摘要: |
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100 000,BiTE相对分子质量约为50 000。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。 |
关键词: 肝癌 磷脂酰肌醇蛋白聚糖3 CD3 双特异性抗体 单域抗体 |
DOI:10.12025/j.issn.1008-6358.2021.20210990 |
分类号:R735.7 |
基金项目:上海市科学技术委员会自然科学基金(21ZR1477600). |
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Preparation of anti-GPC3/CD3 bispecific heavy chain antibody and evaluation of its anti-liver cancer effect |
LI Yong-cheng1, ZHU Qiang-qiang1, WANG Yan-ting1, WANG Yang2, SUN Ya-qi3, LU Bin1
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1.Department of Biochemical Pharmacy, School of Pharmacy, Naval Medical University, Shanghai 200433, China;2.Department of Pathology, Shanghai Fourth People's Hospital, Shanghai 200434, China;3.Department of Pharmacy, Changhai Hospital, Naval Medical Univerisity, Shanghai 200433, China
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Abstract: |
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 100 000. The molecular weight of BiTE was about 50 000. 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. |
Key words: liver cancer glypican-3 CD3 bispecific antibody single domain antibody |