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Endocrinology Vol. 148, No. 5 2375-2382
Copyright © 2007 by The Endocrine Society

Suppression of Thyrotropin Receptor Constitutive Activity by a Monoclonal Antibody with Inverse Agonist Activity

Chun-Rong Chen, Sandra M. McLachlan and Basil Rapoport

Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California, Los Angeles, School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Basil Rapoport, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: rapoportb{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TSH binding to the TSH receptor (TSHR) induces thyrocyte growth and proliferation primarily by activating the adenylyl cyclase signaling pathway. Relative to the other glycoprotein hormone receptors, the TSHR has considerable ligand-independent (constitutive) activity. We describe a TSHR monoclonal antibody (CS-17) with the previously unrecognized property of being an inverse agonist for TSHR constitutive activity. This property is retained, even when constitutive activity is extremely high consequent to diverse TSHR extracellular region mutations. A similar effect on an activating mutation at the base of the sixth transmembrane helix (not accessible to direct CS-17 contact) 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 a significant portion lies in the C-terminal region of the TSHR leucine-rich domain (residues 260–289). By interacting with the large TSHR extracellular domain, CS-17 is, to our knowledge, the first antibody reported to be an inverse agonist for a member of the G protein receptor superfamily. 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TSH RECEPTOR (TSHR), a member of the G protein-coupled receptor (GPCR) superfamily, is a key regulator of thyroid function. Through the TSHR, the natural ligand (TSH) and pathological autoantibodies (primarily stimulatory but occasionally blocking) modify cAMP generation by adenylyl cyclase and, consequently, many aspects of thyroid hormone synthesis and secretion (reviewed in Refs. 1, 2). TSH also induces thyrocyte growth and proliferation (1). The TSHR is structurally similar to the receptors for the other anterior pituitary glycoprotein hormones (reviewed in Ref. 3) yet is functionally different in possessing relatively high constitutive activity in the absence of ligand (4). This activity is partially constrained by the TSHR ectodomain (5) that therefore functions as an inverse agonist (6). Although other activation pathways such as BRAF play an important pathogenic role in thyroid carcinoma (7), significant TSHR constitutive activity is a clinically relevant phenomenon in the treatment of thyroid carcinoma. After thyroidectomy, suppression of endogenous TSH secretion is a therapeutic goal to prevent or retard the proliferation or metastasis of residual thyroid carcinoma cells. However, even complete TSH suppression with supraphysiological doses of thyroxine cannot eliminate potentially harmful TSHR activity. Also, perhaps because of its inherent noisiness, the TSHR is highly susceptible to activation by a large variety of disease-inducing mutations, particularly within its serpentine region, as documented in a recent database (8).

In recent years, understanding of TSHR structure and function has been facilitated by the generation of murine (9, 10, 11, 12, 13, 14, 15, 16, 17, 18), hamster (19), and human (20, 21) monoclonal antibodies (mAbs). Of particular interest and importance are those mAbs that are potent activators of the TSHR (15, 17, 18, 19, 21). mAbs that function as competitive antagonists for thyroid-stimulating autoantibodies (TSAbs) have also received attention as possible therapeutic agents in Graves’ disease (22, 23), although competition for TSH binding, a universal property of these blocking antibodies, will lead to hypothyroidism.

In the course of generating a diverse panel of TSHR mAbs in our own laboratory, we noted that a mAb possessed a novel feature not described previously, namely strong inverse agonist activity. We now report the in vitro characteristics of this mAb (CS-17) and also demonstrate its in vivo activity. Therefore, CS-17 has the potential, after humanization, to be developed as an adjunctive therapeutic agent in thyroid cancer as well as other selected hyperthyroidism states.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TSHR mAbs
Monoclonal antibody CS-17 is one of a panel of TSHR mAbs generated in our laboratory from seven fusions over a 3-yr period. The classification and details of these mAbs will be reported elsewhere. In brief, 6- to 8-wk-old female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were injected im with adenovirus expressing the human TSHR A subunit, as reported previously (24, 25). Three days before fusion, mice were boosted iv with 50 µg of affinity-purified TSHR A subunit protein generated in Chinese hamster ovary (CHO) cells (26). Mouse splenocytes were fused to murine SP-2/0 cells (American Type Culture Collection, Manassas, VA) using 50% polyethylene glycol (Sigma, St. Louis MO). Hybridoma selection was by standard techniques using hypoxanthine, aminopterin, and thymidine in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum, gentamicin, L-glutamine, and sodium pyruvate. Approximately 2 wk after fusion, culture supernatants secreting IgG (ELISA from Bethyl, Montgomery, TX) were screened by flow cytometry using TSHR-expressing CHO cells. Hybridomas of interest were recloned three times by limiting dilution to obtain monoclonal cell lines. For IgG purification, cells were cultured in serum-free medium and the latter applied to Protein G Hi-Trap columns (Pharmacia, now GE Healthcare, Piscataway, NJ). Nonfunctional murine mAb 4C1 (27) was purchased from Serotec (Oxford, UK).

