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A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies
Department of Biotechnology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany
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Received: 1 March 2012; in revised form: 28 March 2012 / Accepted: 30 March 2012 / Published: 11 April 2012
Abstract: Bivalent single chain (sc)Fv-Fc antibodies have been used for years as recombinant alternatives of natural immunoglobulins. We have extended this approach to the scFv-Fc-scFv antibody format to obtain tetravalent antigen binding and the possibility to generate bispecific antibodies. We developed a mammalian expression vector system to construct tetravalent scFv-Fc-scFv antibodies with two NcoI+NotI compatible cloning sites flanking the Fc gene fragment. We demonstrated direct cloning from single chain antibody gene libraries and tested various scFv combinations. Transient production of scFv-Fc-scFv antibodies in human embryonic kidney (HEK) 293T cells achieved volumetric yields of up to 10 mg/L. However, expression levels were strongly dependent on the carboxyterminal scFv and the scFv combination. All scFv-Fc-scFv antibodies exclusively formed disulfide-linked homodimers. Antigen binding studies revealed dual specificity for all scFv-Fc-scFv employing different scFv fragments. Comparison of C-reactive protein (CRP) specific monovalent scFv LA13-IIE3, bivalent scFv-Fc and Fc-scFv LA13-IIE3, and tetravalent scFv-Fc-scFv (scFv LA13-IIE3 in combination with scFvs LA13-IIE3, TOB4-B11, or TOB5-D4) revealed an up to 500-fold increased antigen binding. This novel scFv-Fc-scFv antibody expression system allows simple and fast testing of various scFv combinations.
Keywords: recombinant antibody; single chain Fv; scFv-Fc-scFv; bispecific antibody; tetravalent antibody, dual specificity; mammalian expression
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Pohl, S.C.; Schwarz, S.; Frenzel, A.; Schirrmann, T. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies. Antibodies 2012, 1, 19-38.
Pohl SC, Schwarz S, Frenzel A, Schirrmann T. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies. Antibodies. 2012; 1(1):19-38.
Pohl, Stefanie Claudia; Schwarz, Steffi; Frenzel, André; Schirrmann, Thomas. 2012. "A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies." Antibodies 1, no. 1: 19-38.