| Autor: |
Stefanie Claudia Pohl, Steffi Schwarz, André Frenzel, Thomas Schirrmann |
| Jazyk: |
angličtina |
| Rok vydání: |
2012 |
| Předmět: |
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| Zdroj: |
Antibodies, Vol 1, Iss 1, Pp 19-38 (2012) |
| Druh dokumentu: |
article |
| ISSN: |
2073-4468 |
| DOI: |
10.3390/antib1010019 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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