Composition

Abstract

The present invention provides a composition comprising the following thyroid stimulating hormone receptor (TSHR) peptides: (i) all or a portion of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1), or a portion thereof, or a sequence having at least 60% sequence identity to SEQ ID NO: 1; and (ii) all or a portion of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2), or a portion thereof, or a sequence having at least 60% sequence identity to SEQ ID NO: 2. The invention also relates to the use of such compositions in the prevention or inhibition of activated autoantibody formation in Graves's disease.

Description

[0001] This application is a divisional application of Chinese application No. 201580076154.1, filed on December 23, 2015, entitled "Composition", the entire contents of which are incorporated herein by reference.

[0002] Invention Field This invention relates to compositions comprising peptides derived from the thyroid-stimulating hormone receptor (TSHR). The compositions or peptides may be useful in preventing and / or inhibiting the production of TSHR autoantibodies, which is useful in treating and / or inhibiting Graves' disease. Background of the Invention Graves' disease is an autoimmune disorder affecting the thyroid gland. It is characterized by an overactive thyroid gland, which leads to the production of excessive thyroid hormones and an enlarged thyroid gland (goiter). The state caused by hyperthyroidism can cause a wide range of neuropsychological and physical symptoms. Graves' disease is the most common cause of hyperthyroidism (60-90% of all cases) and typically manifests itself in middle age, but it can also occur in children, adolescents, and the elderly. It affects up to 2% of the female population and is generally five to ten times more common in women than in men. Graves' disease is also the most common cause of severe hyperthyroidism, accompanied by more clinical signs and symptoms and laboratory abnormalities compared to the milder forms of hyperthyroidism.

[0004] There is a strong genetic component associated with Graves' disease. No recent population studies on Graves' disease exist; however, several quasi-population studies on hyperthyroidism do exist, and therefore all estimates of the incidence and prevalence of Graves' disease are approximate. The incidence of hyperthyroidism ranges from 26:100,000 to 93:100,000, and the overall prevalence is estimated at 1.3%, with 40% of cases being overt and 60% subclinical.

[0005] Approximately 30-50% of people with Graves' disease also have Graves' ophthalmopathy (also known as Graves' orbitopathy or thyroid eye disease) (GO), a bulging condition in one or both eyes. Many cases of GO are mild and self-limiting; however, 20% of cases have significant / moderate to severe disease, at least half of which require steroids. 3-5% of GO patients have painful, vision-threatening disease accompanied by dysthyroid optic neuropathy. Because the eyelids cannot close at night, bulging can lead to severe corneal dryness. Increased optic nerve pressure can cause visual field defects and vision loss. GO may also be associated with pretibial myxedemia.

[0006] The symptoms and signs of Graves' disease almost entirely stem from the direct and indirect effects of hyperthyroidism, with major exceptions including goiter (GO), thyroid enlargement, and pretibial myxedema. Symptoms of hyperthyroidism may include insomnia, hand tremors, hyperactivity, hair loss, excessive sweating, heat intolerance, and weight loss despite an increased appetite. Other common signs include diffusely enlarged (usually symmetrical) non-tender thyroid, lid lag, excessive tearing due to GO, cardiac arrhythmias, and hypertension. Patients with thyrotoxicosis may experience behavioral and personality changes such as psychosis, irritability, and depression. In milder cases of hyperthyroidism, patients may experience less pronounced symptoms such as anxiety, agitation, restlessness, and mood instability.

[0007] Currently, there is no cure for Graves' disease, and therefore current treatments target the symptoms. There are three main treatment options for Graves' disease: oral antithyroid drugs (ATD), radioactive iodine (RAI), and thyroidectomy. The latter two methods result in lifelong thyroid hormone replacement therapy. RAI is the most common treatment in the United States, while ATD is a first-line treatment in Europe, Japan, and much of the rest of the world.

[0008] ATD therapy is associated with some rare side effects and has a 50-60% remission rate. There is a growing recognition that RAIs can contribute to or worsen active GO, and the number of patients treated with ATDs is increasing in the United States.

[0009] Because each treatment option yields varying degrees of success, patients often undergo more than one treatment if the first attempt does not prove entirely successful. The risk of relapse or subsequent hypothyroidism is significant, and the general efficacy of available treatments for Graves' disease is less than expected.

