantharidin and its targeting killing effect in vitro

目的采用本院自行研制的ZCH-4—2E8细胞分泌的抗人CD19单克隆抗体与去甲斑蝥素（NCTD）进行连接制备免疫毒素，研究其在体外的靶向杀伤作用。方法通过凝胶过滤层析法纯化2E8单抗，SDS-PAGE电泳鉴定抗体的纯度；活泼酯法制备免疫毒素2E8-NCTD；流式细胞仪测定免疫毒素的抗体活性及Nalm-6细胞和K562细胞上CD19抗原表达情况；台盼蓝计数法比较2E8抗体、NCTD、2E8-NCTD免疫毒素对Nalm-6细胞和K562细胞的杀伤作用。结果纯化后的2E8单抗经SDS-PAGE鉴定纯度达99．00％以上；2E8培养上清在Nalm-6细胞上的阳性率为99．34％，而在K562细胞上则为0．98％，2E8-NCTD免疫毒素与Nalm-6细胞的阳性反应率为99．90％；培养96h时，与对照组相比，2E8-NCTD（10μs／ml）对CD19^＋的Nalm-6细胞的生长影响有统计学意义（96h抑制率71．25％，P〈0．001），而纯化的2E8抗体（10μg/ml）对Nalm-6细胞生长则无明显抑制作用（96h抑制率为30．29％，P〉0．05）；10μg/ml 2E8抗体、10μg/ml 2E8-NCTD对CD19^-的K562细胞（96h抑制率分别为4．17％和15．41％）则无明显抑制作用（P〉0．05）。结论成功制备2E8-NCTD免疫毒素，该免疫毒素在体外具有明显的靶向杀伤白血病细胞Nalm-6的作用。

Objective Monoclonal antibody （mAb） conjugated with certain toxin to generate immunotoxin bears an important and promising effect as a new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated with antibody proteins reported in the literature were toxic proteins which presented immunogenieity to patients capable of inducing anti-toxin antibody. Norcantharidin （NCTD） is a small molecule toxin. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19^＋ lymphoid malignant Nalm-6 cells in vitro to provide the experimental data for the further development of this new targeting agent. Methods A monoclonal antibody named 2E8 was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoeonjugate （2E8-NCTD） against CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometery. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, norcantharidln and immunotoxin 2E8-NCTD groups on cell growth of either Nalm-6 cells or K562 cells were made. Results The purity of the purified 2E8 antibody was higher than 99.00% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99. 34% of Nalm-6 cells, while only 0. 98% of K562 cells reacted with this antibody. The newly generated immunotoxin （2E8-NCTD） had a positive rate of 99.90% on Nalm-6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and norcantharidin were shown to have significant inhibitory effects on the growth of CD19^＋ Nalm-6 cells （P 〈0. 001 ） , while the purified 2E8 antibody did not show any significant influences on the growth of Nalm-6 cells. Since no significant inhibitory effects were identified among immunotoxin 2E8- NCTD, 2E8 antibody and control groups on CD19- K562 cells, a significant targeting effect of the 2E8- NCTD against Nalm-6 cells was confirmed. Conclusions The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing effect on CD19^＋ Nalm-6 leukemia cells in vitro, which provides some experimental data for the further development of this new targeting agent.