Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments

E. Boel, S. Verlaan, M.J.J.G. Poppelier, N.A.C. Westerdaal, J.A.G. van Strijp, T. Logtenberg

Research output: Contribution to journalArticleAcademicpeer-review

57 Citations (Scopus)

Abstract

We have constructed a series of eukaryotic expression vectors that permit the rapid conversion of single chain (sc) Fv antibody fragments, derived from semi-synthetic phage display libraries, into intact fully human monoclonal antibodies (mAb) of each isotype. As a model, a scFv fragment specific for sheep red blood cells (SRBC) was isolated from a semi-synthetic phage antibody (Ab) display library, and used to produce human mAbs of IgM, IgG1-IgG4, IgA1, IgA2m(1) and IgE isotype in vitro in stably transfected cells. N-terminal protein sequence analysis of purified immunoglobulin heavy (H) and light (L) chains revealed precise proteolytic removal of the leader peptide. Biochemical analysis of purified recombinant human mAbs demonstrated that properly glycosylated molecules of the correct molecular size were produced. The IgG and IgA mAbs retained SRBC-binding activity, interacted with different Fc receptor-transfectants, and induced complement-mediated hemolysis and Ab-dependent phagocytosis of SRBC by neutrophils in a pattern consistent with the immunoglobulin (Ig) H chain isotype. We conclude that in vitro produced recombinant human mAbs constructed from phage display library-derived scFv fragments mirror their natural counterparts and may represent a source of mAbs for use in human therapy. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)153-166
Number of pages14
JournalJournal of Immunological Methods
Volume239
Issue number1-2
DOIs
Publication statusPublished - 26 May 2000

Keywords

  • Cloning vectors
  • Complement-mediated lysis
  • Isotype switching
  • Phagocytosis
  • Transient and stable expression

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