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

Antibodies

Stefanie Claudia Pohl¹  Steffi Schwarz¹  André Frenzel¹ 

[1] Department of Biotechnology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany;

关键词: recombinant antibody;    single chain Fv;    scFv-Fc-scFv;    bispecific antibody;    tetravalent antibody;    dual specificity;    mammalian expression;   

DOI  :  10.3390/antib1010019

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2012 by the authors; licensee MDPI, Basel, Switzerland.