Construction and expression of TSHR mutants
Construction of TSHR mutations C24,29S, C24,31S, and C29,31S in the mammalian expression vector pECE-NEO (28) has been reported previously (29). Chimeric TSH-LH receptors (TSH-LHRs) in the vector pECE-neo were also reported previously (30). Of these, TSH-LHR-6, and TSH-LHR-10 (depicted schematically below) required additional modification to convert the histidine 601 polymorphism (28) to tyrosine and delete the 5'- and 3'-untranslated ends (31). This modification is necessary because the H601 polymorphism, unlike the much more common Y601, lacks significant constitutive activity (32, 33). Gain-of-function TSHR mutants S281I (34), I486F and I568T (35), A623I (36), and V656F (37) were introduced into the wild-type TSHR in the same vector using the QuickChange site-directed mutagenesis kit (Stratagene, San Diego, CA). Plasmids were transiently expressed in Cos-7 cells using FuGENE6 (Roche, Indianapolis, IN). Cells were cultured in DMEM supplemented with 10% fetal calf serum, penicillin (100 U/ml), gentamicin (50 µg/ml), and Fungizone (2.5 µg/ml) and were tested approximately 48 h after transfection.

Cultured cell cAMP assays
COS-7 cells expressing the wild-type TSHR and TSHR mutants were transferred into 96-well plates approximately 24 h after transfection and 24 h before assay. Cells from the same transfection were also plated in 6-cm culture dishes to monitor the transfection efficiency by flow cytometry (see below). For bioassay, the culture medium described above was replaced with DMEM supplemented with 1 mM isobutyl methylxanthine and 10 mM HEPES. Where indicated in the text, media also contained purified mAb CS-17 or bovine TSH (Sigma). Purified normal mouse IgG and mock-transfected COS-7 cells were included as controls. After 60 min at 37 C, the medium was aspirated and intracellular cAMP was extracted with 0.2 ml 95% ethanol. The extracts were evaporated to dryness, resuspended in 0.1 ml of Dulbecco’s PBS (pH 7.5), and samples (20 µl) assayed using the LANCE cAMP kit according to the protocol of the manufacturer (PerkinElmer, Shelton, CT).

Flow cytometry
Transiently transfected COS-7 cells were harvested from 6-cm-diameter dishes using 1 mM EDTA and 1 mM EGTA in PBS. After washing twice with PBS containing 10 mM HEPES (pH 7.4), 2% fetal bovine serum, and 0.05% NaN3, the cells were incubated for 30 min at room temperature in 100 µl of the same buffer containing 1 µg of either normal mouse IgG, mAb CS-17, or mAb 2C11. After rinsing, the cells were incubated for 45 min with 100 µl fluorescein isothiocyanate-conjugated goat antimouse IgG (1:100) (Caltag, Burlingame, CA), washed, and analyzed using a FACScan flow cytofluorimeter (Becton-Dickinson, San Jose, CA). Cells stained with propidium iodide (1 µg/ml final concentration) were excluded from analysis. For determining CS-17 blood concentrations after mAb injections in vivo (see below), we performed flow cytometry using intact CHO cells stably expressing the wild-type TSHR and, as standards, normal mouse serum supplemented with different amounts of CS-17.

