抗GPC3/CD3双特异性重链抗体的制备及其抗肝癌作用评价

Preparation of anti-GPC3/CD3 bispecific heavy chain antibody and evaluation of its anti-liver cancer effect

目的:设计并制备抗GPC3/CD3双特异性重链抗体(BiHcAb),分析其与双特异性T细胞衔接子(BiTE)在体内外抗肿瘤活性的差异。方法:通过全基因合成抗GPC3和抗CD3单域抗体(sdAb)序列,克隆至携带IgG4 Fc段的表达载体中,同时利用KIH技术形成稳定的双特异性重链抗体。2种质粒共转染HEK-293F细胞,悬浮摇瓶培养获得目的抗体。SDS-PAGE和考马斯亮蓝染色确定相对分子质量。通过流式细胞术检测与GPC3阳性肿瘤细胞和外周血单个核细胞(PBMC)的结合能力。通过共培养检测BiHcAb介导的细胞杀伤效果及细胞因子释放;采用裸鼠异体移植模型实验检测BiHcAb的体内抑制肿瘤活性。结果:成功构建并表达抗GPC3/CD3 BiHcAb,相对分子质量约为100000,BiTE相对分子质量约为50000。抗GPC3/CD3 BiHcAb较BiTE对表达GPC3的肿瘤细胞具有更强的杀伤活性,同时促进释放更多的细胞因子(IFN-γ、TNF-α、IL-2、IL-6),差异有统计学意义(P<0.05)。裸鼠体内实验显示抗GPC3/CD3 BiHcAb比BiTE具有更强的体内抑制肿瘤生长的作用,差异有统计学意义(P<0.05)。结论:成功制备的抗GPC3/CD3 BiHcAb较BiTE在体内外具有更强的抗肝癌作用。

Objective:To design and prepare anti-GPC3/CD3 bispecific heavy chain antibody(BiHcAb)and analyze the difference of in vivo and in vitro anti-tumor activity between BiHcAb and bispecific T cell engager(BiTE).Methods:The sequences of anti-GPC3 and anti-CD3 single domain antibodies(sdAb)were obtained by gene synthesis,then cloned into an expression vector carrying IgG4 Fc segment with knob-into-hole(KIH)technology to produce stable bispecific heavy chain antibodies.Two plasmids were co-transfected into HEK-293F cells and cultured in suspension shake flasks to produce the target antibody.The molecular weight of productions was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Coomassie brilliant blue staining.The combination of antibodies and GPC3-positive tumor cells and peripheral blood mononuclear cells(PBMC)were analyzed by flow cytometry.Cell cytotoxicity and cytokine release mediated by BiHcAb were measured under co-culture conditions.The tumor-inhibiting activity of BiHcAb in vivo was also tested in nude mouse xenograft model experiment.Results:The anti-GPC3/CD3 BiHcAb was successfully constructed and expressed,with a molecular weight of about 100000.The molecular weight of BiTE was about 50000.Compared with BiTE,anti-GPC3/CD3 BiHcAb has stronger killing activity on GPC3-positive tumor cells,and promotes the release of cytokines(IFN-γ,TNF-α,IL-2,and IL-6).It is shown that anti-GPC3/CD3 BiHcAb has a stronger tumor growth inhibition effect in vivo than BiTE in nude mice.Conclusions:We designed an anti-GPC3/CD3 bispecific heavy chain antibody with a stronger anti-liver cancer effect than BiTE in vivo and in vitro.