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Antibodies 2012, 1(1), 19-38; doi:10.3390/antib1010019
Article

A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies

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Received: 1 March 2012; in revised form: 28 March 2012 / Accepted: 30 March 2012 / Published: 11 April 2012
(This article belongs to the Special Issue Bispecific Antibodies for Dual Targeting Strategies)
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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 recombinant antibody; single chain Fv; scFv-Fc-scFv; bispecific antibody; tetravalent antibody, dual specificity; mammalian expression
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

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.

AMA Style

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.

Chicago/Turabian Style

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.

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