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High Level Escherichia coli Expression and Production of a Bivalent Humanized Antibody Fragment

Bio/Technology volume 10, pages 163–167 (1992)Cite this article

Abstract

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab′)2 fragments by secretion from E. coli. Titers of 1-2 g l−1 of soluble and functional Fab′ fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab′ in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab′ with the single hinge cysteine in the free thiol state, allowing F(ab′)2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab′)2 fragment is indistinguishable from F(ab′)2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

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Author information

Author notes

  1. Mark D. Velligan

    Present address: Gene Labs, 505 Penobscot Drive, Redwood City, CA, 94063

  2. Paul Carter: Corresponding author.

Authors and Affiliations

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

    Paul Carter, Robert F. Kelley & Maria L. Rodrigues

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

    Brad Snedecor, Michael Covarrubias & Mark D. Velligan

  3. Department of Immunological Research and Assay Technology, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Wai Lee T. Wong & Ann M. Rowland

  4. Department of Medicinal and Analytical Chemistry, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Claire E. Kotts & Monique E. Carver

  5. Department of Cell Genetics, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    Maria Yang & Dennis Henner

  6. Department of Protein Chemistry, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    James H. Bourell

  7. Department of Cell Biology, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080

    H. Michael Shepard

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Carter, P., Kelley, R., Rodrigues, M. et al. High Level Escherichia coli Expression and Production of a Bivalent Humanized Antibody Fragment. Nat Biotechnol 10, 163–167 (1992). https://doi.org/10.1038/nbt0292-163

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