PURPOSE: Although considerable progress has been made with autologous T cell-based therapy in B cell malignancies, application in chronic lymphocytic leukemia (CLL) lags behind due to disappointing response rates as well as substantial toxicity that is of particular concern in the elderly CLL population. Vγ9Vδ2-T cells form a conserved T cell subset with strong intrinsic immunotherapeutic potential, largely because of their capacity to be triggered by phosphoantigens that are overproduced by CLL and many other malignant cells. Specific activation of Vγ9Vδ2-T cells by a bispecific antibody may therefore improve the efficacy and toxicity of autologous T cell-based therapy in CLL. EXPERIMENTAL DESIGN: We evaluated CD1d expression in a cohort of 78 untreated CLL patients and generated a CD1d-specific Vγ9Vδ2-T cell engager based on single-domain antibodies (VHHs). RESULTS: We identified CD1d as a suitable target for antibody-based therapy in the majority of CLL patients, particularly in patients with advanced disease. The CD1d-specific Vγ9Vδ2-T cell engager induces robust activation and degranulation of Vγ9Vδ2-T cells, enabling Vγ9Vδ2-T cells from CLL patients to lyse autologous leukemic cells at low effector to target ratios. The expression of CD1d on CLL cells is upregulated by all-trans retinoic acid, and sensitizes the malignant cells to bispecific VHH-induced lysis. Furthermore, we provide evidence that the Vγ9Vδ2-T cell receptor retains its responsiveness to phosphoantigens when the bispecific VHH is bound, and aminobisphosphonates can therefore enhance bispecific Vγ9Vδ2-T cell engager-mediated tumor-specific killing. CONCLUSION: Collectively, our data demonstrate the immunotherapeutic potential of this novel CD1d-specific Vγ9Vδ2-T cell engager in CLL.