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Background Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19+ malignancies. Methods CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)–derived CIKs. Results CB-CIKs, like PB-CIKs, were mostly CD3+ T cells with mean 45% CD3+CD56+ and expressing mostly TCR(T cell receptor)αβ with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4+ cells, mostly CD56−, as well as a greater proportion of naive CCR7+CD45RA+ and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8+ cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19+ tumor cells in the presence of blinatumomab, with 30–60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph+ CD19+ acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 106 CIK/kg. Discussion We conclude that CB-CIKs, combined with bispecific T-cell–engaging antibodies, offer a novel, effective treatment strategy for leukemia.