GRAVES DISEASE (GD) is characterized by the presence

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1 /01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism 86(8): Printed in U.S.A. Copyright 2001 by The Endocrine Society Epitope Mapping of TSH Receptor-Blocking Antibodies in Graves Disease That Appear during Pregnancy A. W. C. KUNG, K. S. LAU, AND L. D. KOHN Department of Medicine, University of Hong Kong, Queen Mary Hospital (A.W.C.K., K.S.L.), Hong Kong, China; and Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (L.D.K.), Bethesda, Maryland GRAVES DISEASE (GD) is characterized by the presence of autoantibodies to the TSH receptor (TSHR), which are pathogenic and responsible for disease activity. It is well recognized that the autoantibodies are heterogeneous, recognizing different epitopes on the TSHR, and that the functional status of the thyroid and the clinical picture of the disease are dependent on the heterogeneity of the TSH receptor antibodies (TRAb) (1). In the last few years, epitope analysis of Graves IgG has been performed extensively using site-directed mutagenesis, chimeras of human TSHR (htshr) and LH-hCG receptor (LH/CGR), and receptor peptides (2 9). As a result, it is now recognized that there are multiple forms of functional TSHR autoantibodies: stimulating Abs that activate the TSHR, and nonstimulating antibodies that block TSH binding and action or block thyroidstimulating antibody (TSAb) as well as TSH action. The epitope for TSAbs is biased more toward the N-terminus of the TSHR ectodomain in 95% of patients (5, 10 17). The epitope for blocking Abs that inhibit TSAb and TSH function and that are associated with hypothyroidism in idiopathic myxedema or some Hashimoto s patients is largely on the Abbreviations: CGR, hcg receptor; ft 4, free T 4 ; htsh, human TSH; GD, Graves disease; PP, postpartum; T1, first trimester; T2, second trimester; T3, third trimester; TBAb, thyroid stimulating-blocking antibody; TBII, TSH binding inhibitory Ig; TRAb, TSH receptor antibody; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor; WT, wild type. Spontaneous remission of Graves disease during pregnancy is thought to be due to a reduction of thyroid-stimulating antibody activity. We suspected, however, that a broader change in TSH receptor antibody characteristics might play an important role in modulating disease activity during pregnancy. We measured TSH binding inhibitory Ig, thyroid-stimulating antibody, and thyroid stimulating-blocking antibody activities in 13 pregnant Graves disease patients at first, second, and third trimesters and 4 months postpartum. To measure and epitope-map thyroid-stimulating antibody and thyroid stimulating-blocking antibody activities, we used CHO cells transfected with wild-type human TSH receptor or with several TSH receptor-lh/hcg receptor chimeras: Mc1 2, Mc2, and Mc4. These chimeric cells have their respective TSH receptor residues 9 165, , and substituted with equivalent residues of the LH/hCG receptor. Overall thyroidstimulating antibody decreased, whereas thyroid stimulating-blocking antibody increased progressively during pregnancy. TSH binding inhibitory Ig fluctuated in individual patients, but overall the activities remained statistically unchanged. Thyroid stimulating-blocking antibody appeared in subjects who were either negative for thyroid-stimulating antibody or whose thyroid-stimulating antibody activity increased or decreased during pregnancy. Epitope mapping showed that the thyroid-stimulating antibodies were mainly directed against residues of the N-terminus of the TSH receptor extracellular domain. All thyroid stimulating-blocking antibodies had blocking activities against residues of the C-terminus of the ectodomain. However, the majority of the thyroid stimulating-blocking antibodies had a hybrid conformational epitope directed against N-terminal residues 9 89 or as well. Despite a change in the activity level, we did not observe any change in the epitope of either the stimulatory or blocking Abs as pregnancy advanced. In conclusion, a change in the specificity of TSH receptor antibody from stimulatory to blocking activity was observed during pregnancy, and the appearance of thyroid stimulating-blocking antibody may