Delineation of the Discontinuous-Conformational Epitope of a Monoclonal Antibody Displaying Fullin Vitroandin VivoThyrotropin Activity

Abstract

An experimental murine model of Graves' disease was used to produce monoclonal antibodies (mAbs) with thyroid stimulating activity. Two of these, IRI-SAb2 and IRI-SAb3, showed particularly high potency (in the low nanomolar range) and efficacy. IRI-SAb2 behaved as a full agonist of the human TSH receptor (TSHr), even when tested in physiological salt concentrations. Both IRI-SAb2 and IRI-SAb3 were displaced from the TSHr by autoantibodies from patients with Graves' disease or harboring thyroid-blocking antibodies, but not from control subjects or patients with Hashimoto thyroiditis. The epitopes of IRI-SAb2 and IRI-SAb3 were precisely mapped, at the amino acid level, to the amino-terminal portion of the concave portion of the horseshoe structure of TSHr ectodomain. They overlap closely with each other and, surprisingly, with the epitope of a mAb with blocking activity. When injected iv in mice, both mAbs caused biological and histological signs of hyperthyroidism. Unexpectedly, they also triggered an inflammatory response in the thyroid glands. Delineation of the conformational epitopes of these stimulating antibodies opens the way to the identification of the molecular mechanisms implicated in the activation of the TSHr.

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease

“…This finding of restricted L chain usage for TSAb activity after SHM events suggests that agonistic activity to the TSHR is mediated by a particular conformation of Ab that can perhaps be attained by a few combinations of IGH and IGK rearrangements. These observations are in line with tentative findings on the common usage of certain gene rearrangements in monoclonal TSAbs from experimental Graves' disease (7,8,30) and the human monoclonal TSAbs from patients with autoimmune hyperthyroidism (11,12). Although limited information is available, there appears to be notable similarity in the gene usage by mouse and human monoclonal TSAbs (Supplemental Table I).…”

“…Although limited information is available, there appears to be notable similarity in the gene usage by mouse and human monoclonal TSAbs (Supplemental Table I). Among the murine monoclonal TSAbs generated in independent laboratories using different genetic delivery immunization procedures (7)(8)(9)30), there is considerable restriction in the both H and L chain gene usage. For example, usage of H chain genes, IGHV1S135, IGHDQ52, and IGHJ2, and L chain genes, IGKV6-13 and IGKJ2 or IGKJ5, is common among the mouse TSAbs.…”

“…Recent genetic studies in inbred mice strains have implicated the IGH V region gene locus in TSAb development (5,6). Monoclonal TSAbs with powerful agonist activity have been characterized from mice undergoing experimental Graves' disease following immunization by genetic delivery of plasmid or adenovirus encoding TSHR or the extracellular region of the receptor, known as TSHR A-subunit (7)(8)(9). The monoclonal TSAbs derived from experimental models of Graves' disease have been shown to be pathogenic in vivo, confirming their role in disease pathogenesis (8)(9)(10).…”

“…Monoclonal TSAbs with powerful agonist activity have been characterized from mice undergoing experimental Graves' disease following immunization by genetic delivery of plasmid or adenovirus encoding TSHR or the extracellular region of the receptor, known as TSHR A-subunit (7)(8)(9). The monoclonal TSAbs derived from experimental models of Graves' disease have been shown to be pathogenic in vivo, confirming their role in disease pathogenesis (8)(9)(10). Importantly, human monoclonal TSAbs from patients with Graves' disease have also been derived, providing detail into their molecular and biochemical properties, and pathogenicity in vivo (11)(12)(13).…”

Thyroid stimulating autoantibody M22 mimics TSH binding to the TSH receptor leucine rich domain: a comparative structural study of protein–protein interactions

“…Also a monoclonal antibody with weak thyroid stimulating activity has been shown to interact with TSHR amino acids 247-266 (Nú ñez . Furthermore, some of the TSHR residues important for interactions with TSH and M22 were found to be important for binding and/or stimulating activity of experimentally produced mouse or hamster TSHR monoclonal antibodies (Ando et al 2002, Costagliola et al 2004.…”