Preparation And Functional Assay Of Immune Checkpoint TIM-3 Monoclonal Antibody

In recent decades,cancer has always been one of the most lethal diseases and has become a global public health issue of great concern.In order to cure cancer,researchers from various countries have tried various anti-tumor strategies.In the past decade,with gradual deepening of research on human immune system,immunological checkpoint therapy has gradually studied.Especially after 2014,with the official listing of PD-1 monoclonal antibody,immune checkpoint proteins were employed to block signaling pathways and up-regulate or down-regulate the activity of T cells.However,with the development of resistance to PD-1 monoclonal antibody and autoimmune system complications,TIM-3,a negative regulator of T cell,has gradually gained attention as a new immune checkpoint molecule.Firstly,this article introduced the structural features of TIM-3 molecule and its three important ligands.Secondly,it described the interaction between TIM-3 and its ligands.Binding with TIM-3 through ligands such as Galectin-9 led to the production of Th1 cells,functional depletion and eventual apoptosis,and also regulated cytokine secretion in Th1 and Th17 cells.Therefore,a recombinant plasmid expressing the TIM-3 sequence was constructed.Afterwards,BALB/c mice were directly immunized with DNA by electroporation.After the immunization cycle,spleen cells were fused with SP2/0myeloma cells.The clone number of the TIM-3 positive reaction was further screened by ELISA to obtain multiple hybridoma cell clones secreting TIM-3 monoclonal antibody.Finally,hybridoma cells were injected into the abdominal cavity of BALB/c mice to prepare ascites and further purified to obtain monoclonal antibody.The ability of antibody specific recognition and affinity was identified by western blot,ELISA,EC₅₀,immunofluorescence,flow cytometry and other methods.After the purified TIM-3 mAb was incubated with T cells such as PBMC,DC,DC-CIK and CD4⁺T,and specifically bound to TIM-3 molecule on the surface of T cells and blocked TIM-3.Via the Galectin-9 pathway,by measuring the levels of cytokines such as IL-2 and IFN-?secreted in the supernatant after co-culture,the combination of TIM-3 mAb and each T cell could effectively kill U937,MDA-MB-231 and other tumor cell lines.Finally,in the BALB/c nude mice model,the PD-1 and TIM-3 monoclonal antibody combination treatment group was more effective than the PD-1 and TIM-3monoclonal antibody group and the control group.It could effectively inhibit the growth and metastasis of tumors,improve the quality of mice life,and prolong the survival time of mice.The TIM-3 monoclonal antibody obtained by DNA immunization can specifically recognize and bind to TIM-3 molecules,effectively block the pathway between TIM-3/Galectin-9,reverse the depletion of T cells,and promote the proliferation of T cells,and activate its ability to secrete cytokines.TIM-3 monoclonal antibody can effectively block the activation of TIM-3 molecules on the surface of T cells effectively killing tumor cells in vivo and in vitro experiments.In conclusion,it will be beneficial for us to use the TIM-3 antibody on targeting treatment of various diseases related to the immune system and open up a new era for tumor immunotherapy.