title: The effect of thyrotropin-receptor blocking antibodies on stimulating autoantibodies from patients with Graves' disease.
abstract: The hyperthyroidism of Graves' disease (GD) is caused by thyrotropin-receptor (TSHR) stimulating autoantibodies (TSAb), which lead to overproduction of thyroid hormones. In this study we tried to block the stimulatory effect of patients' TSAb to the TSHR with monoclonal antibodies (mAbs) and sera from hypothyroid patients. Two groups of blocking mAbs raised by different methods from two independent groups were tested for their ability to inhibit TSH binding to the TSHR, and also the binding of TSAb from the serum of patients with GD. Group 1 mAbs (7E3, 3H10, 4C1, 1B1, 4E9) bind to amino acids 378-387 and group 2 mAbs (23.1 and 31.7) to amino acids 382-415 of the human TSHR. These results were compared to the TSH- and TSAb-inhibiting effect of sera from hypothyroid patients containing bona fide thyroid blocking antibodies (TBAb) without agonistic activity. All studies were done in a conventional cyclic adenosine monophosphate (cAMP) or a modified luciferase reporter gene bioassay. TSH-induced cAMP/luciferase signal was reduced (> 70% inhibition) by all 7 mAbs, verifying the blocking nature. Comparable results (82.2%-96.3% inhibition) were seen when cells were preincubated with 8 TBAb sera. These TBAb sera also inhibited cAMP/luciferase induction of TSAb-positive sera from patients with GD (median of 27 experiments 62.2% inhibition; range, 26.8%-93.9%), and maintained inhibition greater than 20% even when diluted 1:150. However, when mAbs were incubated with these sera, results were heterogeneous: 17 of 30 sera (57%) incubated with mAb 31.7 caused reduced cAMP production compared to incubation with the control antibody, as did 18 of 34 sera (53%) incubated with mAb 7E3, 17 of 33 sera (52%) incubated with mAb 3H10, and 16 of 31 (52%) with mAb 23.1. Mixing all four mAbs did not enhance the cAMP-reductive effect (16/27 sera; 59% inhibited). Inhibition was less pronounced than with TBAb sera (0%-76% of a control antibody) and only present at antibody concentrations greater than 10 microg/mL. We conclude that despite the strong TBAb activity of the mAbs, their effect on TSAb-induced TSHR activation of sera from patients with GD was weaker than that of human TBAb autoantibodies. Thus, the latter are not only strong inhibitors of TSH activity, but also block the stimulatory effect of autoantibodies from patients with GD. However, this effect could not be reproduced by experimental mAbs to the same extent, because it may be the result of a broader spectrum of antibodies present in the TBAb sera, interacting with or in the vicinity of TSAb epitopes. Also of interest, when a TBAb serum was added to a TSAb serum, the TBAb effect was predominant even at high dilutions.
authors.affiliation: Bioassays GmbH, Biotechnology Center Hennigsdorf bei Berlin, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Lenzner
authors.forename: Nils G
authors.initials: NG
authors.lastname: Morgenthaler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001681:Biological Assay; D016466:CHO Cells; D006224:Cricetinae; D006111:Graves Disease / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D018828:Immunoglobulins, Thyroid-Stimulating / Q000187:drug effects*; D009994:Osmolar Concentration; D011989:Receptors, Thyrotropin / Q000037:antagonists & inhibitors* / Q000276:immunology*; D013972:Thyrotropin / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D007106:Immune Sera; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D013972:Thyrotropin