抗EGFR/CD3双特异性抗体的制备与功能的初步研究

Preparation and Preliminary Investigation of Function of Anti-EGFR/CD3 Bispecific Antibody

目的构建抗表皮生长因子受体(anti-EGFR)和抗CD3(anti-CD3)的双特异性抗体(anti-EGFR/CD3),并在体外初步评价其杀伤肿瘤的功能。方法通过基因工程技术,利用knobs-into-holes模式分别构建表达anti-EGFR-HC-knob、anti-EGFR-LC和anti-CD3-scFV-HC-hole的载体,共同转染Expi 293F细胞,表达双特异性抗体,依次用抗体特异的亲和层析和分子排阻层析进行纯化,再用流式细胞仪和实时无标记细胞功能分析仪评价其体外结合能力和体外肿瘤杀伤功能。结果成功获得较高纯度的抗体。流式细胞仪体外结合实验结果显示,双特异性抗体能够同时与EGFR+的U87Ⅷ脑胶质瘤细胞系、HCT-116结肠癌细胞系和CD3+的Jurkat T淋巴瘤细胞系结合,具有2种抗体的功能,同时还能很好地激活T细胞(P<0.000 1);在体外杀伤效果方面,双特异性抗体作用后杀伤效果明显高于其他抗体和对照组(均为P<0.000 1),具有很好的杀伤效果。结论用knobs-intoholes技术获得的anti-EGFR/CD3具有较好的生物活性,为双特异性抗体的肿瘤免疫治疗奠定了一定的基础。

Objective To construct the bispecific antibody anti-EGFR/CD3 （BsAb）, which would be the anti epidermal growth factor receptor （anti-EGFR） and anti CD3 antibody （anti-CD3）, and to carry out the functional evaluation. Methods First, constructing the anti-EGFR-knob and anti-CD3-scFV-hole plasmid by engineering technology. The anti-EGFR and anti-CD3-scFV were cross-linked to prepare the BsAb by knobs-into-holes model. Then, we transfected these plasmids into Expi 293 cells for expression and to assemble into bispecific antibodies. Finally, we used the flow cytometry and real time marker free cell function analyzer to measure their binding capacity and killing function in vitro after purification using the affinity chromatography and superdex 200 increase. Results We were able to produce high purity an- tibodies after purification. This BsAb could simultaneously bind with the U87 Ⅷ glioma cell lines and HCT-116 colon cancer cell line, which would express EGFR on the cell surface and Jurckat T lymphoma cell lines, which would then express CD3. At the same time, the BsAb could activate the T cell very well （P〈0. 000 1）. The results of killing experiment in vitro showed that the BsAb had a higher killing effect than other antibodies （all the P value 〈0. 000 1） and the control group （P〈0. 000 1） significantly. Con- clusion The anti-EGFR/CD3 created by knobs-into-holes model has good biological activities. And this can be laying a foundation for tumor immunotherapy by bispecific antibody in the future.