In vitro antibody generation by panning a large universal gene library with phage display was employed to generate antibodies to more than 60 different antigens. Of particular interest was a comparison of pannings on 20 different SH2 domains provided by the Structural Genomics Consortium (SGC). Streamlined methods for high throughput antibody generation developed within the 'Antibody Factory' of the German National Genome Research Network (NGFN) were demonstrated to minimise effort and provide a reliable and robust source for antibodies. For the SH2 domains, in two successive series of selections, 2668 clones were analysed, resulting in 347 primary hits in ELISA. Half of these hits were further analysed, and more than 90 different scFv antibodies to all antigens were identified. The validation of selected antibodies by cross-reactivity ELISA, western blot and on protein microarrays demonstrated the versatility of the in vitro antibody selection pipeline to generate a renewable resource of highly specific monoclonal binders in proteome scale numbers with substantially reduced effort and time.

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Four completely human antibody derivatives [single-chainantibody 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.

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The fibroblast activation protein (FAP) is selectively expressed on activated fibroblasts of the tumor stroma on more than 90% of lung, breast and colon carcinomas. The high prevalence and abundance of FAP stroma make it a promising target for in vivo diagnosis and therapy of a variety of carcinomas. We describe the humanization of the murine FAP-specific MAb, F19, which has already been clinically used for in vivo diagnostic purposes. Using phage display technology and human V-repertoires, VL and VH regions of F19 were replaced by analogous human V-regions while retaining the original HCDR3 sequence in order to maintain F19 epitope specificity. The resulting human single-chain fragments of immunoglobulin variable regions (scFv 34, scFv 18) showed affinities of 6 nM on cell membrane-bound FAP. scFv 34 was expressed as a bivalent minibody (Mb 34). The antigen-binding characteristics of Mb 34 were comparable to the parental and a complementarity-determining region (CDR)-grafted version of F19. This was revealed by binding competition studies, FACS analyses and immunohistochemistry on various tumor samples including breast, colon and lung carcino-mas. Importantly, compared with the CDR-grafted human-ized scFv version of F19, the V-regions of the selected human scFv 34 showed sequence identity with the parental antibody (Ab) only over the short, 15-amino acid long HCDR3. Thus, a largely reduced xenoantigenic potential is expected. These human Ab derivatives are suitable to develop novel therapeutic concepts with broad applicability for a wide variety of histological carcinomas based on tumor stroma targeting. The growth of tumors (diameter 1-2 mm) is dependent on angiogenesis and blood supply, postulated already by Folkman in 1971 1 and experimentally proven in later years. 2,3 Moreover, a correlation between the degree of tumor vascularization and the formation of metastases has been observed. 4 Cells of the tumor-supplying tissue, in particular the endothelial cells of the tumor vasculature and stromal fibroblasts, are regarded as expedient targets. This is because they are non-transformed and thus considered as genetically stable and shared by practically all solid tumors. Markers selectively expressed on these cells are exploited for in vivo diagnostic and novel therapeutic approaches based on specific antibodies (tumor stroma targeting). The feasibility of this strategy is supported by studies with animal tumor models. 5,6 The FAP is considered as a suitable Ag for a stroma-targeting approach for several reasons: (1) in more than 90% of all epithelial tumors including lung, colorectal and breast carcinomas (primary tumors and metastases), the tumor stroma is FAP ; in post-natal non-tumor tissues, FAP is expressed during wound healing and on pancreatic cells; (2) stromal fibroblasts are non-transformed cells, making the rapid emergence of Ag loss variants and therapy resistance unlikely; (3) the FAP stromal compartment, commonly contributing approximately 50% to 90% of the tumor mass, presents an abundant target...