Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.
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Background: Tumor-associated extracellular matrix molecules are isoforms of proteins with a wide distribution in normal adult tissues, such as fibronectin and tenascin. Large Tenascin-C isoforms are present in almost all normal adult tissues but are upregulated in fetal, regenerating, and neoplastic tissues. In this study we investigated the expression of three tenascin isoforms in tumor tissue samples from lung cancer patients to evaluate the diagnostic and therapeutic value for clinical applications. Methods: In total, 35 corresponding tissue samples (tumor and normal lung tissue of the same patient) have been analyzed by immunohistochemistry using three different human monoclonal antibodies to domains A1 (F16), C (G11) and D (P12). All tumor specimens have been non-small cell lung cancer types. Results: Three isoforms G11, F16 and P12 have exhibited a very intense staining of different histological types of lung cancer. More than 80% of squamous cell carcinomas, adenocarcinomas and large cell carcinoma samples have been positively stained. None of the corresponding normal lung tissue specimens showed an expression of either tenascin domains. Conclusions:The results show that tenascin-C isoforms are highly expressed around the neovasculature and in the stroma of the majority of non-small cell lung cancers but is undetectable in the normal lung tissue. Therefore these isoforms could represent valuable candidates for the development of antibody based biopharmaceuticals for the treatment of lung cancer.
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