TAILIEUCHUNG - Báo cáo khoa học: Application of the Fc fusion format to generate tag-free bi-specific diabodies

We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy. Bacterial expression can be used to express small recombi-nant antibodies on a large scale; however, their overexpression often results in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography. | Application of the Fc fusion format to generate tag-free bi-specific diabodies l . A 1 I I II 111 I I I 1 X z II 1 1 I I A I I I 1 Ryutaro Asano Keiko Ikoma Hiroko Kawaguchi Yuna Ishiyama Takeshi Nakanishi Mitsuo Umetsu1 Hiroki Hayashi2 Yu Katayose2 Michiaki Unno2 Toshio Kudo3 and Izumi Kumagai1 1 Department of Biomolecular Engineering Graduate Schoolof Engineering Tohoku University Sendai Japan 2 Division of GastroenterologicalSurgery Department of Surgery Graduate Schoolof Medicine Tohoku University Sendai Japan 3 CellResource Center for BiomedicalResearch Institute of Development Aging and Cancer Tohoku University Sendai Japan Keywords bi-specific diabody Fc fusion format preparation method smalltherapeutic antibody tag-free protein Correspondence I. Kumagai Aoba 6-6-11-606 Aramaki Aoba-ku Sendai 980-8579 Japan Fax 81 22 795 6164 Tel 81 22 795 7274 E-mail kmiz@ Received 1 May 2009 revised 9 November 2009 accepted 17 November 2009 doi We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 hEx3-Db for cancer immunotherapy. Bacterial expression can be used to express small recombinant antibodies on a large scale however their overexpression often results in the formation of insoluble aggregates and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography. Here we propose a novel method for preparing refined functional tag-free bi-specific diabodies from IgG-like bi-specific antibodies BsAbs in a mammalian expression system. We created an IgG-like BsAb in which bi-specific diabodies were fused to the human Fc region via a designed human rhinovirus 3C HRV3C protease recognition site. The BsAb was purified by protein A affinity chromatography and the refined tag-free hEx3-Db was efficiently produced from the Fc fusion format by protease digestion. The tag-free hEx3-Db from the Fc .

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