[0010] The development of alternative therapies for Graves' disease is limited by the lack of relevant models (especially animal models) for evaluating the potential efficacy of candidate treatments. Known BALB / c mouse models of Graves' disease have not yet been tested for efficacy with approved Graves' disease drugs such as methimazole and methylprednisolone.

[0011] Therefore, alternative therapies are needed for the treatment and prevention of diseases associated with the production of TSHR autoantibodies, such as Graves' disease. Similarly, alternative therapies that effectively treat Graves' disease and alleviate or reduce its symptoms are needed. Alternative models are also required to evaluate the potential efficacy of candidate Graves' disease therapies. Invention Overview The inventors have discovered that a "cocktail" of two TSHR peptides is particularly effective in inhibiting or preventing the production of TSHR autoantibodies in the body and in the treatment of Graves' disease.

[0013] Therefore, in a first aspect, the present invention provides a composition comprising the following TSHR peptide: (i) All or part of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO:1), or a sequence having at least 60% sequence identity with SEQ ID NO:1; and (ii) All or part of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO:2), or a sequence having at least 60% sequence identity with SEQ ID NO:2.

[0014] In this paper, KKKKYVSIDVTLQQLESHKKK (SEQ ID NO:1) is also referred to as RNB-5D-K1. In this paper, GLKMFPDLTKVYSTD (SEQ ID NO:2) is also referred to as RNB-9B.

[0015] The compositions according to the invention can be used in the therapeutic aspects of the invention as described herein.

[0016] In a second aspect, the present invention provides the use of the peptides of the present invention as described herein in vivo in inhibiting or preventing the production of TSHR autoantibodies.

[0017] In a third aspect, the present invention provides the use of the peptides of the present invention as described herein in the treatment and / or prevention of Graves' disease in subjects.

[0018] In a fourth aspect, the present invention provides the use of the peptides of the present invention as described herein in the preparation of medicaments for inhibiting or preventing the production of TSHR autoantibodies in vivo.

[0019] In a fifth aspect, the present invention relates to the use of the peptides of the invention as described herein in the preparation of medicaments for the treatment and / or prevention of Graves' disease.

[0020] In a sixth aspect, the present invention relates to a method for inhibiting or preventing the production of TSHR autoantibodies in a subject, comprising the steps of administering to the subject all or part of the peptide of SEQ ID NO:1 or a peptide having at least 60% sequence identity with it, and all or part of the peptide of SEQ ID NO:2 having at least 60% sequence identity with it.

[0021] In a seventh aspect, the present invention relates to a method of treating Graves' disease in a subject, comprising the steps of administering to the subject all or part of the peptide of SEQ ID NO:1 or a peptide having at least 60% sequence identity with it, and all or part of the peptide of SEQ ID NO:2 having at least 60% sequence identity with it.

[0022] On the one hand, the composition does not contain or contains the following peptides: RNB-4K-GKK: KKGNLPNISRIYVSIDVTGKK The peptide compositions according to the invention may comprise the amino acid sequence as described herein. Alternatively, the peptide compositions may comprise only the amino acid sequence as described herein.

[0023] The object can be HLA-DR3. The object can be HLA-DR4.

[0024] The peptides of the present invention as defined herein, or the compositions of the present invention, may be administered according to a dose-escalation regimen.

[0025] In an eighth aspect, the present invention relates to a kit comprising the following TSHR peptides: (i) All or part of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO:1), or a sequence having at least 60% sequence identity with SEQ ID NO:1; and (ii) All or part of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO:2), or a sequence having at least 60% sequence identity with SEQ ID NO:2; Used for simultaneous, separate, or sequential application.

[0026] This kit can be used to treat or prevent conditions involving the production of TSHR autoantibodies, such as Graves' disease.

[0027] In a ninth aspect of the invention, the peptide or composition of the invention is combined with another therapeutic agent for treating, preventing, or managing Graves' disease. For example, the peptide or composition may be combined with an antithyroid drug or a beta-blocker.

[0028] In a ninth aspect, the present invention relates to an animal model for a disease associated with the production of TSHR antibodies, wherein the animal is transgenic for human HLA-DR3 and wherein, relative to a suitable control animal, the level of TSHR in the animal is elevated. In one aspect, the level of TSHR is elevated by administration of a viral vector containing a nucleic acid molecule encoding a TSHR peptide.

[0029] Diseases associated with the production of TSHR antibodies include Graves' disease.