contribute to the remission of Graves disease during pregnancy. (J Clin Endocrinol Metab 86: , 2001) C-terminus of the TSHR ectodomain (10, 11, 17 19). The epitope for blocking Abs in Graves patients that inhibit TSH, but not TSAb, activity is largely on the N-terminus of the TSHR ectodomain, overlapping, but not identical, to the TSAb sites (17 19). Subtyping the epitopes of the autoantibodies found in GD patients may have clinical relevance, e.g. GD patients with heterogeneous epitope distribution are more likely to become euthyroid after antithyroid during treatment, whereas those with homogeneous epitope directed mainly to the N- terminus of the TSHR ectodomain are less responsive to medical treatment (15, 16). Spontaneous fluctuation of stimulatory and blocking Abs is associated with a change in the clinical profile from hyperthyroidism to hypothyroidism or vice versa (20 22). Remission of GD during pregnancy, with recrudescence after delivery, is commonly observed. It is currently believed that remission of GD during pregnancy is primarily due to a reduction in levels of TSAb that results from the generalized immunosuppression needed to accommodate the fetal graft as pregnancy advances (23). However, we recently observed that there was persistent B cell activation in GD patients throughout pregnancy together with appearance of blocking antibodies (24). To define the epitopes and clinical significance of the thyroid stimulating-blocking antibodies (TBAbs) that appeared during pregnancy, we measured TSH binding inhibitory Ig 3647

2 3648 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): Kung et al. Epitope Mapping of TBAb in Graves Disease (TBII) activity, thyroid-stimulating antibodies (TSAb) and TBAb in each patient s serum at various stages of pregnancy. The stimulating and blocking TRAbs were measured using TSHR/LH-CGR chimeric cell lines that were engineered to remove selective epitopes for the different TRAb. We documented the appearance of blocking antibodies whose epitope on the C-terminus of the TSHR ectodomain is associated with hypothyroidism in idiopathic myxedema or some Hashimoto s patients. The presence of blocking TRAb may contribute in part to the remission of GD during pregnancy. Subjects and methods Thirteen women with GD (aged 29 2 yr) were studied prospectively as soon as pregnancy was confirmed. To avoid possible immunomodulatory action of antithyroid drugs, only patients with mild disease taking no medications or those taking less than 10 mg carbimazole or 100 mg propylthiouracil daily for the preceding 6 months were recruited. Treatment was stopped in the seven patients who were taking antithyroid drugs in the second trimester. Fasting blood samples were obtained in the first trimester (T1; wk), the second trimester (T2; 24th wk), and the third trimester (T3; 32 34th wk) and 4 months postpartum. Ten healthy controls were studied at similar stages of pregnancy. Informed consent was obtained from all subjects, and the protocol was approved by the ethics committee of the University of Hong Kong. IgG preparation Materials and Methods Sera were stored at 70 C until IgG preparation. IgGs were extracted by affinity chromatography using protein A-Sepharose CL-4B columns (Amersham Pharmacia Biotech, Piscataway, NJ), followed by dialysis. The purity of the IgG preparation was confirmed by documentation of unmeasureable TSH levels and hcg levels of less than 5 ng/ml. Thyroid function test Serum free T 4 (ft 4 ) was determined by a competitive chemiluminometric immunoassay; TSH was determined by a two-site immunochemiluminometric assay using an Automated Chemiluminescence System (ACS 180, Ciba Corning, Inc., Medfield, MA). The normal range of ft 4 was pmol/l. The inter- and intraassay coefficients of variance for ft 4 and TSH were 4.9% and 3.6%, and 4.8% and 3.6%, respectively. As ft 4 assays may give spuriously low results during pregnancy, an abnormal ft 4 reading was confirmed with a free T 4 index measurement (Abbott Laboratory, North Chicago, IL). The normal range of free T 4 index was Thyroid autoantibody assays The stimulating and blocking TRAbs were measured using CHO cells transfected with wild-type human TSHR (WT cells) or transfected with several TSHR-LH/CGR chimeras: Mc1 2, Mc2, and