a cassette vector system for the rapid cloning and production of bispecific tetravalent antibodies盒式向量系统双特异性四价的快速克隆和生产抗体.pdfVIP

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a cassette vector system for the rapid cloning and production of bispecific tetravalent antibodies盒式向量系统双特异性四价的快速克隆和生产抗体.pdf

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a cassette vector system for the rapid cloning and production of bispecific tetravalent antibodies盒式向量系统双特异性四价的快速克隆和生产抗体

Antibodies 2012, 1, 19-38; doi:10.3390/antib1010019 OPEN ACCESS antibodies ISSN 2073-4468 /journal/antibodies Article A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies Stefanie Claudia Pohl, Steffi Schwarz, André Frenzel and Thomas Schirrmann * Department of Biotechnology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany; E-Mails: stefanie.pohl@twincore.de (S.C.P.); steffischwarz2@gmx.de (S.S.); andre.frenzel@tu-bs.de (A.F.) * Author to whom correspondence should be addressed; E-Mail: th.schirrmann@tu-bs.de; Tel.: +49-531-391-5760; Fax: +49-531-391-5763. 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