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A hematopoietic stem cell shield for AML-directed immunotherapy

ABSTRACT: One major challenge in the development of successful chimeric antigen receptor (CAR) T cells for patients with acute myeloid leukemia (AML) has been that most well-studied AML-associated antigens are also expressed on vital normal hematopoietic stem cells (HSCs) or mature myeloid cells. This challenge in optimal antigen selection leads to deleterious on-target toxicity, particularly in the context of CAR T cell persistence. CAR T cells targeting CD371, a type II transmembrane glycoprotein, have shown potent anti-leukemic activity in phase I trials to date; however, CD371 is wildly expressed throughout the normal myeloid compartment. Here we identify that CD371 is dispensable for normal human HSC and myeloid cell function, map the epitope of a CD371 CAR T cell currently in phase I trials, develop a base-editing strategy to shield normal hematopoiesis from CD371-directed immunotherapy, and describe an off-the-shelf approach to target AML while sparing normal hematopoiesis through the use of CD371 CAR T cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE317101 | GEO | 2026/04/08

REPOSITORIES: GEO

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Project description: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.

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A hematopoietic stem cell shield for AML-directed immunotherapy

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