Study The Efficacy Of ITK Gene-Edited CD19 CAR T Cells On Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia(CLL)is a lymphoproliferative disorder that is characterized by the expansion of monoclonal mature B cells.Ibrutinib,an inhibitor of Bruton tyrosine kinase(BTK)is currently the main therapeutic drug for CLL.The chimeric antigen receptor(CAR)T cell immunotherapy is an alternative treatment for CLL patients who resisted BTK inhibitors,however,the therapeutic efficacy of autologous CD19 CAR T cells is relatively poor in CLL as there is acquired T cell dysfunction in CLL patients,characterized by decreased proliferation and increased exhaustion.Studies have reported that Ibrutinib treatment significantly increases the number of T cells and reduces T cell exhaustion levels in CLL patients.Moreover,the combination of Ibrutinib and anti-CD19 CAR T cells improves the anti-CLL activity of CD19 CAR T cells.Nevertheless,the exact molecular mechanism of Ibrutinib in regulating the function of CD19 CAR T cells has not been fully elucidated.It is speculated that Ibrutinib exerts the above function via inhibiting interleukin-2 inducible T cell kinase(ITK).These results imply that reducing the expression of ITK in CD19 CAR T cells might improve the efficacy of CD19 CAR T cells in CLL.Objective: To study the effect and underlying mechanism of ITK gene knockout on cell cytotoxicity,activation level,exhaustion level,cytokine release,cell expansion,cell apoptosis,cell proliferation,and anti-CLL potency of CD19 CAR T cells.To optimize the efficacy and persistent response rate of CD19 CAR T cells in treating CLL.Methods: In vitro experiments: The lentivirus vector was used to produce CD19 CAR T cells.We utilized CRISPR/Cas9 gene-editing technique to specifically knock out the ITK gene in CD19 CAR T cells and the knockout efficiency was assessed by Sanger sequencing of the singleguide RNA target DNA region.The control and ITK-KO CD19 CAR T cells were co-cultured with CLL cells separately in vitro to detect the effect of ITK knockout on the cytotoxicity of CD19 CAR T cells.The effects of ITK knockout on the activation level,exhaustion level,cytokine release,cell apoptosis,and cell proliferation of CD19 CAR T cells were evaluated by flow cytometry under unstimulated conditions or stimulated with MEC1 cells.RNA sequencing was used to investigate the related mechanisms of the effect of ITK knockout on CD19 CAR T cells.In vivo experiments:MEC1 cell-derived xenograft mice were divided into three groups:negative control group(injecting sterile phosphate solution),control CD19 CAR T cell group,and ITK-KO CD19 CAR T cell group.The peripheral blood of mice was harvested regularly for flow cytometry tests to analyze the effect of ITK knockout on the expansion ability and exhaustion level of CD19 CAR T cells.The impact of ITK knockout on the anti-CLL potency of CD19 CAR T cells was evaluated by measuring tumor volume.Results: The production efficiency of CD19 CAR T cells exceeds 80%and the ITK gene knockout efficiency was as high as 86% to 96% with no obvious off-target effect.In vitro cytotoxicity assay indicated that ITK gene knockout slightly decreased the cytotoxic activity of CD19 CAR T cells,but still retained strong cytotoxicity.Flow cytometry tests showed that ITK knockout decreased the proportion of CD19 CAR T cells expressing activation marker CD69 as well as exhaustion markers LAG-3 and PD-1.ITK knockout slightly reduced the interferon-γ release of CD19 CAR T cells while significantly decreasing the expression of Th2 cytokines.Continuous cell counting discovered that ITK gene knockout significantly improved the long-term expansion ability of CD19 CAR T cells in vitro.Further proliferation and apoptosis assays revealed that ITK knockout does not affect proliferation but significantly reduces apoptosis of CD19 CAR T cells.ITK knockout decreased the activation level of the T cell receptor pathway in CD19 CAR T cells.The analysis of RNA sequencing indicated that ITK gene knockout mainly affected cytokine-related pathways of CD19 CAR T cells.In vivo experiments in MEC1 cell-derived xenograft mice demonstrated that ITK knockout dramatically decreased the exhaustion level of CD19 CAR T cells and improved the expansion speed and extent of CD19 CAR T cells.Importantly,the ITK-KO CD19 CAR T cell outcompeted control CD19 CAR T cells in persistent anti-CLL ability.Conclusion: ITK gene knockout reduced the exhaustion level while improved the long-term expansion and survival ability,and enhanced the anti-CLL potency of the CD19 CAR T cells.