Tim-3 In Acute Myeloid Leukemia Immunotherapy

Background:Acute myeloid leukemia（AML）is a malignant clonal disease originating from hematopoietic stem cells.It is highly heterogeneous and has a low overall survival rate.At present,it is believed that the persistence of leukemia stem cells is the main reason for the recurrence and drug resistance of leukemia.Like hematopoietic stem cells,leukemia stem cells can self-renew and differentiate into leukemia cells,so they are an ideal target for high-efficiency and low-toxicity treatment of leukemia and prevention of recurrence.T cells immunoglobulin mucin 3（Tim-3）is a cell surface glycoprotein expressing on normal T cells,NK cells,and monocytes.Its primary function is to participate in the regulation of Th1-mediated immune response,induce immune tolerance,and promote depletion of CD8⁺T cells.The Tim-3 monoclonal antibody,as another immune checkpoint inhibitor besides CTLA-4monoclonal antibody and PD-1 monoclonal antibody,can relieve the suppression of T cells and has recently attracted attention in tumor immunity research.Moreover,existing studies have found that Tim-3 is expressed explicitly in AML leukemia stem cells and bulk cells,which is regarded as a marker of leukemia stem cells and may be used as a potential treatment target.Purpose:This project aims to study the molecular mechanism of the Tim-3 monoclonal antibody inhibiting the proliferation of AML cells and blocking the cell cycle,providing a basis for Tim-3 to become a target of AML immunotherapy,treating relapsed AML.Furthermore,we aim to explore the therapeutic effect of Tim-3 Chimeric Antigen Receptor T-Cell Immunotherapy（CAR-T）on AML.Methods:This study used bioinformatics methods to analyze The Cancer Genome Atlas database and Genotype-Tissue Expression database to study the expression of Tim-3 in AML patients and its impact on the survival and prognosis of AML patients.Cell Counting Kit-8 and flow cytometry were used to detect the effect of the Tim-3 monoclonal antibody on the proliferation,apoptosis,and cell cycle of AML cell lines.Western blotting was used to observe the protein expression levels of cyclin D1,p21,and p27^KIP,which were function to the cell cycle,in the AML cell lines after being treated by the Tim-3 monoclonal antibody.Then BALB/c mice were used for in vivo experiments to observe the in vivo effects of the Tim-3monoclonal antibody on AML model mice with WEHI-3.We also designed the Tim-3 CAR sequence to construct Tim-3 CAR-T cells by lentiviral infection.Flow cytometry was used to detect the CAR-T cells’targeted killing efficiency against AML cells with high Tim-3expression and the level of killing-related cytokines released and secreted.Moreover,NCG immunodeficient mice were used for in vivo experiments to observe the in vivo effects of Tim-3 CAR-T cells on AML model mice with K562-Tim-3⁺-Luc.Results:Analyzing the difference in Tim-3 m RNA expression in bone marrow samples of AML patients,it was found that the expression of Tim-3 in AML patients was significantly higher than that of healthy controls（p<0.001）.Divided AML patients into high and low groups according to Tim-3 expression（3/4 high expression and 1/4 low expression）,the overall survival of Tim-3^high AML patients was significantly shortened（p=0.0021）.The Tim-3monoclonal antibody inhibited the proliferation of K562,U937,THP-1,and WEHI-3 cell lines in vitro（p<0.0001）,promoted the apoptosis of K562 and U937 cell lines,and led to the cell cycle arrest of K562 and U937 in G₀/G₁ phase.The cell cycle of the cell line was arrested in the G₀/G₁ phase.Western blot experiments showed that the Tim-3 monoclonal antibody up-regulated the expression of p21 and p27^KIP proteins of K562 and U937 and down-regulate the protein expression of cyclin D1.In vivo experiments found that the Tim-3 monoclonal antibody prolonged the survival of BALB/c mice with WEHI-3（p=0.0068）and significantly reduced the number and volume of extramedullary tumor infiltration in the livers and spleens of AML mice.The weight of the livers and spleens（p=0.0466）of AML mice in the Tim-3 monoclonal antibody group was lighter than that in the control group.Histopathological immunofluorescence staining showed that the Tim-3 monoclonal antibody increased CD4⁺and CD8⁺T cells in AML mice’s spleens.The Tim-3 CAR-T cells constructed in this study can correctly bind to the Tim-3 protein,and the infection rate of Tim-3 CAR-T obtained by lentiviral vector infection exceeds 60%.Tim-3 CAR-T cells can kill K562-Tim-3⁺-Luc and Jurkat-Tim-3⁺cell lines and THP-1,which naturally highly expressed Tim-3 in vitro,releasing the cytokines IFN-γand TNF-α.And with the increase of the effective target ratio,the killing efficiency and the release of cytokines continue to increase.In contrast,the K562 and Jurkat cell lines with low Tim-3 expression cannot be efficiently killed by Tim-3 CAR-T.In mice experiments,Tim-3 CAR-T can extend the median survival time of NCG mice with K562-Tim-3⁺-Luc from 30 days to 51 days（p=0.0018）.Conclusion:1.The prognosis of AML patients with high Tim-3 expression is poor.2.Tim-3monoclonal antibody can inhibit the proliferation of AML cells in vivo and in vitro by blocking the cell cycle,and treat AML.3.Tim-3 CAR-T cells can target Tim-3 in vivo and in vitro to kill AML cells and treat AML.4.Tim-3 may become a new target for AML immunotherapy.