Background: CD44v6 chimeric antigen receptor T (CD44v6 CAR-T) cells demonstrate strong anti-tumor ability and safety in acute myeloid leukemia (AML). However, the expression of CD44v6 on T cells leads to transient fratricide and exhaustion of CD44v6 CAR-T cells, which affect the application of CD44v6 CAR-T. The exhaustion and function of T cells and CD44v6 expression of AML cells are associated with DNA methylation. Hypomethylating agents (HAMs) decitabine (Dec) and azacitidine (Aza) have been widely used to treat AML. Therefore, there may be synergy between CD44v6 CAR-T cells and HAMs in the treatment of AML.Methods: CD44v6 CAR-T cells pretreated with Dec or Aza were co-cultured with CD44v6+ AML cells. Dec or aza pretreated AML cells were co-cultured with CD44v6 CAR-T cells. The cytotoxicity, exhaustion, differentiation and transduction efficiency of CAR-T cells, and CD44v6 expression and apoptosis in AML cells were detected by flow cytometry. The subcutaneous tumor models were used to evaluate the anti-tumor effect of CD44v6 CAR-T cells combined with Dec in vivo. The effects of Dec or Aza on gene expression profile of CD44v6 CAR-T cells were analyzed by RNA-seq.Results: Our results revealed that Dec and Aza improved the function of CD44v6 CAR-T cells through increasing the absolute output of CAR+ cells and persistence, promoting activation and memory phenotype of CD44v6 CAR-T cells, and Dec had a more pronounced effect. Dec and Aza promoted the apoptosis of AML cells, particularly with DNA methyltransferase 3A (DNMT3A) mutation. Dec and Aza also enhanced the CD44v6 CAR-T response to AML by upregulating CD44v6 expression of AML cells regardless of FMS-like tyrosine kinase 3 (FLT3) or DNMT3A mutations. The combination of Dec or Aza pretreated CD44v6 CAR-T with pretreated AML cells demonstrated the most potent anti-tumor ability against AML.Conclusion: Dec or Aza in combination with CD44v6 CAR-T cells is a promising combination therapy for AML patients.

Chimeric antigen receptor T cell (CAR-T) therapy in hematologic malignancies has made great progress, but there are still some problems. First, T cells from tumor patients show an exhaustion phenotype; thus, the persistence and function of the CAR-Ts are poor, and achieving a satisfactory curative effect is difficult. Second, some patients initially respond well but quickly develop antigen-negative tumor recurrence. Thirdly, CAR-T treatment is not effective in some patients and is accompanied by severe side effects, such as cytokine release syndrome (CRS) and neurotoxicity. The solution to these problems is to reduce the toxicity and enhance the efficacy of CAR-T therapy. In this paper, we describe various strategies for reducing the toxicity and enhancing the efficacy of CAR-T therapy in hematological malignancies. In the first section, strategies for modifying CAR-Ts using gene-editing technologies or combining them with other anti-tumor drugs to enhance the efficacy of CAR-T therapy are introduced. The second section describes some methods in which the design and construction of CAR-Ts differ from the conventional process. The aim of these methods is to enhance the anti-tumor activity of CAR-Ts and prevent tumor recurrence. The third section describes modifying the CAR structure or installing safety switches to radically reduce CAR-T toxicity or regulating inflammatory cytokines to control the symptoms of CAR-T-associated toxicity. Together, the knowledge summarized herein will aid in designing better-suited and safer CAR-T treatment strategies.