title: Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments.
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.
authors.affiliation: Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Utrecht University Hospital, Rm G04.614, P.O. Box 85500, NL-3508 GA, Utrecht, The Netherlands. eboel@wxs.nl
authors.forename: E
authors.initials: E
authors.lastname: Boel
authors.forename: S
authors.initials: S
authors.lastname: Verlaan
authors.forename: M J
authors.initials: MJ
authors.lastname: Poppelier
authors.forename: N A
authors.initials: NA
authors.lastname: Westerdaal
authors.forename: J A
authors.initials: JA
authors.lastname: Van Strijp
authors.forename: T
authors.initials: T
authors.lastname: Logtenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary; D004912:Erythrocytes; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012756:Sheep
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007070:Immunoglobulin A; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D007073:Immunoglobulin E