The epidemiology of benzodiazepine-related toxicity in Ontario, Canada: a population-based descriptive study.

OBJECTIVES: Despite the widespread use of prescription benzodiazepines, there are few studies examining trends and patterns of benzodiazepine-related toxicity. We describe the epidemiology of benzodiazepine-related toxicity in Ontario, Canada. METHODS: We conducted a population-based, cross-sectional study of Ontario residents who had an emergency department visit or hospitalization for benzodiazepine-related toxicity between January 1, 2013 and December 31, 2020. We reported annual crude and age-standardized rates of benzodiazepine-related toxicity overall, by age, and by sex. In each year, we characterized the history of benzodiazepine and opioid prescribing among people who experienced benzodiazepine-related toxicity, and reported the percentage of encounters with opioid, alcohol, or stimulant co-involvement. RESULTS: Between 2013 and 2020, there were 32,674 benzodiazepine-related toxicity encounters among 25,979 Ontarians. During this period, the crude rate of benzodiazepine-related toxicity declined overall, from 28.0 to 26.1 per 100,000 population (age-standardized rate: 27.8 to 26.4 per 100,000), but increased among young adults aged 19 to 24 (39.9 to 66.6 per 100,000 population). Moreover, by 2020, the percentage of encounters associated with active benzodiazepine prescriptions had declined to 48.9%, while the percentage of encounters that had opioid, stimulant, or alcohol co-involvement rose to 28.8%. CONCLUSION: Benzodiazepine-related toxicity has declined in Ontario overall, but has increased among youth and young adults. Furthermore, there is growing co-involvement of opioids, stimulants, and alcohol, which may reflect the recent emergence of benzodiazepines in the unregulated drug supply. Multifaceted public health initiatives comprising harm reduction, mental health supports, and promotion of appropriate prescribing are needed to reduce benzodiazepine-related harm.

RéSUMé: OBJECTIFS: Malgré l'utilisation généralisée des benzodiazépines sur ordonnance, peu d'études portent sur les tendances et les schémas de toxicité de ces médicaments. Nous décrivons l'épidémiologie de la toxicité liée aux benzodiazépines en Ontario, au Canada. MéTHODE: Nous avons mené une étude populationnelle transversale des résidentes et résidents de l'Ontario ayant visité le service des urgences ou été hospitalisés pour toxicité liée aux benzodiazépines entre le 1er janvier 2013 et le 31 décembre 2020. Nous avons rapporté globalement, par âge et par sexe les taux annuels de toxicité liée aux benzodiazépines, bruts et standardisés pour l'âge. Pour chaque année, nous avons caractérisé les antécédents de prescription de benzodiazépines et d'opioïdes chez les personnes ayant présenté une toxicité liée aux benzodiazépines, et rapporté le pourcentage de rencontres présentant une co-implication avec les opioïdes, l'alcool ou les stimulants. RéSULTATS: Entre 2013 et 2020, il y a eu 32 674 rencontres pour toxicité liée aux benzodiazépines avec 25 979 Ontariens et Ontariennes. Durant cette période, le taux brut de toxicité liée aux benzodiazépines a baissé dans l'ensemble, passant de 28 à 26,1 pour 100 000 habitants (taux standardisé pour l'âge : 27,8 à 26,4 p. 100 000), mais il a augmenté chez les jeunes adultes de 19 à 24 ans (de 39,9 à 66,6 p. 100 000). De plus, en 2020, le pourcentage de rencontres associées à des ordonnances actives de benzodiazépines avait baissé à 48,9 %, tandis que le pourcentage de rencontres présentant une co-implication avec les opioïdes, les stimulants ou l'alcool avait augmenté à 28,8 %. CONCLUSION: La toxicité liée aux benzodiazépines a diminué en Ontario dans l'ensemble, mais elle a augmenté chez les jeunes et les jeunes adultes. De plus, cette toxicité présente une co-implication croissante avec les opioïdes, les stimulants ou l'alcool, ce qui peut refléter l'émergence récente des benzodiazépines dans l'approvisionnement non réglementé en drogues. Des initiatives de santé publique multidimensionnelles incluant la réduction des méfaits, le soutien en santé mentale et la promotion de la prescription appropriée sont nécessaires pour réduire les méfaits liés aux benzodiazépines.

Structure of full-length TSH receptor in complex with antibody K1-70™.

Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

TSH receptor specific monoclonal autoantibody K1-70TM targeting of the TSH receptor in subjects with Graves' disease and Graves' orbitopathy-Results from a phase I clinical trial.

