Tandab(CD3/CD19)与CD19^+、CD3^+血液病细胞结合力及对PBMC的CD19^+血液病细胞杀伤力观察

Adhesion of Tandab(CD3/CD19) binding to hematological cells CD19^+ and CD3^+ and its effect on PBMCs killing CD19^+ cells

目的观察串联四价双特异性抗体Tandab(CD3/CD19)与CD19^+、CD3^+血液病细胞结合力及对外周血单个核细胞(PBMC)的CD19^+血液病细胞杀伤力。方法采用PCR、overlap PCR、酶切、连接等方法构建慢病毒表达载体p LentiR.SV40-Tandab(CD3/CD19),用p LentiR.SV40-Tandab(CD3/CD19)瞬时转染293T细胞,提取并纯化Tandab(CD3/CD19)。采用FACS法检测Tandab(CD3/CD19)与CD19^+(Raji、Daudi、BJAB)、CD3^+(Jurkat)血液病细胞的直接结合力,检测Tandab(CD3/CD19)和抗CD19单克隆抗体HIT19a(或抗CD3单克隆抗体HIT3a)对CD19^+、CD3^+血液病细胞的竞争结合力。采用乳酸脱氢酶释放法观察Tandab(CD3/CD19)对外周血单个核细胞(PBMC)CD19^+血液病细胞杀伤力(以细胞毒性百分比表示)的影响。结果 Tandab(CD3/CD19)可与Raji、Daudi、BJAB、Jurkat直接结合,也可与HIT19a(或HIT3a)竞争性结合Raji、Daudi、BJAB、Jurkat。随Tandab(CD3/CD19)浓度增加,PBMC细胞对Raji、Daudi、BJAB细胞的细胞毒性百分比增加,呈剂量依赖性(P均<0.05)。结论 Tandab(CD3/CD19)与CD19^+、CD3^+血液病细胞具有直接结合力和竞争结合力,并可促进PBMC对CD19^+血液病细胞的杀伤力。

Objective To observe the adhesion of the tetravalent bispecific antibody named Tandab（CD3/CD19）with hematological cells CD19＋and CD3＋,and its effect on peripheral blood mononuclear cells（PBMCs） killing CD19＋cells. Methods The methods of PCR,overlap PCR,restriction enzyme cleavage and linkage were used to construct the lentiviral expression vector p LentiR. SV40-Tandab（CD3/CD19）,which was used to transiently transfect 293 T cells. The Tandab（CD3/CD19） was extracted and purified. The direct binding ability of Tandab（CD3/CD19） to CD19＋（Raji,Daudi,BJAB） or CD3＋（Jurkat） was determined by FACS,respectively. The competition binding ability of Tandab（CD3/CD19） and anti-CD19 monoclonal antibody HIT19 a（or anti-CD3 monoclonal antibody HIT3 a） to hematological cells CD19＋and CD3＋was examined. Lactic dehydrogenase（LDH） release assay was employed to detect the cytotoxicity of T cells induced by Tandab（CD3/CD19）. Results Tandab（CD3/CD19） could specifically bind to both CD19-positive cells（Raji,Daudi,and BJAB） and CD3-positive cells（Jurkat）. Tandab（CD3/CD19） could competitively bind to Raji,Daudi,BJAB,and Jurkat with HIT19 a（HIT3 a）. With the increased concentrations of Tandab（CD3/CD19）,the cytotoxicity index of PBMC to Raji,Daudi,and BJAB increased in a dose-dependent manner（P〈0. 05）. Conclusion Tandab（CD3/CD19） has specific combining capacity with CD19＋and CD3＋cells and can increase the lethality of PBMC to CD19＋cells.