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High Level Secretion of a Humanized Bispecific Diabody from Escherichia coli

Bio/Technology volume 14, pages 192–196 (1996)Cite this article

Abstract

Clinical development of bispecific antibodies (BsAb) has been effectively stymied by the lack of efficient production methods. We therefore attempted to produce a humanized BsAb fragment using an expression system that has proved very successful for secretion of monospecific Ab fragments from E. coli. An anti-p185HER2/anti-CD3 BsF(ab′)2 was first recast into the diabody format and then periplasmically secreted from E. coli grown to high cell density in a fermentor. The diabody was recovered in very high yield (up to 935 mg/1) after protein A purification and predominantly (≥ 80 %) as a dimer as judged by size exclusion chromatography. Diabody dimers were found to be mainly functional heterodimers (∼75%) by titration with p185HER2 extracellular domain. The diabody binds p185HER2 extracellular domain and human T lymphocytes with affinities close to those of the parent BsF(ab′)2. Furthermore, the diabody is capable of simultaneous binding to tumor cells overexpressing p185HER2 and CD3 on T cells as shown by cellular rosetting. The diabody is equally potent as the parent BsF(ab′)2 in retargeting IL-2 activated T-enriched peripheral blood lymphocytes to lyse tumor cells overexpressing p185HER2.

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Author notes

  1. Paul Carter: email: pjc@gene.com.

Authors and Affiliations

  1. Department of Molecular Oncology, Genentech Inc., 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Zhenping Zhu & Paul Carter

  2. Department Process Sciences, Genentech Inc., 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Gerardo Zapata

  3. Department Bioanalytical Technology, Genentech Inc., 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Refaat Shalaby

  4. Department Fermentation and Cell Culture Research and Development, Genentech Inc., 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Brad Snedecor & Han Chen

Authors
  1. Zhenping Zhu
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  2. Gerardo Zapata
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  3. Refaat Shalaby
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  4. Brad Snedecor
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  5. Han Chen
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  6. Paul Carter
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Zhu, Z., Zapata, G., Shalaby, R. et al. High Level Secretion of a Humanized Bispecific Diabody from Escherichia coli. Nat Biotechnol 14, 192–196 (1996). https://doi.org/10.1038/nbt0296-192

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