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.

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.

Abstract 215: An Agonistic Human Monoclonal Autoantibody to the β2-adrenergic Receptor: Implications in Postural Hypotension

Functional autoantibodies directed to the autonomic receptors have been increasingly recognized to contribute to the pathophysiology of various cardiovascular diseases. However, to date, no human monoclonal autoantibodies to these receptors with agonist activity have been available. We have recently reported a subgroup of patients with idiopathic postural hypotension who have no known etiology are associated with vasodilatory autoantibodies to the β2-adrenergic receptor (β2AR). Using standard hybridoma techniques, we produced a β2AR-activating monoclonal autoantibody C5F2 from the peripheral blood lymphocytes of a patient with idiopathic postural hypotension and high ELISA antibody titers to a peptide corresponding to the second extracellular loop (ECL2) of β2AR. C5F2 was of the IgG3 isotype and recognized an epitope located at the N-terminus of β2AR ECL2 as determined by epitope mapping using multipin peptide synthesis. Confocal immunofluorescence revealed C5F2 binding to the β2AR in Chinese hamster ovary (CHO) cells transfected with human β2AR and rat cremaster arterioles, both of which were inhibited by presabsorption with the β2AR ECL2 peptide. Functionally, the C5F2 IgG stimulated cyclic AMP production in β2AR-expressing CHO cells in a dose-dependent manner (0.005-5 mg/ml). C5F2-induced cyclic AMP activation was specifically blocked by both the β2AR ECL2 peptide and β2AR selective antagonist ICI-118551. In an isolated rat cremaster arteriole assay, infusion of the C5F2 IgG produced a potent vasodilation which was inhibited by the β2AR ECL2 peptide and β2AR blocker ICI-118551 (from 67.8±5.8% to 9.3±5.1% and 4.4±2.1% of maximal dilatory response, respectively), indicating that antibody activation of vascular β2AR may contribute to systemic vasodilation. These data support the concept that circulating agonistic autoantibodies serve as vasodilators and may cause or exacerbate orthostasis by altering the compensatory postural vascular response. The availability of this first human monoclonal autoantibody to the β2AR provides opportunities to identify previously unrecognized causes and new pharmacological management of postural hypotension.

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-β chain. By contrast, M22 HC mimics the interactions of TSH-α while M22 LC mimics TSH-β 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.