The Prostate 71:588 ^596 (2011) EffectiveTargeting of Prostate Cancer by Lymphocytes Redirected by a PSMA  CD3 Bispecific Single-Chain Diabody Kerstin Fortmu ¨ ller, 1,2 Karen Alt, 1,2 Dorothee Gierschner, 1 Philipp Wolf, 1 Volker Baum, 1,2 Nikolaus Freudenberg, 3 Ulrich Wetterauer, 1 Ursula Elsa ¨sser-Beile, 1 * and Patrick Bu ¨ hler 1 1 Department of Urology, Experimental Urology,University Hospital Freiburg, Freiburg,Germany 2 Faculty of Biology,University of Freiburg, Freiburg,Germany 3 Department of Pathology,University Hospital Freiburg, Freiburg,Germany BACKGROUND. For redirecting T-lymphocytes to induce prostate cancer cell lysis, we constructed a novel bispecific single-chain (bsc) diabody directed to the prostate specific membrane antigen (PSMA) and the T-cell receptor (TCR)-associated CD3 molecule on T-cells. METHODS. The PSMA  CD3 bsc diabody was generated from an anti-CD3 single chain Fv fragment (scFv) and the anti-PSMA scFv D7. It was expressed in E. coli and purified from the periplasmic extract and culture supernatant by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST-1) was used and activation of T-cells was determined by measuring the surface marker expression of CD25 and CD69. For in vivo evaluation, the diabody was administered in combination with human peripheral blood lymphocytes (Ly) in a C4-2 xenograft-SCID mouse model. RESULTS. Specific binding of the PSMA  CD3 bsc diabody both to CD3-positive Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the PSMA  CD3 bsc diabody proved to be a potent agent for retargeting CD4 þ and CD8 þ human lymphocytes to lyse C4-2 prostate cancer cells. Treatment of SCID mice bearing C4-2 tumor xenografts with the diabody and human lymphocytes efficiently inhibited tumor growth. CONCLUSIONS. The PSMA  CD3 bsc diabody bears a high potential for the immuno- therapy of prostate cancer. Prostate 71: 588–596, 2011. # 2010 Wiley-Liss, Inc. KEY WORDS: PSMA  CD3 bsc diabody; prostate cancer; T-cell redirecting; bispecific diabody; immunotherapy INTRODUCTION Prostate cancer remains a leading cause of death for men in the Western civilization [1]. Despite the effectiveness of prostatectomy, radiotherapy, and hormonal therapy, a significant proportion of patients progresses to advanced metastatic disease for which there are currently no effective treatment options. This necessitates the development of new and targeted therapies. Among the different identified prostate antigens, the prostate specific membrane antigen (PSMA) has been shown to represent an excellent target for immunotherapy because it is (i) selectively expressed in the prostate, (ii) abundantly expressed as protein at all stages of prostate cancer and even upregulated in androgen-insensitive metastatic Grant sponsor: Deutsche Krebshilfe. *Correspondence to: Ursula Elsa ¨sser-Beile, Department of Urology, Experimental Urology, University of Freiburg, Breisacher Str. 117, 79106 Freiburg, Germany. E-mail: ursula.elsaesser@uniklinik-freiburg.de Received 15 July 2010; Accepted 24 August 2010 DOI 10.1002/pros.21274 Published online 14 October 2010 in Wiley Online Library (wileyonlinelibrary.com). ß 2010 Wiley-Liss, Inc.