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.