OBJECTIVES: In Graves' disease (GD), autoantibodies to the thyroid stimulating hormone receptor (TSHR) cause hyperthyroidism. The condition is often associated with eye signs including proptosis, oedema, and diplopia (collectively termed Graves' orbitopathy [GO]). The safety profile of K1-70TM (a human monoclonal TSHR specific autoantibody, which blocks ligand binding and stimulation of the receptor) in patients with GD was evaluated in a phase I clinical trial. PATIENTS AND STUDY DESIGN: Eighteen GD patients stable on antithyroid drug medication received a single intramuscular (IM) or intravenous (IV) dose of K1-70TM during an open label phase I ascending dose, safety, tolerability, pharmacokinetic and pharmacodynamic (PD) study. Immunogenic effects of K1-70TM were also determined. RESULTS: K1-70TM was well-tolerated in all subjects at all doses and no significant immunogenic response was observed. There were no deaths or serious adverse events. Increased systemic exposure to K1-70TM was observed following a change to IV dosing, indicating this was the correct dosage route. Expected PD effects occurred after a single IM dose of 25 mg or single IV dose of 50 mg or 150 mg with fT3, fT4, and TSH levels progressing into hypothyroid ranges. There were also clinically significant improvements in symptoms of both GD (reduced tremor, improved sleep, improved mental focus, reduced toilet urgency) and GO (reduced exophthalmos measurements, reduced photosensitivity). CONCLUSIONS: K1-70TM was safe, well tolerated and produced the expected PD effects with no immunogenic responses. It shows considerable promise as a new drug to block the actions of thyroid stimulators on the TSHR.

Social Work Practice During COVID-19: Client Needs and Boundary Challenges.

While information and communication technologies (ICTs) permeated social work practice long before the onset of COVID-19, the abrupt need to close non-essential workplaces resulted in an unparalleled incorporation of digital technology into practice across the globe. The onset of COVID-19 occurred during phase two of research in which we were investigating social workers' informal use of ICT with clients. Prior to COVID-19, we were conducting interviews with practitioners and clients from four agencies serving diverse client populations in a large city in Canada. With the onset of COVID-19, we adapted to the COVID-19 context and amended the questions to investigate ICT use during the pandemic. In addition, with ethics approval, we conducted second interviews with practitioners interviewed prior to COVID-19 with a revised guide to address the pandemic context; and we continued to recruit and interview practitioners and clients using an amended interview guide incorporating pandemic-related questions. The sample comprised 27 practitioners and 22 clients. Eleven practitioners participated in interviews prior to and during COVID-19. Analysis of transcribed interviews revealed that the COVID-19 context had led to a paradigm shift in practitioners' ICT use, with two key themes identified: (1) boundary challenges and (2) clients' diverging ICT needs. We discuss these themes and present implications for policy and practice in a post-COVID-19 world.

Blocking the Thyrotropin Receptor with K1-70 in a Patient with Follicular Thyroid Cancer, Graves' Disease, and Graves' Ophthalmopathy.

Background: We report the therapeutic use of K1-70™, a thyrotropin receptor (TSHR) antagonist monoclonal antibody, in a patient with follicular thyroid cancer (FTC), Graves' disease (GD), and Graves' ophthalmopathy (GO). Methods: A 51-year-old female patient, who smoked, presented in October 2014 with FTC complicated by GD, high levels of TSHR autoantibodies with high thyroid stimulating antibody (TSAb) activity, and severe GO. K1-70 was administered at 3 weekly intervals with the dose adjusted to block TSAb activity. Her cancer was managed with lenvatinib and radioiodine therapy. Results: Following initiation of K1-70 therapy, TSAb activity measured in serum decreased and GO (proptosis and inflammation) improved. On K1-70 monotherapy during the pause in lenvatinib, several metastatic lesions stabilized while others showed progression attenuation compared with that before lenvatinib therapy. Conclusions: These observations suggest that blocking TSHR stimulation with K1-70 can be an effective treatment for GO and may also benefit select patients with FTC and GD.

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.

Preclinical studies on the toxicology, pharmacokinetics and safety of K1-70TM a human monoclonal autoantibody to the TSH receptor with TSH antagonist activity.

BACKGROUND: The human monoclonal autoantibody K1-70™ binds to the TSH receptor (TSHR) with high affinity and blocks TSHR cyclic AMP stimulation by TSH and thyroid stimulating autoantibodies. METHODS: The preclinical toxicology assessment following weekly intravenous (IV) or intramuscular (IM) administration of K1-70™ in rats and cynomolgus monkeys for 29 days was carried out. An assessment of delayed onset toxicity and/or reversibility of toxicity was made during a further 4 week treatment free period. The pharmacokinetic parameters of K1-70™ and the effects of different doses of K1-70™ on serum thyroid hormone levels in the study animals were determined in rats and primates after IV and IM administration. RESULTS: Low serum levels of T3 and T4 associated with markedly elevated levels of TSH were observed in the study animals following IV and IM administration of K1-70™. The toxicological findings were attributed to the pharmacology of K1-70™ and were consistent with the hypothyroid state. The no observable adverse effect level (NOAEL) could not be established in the rat study while in the primate study it was 100 mg/kg/dose for both males and females. CONCLUSIONS: The toxicology, pharmacodynamic and pharmacokinetic data in this preclinical study were helpful in designing the first in human study with K1-70™ administered to subjects with Graves' disease.

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.

Structure and activation of the TSH receptor transmembrane domain.

PURPOSE: The thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves' disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD-M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown. METHODS: We have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available. RESULTS: Most conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs. CONCLUSIONS: Insight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves' disease.

