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CD21 is expressed in 50% of T-ALL cases at diagnosis but in fewer than 10% of mature T cells. We observed that CAR-T cells targeting membrane-distal CD21 epitopes were ineffective, likely because of the bulky, glycosylated nature of the antigen. However, when we engineered CAR-T cells to target membrane-proximal CD21 epitopes using an antigen-binding fragment (Fab)\u2013CAR design, we demonstrated robust activity against T-ALL cell lines, primary tumors, and patient-derived xenografts in both in vitro and in vivo models. The enhanced efficacy of this Fab-CAR design was driven by its high stability and reduced surface expression, addressing limitations of traditional CAR constructs. In addition, pharmacological inhibition of the phosphatidylinositol 3-kinase axis up-regulated CD21 expression in T-ALL, further enhancing the potency of anti-CD21 CAR-T cells in vitro and in a patient-derived xenograft in vivo model. This study establishes CD21 as a viable CAR-T target and highlights advances in CAR design for bulky antigens, as well as the potential for pharmacological strategies to augment target expression. Anti-CD21 CAR-T cells represent a promising therapeutic option for improving outcomes for patients with T-ALL.<\/jats:p>","DOI":"10.1126\/scitranslmed.adr1476","type":"journal-article","created":{"date-parts":[[2025,4,16]],"date-time":"2025-04-16T18:00:51Z","timestamp":1744826451000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":1,"title":["Preclinical development of anti-CD21 chimeric antigen receptor T cells to treat T cell acute lymphoblastic leukemia"],"prefix":"10.1126","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3865-3723","authenticated-orcid":true,"given":"Nicola","family":"Maciocia","sequence":"first","affiliation":[{"name":"Department of Haematology, Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, 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