TSH binding to transfected cells
COS-7 cells transiently transfected with plasmids expressing the wild-type TSHR or TSHR mutants were grown to confluence in 24-well plates. Medium was aspirated and replaced with 250 µl binding buffer (Hanks’ buffer with 250 mM sucrose substituting for NaCl to maintain isotonicity and 0.25% BSA) containing approximately 8000 cpm 125I-TSH (Kronus, Boise, ID). After incubation for 1–2 h at room temperature, cells were rapidly rinsed three times with binding buffer (4 C), solubilized with 0.5 ml 1 N NaOH, and radioactivity was then measured in a {gamma}-counter. Nonspecific binding was determined using COS-7 cells transfected in parallel with the vector alone. In some experiments, cells were preincubated for 1 h at 37 C in DMEM containing 10% fetal calf serum and the indicated concentrations of CS-17 before replacement of the medium with binding buffer containing 125I-TSH and the same CS-17 concentration.

In vivo study of TSHR monoclonal antibody CS-17
Purified CS-17 or normal mouse IgG (250 µg) in sterile PBS was injected ip into 6- to 8-wk-old female BALB/c mice (Jackson Laboratory). Injections were administered on d 2 and 5, and blood was collected on d 1, 4, and 7. Serum total T4 levels were measured in undiluted serum (25 µl) by RIA using a kit (Diagnostic Products Corp., Los Angeles, CA). Sera were also used to estimate CS-17 concentrations by flow cytometry (see above). These animal studies were approved by the Institutional Animal Care and Use Committee and performed with the highest standards of animal care in a pathogen-free facility.

Statistical analyses
Student’s t test was used to determine the significance of differences in intracellular cAMP levels in cells treated with or without CS-17 as well as the significance of differences of T4 levels in mice injected with either normal mouse IgG or TSHR mAb CS-17.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of TSHR monoclonal antibody CS-17
Among the TSHR mAbs that we isolated, CS-17, an IgG2a, recognized the wild-type TSHR expressed on COS-7 cells as detected by flow cytometry (Fig. 1AGo). Although CS-17 lacked thyroid stimulating activity (see below), we noted a novel property for a TSHR mAb, namely inverse agonist activity. The TSHR is noisy in the absence of ligand (TSH), as reflected by increased intracellular cAMP levels in TSHR-expressing relative to mock-transfected COS-7 cells (Fig. 1BGo). TSHR mAb CS-17, at a concentration of 10 µg/ml, significantly reduced this constitutive activity (P = 0.0052). As a control, another TSHR mAb (4C1) (27) had no effect on TSHR constitutive activity. CS-17 did not alter cAMP levels in mock-transfected cells (Fig. 1CGo). Inverse agonist activity on TSHR constitutive activity was evident at a concentration of 0.1 µg/ml and was near complete at 100 µg/ml (Fig. 1CGo). At intermediate concentrations, CS-17 inverse agonist activity varied in different experiments but was typically 60–90% at 10 µg/ml (see also Figs. 4Go and 5Go).


Figure 1
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  		FIG. 1. TSHR mAb CS-17 has inverse agonist activity. A, Flow cytometric recognition by CS-17 (10 µg/ml) of the wild-type TSHR on the cell surface. COS-7 cells were transiently transfected with a plasmid expressing the wild-type (wt) TSHR. As controls, cells were subjected to mock transfection, and flow cytometry was performed using purified normal mouse IgG (NmIgG) at the same concentration (10 µg/ml). B, Purified CS-17 and control mAb (4C1), both at 10 µg/ml, were incubated for 60 min with aliquots of the same COS-7 cells used for flow cytometry (A). The contribution of the TSHR to intracellular cAMP levels (TSHR transfected vs. mock transfected cells) indicates constitutive TSHR activity. CS-17, but not 4C1, suppressed TSHR constitutive activity (*, P = 0.0052; Student’s t test). Data shown are the mean + range of values from duplicate wells and are representative of at least 10 different experiments in which CS-17 suppressed constitutive activity. C, Dose-effect relationship of TSHR mAb CS-17 on TSHR constitutive activity. COS-7 cells transiently transfected with the wild-type TSHR were incubated for 60 min with the indicated CS-17 concentrations. Mock-transfected cells were included as controls. Intracellular cAMP was measured in duplicate wells of cells. Values indicate the mean ± range. Similar data were obtained in a separate experiment.

