PhD student University of Calgary Calgary, Alberta, Canada
Disclosure(s):
Shanshan Wang, MSc: No financial relationships to disclose
Introduction/Rationale: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Current Immunotherapies, including chimeric antigen receptor (CAR) T cell strategies, are limited by the scarcity of antigens that are present on AML cells but absent from hematopoietic stem and progenitor cells (HSPCs). To identify a potential therapeutic target, we analyzed two publicly available single-cell RNA sequencing datasets and found that CD4 is frequently expressed on AML blasts but absent from HSPCs, supporting its potential as a novel CAR target.
We developed a CD4-targeted CAR using CD3⁺CD4⁻CD8⁻ double-negative T cells (CAR4-DNTs). DNTs offer a unique therapeutic platform: they naturally lack CD4, preventing fratricide, and elicit endogenous anti-leukemic activity. Their feasibility and safety as an allogeneic cell therapy platform was demonstrated in a Phase I trial. This work aims to establish CAR4-DNTs as a next-generation, off-the-shelf CAR-T cell therapy for AML.
Methods: Donor-derived DNTs were engineered to express a CD4-CAR. Cytotoxicity, persistence, and cytokine secretion were evaluated against AML cell lines, CD4⁺ and CD4-knockout AML variants, and primary AML samples. NSG-SGM3 mice engrafted with luciferase⁺ MV4-11 cells received CAR4-DNTs, untransduced (UT) DNTs, or PBS. Disease progression was monitored by bioluminescence imaging and survival analysis.
Results: CAR4-DNTs were successfully manufactured without fratricide. They exhibited significantly enhanced and more durable cytotoxicity than UT-DNTs against AML cell lines and patient samples. Importantly, CAR4-DNTs retained endogenous cytotoxicity towards CD4-negative AML cells. In vivo, CAR4-DNT achieved durable leukemia clearance and prolonged survival, whereas PBS-treated mice rapidly progressed and UT-DNT recipients showed transient disease control before relapse.
Conclusion: These findings establish CAR4-DNTs as a promising immunotherapy candidate for AML, supporting their further preclinical and translational development.
