Targeting CD28 on T lineage malignancies with chimeric antigen receptor T cells

Currently, no immunotherapy is approved for T cell acute lymphoblastic leukemia (T‑ALL). High initial response rates to CD7‑directed chimeric antigen receptor (CAR) T cells are limited by profound T cell aplasia and CD7‑negative immune escape, underscoring the need for alternative targets. Here, we show that CD28 is overexpressed on T‑ALL blasts from children and adolescents compared with lymphoid progenitors from healthy donors and is uniformly upregulated in nodal T-follicular helper cell lymphomas (nTFHL-AI, nTFHL-F). Using CRISPR/Cas9-mediated CD28 knockout to prevent fratricide, we generate highly functional anti‑CD28 CAR T cells with selective cytotoxicity against CD28⁺ T‑ALL. Anti‑CD28 CAR T cells match anti‑CD7 CAR T cells in vitro and in vivo, but cause markedly less lymphodepletion, largely sparing CD8 T cells and NK cells while preferentially depleting CD4 T cells, particularly TH2 and TH17 subsets. These findings establish CD28 as an actionable target for CAR T cell therapy of T cell malignancies.