 

Figure 4
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  		FIG. 4. Effect of CS-17 on TSH binding to, and activation of, the TSHR. A, Cell monolayers expressing the wild-type TSHR were preincubated for 1 h at 37 C with the indicated concentrations of CS-17 or purified normal mouse IgG (NmIgG) before the addition of 125I-TSH (see Materials and Methods). After a further 2-h incubation at room temperature, cells were rinsed, and radioactivity was measured in solubilized cells. The values shown are net after subtraction of binding to cells not expressing the TSHR. Each point represents the mean ± SD of values obtained in duplicate dishes of cells. Similar data were obtained in a separate experiment (total of ~8000 cpm 125I-TSH added per dish with 600–850 cpm subtracted to provide the net data). B, Influence of CS-17 on the cAMP response to low-dose TSH stimulation. COS-7 cells transiently transfected with the wild-type TSHR were incubated for 60 min in the indicated concentrations of bovine TSH in the absence or presence of CS-17 (10 and 100 µg/ml). Intracellular cAMP levels in mock-transfected cells (empty expression vector) were unaffected by either concentration of CS-17. Each point represents the mean of cAMP values determined in duplicate wells of cells. C, Data similar to those for CS-17 but with a control TSHR mAb, 4C1.

 

Figure 5
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  		FIG. 5. CS-17 inverse agonist activity is unrelated to the TSHR N-terminal cysteine cluster important for TSAb responsiveness. A, Schematic representation of three selected TSHR mutant in which pairs of Cys residues in the N terminus cluster (C24, C29, C31, and C41) were converted to Ser (arrows), leaving only the remaining two Cys residues available for disulfide bridging (29 ). TSHR mutants C24,31S and C24,29S are fully responsive to TSAb, similar to the wild-type TSHR. In contrast, TSHR C29,31S (forcing a C41-C24 disulfide bond) is poorly responsive to TSAb while retaining a normal response to TSH stimulation (29 ). B, Constitutive intracellular cAMP levels in aliquots of the same cells used for flow cytometry. Cells were incubated for 60 min in medium supplemented with mAb CS-17 or purified normal mouse IgG (both at 10 µg/ml) before cAMP extraction (see Materials and Methods). Bars indicate the mean + range of values obtained in duplicate wells of cells. Similar data were observed in a separate experiment. C, Flow cytometry (FACS) of mock-transfected cells and TSHR-transfected cells subjected to flow cytometry with mAb CS-17 and purified normal mouse IgG (both at 10 µg/ml).

 
CS-17 suppresses gain-of-function TSHR mutations
TSHR gain-of-function mutations are associated with autonomously functioning thyroid adenomas (36) and nonautoimmune autosomal dominant hyperthyroidism (38). Because these mutant TSHRs have far greater constitutive activity than the wild-type TSHR, we examined whether mAb CS-17 maintained its suppressive property with the former. We generated and tested the effect of CS-17 on gain-of-function mutations known to involve different regions of the TSHR ectodomain accessible to antibody in intact cells, namely the hinge region (S281I) and all three extracellular loops (I486F, I568T, and V656F). As a control, we included a gain-of-function mutation at the junction of the third intracellular loop and sixth transmembrane domain (A623I) that would not be accessible to antibody.

In confirmation of previous reports (34, 35, 36, 37), all of these mutations greatly increased TSHR constitutive activity, the most potent of these being S281I and A623I (Fig. 2Go). Despite these high levels of activity, CS-17 partially suppressed all mutant receptors including A623I that cannot be directly contacted by CS-17. CS-17 concentrations producing half-maximal inhibition (IC50) were similar to those for the wild-type TSHR (Fig. 1CGo), approximately 1 µg/ml, with the exception of V656F, which was less sensitive to CS-17 inhibition.


Figure 2
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  		FIG. 2. CS-17 suppresses gain-of-function TSHR mutations. Plasmids expressing the wild-type TSHR or the indicated TSHR activating mutations were transiently transfected into COS-7 cells. After 2 d, cells were incubated for 1 h in control medium or in medium supplemented with the indicated CS-17 concentrations. Intracellular cAMP levels were determined in duplicate wells of cells. Bars indicate the range of duplicate values. These data are representative of three experiments. Note that because of the high degree of activity of the mutants, suppression of wild-type TSHR constitutive activity is barely perceptible.

