%0 Journal Article
%T A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies
%A Stefanie Claudia Pohl
%A Steffi Schwarz
%A Andr¨¦ Frenzel
%A Thomas Schirrmann
%J Antibodies
%D 2012
%I MDPI AG
%R 10.3390/antib1010019
%X 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.
%K recombinant antibody
%K single chain Fv
%K scFv-Fc-scFv
%K bispecific antibody
%K tetravalent antibody
%K dual specificity
%K mammalian expression
%U http://www.mdpi.com/2073-4468/1/1/19