Journal of Immunological Methods 237 (2000) 131–145 www.elsevier.nl / locate / jim Recombinant Technology A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions a, b c d * Wijnand Helfrich , Hidde J. Haisma , Victor Magdolen , Thomas Luther , a,e a a a Victor J.J. Bom , Jantine Westra , Renske van der Hoeven , Bart Jan Kroesen , a a Grietje Molema , Lou de Leij a Groningen University Institute for Drug Exploration ( GUIDE) at the University Hospital Groningen, Department of Pathology and Laboratory Medicine, Medical Biology Branch, Laboratory for Tumor Immunology, Hanzeplein 1, 9713 GZ Groningen, The Netherlands b Department of Medical Oncology, University Hospital Free University, Amsterdam, The Netherlands c ¨ ¨ ¨ Frauenklinik der Technischen Universitat Munchen, Munchen, Germany d ¨ ¨ Universitatsklinikum der Technischen Universitat Dresden, Dresden, Germany e Groningen University Institute for Drug Exploration ( GUIDE) at the University Hospital Groningen, Division of Hemostasis and Thrombosis, Hanzeplein 1, 9713 GZ Groningen, The Netherlands Received 17 May 1999; accepted 29 October 1999 Abstract A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique Sfi I and Not I restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv) (2C11-MOC31) was constructed for retargeted T-cell 2 cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and / or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low Abbreviations: ScFv, single chain variable fragment; bs-(scFv) , bispecific scFv; bsAb, bispecific antibody; mAb, monoclonal antibody; 2 AS, angiostatin; tTF, truncated tissue factor; CD, cytosine deaminase; MCS, multiple cloning site; EGP2, epithelial glycoprotein 2; ADCC, antibody dependent cellular cytotoxicity; ADEPT, antibody directed enzyme prodrug therapy; 5-FU, 5-fluorouracil; Amp, ampicillin resistance gene *Corresponding author. Room T2.212, Building CMC VII, GUIDE at the University Hospital Groningen, Department of Pathology and Laboratory Medicine, Laboratory for Tumor Immunology, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Tel.: 131-50-361-3733; fax: 131-50-312-1576. E-mail address: w.helfrich@med.rug.nl (W. Helfrich) 0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759(99)00220-3