Tetravalent Bispecific Single-Chain Fv Antibodies for Lysis of Leukemia Cells by Autologous T Cells

Abstract

Activation of host immune mechanisms using bispecific antibodies is one of a few alternative strategies to address the limitations of the conventional monoclonal antibodies and to improve the efficacy of the targeted cancer therapy. Two single‐chain fragment variable (scFv)-based bispecific antibody formats have been intensively studied preclinically and in clinical trials: tandem scFv molecules [or (scFv)2] and diabodies. Both are small in size and are able to bind the effector and target cells only monovalently. To circumvent the drawbacks of both formats, we have designed a single-chain molecule that is composed solely of the variable (VH and VL) domains from two different specificities and has a modular structure, where a diabody module plays a role of oligomerization motif due to interchain pairing of the cognate VH and VL domains. The resulting bispecific scFvA–diabodyB–scFvA tandem molecules (where A and B are different specificities), which we called ‘flexibodies’, possess structural flexibility, bind bivalently to both effector and target cell, and are able to cross-link spatially distinct antigens. Here, we report effective killing of malignant primary B cells from patients with B-cell chronic lymphocytic leukemia mediated by bispecific CD19×CD3 flexibodies at very low effector-to-target cell ratios. Use of tetravalent flexibodies caused almost quantitative elimination of leukemia cells from the blood samples of B‐cell chronic lymphocytic leukemia patients, while the structurally similar but bivalent (scFv)2 molecules showed no antitumor activity. These data demonstrate that the novel oligovalent flexibody format might be a promising tool for treatment of human malignancies.