A complex of the TSH receptor extracellular domain (amino acids 22-260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1 . 9 Å resolution. complexes show a root mean square deviation on all C a atoms of only 0 . 51 Å . These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.

This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.

PurposeTo study in vivo effects of the human monoclonal TSH receptor (TSHR) autoantibodies M22 (stimulating type) and K1-70 (blocking type) on thyroid hormone levels in rats.MethodsSerum levels of total T4, free T4, M22 and K1-70 were measured following intramuscular injection of M22 IgG (2–4 μg/animal), K1-70 IgG (10–200 μg/animal) or both into rats. Thyroid pathology was assessed in M22-injected rats.ResultsSerum levels of total T4 and free T4 increased in a dose-dependent manner following injection of M22 IgG. Thyroid follicular cell hypertrophy was dependent on the dose of M22 IgG. K1-70 IgG caused a dose dependent decrease of total T4 and free T4 levels in rats receiving K1-70 only. The stimulating effects of M22 IgG on T4 levels in rats were completely inhibited by K1-70 IgG.ConclusionM22 is a potent stimulator of thyroid hormone secretion in vivo. In contrast, K1-70 inhibits thyroid hormone secretion in vivo. Furthermore, K1-70 has the ability to inhibit the stimulating activity of M22 in vivo and as such has potential as a new drug to block TSHR stimulation by autoantibodies in Graves’ disease.

The reasons for an immunological paradigm shift in this elderly woman remain speculative. We believe that de-novo TSAb synthesis occurred converting her long-standing HT to GD although the mechanisms responsible remain unexplained. TRAb analysis confirmed stable autoantibody characteristics over 1 year and variable effects of TSHR mutations on TRAb and M22 function.

Binding of a new thyroid-stimulating human monoclonal autoantibody (MAb) K1-18 to the TSH receptor (TSHR) leucinerich domain (LRD) was predicted using charge-charge interaction mapping based on unique complementarities between the TSHR in interactions with the thyroid-stimulating human MAb M22 or the thyroid-blocking human MAb K1-70. The interactions of K1-18 with the TSHR LRD were compared with the interactions in the crystal structures of the M22-TSHR LRD and K1-70-TSHR LRD complexes. Furthermore, the predicted position of K1-18 on the TSHR was validated by the effects of TSHR mutations on the stimulating activity of K1-18. A similar approach was adopted for predicting binding of a mouse thyroid-blocking MAb RSR-B2 to the TSHR. K1-18 is predicted to bind to the TSHR LRD in a similar way as TSH and M22. The binding analysis suggests that K1-18 light chain (LC) mimics binding of the TSH-a chain and the heavy chain (HC) mimics binding of the TSH-b chain. By contrast, M22 HC mimics the interactions of TSH-a while M22 LC mimics TSH-b in interactions with the TSHR. The observed interactions in the M22-TSHR LRD and K1-70-TSHR LRD complexes (crystal structures) with TSH-TSHR LRD (comparative model) and K1-18-TSHR LRD (predictive binding) suggest that K1-18 and M22 interactions with the receptor may reflect interaction of thyroid-stimulating autoantibodies in general. Furthermore, K1-70 and RSR-B2 interactions with the TSHR LRD may reflect binding of TSHR-blocking autoantibodies in general.