Glycosylation pattern analysis of glycoprotein hormones and their receptors.

We have studied glycosylation patterns in glycoprotein hormones (GPHs) and glycoprotein hormone receptor (GPHR) extracellular domains (ECD) from different species to identify areas not glycosylated that could be involved in intermolecular or intramolecular interactions. Comparative models of the structure of the TSHR ECD in complex with TSH and in complex with TSHR autoantibodies (M22, stimulating and K1-70, blocking) were obtained based on the crystal structures of the FSH-FSHR ECD, M22-TSHR leucine-rich repeat domain (LRD) and K1-70-TSHR LRD complexes. The glycosylation sites of the GPHRs and GPHs from all species studied were mapped on the model of the human TSH TSHR ECD complex. The areas on the surfaces of GPHs that are known to interact with their receptors are not glycosylated and two areas free from glycosylation, not involved in currently known interactions, have been identified. The concave faces of GPHRs leucine-rich repeats 3-7 are free from glycosylation, consistent with known interactions with the hormones. In addition, four other non-glycosylated areas have been identified, two located on the receptors' convex surfaces, one in the long loop of the hinge regions and one at the C-terminus of the extracellular domains. Experimental evidence suggests that the non-glycosylated areas identified on the hormones and receptors are likely to be involved in forming intramolecular or intermolecular interactions.

Blocking type TSH receptor antibodies.

TSH receptor (TSHR) autoantibodies (TRAbs) play a key role in the pathogenesis of Graves' disease. In the majority of patients, TRAbs stimulate thyroid hormone synthesis via activation of the TSHR (stimulating TRAbs, TSHR agonists). In some patients, TRAbs bind to the receptor but do not cause activation (blocking TRAbs, TSHR antagonists). Isolation of human TSHR monoclonal antibodies (MAbs) with either stimulating (M22 and K1-18) or blocking activities (5C9 and K1-70) has been a major advance in studies on the TSHR. The binding characteristics of the blocking MAbs, their interaction with the TSHR and their effect on TSHR constitutive activity are summarised in this review. In addition, the binding arrangement in the crystal structures of the TSHR in complex with the blocking MAb K1-70 and with the stimulating MAb M22 (2.55 Å and 1.9 Å resolution, respectively) are compared. The stimulating effect of M22 and the inhibiting effect of K1-70 on thyroid hormone secretion in vivo is discussed. Furthermore the ability of K1-70 to inhibit the thyroid stimulating activity of M22 in vivo is shown. Human MAbs which act as TSHR antagonists are potentially important new therapeutics. For example, in Graves' disease, K1-70 may well be effective in controlling hyperthyroidism and the eye signs caused by stimulating TRAb. In addition, hyperthyroidism caused by autonomous TSH secretion should be treatable by K1-70, and 5C9 has the potential to control hyperthyroidism associated with TSHR activating mutations. Furthermore, K1-70 has potential applications in thyroid imaging as well as targeted drug delivery to TSHR expressing tissues.

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.

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.

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.

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.

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.

A human monoclonal autoantibody to the thyrotropin receptor with thyroid-stimulating blocking activity.

BACKGROUND: Human monoclonal autoantibodies (MAbs) are valuable tools to study autoimmune responses. To date only one human MAb to the thyrotropin (TSH) receptor (TSHR) with stimulating activity has been available. We now describe the detailed characterization of a blocking type human MAb to the TSHR. METHODS: A single heterohybridoma cell line was isolated from the peripheral blood lymphocytes of a patient with severe hypothyroidism (TSH 278 mU/L) using standard techniques. The line stably expresses a TSHR autoantibody (5C9; IgG1/kappa). Ability of 5C9 to bind and compete with 125I-TSH or TSHR antibodies binding to the TSHR was tested using tubes coated with solubilized TSHR. Furthermore, the blocking effects of 5C9 on stimulation of cyclic AMP production was assessed using Chinese hamster ovary (CHO) cells expressing the wild-type human TSHR or TSHRs with amino acid mutations. MAIN OUTCOME: 5C9 IgG bound to the TSHR with high affinity (4 x 10(10) L/mol) and inhibited binding of TSH and a thyroid-stimulating human monoclonal autoantibody (M22) to the receptor. 5C9 IgG preparations inhibited the cyclic AMP-stimulating activities of TSH, M22, serum TSHR autoantibodies and thyroid-stimulating mouse monoclonal antibodies. Furthermore 5C9 reduced the constitutive activity of wild-type TSHR and TSHR with some activating mutations. The effect of different amino acid mutations in the TSHR on 5C9 biological activity was studied and TSHR Lys129Ala or Asp203Ala completely abolished the ability of 5C9 to block TSH-mediated stimulation of cyclic AMP production. CONCLUSIONS: The availability of 5C9 provides new opportunities to investigate the binding and biological activity of TSHR blocking type autoantibodies including studies at the molecular level. Furthermore, monoclonal antibodies such as 5C9 may well provide the basis of new drugs to control TSHR activity including applications in thyroid cancer and Graves' ophthalmopathy.