融合C3Fab抗PD-L1纳米抗体的制备及其对血浆半衰期的影响

Preparation of anti-PD-L1 nanobodies fused with C3Fab and their effect on plasma half-life

目的制备融合C3Fab的抗程序性死亡-配体1(PD-L1)纳米抗体P3C8-C3Fab,并研究其对血浆半衰期的影响。方法将来源于蛋白G的C3Fab肽段,通过DNA重组技术与纳米抗体P3C8进行分子融合,并构建重组质粒pET21a-P3C8-C3Fab,在大肠杆菌BL21菌株中进行诱导表达和纯化,并采用酶联免疫吸附试验(ELISA)检测P3C8-C3Fab与PD-L1蛋白、小鼠IgG和表达PD-L1肿瘤细胞的结合,利用双抗夹心ELISA检测不同时间小鼠血清中残留P3C8-C3Fab,以评估C3Fab对PD-L1纳米抗体血浆半衰期的延长作用。结果成功构建了融合C3Fab的抗PD-L1纳米抗体P3C8-C3Fab,其在BL21菌中以可溶形式高效表达,经纯化获得质量分数约为90%的NbP3C8-C3Fab蛋白,得率为7.18 mg/L。P3C8-C3Fab随着质量浓度的增加,对PD-L1蛋白和小鼠IgG亲和力也逐渐升高,且呈现浓度相关性;P3C8-C3Fab与肺癌A549细胞的结合呈浓度相关性。小鼠血清中P3C8-C3Fab的浓度标准曲线呈典型的S型浓度相关性;P3C8血浆半衰期仅为0.44 h,而P3C8-C3Fab的血浆半衰期达到9.36 h,血浆半衰期延长了21.27倍。结论C3Fab与纳米抗体P3C8连接可以显著延长其血浆半衰期,对改善纳米抗体的体内作用具有应用价值。

Objective To prepare the anti-programmed death-ligand 1(PD-L1)nanoantibody P3C8-C3Fab by ligating with C3Fab and to investigate its role in plasma half-life.Methods The C3Fab peptide derived from protein G was molecularly fused with the nanobody P3C8 by DNA recombination technology.The nanoantibody P3C8-C3Fab was inducibly expressed and purified in the E.coli BL21 strain,and the binding of it to PD-L1 protein,mouse IgG,and PD-L1-expressing tumor cells was detected by enzyme-linked immunosorbent assay(ELISA).The residual P3C8-C3Fab was detected in mouse serum at different times using double-antibody sandwich ELISA to assess the prolongation of the plasma half-life of PD-L1 nanobodies by C3Fab.Results The nanoantibody P3C8-C3Fab was successfully constructed,and it could efficiently express itself in soluble form in BL21.The purified NbP3C8-C3Fab protein was obtained with a mass fraction of about 90%at a yield of 7.18 mg/L.The affinity of P3C8-C3Fab for PD-L1 protein and mouse IgG gradually increased with increasing mass concentration and showed a concentration correlation.The binding of P3C8-C3Fab to lung cancer A549 cells showed a concentration correlation.The concentration standard curve of P3C8-C3Fab in mouse serum showed a typical S-shape with a concentration correlation.The plasma half-life of P3C8 was only 0.44 h,while the plasma half-life of P3C8-C3Fab was 21.27-fold higher,up to 9.36 h.Conclusions The linkage of C3Fab to the nanobodies of P3C8 can significantly prolong the plasma half-life of P3C8,which is valuable for the improvement of in vivo nanobody effects.