Combining CD33-Targeted Chimeric Antigen Receptor T cells with Decitabine for Acute Myeloid Leukemia
Background: Relapsed and/or refractory (r/r) acute myeloid leukemia (AML) is a challenging disease with poor prognosis and limited treatment options. CD33, a cell surface antigen expressed on over 85% of AML blasts, is a promising target for chimeric antigen receptor (CAR) T-cell therapy. Decitabine (DAC), a hypomethylating agent (HMA), is a well-tolerated treatment for AML patients. Combining anti-CD33 immunotherapy with HMAs has shown benefits in AML patients unfit for intensive chemotherapy. Our goal is to develop optimal CD33-CAR T cells and combine them with DAC to provide an effective treatment for AML. Methods: Flow cytometry and antibody-binding capacity analysis were used to measure cell surface marker expression. Degranulation, intracellular cytokine expression, and tumor cell killing assays were used to assess CAR-T cell function in vitro. In vivo antileukemic activity was examined in AML xenografted NSG mice, with leukemic burden monitored via bioluminescence imaging. colony-forming-unit assay assessed the impact of CD33-CAR T cells on normal hematopoiesis. Public dataset analysis assessed correlation between two variables, such as CD33 mRNA expression and DNA methylation. Pairwise Controlled Manifold Approximation (PaCMAP) was used to visualized T cell-patient AML blast microenvironment after killing assay. Bulk RNA-seq analyzed mRNA and signaling pathways changes in AML cells after exposure to DAC. Results: We generated CD33-CAR T cells using a humanized anti-CD33 scFv and found that the CD8H version was more potent than the IgG4(EQ) variant against CD33? AML cell lines. The CH8H CAR T cells exhibited strong effector functions and significantly reduced AML tumor burden in NSG mice, without impairing HSPC colony formation. In silico analysis revealed an inverse correlation between CD33 methylation and gene expression. DAC pretreatment upregulated CD33 expression, enhancing AML cell elimination by CD33-CAR T cells in vitro and in vivo, especially under conditions with limited CAR T-cells. DAC upregulated the proteasome inhibition-induced apoptosis pathway and downregulated DNA repair and MYC oncogenic pathways, which, together with enhanced CD33 expression, primed AML cells for more effective CAR T-cell targeting.   Conclusions: This study demonstrates the preclinical efficacy and safety of CD33-CAR T cells in treating AML and shows that DAC pretreatment boosts their antileukemic activity. This strategy holds promise for improving clinical outcomes and overall survival in r/r AML patients. Overall design: RNA-seq profiling of KG-1A cells treated with Decitabine (DAC) for 2 days. KG-1A cells treated with DMSO for 2 days were used as control.