 
CS-17 suppresses thyroid function in vivo
We examined whether the in vitro effect of mAb CS-17 would be reflected in vivo. BALB/c mice were injected ip twice at a 3-d interval with 250 µg of either normal mouse IgG or mAb CS-17 (eight mice in each group). CS-17 concentrations in serum were estimated by flow cytometric analysis of CHO cells expressing the wild-type TSHR using a standard curve of known concentrations of CS-17 added to normal mouse serum. Serum CS-17 levels after the first and second injections were 127 ± 5 and 232 ± 20 µg/ml (mean ± SEM; n = 8), respectively. Serum total T4 levels 1 d before the first injection were 5.2 ± 0.17 µg/dl (mean ± SEM; n = 16) (Fig. 3Go). Two days after the second injection, serum T4 was significantly lower in the mice receiving CS-17 (3.3 ± 0.3) than the animals receiving NmIgG (5.2 ± 0.2) (P < 0.001). Similar suppression of serum T4 levels by CS-17 was observed in three separate experiments.


Figure 3
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  		FIG. 3. CS-17 suppresses thyroid function in vivo. BALB/c mice were injected ip on the indicated days with 250 µg of either normal mouse IgG (NmIgG) or mAb CS-17 (eight mice in each group). Serum total T4 levels were determined on serum obtained before the first injection (d 1) and then 2 d after each injection (d 4 and 7). *, P < 0.001, Student’s t test; ns, not significant. The same phenomenon was observed in three separate experiments.

 
CS-17 reduces TSH binding to and activation of the TSHR
In addition to suppressing TSHR ligand-independent activity, CS-17, unlike purified, normal mouse IgG, inhibited 125I-TSH binding to the human TSHR expressed on the surface of cell monolayers (Fig. 4AGo). However, CS-17 was less potent in inhibiting TSH binding than in suppressing constitutive activity. At 10 µg/ml, CS-17 suppressed TSH binding by approximately 35%, whereas the same concentration reduced constitutive activity of the wild-type TSHR by at least 60% (e.g. Fig. 1CGo).

With TSHR expressing cell monolayers, CS-17 at 10 µg/ml also reduced the cAMP response to TSH stimulation, but only at TSH concentrations less than 100 µU/ml (Fig. 4BGo). TSH at 100 µU/ml, a near maximal stimulatory concentration, broke through CS-17 suppression, inducing a cAMP response similar to that in the absence of CS-17. However, a 10-fold increase in CS-17 to 100 µg/ml (approximately the concentration attained in blood in the in vivo experiments), was able to partially suppress the cAMP response to this high TSH concentration. Unlike CS-17, similar concentrations of TSHR mAb 4C1 (10 and 100 µg/ml) used as a control did not suppress the cAMP response to even weak TSH stimulation (10 µU/ml) (Fig. 4CGo).

Site of action of mAb CS-17
Monoclonal antibody CS-17 was generated by immunizing mice with the major component of the TSHR A subunit (amino acid residues 1–289). Consequently, the CS-17 epitope cannot involve TSHR ectodomain residues downstream residue 289, including the hinge region. CS-17 does not recognize an overlapping series of synthetic TSHR ectodomain peptides (data not shown), indicating that its epitope is conformational and possibly discontinuous. We therefore explored CS-17 recognition of selected conformationally intact TSHR mutants expressed on the surface of transfected cells.

Previously we observed that the cysteine-rich N-terminal region of the TSHR ectodomain contributed to thyroid-stimulating autoantibody (but not TSH) binding and function (39, 40). Furthermore, by mutating permutations of cysteine residue pairs among the four cysteines (C24, C29, C31, and C41), we deduced that a disulfide bond involving C41 with either C29 or C31 was necessary for TSAb responsiveness comparable with the wild-type TSHR (schematically represented in Fig. 5AGo) (29). We therefore examined whether CS-17 suppression of TSHR constitutive activity was related to this TSAb-critical region. This was not the case (Fig. 5BGo). Strong, similar CS-17 inverse agonist activity was evident with the TSHR mutation associated with reduced TSAb responsiveness (C29,31S) in comparison with the wild-type TSHR and TSHR mutants fully responsive to TSAb (C24,31S and C24,29S). All TSHR mutants expressed well on the cell surface (Fig. 5CGo).

