论文部分内容阅读
抗体融合蛋白是新一代抗肿瘤抗体药物.CD20在约95%的B细胞非霍奇金淋巴瘤表面过度表达,是治疗B细胞淋巴瘤的理想靶点.力达霉素(LDM)是强效烯二炔抗肿瘤抗生素,目前已进入Ⅱ期临床阶段.采用DNA重组技术,利用大肠杆菌表达体系,制备抗CD20单链抗体与力达霉素辅基蛋白LDP的基因工程融合蛋白scFv-LDP.经纯化和复性后,ELISA和间接免疫荧光实验测定其抗原结合活性,发现抗CD20scFv-LDP能够与表达CD20的肿瘤细胞特异性结合.将其与LDM的活性发色团AE进行分子组装,得到强化融合蛋白scFv-LDP-AE.MTT结果表明,和LDM一样,scFv-LDP-AE对肿瘤细胞有强烈的杀伤作用,CD20+的Raji细胞和Daudi细胞的IC50值分别为1.21×10-11和6.24×10-11mol/L.CD20+B细胞淋巴瘤裸鼠移植模型检测抗肿瘤活性.接种肿瘤后第14和21天分别尾静脉注射给药,共2次.发现scFv-LDP-AE能够显著抑制肿瘤生长,0.3mg/kg剂量组抑瘤率为79.3%,显著强于LDM可耐受剂量0.05mg/kg(68.6%)的抑瘤率.强化融合蛋白scFv-LDP-AE有发展成抗肿瘤抗体靶向药物的潜能.
Antibody fusion proteins are a new generation of anti-tumor antibody drugs.CD20 is overexpressed on about 95% of B cell non-Hodgkin’s lymphomas and is an ideal target for the treatment of B cell lymphomas.Ladamicin (LDM) is potent Enkephalin antitumor antibiotics, has entered the phase Ⅱ clinical stage.Using DNA recombinant technology, using E. coli expression system, preparation of anti-CD20 scFv-LDP engineered fusion protein and lidamycin cofactor protein LDP. After purification and refolding, the antigen-binding activity of the anti-CD20 scFv-LDP was determined by ELISA and indirect immunofluorescence assay, and it was found that anti-CD20 scFv-LDP could specifically bind with CD20-expressing tumor cells and molecular assembly with the active chromophore AE of LDM The results of MTT showed that scFv-LDP-AE had a strong killing effect on tumor cells, like LDM, and the IC50 values of Raji cells and Daudi cells were 1.21 × 10-11 and 6.24 × 10-11mol / L. The anti-tumor activity of CD20 + B cell lymphoma xenograft model was tested in nude mice.The tumor cells were inoculated through the tail vein intravenously on the 14th and 21th day after inoculation.2 The results showed that scFv-LDP-AE could significantly inhibit Tumor growth, 0.3mg / kg dose group inhibition rate was 79.3% LDM stronger than the tolerable dose 0.05mg / kg (68.6%) of the tumor growth rate on the Fusion Protein scFv-LDP-AE has developed into anti-tumor antibody targeted drug potential.