Stimulating and blocking antibodies to the TSH-receptor have similar affinities and only subtle differences in their epitope recognition

TSH-receptor (TSH-R) autoantibodies (TRAb) in autoimmune thyroid disease have been known for their heterogeneous effect with either stimulation (TSAb) or blocking (TBAb) of their target, resulting in the clinical symptoms of hyperthyroidism or hypothyroidism. Over years these distinct properties were attributed to either different affinities or binding sites to the TSH-R. Aim of this study was the verification of this concept using for the first time both affinity purified TRAb from the serum of patients and monoclonal antibodies (moabs) to the TSH-R with TSAb or TBAb activity. Affinity purification of TRAb was performed from 11 patients with Graves' disease having strong TSAb activity and 11 hypothyroid autoimmune thyroid patients with strong TBAb activity. In parallel, murine moAbs were generated by genomic vaccination with TSH-R DNA, of which IRI-Sab2 and IRI-Sab3 showed particular high stimulating potency (in the nanomolar range), whilst 1H7 revealed strong blocking activity. After labelling and saturation studies on TSH-R the Kd of individual purified TRAb was between 0.6 and 4.4 10^–10 M with no difference between TSAb and TBAb patients. A similar Kd in the 10^–10 M range was seen for both the TSAb and TBAb moabs and also bovine TSH. Competition studies with TSAb sera from patients with Graves' disease (n=100), or having TBAb (n=8) or controls (n=104) for binding to the TSH-R revealed virtually identical patterns for all purified TRAb and all moabs, irrespective of their TSAb or TBAb properties. A subsequent mapping of the epitopes of the moabs revealed an overlap of the stimulating IRI-Sab2 and IRI-Sab3 with the blocking 1H7 at the concave horse shoe part of the TSH-R ectodomain, which is also the likely side of TSH binding. These two approaches challenge the notion that stimulating and blocking antibodies would recognize structurally distant epitopes on the TSH-R, or that they have different affinities. Their different clinical effects seem to be the result of subtle differences in aminoacid activation.