Mc4 (15 19, 25). The Mc1 2, Mc2, and Mc4 chimeras have, respectively, TSHR residues 9 165, , and substituted with equivalent residues of the LH/CGR. Using this panel, the following functional TRAbs can be separately measured. TSAbs in 95% of Graves patients whose functional epitopes are on the N-terminal portion of the extracellular domain, residues 9 165, can be measured by comparing the activity of wild-type TSHR transfected cells vs. the activity of the Mc1 2 and Mc2 chimeras. TBAbs whose epitope is on the C-terminal portion of the extracellular domain, residues , and which block stimulating TRAb as well as TSH activity are readily measured comparing Mc1 2, Mc2, and Mc4 activities with wild-type TSHR transfectants. TBAbs whose epitope on the N-terminus are similarly detected with this comparison. Thyroid-stimulating antibody (TSAb) was determined in purified IgG by measuring camp released from CHO-hTSHR cells (WT cells). In brief, assays were performed in NaCl-free HBSS containing 20 mmol/ liter HEPES (ph 7.4), 1% BSA, 0.5 mmol/liter 3-isobuty1-1-methylxanthine, and 222 mmol/liter sucrose to make incubations isotonic. Bovine TSH (1 mu/ml; Sigma, St. Louis, MO) or purified IgG (1.5 mg/ml) was dissolved in 300 l incubation medium and incubated with cells for 2 h at 37 C. Supernatants were aspirated and stored at 20 C. camp released into the medium was measured by RIA. TSAb activity was expressed as the percent increase in camp levels in the experimental IgG relative to pooled IgG from 20 normal individuals. TBAb was determined by IgG-induced inhibition of the TSH-stimulated camp response in CHO-hTSHR cells (WT cells). To calculate TBAb activity, the following formula was used: 100% (1 [(camp accumulation in the presence of TSH and test IgG)/cAMP accumulation in the presence of TSH (1 miu/ml) and normal IgG)]). To further characterize the blocking activity of the IgG, the Igs were also evaluated using Mc1 2, Mc2, and Mc4 cells. Mc1 2 cells are CHO cells transfected with an htshr-lh/cgr chimera in which residues of TSHR were replaced by residues of the LH/CGR. In Mc2 cells, TSHR residues were replaced by residues of the LH/CGR. In Mc4 cells, TSHR residues were replaced by residues of the LH/CGR. In Mc4 cells TBAb activity was measured using a TSAb as the stimulant rather than TSH, with otherwise identical calculations (15, 16). TBII was determined in patients sera by inhibition of specific [ 125 I]bovine TSH binding to porcine thyroid membranes in an RRA (PSR, Cardiff, UK). Assay results were expressed in terms of inhibition of TSH binding, calculated as follows: 100% [1 (labeled TSH specifically bound in the presence of test serum/labeled TSH specifically bound in the presence of normal serum)]. Triplicate assays were performed on all samples for antibody assays. The camp RIA assays (INCSTAR Corp., Stillwater, MN) were performed in duplicate. The normal ranges in 20 healthy subjects for TBII, TSAb, and TBAb were 15% to 15%, %, and 15% to 20%, respectively. The determination of TSAB and TBAb in the same sample was performed in the same assay. The intra- and interassay coefficients of variance for TBII, TSAb, and TBAb were 5.0% and 8.6%, 6.0% and 8.9%, and 9.7% and 11.6% respectively. Statistics Comparisons within GD patients at various stages of pregnancy were made by ANOVA using two-sample t tests or Wilcoxon ranks test depending on the distribution of the data. Results Thyroid function tests Five patients were biochemically hyperthyroid at T1. Two patients were in remission before pregnancy and were not taking any medications. All other patients were taking either 5 mg carbimazole or 50 mg propylthiouracil daily at T1. At T2, all patients were biochemically euthyroid, and all antithyroid drugs were stopped. ft4 levels were significantly lowered by T2 and T3 compared with T1 levels [T1, 18 5 pmol/liter; T2, 13 5 pmol/liter; T3, 13 3 pmol/liter; postpartum (PP), 15 5 pmol/liter; mean sd; by ANOVA, P 0.05]. During T3, two patients had subnormal ft 4 levels (both 10 pmol/l). Their ft 4 indexes were at the lower limit of normal (76 and 78), but their TSH levels remained within the normal range. One patient relapsed at 4 months PP. All healthy pregnant controls had normal ft 4 levels throughout pregnancy.

