Generation of human high-affinity antibodies specific for the fibroblast activation protein by guided selection.
Four completely human antibody derivatives [single-chain-antibody fragments (scFvs)] with specificity for the general tumor stroma marker fibroblast activation protein (FAP) were isolated by guided selection. Highly diverse IgG, IgM and IgD isotypes comprising heavy-chain variable domain libraries were generated using cDNAs derived from diverse lymphoid organs of a multitude of donors. Three of the human scFvs were converted into bivalent minibodies and expressed in eukaryotic cells for further functional characterization. Binding-competition studies and analysis by fluorescence-activated cell sorting showed high-affinity binding (10--20 nM) for two clones and recognition of the same epitope as the murine guiding antibody. The minibodies were successfully used for immunohistology of a variety of human carcinoma biopsies, revealing specific staining of stromal fibroblasts. Therefore, they should be suitable for in vivo diagnostic and tumor-targeting studies and, because of their completely human origin, be superior to murine or humanized antibody derivatives.
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
A
A
Schmidt
D
D
Müller
M
M
Mersmann
T
T
Wüest
E
E
Gerlach
P
P
Garin-Chesa
W J
WJ
Rettig
K
K
Pfizenmaier
D
D
Moosmayer
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000951:Antigens, Neoplasm; D001483:Base Sequence; D001667:Binding, Competitive; D014408:Biomarkers, Tumor; D017931:DNA Primers; D018076:DNA, Complementary; D010450:Endopeptidases; D018093:Gelatinases; D006133:Growth Substances / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000276:immunology*; D000099167:Fibroblast Activation Protein Alpha
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D017931:DNA Primers; D018076:DNA, Complementary; D010450:Endopeptidases; D018093:Gelatinases; D006133:Growth Substances; D008565:Membrane Proteins; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha