提高抗人膀胱癌人-鼠嵌合抗体的表达和抗体的功能鉴定

Expression of human-mouse chimeric antibody ch-BDl and its affinity to human bladder cancer in vitro and in vivo

目的 探讨人-鼠嵌合抗体ch-BD1体内和体外的表达和功能。方法 用分子生物学方法得到系列弱化表达的双氢叶酸还原酶(DHFR)基因片段,将其克隆入载体pDHL-BD1中构建得系列弱化表达DHFR基因的人-鼠嵌合抗体pWSD1-BD1,pWSD2-BD1,和pWSD3-BD1。将这些嵌合抗体以及pDHL-BD1分别转染缺陷表达DHFR基因的中国仓鼠卵巢细胞(CHO)/DHFR-细胞,以Northern印迹鉴定其DHFR基因表达水平。将不同浓度的氨甲蝶呤(MTX)加入转染细胞的培养液,测定上清中的抗体水平。用 ^(99m)标记纯化的ch-BD1,加入系列稀释的人膀胱癌EJ细胞溶液中,测定结合在细胞上的抗体放射活性,计算标记抗体的亲和常数。将人膀胱癌EJ细胞注射入3只Balb/C小鼠后肢根部,4周后对肿瘤进行放射免疫显像。将标记抗体注射入小鼠尾静脉,分别于2 h,6 h,22 h和24 h后进行放射显像。结果 构建载体的 DHFR基因表达活性由强至弱的顺序为 pDHL-BD1>pWS1-BD1>pWS3-BD1>pWS2-BD1。MTX浓度为10^(-6)mol/L时ch-BD1的表达水平与DHFR基因的基础表达水平明显相关,DHFR基础水平越高抗体的表达水平亦越高。EJ细胞与标记抗体孵育后,ch-BD1的免疫活性分数为76％,亲和常数为3.56×10~9 M^(-1);鼠单抗BD1的免疫活性分数为81％,亲和常数为1.22×10~9M^(-1)。^(99m)标记的ch-BD1经尾静脉?

Objective To investigate the expression of human-mouse chimeric antibody ch-BDl against human bladder cancer and its affinity to human bladder cancer in vitro and in vivo. Methods Three kinds of mutated fragments of dihydrofolate reductase (DHFR) gene were created by techniques of molecular biology to decrease the transcriptional activities and then cloned into pDHL-BDl so as to construct the vectors pWSD1-BD1, pWSD2-BD1, and pWSD3-BD1 expressing the human-mouse chimeric antibody ch-BDl against human bladder cancer with decreased expression of DHFR gene. These vectors and pDHL-BDl were transfected into Chinese hamster ovary cell (CHO) /DHFR- cell respectively. 72 hours later Northern blotting was used to examine their DHFR gene expression. Methotrexate (MTX) of increasing concentrations was added into the culture of transfected CHO/DHFR- cells. The ch-BD1 levels in the supernatants were measured. Purified ch-BDl was labeled by 99mTcO4- and added into the serially diluted solutions of human bladder cancer EJ cells to examine their radioactivities and calculate their affinity constants. EJ cells were injected into the roots of hind limb of 3 Balb/C mice. Four weeks later, 99m TcO4- -labeled antibody ch-BDl were injected into the rats' caudal veins. Radioimmunoimaging was conducted to examine the distribution of the antibody. Results The sequence of DHFR gene expression levels from strong to weak in the constructed vectors was as follows: pDHL-BD1 > pWS1-BD1 > pWS3-BD1 > pWS2-BD1. When the concentration of MTX was 10-6 mol/L the expression level of ch-BDl was significantly correlated to the expression level of DHFR gene, the lower the baseline expression level of DHFR gene the higher the expression level of ch-BDl. After the serially diluted EJ cells were co-incubated with the 99mTcO4- -labeled antibody ch-BDl the immunoacitivity ratio of ch-BDl was 76% , and that of murine monoclonal antibody was 81 % ; the affinity constant of ch-BDl was 3 .56 × 109M -, and that of murine monoclonal antibody BD1 was 1 .22 × 109M-1. 6 hours after injection of the 99mTcO4- -labeled antibody ch-BDl into the mice body it was mainly distributed in the tumor, 22 hours later, it was specifically distributed in tumor, and 24 hours later it was still concentrated here. Conclusion Decrease of the baseline expression level of DHFR gene effectively increases the amplification of MTX to exogenous gene. The human-mouse chimeric antibody ch-BDl shows an ideal affinity activity to human bladder cancer in vivo and in vitro and has a certain clinical prospect.