3 Kung et al. Epitope Mapping of TBAb in Graves Disease The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): TSHR antibodies Two patients (patients 1 and 2; Table 1) were negative for all TSHR antibodies throughout pregnancy. Five patients (patients 3, 5, 8, 10, and 13) had TSAb activity at T1. Their TSAb activities decreased progressively in all but 1 patient (patient 8) as pregnancy advanced. Three patients (patients 4, 5, and 9; Table 1) were positive for TBAb activity at T1. By T2 and T3, the number of patients positive for TBAb increased to 8 and 9, respectively. TBAb activity remained positive in seven patients at 4 months PP. Six patients (patients 3, 4, 8, 9, 10, and 13; Table 1) were positive for TBII at T1. The TBII activity fluctuated in individual patients and showed no correlation with TSAb or TBAb activity at any time during pregnancy. However, TBII was noted to correlate with TSAb (r 0.76; P 0.01), but not with TBAb, activity after delivery. The healthy controls were all negative for TSAb, TBAb, and TBII throughout pregnancy. Characteristics of the stimulating TSAbs We characterized the stimulating TSAb epitopes during the different trimesters of pregnancy. Among the five patients positive for TSAbs at T1, two patients (patients 3 and 10; Table 2) had a complete loss of the ability of the stimulating TSAb to increase camp levels in Mc2 cells (Table 2, column 3 vs. column 1). Mc2 cells have TSHR residues substituted by the homologous LH/CGR residues. All five patients had a complete loss of TSAb activity in Mc1 2 cells, which had TSHR residues substituted (Table 2, column 2 vs. column 1). There was no comparable loss when TSAb activity was measured in Mc4 cells, where TSHR residues are replaced (Table 2, column 4 vs. column 1). Overall, therefore, all of these stimulating antibodies had the major portion of their functional epitopes directed against the N-terminal portions of the extracellular domain of TSHR residues 9 165, with two patients directed against residues for their activity. Although the TSAb activity changed during pregnancy, epitope mapping did not reveal any change in the character of the TSAb in individual patients. Subjects negative for TSAb in the WT cells were all negative for camp release in either Mc2, Mc1 2, or Mc4 cells. Similarly, healthy pregnancy women were all negative for TSAb in these cell lines. Characteristics of blocking TBAbs To characterize the blocking activity, IgGs were incubated with purified bovine TSH (1 mu/ml) in WT, Mc1 2, and Mc2 cells or with a standard TSAb in WT cells and Mc4 cells. The ability of the IgG to inhibit the stimulating activity of TSH was lost in six patients (patients 4, 6, 7, 8, 10, and 13; Table 3) when their IgGs were incubated with Mc1 2 cells (Table 3, column 2 vs. column 1), but retained the inhibitory activity in Mc2 cells (Table 3, column 3 vs. column 1). The ability of these TBAb to inhibit the stimulating activity of Graves IgG was also lost in Mc4 cells (Table 3, column 5 vs. column 4). This pattern is characteristic of a TBAb that has its functional epitope directed against residues 9 89 of the TABLE 1. Stimulating TSAb activity (percentage) and blocking TBAb activity (percentage) measured in CHO cells transfected with wild-type human TSHR and TBII activity measured in a commercial assay TSAb TBAb TBII Normal T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP Underline signifies positive antibody activity.

