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Functional Epitope Mapping of Insulin-Like Growth Factor I (IGF-I) by Anti-IGF-I Monoclonal Antibodies¹

Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autonoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain

Address all correspondence and requests for reprints to: Dr. Santos Mañes, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autonoma, Campus de Cantoblanco, E-28049, Madrid, Spain. E-mail: smanes{at}samba.cnb.uam.es

Based on a collection of monoclonal antibodies (mAb) against insulin-like growth factor I (IGF-I), we have defined the IGF-I epitopes involved in the interaction with IGF-binding proteins (IGFBP) and IGF-I receptors. We have also characterized the ability of these antibodies to block IGF-I-induced survival of the IL-3-dependent Ba/F3 cell line. More than 140 hybridomas secreting IGF-I-specific mAb were characterized, of which 28 were studied in detail. They display apparent affinity constants ranging from less than 10⁶ to 10¹⁰ M^-1 and varying crossreactivity with IGF-II, including 2 mAb with higher affinity for IGF-II than for IGF-I. None crossreact with insulin or any other growth factor tested. Using both enzyme immunoassays and real-time biospecific interaction analysis, we have identified 8 epitopic clusters related to the primary structure of IGF-I, according to mAb reactivity to synthetic peptides, proteolytic fragments of IGF-I, and various IGF-I mutants. The mAb panel also was used to map the IGF domains implicated in the interaction with IGFBP and IGF-I receptors. An IGF-I domain has been identified that remains exposed after IGF-I binding to IGFBP-1 or to IGFBP-3, which is recognized by 6 different mAb. The mAb in this group also bind IGF-I, when complexed to the type-1 IGF receptor on the murine pro-B cell line Ba/F3, and BALB/c 3T3 fibroblasts overexpressing the human receptor. Finally, IGF-I-promoted survival can be blocked with mAb specific for target epitopes, and their potential use in tumor cell growth control is discussed.

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