T cell immunoglobulin and ITIM domain(TIGIT)is a novel immune checkpoint that has been considered as a target in cancer immunotherapy.Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable.Here,we designed a reporter gene assay comprising two cell lines,namely,CHO-CD112-CD3 scFv,which stably expresses CD112(PVRL2,nectin-2)and a membrane-bound anti-CD3 single-chain fragment variable(scFv)as the target cell,and Jurkat-NFAT-TIGIT,which sta-bly expresses TIGIT as well as the nuclear factor of activated T-cells(NFAT)response element-controlled luciferase gene,as the effector cell.The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell,whereas interactions be-tween CD112 and TIGIT prevent activation.The presence of anti-TIGIT mAbs disrupts their interaction,which in turn reverses the inactivation and luciferase expression.Optimization and validation studies have demonstrated that this assay is superior in terms of specificity,accuracy,linearity,and precision.In summary,this reliable and effective reporter gene assay may potentially be utilized in lot release control,stability as-says,screening,and development of novel TIGIT-targeted therapeutic antibodies.