4 3650 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): Kung et al. Epitope Mapping of TBAb in Graves Disease TABLE 2. Stimulating TSHRAb activity measured in CHO cells transfected with wild-type (WT) human TSHR or the Mc2, Mcl 2, and Mc4 TSHR/LH-CGR chimeras Patient IgG WT CHO-TSHR cells Mcl 2, CHO-TSHR cells Mc2 CHO-TSHR cells Mc4 CHO-TSHR cells Normal IgG T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP Underline signifies positive antibody activity.

5 Kung et al. Epitope Mapping of TBAb in Graves Disease The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): TABLE 3. Blocking TSHRAb activity measured in CHO cells transfected with wild-type (WT) human TSHR or the Mc2, Mcl 2, and Mc4 TSHR/LH-CGR chimeras Patient IgG TSH-blocking WT CHO-TSHR TSH-blocking Mcl 2 CHO-TSHR TSH-blocking Mc2 CHO-TSHR Graves IgG-blocking WT CHO-TSHR Graves IgG-blocking Mc4 CHO-TSHR Normal IgG T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP T T T PP Underline signifies positive antibody activity.

6 3652 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): Kung et al. Epitope Mapping of TBAb in Graves Disease N-terminus as well as residues of the C-terminus of the TSHR ectodomain. IgG from patient 5 (Table 3) lost its inhibitory activity when incubated with Mc2 and Mc4 cells, but not Mc1 2 cells, suggesting that its functional epitope was directed against residuals as well as residues of the TSHR. For the other three patients (patients 9, 11, and 12; Table 3) the ability of the IgGs to inhibit the stimulatory activity of TSH was retained in Mc1 2 cells as well as in Mc2 cells. However, the ability of the IgGs to inhibit the stimulatory activity of Graves IgG was lost in Mc4 cells. This suggests that the functional epitope of these TBAb is directed only against TSHR residues Similar to TSAb, there was no change in the TBAb epitope in these patients despite the change in inhibitory activity as pregnancy progressed. None of the healthy pregnant controls showed blocking activities in any of the cell lines at any stage of pregnancy. Discussion In this study we characterized the antibodies against the TSHR in GD patients during the various stages of pregnancy. We observed the appearance of blocking antibodies in these subjects as pregnancy advanced and the signs of hyperthyroidism waned. This result extends our previous study using human thyrocytes in primary culture (24), where TBAb detection is more problematic. In this study we used CHO-WT TSHR transfectants together with LH/CG-TSHR chimeras, in which TBAb activity can be measured unequivocally even in the presence of TSAb. Thus, we could now show that blocking antibodies were present in subjects who were negative for TSAb throughout pregnancy as well as in subjects whose TSAb activity decreased or increased during pregnancy. Similar to previous observations, we noticed that in a majority of the patients TSAb activity decreased as pregnancy advanced. TBII, however, fluctuated in individual patients and showed no correlation to TSAb activity during pregnancy. The separation of TBII and TSAb activity is consistent with data from monoclonal antibody studies and the Shimojo GD model indicating that TBIIs and TSAbs are distinct autoantibody populations (12, 25). A number of animal models of GD have demonstrated that TBII activity from sera of these animals can exist without TSAb activity (26) or that TBII activity precedes TSAb activity (27). The increase in TBAb activity in the absence of a change in TBII activity in the present study probably reflects the fact that TBIIs measure a multiplicity of antibodies that inhibit TSH binding. In GD patients, most TBAbs have their functional epitopes on the N-terminus of the TSHR extracellular domain (17 19, 28). However, the TBAbs appearing in our pregnant GD patients have a dominant epitope on the C-terminus of the extracellular domain of the TSHR, similar to those found in hypothyroid patients with idiopathic myxedema or some patients with Hashimoto s thyroiditis (16 19). Thus, there is a lack of correlation between the TBAb and TBII activities of these patients as the major epitopes of these antibody activities were directed against different parts of the TSHR, i.e. TBAb predominantly against the C-terminus and TBII against the N-terminus of the ectodomain. In light of the importance of detecting TBAb during pregnancy (29, 30), these data suggest that a functional TBAb assay in the chimeras is more revealing than measuring TBII values. In this report we characterized the epitopes of the stimulatory and blocking