// Claudia C. Roskopf 1 , Christian B. Schiller 2 , Todd A. Braciak 1 , Sebastian Kobold 3 , Ingo A. Schubert 4 , Georg H. Fey 4 , Karl-Peter Hopfner 2 , Fuat S. Oduncu 1 1 Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, Haematology/Oncology, Munich, Germany 2 Ludwig-Maximilians-Universität München, Department of Biochemistry/Gene Center, Munich, Germany 3 Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, Division of Clinical Pharmacology, Munich, Germany 4 Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Biology, Erlangen, Germany Correspondence to: Claudia C. Roskopf, e-mail: Claudia.Roskopf@med.uni-muenchen.de Key Words: immunotherapy, triplebody, cytolytic T cells, antibody-dependent cellular cytotoxicity, leukemia Received: June 06, 2014 Accepted: July 15, 2014 Accepted: July 23, 2014 Abstract Triplebody 19-3-19, an antibody-derived protein, carries three single chain fragment variable domains in tandem in a single polypeptide chain. 19-3-19 binds CD19-bearing lymphoid cells via its two distal domains and primary T cells via its CD3-targeting central domain in an antigen-specific manner. Here, malignant B-lymphoid cell lines and primary cells from patients with B cell malignancies were used as targets in cytotoxicity tests with pre-stimulated allogeneic T cells as effectors. 19-3-19 mediated up to 95 % specific lysis of CD19-positive tumor cells and, at picomolar EC 50 doses, had similar cytolytic potency as the clinically successful agent Blinatumomab TM . 19-3-19 activated resting T cells from healthy unrelated donors and mediated specific lysis of both autologous and allogeneic CD19-positive cells. 19-3-19 led to the elimination of 70 % of CD19-positive target cells even with resting T cells as effectors at an effector-to-target cell ratio of 1 : 10. The molecule is therefore capable of mediating serial lysis of target cells by a single T cell. These results highlight that central domains capable of engaging different immune effectors can be incorporated into the triplebody format to provide more individualized therapy tailored to a patient’s specific immune status.