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Combining CD33-Targeted Chimeric Antigen Receptor T cells with Decitabine for Acute Myeloid Leukemia

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE289928 | GEO | 2026/02/23

REPOSITORIES: GEO

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Project description:Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile than non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with a highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allow the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

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Combining CD33-Targeted Chimeric Antigen Receptor T cells with Decitabine for Acute Myeloid Leukemia

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