An inverse agonist for the TSH receptor

Topic: MECHANISM OF TSH RECEPTOR ACTIVATION

Title: Suppression of thyrotropin receptor constitutive activity by a monoclonal antibody with inverse agonist activity.

Authors: Chen C-R, McLachlan SM, & Rapoport B.

Reference: Endocrinology, 2007

(First published ahead of print on February 1, 2007 as doi:10.1210/en.2006-1754)

Summary

Background

Thyrotropin (TSH) binding to the TSH receptor (TSHR) induces thyrocyte growth and proliferation primarily by activating the adenylyl cyclase signalling pathway. Relative to the other glycoprotein hormone receptors (LH/CG & FSH receptors), the wild type TSHR has readily measurable ligand-independent (constitutive) activity. Numerous mutations of the TSHR resulting in an increase in this basal activity have been shown to cause autonomous thyroid adenomas (when somatic) or hereditary or sporadic non-autoimmune hyperthyroidism (when germline). Recently, monoclonal antibodies have been isolated which, upon binding, are capable of substituting to TSH or TSAbs to activate the TSHR.

Initial Purpose

The objective of the study was to generate monoclonal antibodies with TSHR stimulating or blocking activity.

Material and Methods

Monoclonal antibodies (mAbs) were generated by genetic immunization with adenovirus expressing the ectodomain of the human TSHR (A-subunit).. Approximately two weeks after fusion, hybridoma supernatants were screened by flow cytometry using TSHR-expressing CHO cells. The mAbs of interest were tested for their binding to the TSHR and for ability to stimulate or inhibit cAMP accumulation in cells transfected with wild type or mutant TSHR constructs.

Results

A monoclonal antibody (CS-17) was identified with the previously unrecognised property of being an inverse agonist for TSHR constitutive activity. Beyond its inhibitory effect on the basal activity of wild type TSHR, CS-17 is effective in decreasing strongly the constitutive activity of a series of TSHR mutants. Remarkably, it is equally effective on mutants with mutations in the extracellular or the transmembrane/intracellular portions of the receptor (i.e. not accessible to direct CS-17 contact). This indicates that CS-17 is acting allosterically. Administered to mice in vivo, CS-17 reduces serum T4 levels. The CS-17 epitope is conformational and lies in the C-terminal region of the TSHR leucine-rich domain (residues 260-289).

Conclusions

By interacting with the large TSHR extracellular domain, CS-17 is the first antibody reported to be an inverse agonist for a member of the G protein receptor super family. After humanization of its murine constant region, CS-17 has the potential to be an adjunctive therapeutic agent in athyreotic patients with residual well-differentiated thyroid carcinoma, as well as pending definitive treatment in some selected hyperthyroidism states.

Commentary

Amongst the rhodospin-like G protein-coupled receptors (GPCRs), the glycoprotein hormone receptors display a bipartite structure which parallels a functional dichotomy: a large ectodomain with leucine-riche repeats is responsible for binding of the hormones, while a serpentine domain with seven transmembrane alpha helices resembling rhodopsin transmits the activation signal to the Gsa protein. The mechanisms implicated in the intramolecular transduction of the activation signal, from the hormone-bound ectodomain to the serpentine domain remain unknown. This is not a purely academic problem, as understanding of the molecular mechanisms involved might increase our knowledge, and hence our therapeutic strategies, of the pathophysiology of Graves- disease.

The TSH receptor is peculiar, among the glycoprotein hormone receptors, as it displays readily measurable basal activity, when expressed by transfection in any cell type. The physiological significance of this characteristic, if any, remains unknown. It is very possible that the basal activity of the TSH receptor is there simply-. because it does not harm Pokies. Indeed, a rare non-pathological TSH receptor variant, with the tyrosine in position 601 replaced by histidine, is virtually devoid of constitutive activity. However, it is the existence of the basal activity of the TSH receptor which opened the way to hypotheses regarding the mechanism of intramolecular transduction of the activation signal. The observation that removal by truncation of the ectodomain of the receptor resulted in increase in its constitutive activity led to the notion that the ectodomain would function as an inverse agonist of the serpentine domain. Similarly, the observation that point mutations in a specific residue of the ectodomain (Ser 281) were associated with a strong increase in constitutive activity (in the absence of hormone binding), led to the hypothesis that an -activated- conformation of the ectodomain functioned as the immediate agonist of the serpentine domain. According to this model, binding of TSH, or TSAb, to the ectodomain would activate it, i.e. change its conformation so as to transform it into an agonist of the serpentine domain.

The study by the Rapoport-s group fits nicely in this scheme and adds an important piece to the puzzle. Up to now, we had at our disposal, only receptor agonists (TSH or TSAbs), or neutral antagonists [blocking (auto)antibodies, sometimes called thyroid stimulation blocking antibodies, or TSBAb]. Of note, these blocking antibodies are simple competitors of TSH for receptor binding but do not modify receptor activity (hence their -neutral- denomination). The CS-17 monoclonal antibody described in the article is shown to bind to a conformational epitope of the ectodomain (it was generated by an immunogen devoid of the serpentine domain), and the effect of this binding is to decrease the basal activity of the wild type TSHR, as well as of a panoply of activated mutants. This corresponds to the pharmacological definition of an inverse agonist, a ligand which, upon binding, stabilizes an inactive conformation of a receptor, with the effect of decreasing its basal activity. The fact that the epitope recognized by CS-17 is present in the ectodomain implies that the effect on the serpentine domain is of allosteric nature. It strongly supports the model of the ectodomain being the controlling intramolecular ligand of the serpentine domain. In its unliganded wild-type state, the ectodomain would play the role of an imperfect inverse agonist, allowing some constitutive activity to manifest. As already stated, when bound to TSH (or TSAb), or mutated on residue 281, the ectodomain would activate the serpentine domain. The CS-17 monoclonal antibody completes the picture by showing that it is possible to change the conformation of the ectodomain in a way which increases its efficacy as an inverse agonist of the serpentine domain.

Besides these fundamental considerations, the article shows the feasibility of engineering inverse agonist monoclonal antibodies with therapeutic value. As stated by the authors, potential uses of a humanized version of CS-17 could include the treatment of differentiated thyroid carcinoma (inhibiting the low grade stimulation of growth which may be associated with basal activity in residual differentiated tumor tissue), toxic nodular goiter in which a -cooling off- period may be of value prior to definitive surgical or radio-iodine therapy, non-autoimmune genetic causes of hyperthyroidism and amiodarone-induced, non-autoimmune thyrotoxicosis.

( Summary and commentary prepared by Gilbert Vassart )

Present summary & commentary are related to the Chapter N- 16a of TDM

Full paper obtainable at http://endo.endojournals.org/cgi/rapidpdf/en.2006-1754v1sd