Biological Function Study Of Anti-human TIM-3 Monoclonal Antibody In Vitro

Objective Multiple ligands have been associated with TIM-3.We used TIM-3 mAbs(clones 4E8 and 9H7)generated by us and in vitro assays to determine whether the biological function of antibodies was associated with their abilities to block binding of certain TIM-3ligands.Through these studies,we hope to provide the scientific basis for targeting TIM-3for cancer immunotherapy using monoclonal antibodies.Methods The cell line stably expressing human TIM-3 was obtained by the cell transfection technique.We examined the ability of 4E8 and 9H7 to bind to human TIM-3 by indirect immunofluorescence staining and flow cytometry.In addition,the ability of 4E8 to block TIM-3 binding to Galectin-9(Gal-9)was measured by ELISA.Flow cytometry was used to measure the ability of 4E8 and 9H7 to block TIM-3 binding to CEACAM-1 and PtdSer.Mixed lymphocyte reaction(MLR)and ELISA assays were used to determine the effect of TIM-3 mAbs 4E8 and 9H7 on the IL-2 and IFN-γsecretion by CD4~+T cells.Results Our data indicated that 4E8 and 9H7 could specifically bound to human TIM-3.4E8 could block the binding of TIM-3 to Gal-9.But it could not block the binding of TIM-3 to CEACAM-1or PtdSer.4E8 enhanced the ability of CD4~+T cells to secrete IL-2 and IFN-γin MLR experiment,and the difference was statistically significant(P<0.05).In addition,it had a synergistic effect with PD-1 antibody in increasing IL-2 and IFN-γsecretion in the MLR assay,and the difference was statistically significant(P<0.05).At the same time,9H7 could block the binding of TIM-3 to CEACAM-1 and PtdSer.It also enhanced the ability of CD4~+T cells to secrete IL-2 and IFN-γin MLR experiment,and the difference was statistically significant(P<0.05).9H7 had a synergistic effect with PD-1 antibody in increasing IL-2(P<0.01).Conclusion TIM-3 mAb 4E8 could block the binding of TIM-3 to Gal-9,but could not block the binding of TIM-3 to CEACAM-1 or PtdSer.In addition,4E8 enhanced the ability of CD4~+T cells to secrete IL-2 and IFN-γ,and had a synergistic effect with PD-1 mAb.But this effect of 4E8 on CD4~+T cells did not depend on blocking the binding of TIM-3 to its ligand CEACAM-1 or PtdSer.Another TIM-3 mAb 9H7 could block the binding of TIM-3with CEACAM-1 and PtdSer,and it enhanced CD4~+T cells secreting IL-2 and had a synergistic effect with the PD-1 mAb.9H7 also could enhance CD4~+T cells secreting IFN-γ.Our data indicate that the in vitro biological activities of TIM-3 mAbs are not dependent on their abilities to block binding of certain known TIM-3 ligands.