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

Abstract

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 10(6) to 10(10) 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.

Pitfalls of immunoassay and sample for IGF-I: Comparison of different assay methodologies using various fresh and stored serum samples

“…This observation, suggesting accessibility of IGF-I in ternary protein complexes to proteolysis, is in agreement with the reported accessibility of IGF-I in such complexes to antibody binding [10,29] and thus supports the notion that limited IGF-I proteolysis might be a real in vivo phenomenon as well. It might be foreseeable that regulated proteolysis of IGF-I could result in generation of readily dissociable bioactive IGF-I variants [2].…”

“…Although there is no direct evidence in support of IGF-I proteolysis, particularly when sequestered by IGFBPs, we recently demonstrated that an antigenic domain on IGF-I in its native ternary protein complex configuration remains exposed and accessible for antibody binding [10]. A similar finding of IGF-I interaction with antibody after its in vitro binding to IGFBP-1, IGFBP-3, or type 1 IGF receptor has been also reported [29]. Based on the aforementioned, it might be reasonable to assume that the same antigenic domain could be also available for proteolytic conversion.…”

The High Molecular Weight Insulin-Like Growth Factor-Binding Protein Complex: Epitope Mapping, Immunoassay, and Preliminary Clinical Evaluation

“…This finding is in agreement with the reported identification of an antigenic domain on IGF-I that remains exposed after IGF-I interaction with IGFBP-1, IGFBP-3, or type 1 IGF receptor (41). However, the lack of simultaneous binding of the present anti-IGF-I and anti-ALS antibodies to serum IGFBP-3 complexes may indicate steric hindrance, suggesting close binding proximity of the N-terminal of ALS to the IGF-binding site on IGFBP-3.…”

Differential mRNA Expression of Insulin-like Growth Factor-1 Splice Variants in Patients With Idiopathic Pulmonary Fibrosis and Pulmonary Sarcoidosis

“…Our primers would detect this transcript but we failed to find its expression in any of our samples. The transcripts potentially affect the interaction of IGF-1 with IGF-1 receptor or IGFBP because it has been shown by functional epitope mapping that the carboxy terminus (3Ј end) of IGF-1 is an important determinant of the affinity of the peptide for a particular receptor or binding protein (26). However, the E peptide is translated, and cleaved during processing of IGF-1 prohormone; therefore, E peptides themselves may have distinct biologic roles after being cleaved from the prohormone (27).…”