Background <p>Clonal and pathogenic eosinophil expansion in hypereosinophilic disorders (e.g., refractory hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL)) remains an unmet therapeutic challenge, with current strategies often failing to induce durable remission. While monoclonal antibodies targeting the IL-5/IL-5Rα pathway have shown efficacy in treating eosinophil-driven diseases, a subset of patients experience incomplete responses or relapses, highlighting the need for more durable and comprehensive therapeutic strategies. Chimeric antigen receptor T (CAR-T) cell therapy, with its potential for long-term persistence and durable remission after a single infusion, represents a promising alternative for patients with refractory disease.</p> Methods <p>We performed single-cell RNA sequencing on peripheral blood (PB) and bone marrow (BM) samples from both healthy individuals and the patient with hypereosinophilic disorder, to identify key therapeutic targets for intervention. Based on these findings, we developed a first-in-class CAR-T therapy using human interleukin-5 (hIL-5) as a ligand-based targeting domain, to selectively recognize and eliminate IL-5Rα⁺ eosinophils and precursors. In vitro cytotoxicity and IFN-γ secretion were measured against target cells and patient-derived PB/BM samples. Preclinical safety was evaluated through comprehensive toxicity assessment. Efficacy was evaluated in a hypereosinophilic leukemia mouse model, with tumor burden reduction and survival as the key evaluation indicators.</p> Results <p>Single-cell profiling revealed concurrent expansion of both eosinophil progenitors and mature eosinophils in the BM and PB, highlighting the need for therapies targeting all stages of eosinophil development. IL-5 receptor α (IL-5Rα) was identified as the optimal target due to its high expression across all stages of eosinophil development, with relatively restricted expression on non-eosinophil immune populations. hIL-5 CAR-T cells demonstrated potent in vitro cytotoxicity and IFN-γ secretion against target cells and effectively eliminated eosinophils in patient-derived PB/BM samples. No dose-limiting toxicities were observed, and no evidence of cytokine release syndrome (CRS) was detected in preclinical models. In the hypereosinophilic leukemia mouse model, a single infusion of hIL-5 CAR-T cells significantly reduced tumor burden and extended survival, demonstrating its therapeutic potential.</p> Conclusions <p>IL-5 CAR-T cell therapy represents a promising targeted therapeutic approach for IL-5Rα-expressing hypereosinophilic disorders. Its ability to target eosinophils and their precursors across all developmental stages in BM and PB addresses a critical unmet medical need in refractory HES and CEL.</p>