Abstract Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. Significance: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development. This article is highlighted in the In This Issue feature, p. 1861