摘要
目的构建一种可同时靶向人CD3、CD19、PD-L1的三特异性T淋巴细胞衔接器TriTE-3-19-1,并评估其介导的T淋巴细胞特异性杀伤CD19^(+)PD-L1^(+)淋巴瘤细胞的效果。方法通过分子克隆技术构建慢病毒表达载体pLentiR.SV40-TriTE-3-19-1,将其瞬时转染至293T细胞,收集培养上清并纯化获得三特异性T淋巴细胞衔接器TriTE-3-19-1。采用ELISA法检测TriTE-3-19-1与Jurkat(CD3^(+))、Raji(CD19^(+))、3T3/PD-L1(PD-L1^(+))和Raji-PD-L1(CD19^(+)PD-L1^(+))细胞的结合活性。通过LDH释放实验检测不同浓度TriTE-3-19-1介导的T淋巴细胞对靶细胞(CD19^(+)、PD-L1^(+)、CD19^(+)PD-L1^(+)细胞)的杀伤能力。通过抗PD-L1结构域封闭实验观察TriTE-3-19-1对CD19^(+)PD-L1^(+)细胞的杀伤能力变化,采用ELISA法检测细胞因子白细胞介素2(IL-2)、干扰素γ(IFN-γ)、肿瘤坏死因子α(TNF-α)。结果慢病毒表达载体pLentiR.SV40-TriTE-3-19-1构建成功,纯化后的三特异性T淋巴细胞衔接器TriTE-3-19-1在84 kDa处以单体的形式出现预期条带。TriTE-3-19-1与Jurkat(CD3^(+))、Raji(CD19^(+))、3T3/PD-L1(PD-L1^(+))和Raji-PD-L1(CD19^(+)PD-L1^(+))细胞的结合活性呈现典型的剂量-响应关系。随着TriTE-3-19-1浓度升高,T淋巴细胞对CD19^(+)、PD-L1^(+)、CD19^(+)PD-L1^(+)细胞的杀伤作用呈剂量依赖性增强(P均<0.05);且其介导的T淋巴细胞对CD19^(+)PD-L1^(+)细胞的杀伤作用显著强于CD19^(+)细胞、PD-L1^(+)细胞(P均<0.05)。TriTE-3-19-1的抗PD-L1结构域被封闭后,其介导的T淋巴细胞对Raji-PD-L1(CD19^(+)PD-L1^(+))细胞的杀伤能力减弱(P<0.05),细胞因子IL-2、IFN-γ、TNF-α表达水平也下降(P均<0.05)。结论成功构建三特异性T淋巴细胞衔接器TriTE-3-19-1,可分别与CD3^(+)、CD19^(+)、PD-L1^(+)、CD19^(+)PD-L1^(+)细胞结合,并有效介导T淋巴细胞对CD19^(+)PD-L1^(+)淋巴瘤细胞的杀伤能力。
Objective To construct a tri-specific T-cell engager(TriTE-3-19-1)capable of simultaneously targeting human CD3,CD19,and PD-L1,and to evaluate its ability to mediate T-cell-specific killing of CD19^(+)PD-L1^(+)lymphoma cells.Methods The lentiviral expression vector pLentiR.SV40-TriTE-3-19-1 was constructed using molecular cloning technique,transiently transfected into 293T cells,and the culture supernatant was collected and purified to obtain the trispecific T-cell engager TriTE-3-19-1.ELISA was used to detect the binding activity of TriTE-3-19-1 to Jurkat(CD3^(+)),Ra⁃ji(CD19^(+)),3T3/PD-L1(PD-L1^(+)),and Raji-PD-L1(CD19^(+)PD-L1^(+))cells.LDH release assay was performed to assess the killing ability of T cells mediated by different concentrations of TriTE-3-19-1 against target cells(CD19^(+),PD-L1^(+),and CD19^(+)PD-L1^(+)cells).Blocking experiments targeting the anti-PD-L1 domain were conducted to observe changes in the killing ability of TriTE-3-19-1 against CD19^(+)PD-L1^(+)cells,and ELISA was used to measure the levels of cytokines interleukin-2(IL-2),interferon-γ(IFN-γ),and tumor necrosis factor-α(TNF-α).Results The lentiviral expression vector pLen⁃tiR.SV40-TriTE-3-19-1 was successfully constructed.The purified tri-specific T-cell engager TriTE-3-19-1 showed an ex⁃pected band at approximately 84 kDa in its monomeric form.The binding activity of TriTE-3-19-1 to Jurkat(CD3^(+)),Raji(CD19^(+)),3T3/PD-L1(PD-L1^(+)),and Raji-PD-L1(CD19^(+)PD-L1^(+))cells exhibited typical dose-response relationships.As the concentration of TriTE-3-19-1 increased,the killing effect of T cells on CD19^(+),PD-L1^(+),and CD19^(+)PD-L1^(+)cells showed a dose-dependent enhancement(all P<0.05).Moreover,the T-cell-mediated killing of CD19^(+)PD-L1^(+)cells was significantly stronger than that of CD19^(+)cells or PD-L1^(+)cells(all P<0.05).When the anti-PD-L1 domain of TriTE-3-19-1 was blocked,its ability to mediate T-cell killing of Raji-PD-L1(CD19^(+)PD-L1^(+))cells was reduced(P<0.05),and the ex⁃pression levels of cytokines IL-2,IFN-γ,and TNF-αalso decreased(all P<0.05).Conclusion The tri-specific T-cell engager TriTE-3-19-1 was successfully constructed,demonstrating binding to CD3^(+),CD19^(+),PD-L1^(+),and CD19^(+)PD-L1^(+)cells,and effectively mediating T-cell killing of CD19^(+)PD-L1^(+)lymphoma cells.
作者
段惠
韩建庚
卢杨
张洁
张晓龙
DUAN Hui;HAN Jiangeng;LU Yang;ZHANG Jie;ZHANG Xiaolong(Tianjin Medical University Cancer Institute&Hospital,National Clinical Research Center for Cancer,Tianjin’s Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Tianjin 300060,China;不详)
出处
《山东医药》
2026年第1期54-59,共6页
Shandong Medical Journal
基金
国家自然科学基金项目(82000197)。