We also used flow cytometry to determine CS-17 recognition of selected chimeric receptors involving the substitution of TSHR segments with the LH receptor (Fig. 6AGo) (30). In TSH-LHR-6, the C-terminal portion of the TSHR ectodomain (domains D and E) is substituted with the LH receptor. In TSH-LHR-10, only the middle portion of the TSHR ectodomain (residues 170–360; domains C and D) remain unchanged. As reported previously (30), both chimeras are expressed on the cell surface and bind TSH (Fig. 6BGo). On flow cytometry using aliquots of the same cells, CS-17 recognized the wild-type TSHR and TSH-LHR-10, but not TSH-LHR-6 (Fig. 6CGo). Taken together, these data suggest that a significant portion of the conformational (possibly discontinuous) CS-17 epitope lies between amino acid residues 260 and 289.


Figure 6
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  		FIG. 6. CS-17 recognition of chimeric TSH-LHRs. COS-7 cells were transiently transfected with plasmids expressing the wild-type (wt) TSHR or the indicated TSH-LHR chimeras. Mock represents cells transfected with vector alone. A, Schematic representation of selected chimeric TSH-LHR For these receptors, the TSHR ectodomain was divided into five arbitrary domains (A–E) (30 ). Segments of the rat LHR (black bars) were substituted with the homologous regions of the wild-type (wt)TSHR (white bars). B, Both TSH-LHR-6 and TSH-LHR-10 are expressed on the cell surface and bind 125I-TSH. TSH binding was assessed using cells in monolayer culture (see Materials and Methods) and was expressed (net of mock transfection values) as percent of total 125I-TSH added to the dishes (~10,000 cpm). Bars indicate the mean ± SE of values obtained with triplicate dishes of cells. WT, Wild type. C, CS-17 recognition of chimeric receptors on flow cytometry. Purified, normal mouse IgG (NmIgG) was used as a control (both preparations at 10 µg/ml). Of these chimeric receptors, CS-17 recognized only TSH-LHR-10.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Monoclonal antibody CS-17 possesses a property not noted for any of the numerous TSHR mAbs reported previously. In the absence of TSH, CS-17 reduces TSHR constitutive activity in vitro with a half maximal inhibitory concentration of approximately 1 µg/ml (6.7 x 10–9 M). Moreover, CS-17 suppresses to a similar extent a number of TSHR mutations associated with constitutive activities far higher than the wild-type TSHR. Administered to mice in vivo, CS-17 reduces serum T4 levels. Because CS-17 also has modest TSH blocking activity, it is not possible to determine the relative contributions of TSH blocking and reduced constitutive activities to the decrease in serum T4. However, it must be emphasized that this distinction is not relevant to the most important potential clinical use for an antibody such as CS-17, namely in the treatment of thyroid cancer after thyroid ablation (see below). Moreover, at physiological TSH levels, the CS-17 effect on TSHR constitutive activity predominates over TSH responsiveness (Fig. 4CGo).

In recent years, the realization that many GPCRs have ligand-independent constitutive activity to varying degrees has introduced a new classification of pharmacological agents. Besides agonists and antagonists, inverse agonists and neutral antagonists are now described. Inverse agonists reduce ligand-independent constitutive activity. Many classical competitive antagonists also have inverse agonist properties, unlike neutral antagonists (reviewed in Ref. 41). The great majority of GPCR inverse agonists are small molecules, many used in clinical practice as drugs to reduce activity of receptors such as those for epinephrine, histamine, dopamine, and angiotensin. In general, these agents bind to a pocket within the transmembrane helices. However, in a few cases and not yet in clinical use, large antibody molecules have been generated that function as inverse agonists by binding to the extracellular loops of the ß2-adrenergic (42) and M2-muscarinic acetylcholine (43) receptors.

Turning to the thyroid, the TRH receptors in the pituitary thyrotroph (44, 45, 46) and the TSHR in the thyrocyte (28, 47) are both GPCRs. The former receptor, activated by a small ligand (TRH), has a small extracellular domain. The TSHR has a large ectodomain (397 amino acid residues after signal peptide deletion) consistent with its large (~30 kDa), glycosylated ligand (TSH). Besides their natural ligands, small, synthetic molecules have been sought to modulate receptor function. For example, midazolam has been identified as an inverse agonist for the TRH receptor (48), and another synthetic compound (org41821) is a partial agonist for the TSHR (49). Unlike TSH, org41821 interacts directly with TSHR transmembrane helices (49). A modification of this compound acts allosterically as an antagonist of TSH action (50). However, until the present, no TSHR inverse agonist has been reported.

