Construction and cytotoxic evaluation of tri-specific T-cell engager TriTE-3-19-1 simultaneously targeting human CD3,CD19,and PD-L1
DUAN Hui
HAN Jiangeng
LU Yang
ZHANG Jie
ZHANG Xiaolong
Abstract：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 tri-specific 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 kill-ing 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-ti R.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-α also 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.
Keywords：tri-specific antibodyT-cell engagertri-specific T-cell engagerspecific killingB-cell lymphoma
Publication Date：2026-01-25
Online Publishing Date：2026-03-05(First online date of this platform, not the publication date of the document)
Pages：6( 54-59 )