Abs using TSHR/LH-CGR chimeric cell lines that were engineered to selectively remove different epitopes for TRAbs. The data once again support the existence of a heterogeneous population of TSHR antibodies in GD patients. Similar to previous studies (14 19), the stimulatory Abs were predominantly directed against the N- terminal ectodomain of TSHR, residues However, unlike previous studies of patients treated with antithyroid drugs (16), we did not observe a change in the epitope of the stimulatory Abs in different individuals as pregnancy advanced. It has been reported that in the patients treated with antithyroid drugs, there was a change in the TSAb epitope from antibodies solely directed against the N-terminal ectodomain residues to antibodies recognizing other parts of the TSHR (16). With respect to the blocking TSHRAbs, all patients had blocking activities requiring the C-terminus of the extracellular domain of the TSHR residues However, the majority of these TBAb had a hybrid conformational epitope directed against residues as well as the ectodomain N-terminus, residues 9 89 or Thus, all patients had blocking activities against a C-terminal ectodomain epitope that was previously reported to be associated with hypothyroidism in patients with idiopathic myxoedema or Hashimoto s thyroiditis (17 19, 21). We believe that the appearance of this type of TBAb may play a role in modulating thyroid function and contribute toward the remission of thyrotoxicosis in GD during pregnancy, as these TBAbs are able to block TSAb as well as TSH activities, whereas TBAbs directed against the N-terminal epitopes appear to block only TSH activity (18). Although none of our pregnant subjects actually had hypothyroidism, two patients had subnormal ft 4 levels together with borderline low ft 4 index at T3, when the titers of these blocking antibodies were at their highest. The presence of this type of TBAb may counteract the stimulating TSAb circulating in some of these patients and helps explain why these patients develop biochemical euthyroidism despite the lack of medical treatment. Thus, these data continue to support the conclusion that blocking activities against the C-terminus of the extracellular domain are not confined to patients with idiopathic myxoedema or Hashimoto s thyroiditis, but are also present in patients with GD and can influence the disease course as their levels fluctuate (20, 21). The exact mechanism accounting for the change from stimulating to blocking antibodies during pregnancy in GD patients is unknown. Whether this is related to the changes in cytokine production in these patients during pregnancy is uncertain. We observed raised IL-10 levels in GD patients throughout pregnancy compared with control values (31). Also, IL-12 levels were suppressed to a greater extent in controls than in GD patients, especially during the second and third trimesters (31). The ratio of IL-10 to IL-12 in phytohemagglutinin-stimulated cultures was much lower in pregnant patients with GD than in normal pregnancy, sug-

7 Kung et al. Epitope Mapping of TBAb in Graves Disease The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8): gesting that cross-regulation of IL-10 and IL-12 may be deficient in GD during pregnancy. Whether the switch to a type 1 polarized cytokine profile during pregnancy will direct the proliferation of distinct clones of B lymphocytes specific for TBAb instead of TSAB, resulting in the production of predominantly inhibitory antibodies rather than stimulating antibodies, is unclear. Further understanding of this phenomenon during pregnancy may enable development of immunotherapy to induce natural remission of GD. As the appearance of blocking antibodies may be important to fetal development (29, 30), future studies should consider whether this is of potential clinical concern in pregnant women with positive TBAb values in the chimeric assay system. Acknowledgments We thank Ms. Karman Yu for typing the manuscript. Received September 14, Accepted April 3, Address all correspondence and requests for reprints to: Dr. Annie W. C. Kung, Department of Medicine, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. awckung@ hkucc.hku.hk. This work was supported by a Committee of Research and Conference Grant from University of Hong Kong. References 1. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 19: Nagayama Y, Wadsworth HL, Chazenbalk GD, Russo D, Seto P, Rapoport B 1991 Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function. 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