TSH receptor monoclonal antibodies with agonist, antagonist, and inverse agonist activities.

Autoantibodies in autoimmune thyroid disease (AITD) bind to the TSH receptor (TSHR) and can act as either agonists, mimicking the biological activity of TSH, or as antagonists inhibiting the action of TSH. Furthermore, some antibodies with antagonist activity can also inhibit the constitutive activity of the TSHR, that is, act as inverse agonists. The production of animal TSHR monoclonal antibodies (MAbs) with the characteristics of patient autoantibodies and the isolation of human autoantibodies from patients with AITD has allowed us to analyze the interactions of these antibodies with the TSHR at the molecular level. In the case of animal MAbs, advances such as DNA immunization allowed the production of the first MAbs which showed the characteristics of human TSHR autoantibodies (TRAbs). Mouse MAbs (TSMAbs 1-3) and a hamster MAb (MS-1) were obtained that acted as TSHR agonists with the ability to stimulate cyclic AMP production in CHO cells expressing the TSHR. In addition, a mouse TSHR MAb (MAb-B2) that had the ability to act as an antagonist of TRAbs and TSH was isolated and characterized. Also, a mouse TSHR MAb that showed TSH antagonist and TSHR inverse agonist activity (CS-17) was described. Furthermore, a panel of human TRAbs has been obtained from the peripheral blood lymphocytes of patients with AITD and extensively characterized. These MAbs have all the characteristics of TRAbs and are active at ng/mL levels. To date, two human MAbs with TSHR agonist activity (M22 and K1-18), one human MAb with TSHR antagonist activity (K1-70) and one human MAb (5C9) with both TSHR antagonist and TSHR inverse agonist activity have been isolated. Early experiments showed that the binding sites for TSH and for TRAbs with thyroid stimulating or blocking activities were located on the extracellular domain of the TSHR. Extensive studies using TSHRs with single amino acid mutations identified TSHR residues that were important for binding and biological activity of TSHR MAbs (human and animal) and TSH. The structures of several TSHR MAb Fab fragments were solved by X-ray crystallography and provided details of the topography of the antigen binding sites of antibodies with either agonist or antagonist activity. Furthermore stable complexes of the leucine-rich repeat domain (LRD) of the TSHR with a human MAb (M22) with agonist activity and with a human MAb (K1-70) with antagonist activity have been produced and their structures solved by X-ray crystallography at 2.55 and 1.9Å resolution, respectively. Together these experiments have given detailed insights into the interactions of antibodies with different biological activities (agonist, antagonist, and inverse agonist) with the TSHR. Although the nature of ligand binding to the TSHR is now understood in some detail, it is far from clear how these initial interactions lead to functional effects on activation or inactivation of the receptor.