新型抗体偶联药物OKT3-vcMMAE抗CD3^(+)T细胞肿瘤活性的研究

Antibody-drug conjugate OKT3-vcMMAE shows potent antitumor activity against CD3^(+)T cell malignancies

为探究治疗CD3^(+)T细胞肿瘤的新方法,将靶向CD3的单克隆抗体莫罗单抗(orthoclone OKT3,OKT3)与细胞毒性药物单甲基澳瑞他汀E(monomethyl auristatin E,MMAE)通过蛋白酶可裂解型的缬氨酸-瓜氨酸(valine-citrulline,vc)连接子连接,构建出了新型抗体偶联药物OKT3-vcMMAE。在体外试验中,采用SDS-PAGE检测OKT3-vcMMAE的相对分子质量和纯度,用流式细胞术探究其与CD3^(+)T细胞肿瘤细胞系的结合能力,用CCK-8试剂盒验证其体外肿瘤杀伤活性,并用流式微球阵列技术(cytometric bead array,CBA)检测其刺激T细胞释放细胞因子的能力。在体内试验中,利用OKT3-vcMMAE治疗接种T细胞肿瘤细胞系小鼠,并通过生物荧光成像检测肿瘤在小鼠体内的进展。结果显示,OKT3和OKT3-vcMMAE对Jurkat细胞的结合能力相近,半数效应浓度(half maximal effective concentration,EC50)分别为0.36和0.34μg/mL。体外抗肿瘤试验显示,与OKT3组比较,OKT3-vcMMAE组的Jurkat细胞活力在给药浓度为1、10和50μg/mL时显著降低(P<0.001),OKT3-vcMMAE组的HuT78细胞活力在给药浓度为10和50μg/mL时显著降低(P<0.01);与OKT3组比较,在给药浓度为10μg/mL时,OKT3-vcMMAE组T细胞释放的IFN-γ显著降低(P<0.01)。体内抗肿瘤试验显示,与PBS对照组比较,第13天OKT3-vcMMAE组小鼠的肿瘤负荷显著降低(P<0.01)。该研究提示,抗体偶联药物OKT3-vcMMAE可以作为治疗CD3^(+)T细胞肿瘤的潜在药物,这为治疗T细胞肿瘤提供了新思路。

This work aims to develop new treatments for CD3^(+)T cell malignancies.For this purpose,the CD3-targeting monoclonal antibody orthoclone OKT3(OKT3)was conjugated to the cytotoxic drug monomethyl auristatin E(MMAE)through protease cleavable valine-citrulline(vc)linker to generate an antibody-drug conjugate OKT3-vcMMAE.The molecular weight of OKT3-vcMMAE was verified by SDS-PAGE,and the binding ability to CD3^(+)tumor T cell line was verified by flow cytometry.The in vitro cytotoxic activity of OKT3-vcMMAE was verified by CCK-8,and its capacity to stimulate T cells to release cytokines was tested by the cytometric bead array(CBA).To test the in vivo antitumor efficacy,mice inoculated with T-cell tumor cell lines were treated with OKT3-vcMMAE and tumor burden was measured by bioluminescence imaging.The results showed that OKT3 and OKT3-vcMMAE had a similar affinity to Jurkat cells,with half maximal effective concentration(EC50)of 0.36 and 0.34μg/mL,respectively.In vitro antitumor experiments revealed that compared to that of the OKT3 group,the viabilities of Jurkat cells in the 1,10,and 50μg/mL of OKT3-vcMMAE-treatment groups were significantly decreased(P<0.001).Compared to that of the OKT3 group,the viabilities of HuT78 cells with 10 and 50μg/mL of OKT3-vcMMAE treatment were significantly decreased(P<0.01).IFN-γproduction of primary T cells upon 10μg/mL of OKT3-vcMMAE treatment was significantly decreased compared to cells treated with OKT3(P<0.01).In addition,in vivo antitumor experiments showed that compared to that of the PBS control group,the tumor burden of mice in the OKT3-vcMMAE group was significantly reduced on day 13(P<0.01).This study suggests that the antibody-drug conjugate OKT3-vcMMAE may be a potential treatment for CD3^(+)T cell malignancies,thus providing new insights for the development of anti-T-cell-malignancy drugs.