Thyrotrophin receptor antibody concentration and activity, several years after treatment for Graves' disease.

OBJECTIVE: TSH receptor antibodies (TRAb) are responsible for autoimmune hyperthyroid disease (Graves' disease; GD) with TRAb levels tending to decrease following treatment. Measurement of TRAb activity during follow-up could prove valuable to better understand treatment effectiveness. STUDY DESIGN: TRAb concentration and stimulating (TSAb) and blocking (TSBAb) activity of patient serum were assessed following different treatment modalities and follow-up length. METHODS: Sixty-six subjects were recruited following treatment with carbimazole (n = 26), radioiodine (n = 27) or surgery (n = 13). TRAb, TPOAb, TgAb and GADAb were measured at a follow-up visit as well as bioassays of TSAb and TSBAb activity. RESULTS: Forty-five per cent of all patients remained TRAb-positive for more than one year and 23% for more than 5 years after diagnosis, irrespective of treatment method. Overall, TRAb concentration fell from a median (IQR) of 6.25 (3.9-12.7) to 0.65 (0.38-3.2) U/L. Surgery conferred the largest fall in TRAb concentration from 11.4 (6.7-29) to 0.58 (0.4-1.4) U/L. Seventy per cent of TRAb-positive patients were positive for TSAb, and one patient (3%) was positive for TSBAb. TRAb and TSAb correlated well (r = 0.83). In addition, 38/66 patients were TgAb-positive, 47/66 were TPOAb-positive and 6/66 were GADAb-positive at follow-up. CONCLUSIONS: TRAb levels generally decreased after treatment but persisted for over 5 years in some patients. TRAb activity was predominantly stimulatory, with only one patient demonstrating TSBAb. A large proportion of patients were TgAb/TPOAb-positive at follow-up. All treatment modalities reduced TRAb concentrations; however, surgery was most effective.

Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample.

OBJECTIVE: Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes. DESIGN, PATIENTS AND MEASUREMENTS: Two heterohybridoma cell lines secreting TSHR autoantibodies were isolated using standard techniques from the lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies (160 units/l by inhibition of TSH binding). The ability of the two new monoclonal antibodies (MAbs; K1-18 and K1-70) to bind to the TSHR and compete with TSH or TSHR antibody binding was analysed. Furthermore, the effects of K1-18 and K1-70 on cyclic AMP production in Chinese hamster ovary cells (CHO) cells expressing the TSHR were investigated. RESULTS: One MAb (K1-18) was a strong stimulator of cyclic AMP production in TSHR-transfected CHO cells and the other (K1-70) blocked stimulation of the TSHR by TSH, K1-18, other thyroid-stimulating MAbs and patient serum stimulating type TSHR autoantibodies. Both K1-18 (IgG1 kappa) and K1-70 (IgG1 lambda) bound to the TSHR with high affinity (0.7 x 10(10) l/mol and 4 x 10(10) l/mol, respectively), and this binding was inhibited by unlabelled K1-18 and K1-70, other thyroid-stimulating MAbs and patient serum TSHR autoantibodies with stimulating or blocking activities. V region gene analysis indicated that K1-18 and K1-70 heavy chains used the same V region germline gene but different D and J germline genes as well as having different light chains. Consequently, the two antibodies have evolved separately from different B cell clones. CONCLUSIONS: This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain.

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22-260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

In vivo effects of a human thyroid-stimulating monoclonal autoantibody (M22) and a human thyroid-blocking autoantibody (K1-70).

PURPOSE: To 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. METHODS: Serum 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. RESULTS: Serum 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. CONCLUSION: M22 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.

Similarities and differences in interactions of thyroid stimulating and blocking autoantibodies with the TSH receptor.

Binding of a new thyroid-stimulating human monoclonal autoantibody (MAb) K1-18 to the TSH receptor (TSHR) leucine-rich 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-α chain and the heavy chain (HC) mimics binding of the TSH-ß chain. By contrast, M22 HC mimics the interactions of TSH-α while M22 LC mimics TSH-ß 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. Interactions involving the C-terminal part of the TSHR LRD may be important for receptor activation by autoantibodies.

Crystal structure of the TSH receptor (TSHR) bound to a blocking-type TSHR autoantibody.

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 Šresolution. K1-70 Fab binds to the concave surface of the TSHR leucine-rich domain (LRD) forming a large interface (2565 Å(2)) with an extensive network of ionic, polar and hydrophobic interactions. Mutation of TSHR or K1-70 residues showing strong interactions in the solved structure influenced the activity of K1-70, indicating that the binding detail observed in the complex reflects interactions of K1-70 with intact, functionally active TSHR. Unbound K1-70 Fab was prepared and crystallised to 2.22 Šresolution. Virtually no movement was observed in the atoms of K1-70 residues on the binding interface compared with unbound K1-70, consistent with 'lock and key' binding. The binding arrangements in the TSHR260-K1-70 Fab complex are similar to previously observed for the TSHR260-M22 Fab complex; however, K1-70 clasps the concave surface of the TSHR LRD in approximately the opposite orientation (rotated 155°) to M22. The blocking autoantibody K1-70 binds more N-terminally on the TSHR concave surface than either the stimulating autoantibody M22 or the hormone TSH, and this may reflect its different functional activity. The structure of TSHR260 in the TSHR260-K1-70 and TSHR260-M22 complexes show a root mean square deviation on all C(α) 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.