Site-specific modification of ED-B-targeting antibody using intein-fusion technology

BMC Biotechnology

Research Article

Peter Bringmann¹  Sina Möhlmann²  Simone Greven²  Axel Harrenga³ 

[1] Bayer HealthCare LLC, 455 Mission Bay Blvd South, 94158, San Francisco, CA, USA;Bayer Healthcare Research Center, Aprather Weg 18a, 42113, Wuppertal, Germany;Bayer Healthcare, Nattermannallee 1, 50829, Cologne, Germany;

关键词: Mesna;    Nostoc Punctiforme;    Refractive Index;    Interchain Disulfide Bond;    Express Protein Ligation;   

DOI  :  10.1186/1472-6750-11-76

 received in 2010-10-27, accepted in 2011-07-21,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundA promising new approach in cancer therapy is the use of tumor specific antibodies coupled to cytotoxic agents. Currently these immunoconjugates are prepared by rather unspecific coupling chemistries, resulting in heterogeneous products. As the drug load is a key parameter for the antitumor activity, site-specific strategies are desired. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are methods for the specific C-terminal modification of a target protein. Both include the expression as an intein fusion protein, followed by the exchange of the intein for a functionalized moiety.ResultsA full-length IgG specific for fibronectin ED-B was expressed as fusion protein with an intein (Mxe GyrA or Npu DnaE) attached to each heavy chain. In vitro protocols were established to site-specifically modify the antibodies in high yields by EPL or PTS, respectively. Although reducing conditions had to be employed during the process, the integrity or affinity of the antibody was not affected. The protocols were used to prepare immunoconjugates containing two biotin molecules per antibody, attached to the C-termini of the heavy chains.ConclusionFull-length antibodies can be efficiently and site-specifically modified at the C-termini of their heavy chains by intein-fusion technologies. The described protocols can be used to prepare immunoconjugates of high homogeneity and with a defined drug load of two. The attachment to the C-termini is expected to retain the affinity and effector functions of the antibodies.

【 授权许可】

Unknown   
© Möhlmann et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.