CS-17 is therefore a rare example of a large molecule (IgG) that is a GPCR inverse agonist and representing a novel class of these agents that do not insert directly into a transmembrane helix pocket or bind to the extracellular loops. Having been generated by immunization with the TSHR A subunit, the mAb CS-17 epitopes lies within the ectodomain, upstream of amino acid residue 289, as discussed above. It is noteworthy that the TSHR ectodomain is itself a tethered inverse agonist (6). We speculate that mAb CS-17 enhances this suppressive activity and that further understanding of its mechanism of action will provide insight into this property of the TSHR ectodomain. Clearly, suppressing activity of a TSHR with very high constitutive activity consequent to an intracellular mutation (A623I) indicates that CS-17 is acting allosterically.

The search for inverse agonists as therapeutic agents to modulate GPCR expression is of much current interest (reviewed in Ref. 51). In this light, CS-17 has a number of potential clinical applications. Perhaps the most common would be in reducing the risk of recurrence or progression of differentiated thyroid carcinoma after surgery and radioiodine ablation of residual thyroid tissue. Because TSH stimulates thyrocyte growth (1), it is common practice after thyroid ablation to administer L-thyroxine at a supraphysiological dose to partially or completely suppress pituitary secretion of TSH (reviewed in Ref. 52). However, sustained mild elevations of peripheral thyroid hormone levels (subclinical thyrotoxicosis) carry the risk of cardiac arrhythmias and osteoporosis. More important, in well-differentiated thyroid carcinomas retaining TSHR expression, even total TSH suppression may not eliminate TSH-independent constitutive activity. Therefore, reduction in TSHR constitutive activity with an inverse agonist such as CS-17 could be achieved while maintaining TSH and thyroid hormone levels within the physiological range, reducing the risks mentioned above. Even at physiological TSH levels, the CS-17 effect on TSHR constitutive activity predominates over TSH responsiveness (Fig. 4CGo).

Other potential applications for TSHR inverse agonist therapy would be in a situations in which serum TSH levels are not elevated. One example would be in toxic nodular goiter, in which a cooling-off period may be of value before definitive surgical or radioiodine therapy, particularly in elderly patients. The finding that CS-17 can reduce greatly elevated constitutive activity associated with TSHR mutations suggests that a TSHR inverse agonist could also be considered in rare instances of familial, nonautoimmune hyperthyroidism. Finally, because CS-17 appears to act allosterically, it might be of value in treating amiodarone-induced, nonautoimmune thyrotoxicosis, a frequently serious condition in elderly patients with underlying cardiac conditions in whom thionamide drugs or radioiodine therapy are not options for near-term relief (reviewed in Ref. 53). Of course, any future use of a mouse IgG in humans would require humanization by replacing its constant regions with its human counterparts.

In conclusion, we report the generation of a TSHR mAb with the previously unrecognized property of being an inverse agonist for TSHR constitutive activity. CS-17 interacts with the large extracellular domain of the TSHR and is, to our knowledge, unique among GPCR inverse agonists. This mAb is active with TSHR mutations responsible for enhanced constitutive activity. After humanization (splicing of the TSHR-specific variable region of the murine antibody to a human antibody constant region), CS-17 has the potential to be a useful therapeutic agent in a number of thyroid diseases ranging from thyroid cancer to some forms of hyperthyroidism.


   Acknowledgments
 
We are grateful for contributions by Dr. Boris Catz (Los Angeles, CA).


   Footnotes
 
This work was supported by National Institutes of Health Grants DK 19289 (to B.R.) and DK 54684 (to S.M.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 1, 2007

Abbreviations: CHO, Chinese hamster ovary; GPCR, G protein-coupled receptor; mAb, monoclonal antibody; TSAb, thyroid-stimulating autoantibody; TSH-LHR, TSH-LH receptor; TSHR, TSH receptor.

Received December 28, 2006.

Accepted for publication January 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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