[0030] TSHR can be human TSHR. For example, TSHR can be human TSHR A subunit or extracellular domain (ECD).

[0031] The viral vector can be an adenovirus vector or an adenovirus construct.

[0032] Adenovirus vectors or constructs can be administered at three-week intervals. Adenovirus vectors or constructs can be administered in two or three consecutive doses.

[0033] Adenovirus vectors or constructs are administered at doses of 10⁹ to 10¹¹ viral particles. Adenovirus vectors or constructs can be administered via intramuscular injection.

[0034] The animal is likely a mouse. The mouse could be an HLA-BRD1*0301 transgenic mouse. Brief description of the attached diagram Figure 1 --5D-K1 treatment reduced TSHR-induced proliferation in DR3tg mice DR3tg mice (n = 10 / group) were pretreated with 5D-K1 (GLS) or control (HLA-DR3-binding peptide; GLS) according to a dose escalation schedule with a top dose of 100 μg. Mice were immunized with 50 μg 5D (GLS) in CFA and lymph nodes and spleens were harvested 10 days later to assess TSHR-specific proliferation. Data are presented as mean ± standard error of mean (SEM) of stimulation index (SI) values ​​for control-treated mice (red line) and peptide-treated mice (blue line). Two-way ANOVA was used to measure the overall effect of treatment on T cell proliferation and p-values ​​are shown in the figure. Bonferonni post-hoc tests were used and significant differences were shown in the figure (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The mean percentage reduction in T cell proliferation induced by peptide treatment is shown in the figure. SI, stimulation index; LN; lymph node.

[0036] Figure 2 --9B-N treatment reduced TSHR-induced proliferation in DR3tg mice DR3tg mice (n = 10 / group) were pretreated with 9B-N (PPL) or PBS according to a dose-escalation schedule with a top dose of 33 μg. Animals were immunized with 50 μg 9B (GLS) in CFA and, 10 days later, lymph nodes and spleens were harvested to assess TSHR-specific proliferation. Data are presented as mean ± standard error (SEM) of the stimulation index (SI) values ​​for control-treated mice (red line) and peptide-treated mice (blue line). Two-way ANOVA was used to measure the overall effect of treatment on T cell proliferation, and p-values ​​are plotted in the figure. Bonferonni post-hoc tests were used, and significant differences were shown in the figure (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The mean percentage reduction in T cell proliferation induced by peptide treatment is shown in the figure. SI, stimulation index; LN; lymph node.

[0037] Figure 3 --ATX-GD-59 treatment reduced TSHR-induced proliferation in DR3tg mice DR3tg mice (n = 10 / group) were pretreated with either ATX-GD-59 (PPL) or HIP-15F-GKK control peptide (GLS) according to a dose-escalation schedule with a top dose of 15 nmol for each peptide. Animals were immunized with 30 nmol of each parental peptide 5D (Severn) and 9B (PPL) in CFA, and LNs and spleens were harvested 10 days later to assess TSHR-specific proliferation. Data are presented as mean ± standard error (SEM) of the stimulation index (SI) values ​​for control-treated mice (red line) and peptide-treated mice (blue line). Two-way ANOVA was used to measure the overall effect of treatment on T cell proliferation, and p-values ​​are plotted in the graph. Bonferonni post-hoc tests were used, and significant differences are shown in the graph (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The mean percentage reduction in T cell proliferation induced by peptide treatment is shown in the graph. SI, Stimulation Index; LN, Lymph Node. Data represent two independent experiments.

[0038] Figure 4 --ATX-GD-59 treatment reduced TSHR-specific splenocyte proliferation in Ad-TSHR-immunized DR3tg mice. DR3tg mice (n=11 / group) were subcutaneously injected into the lateral region on days -15, -13, and -11 with 22.5 pmol, 225 pmol, and 2250 pmol ATX-GD-59 or control, followed by three injections of 22.5 nmol / ATX-GD-59 peptide or control on days -8, -6, and -4 (dose escalation schedule). Mice were then intramuscularly injected with 10¹⁰ Ad-TSHR or Ad-LacZ at two times (day 0 and day 21) at three-week intervals. The experiment was terminated 5 weeks after the first immunization, and spleens were harvested to assess TSHR-specific proliferation. Data are presented as mean ± standard error (SEM) of absolute counts per minute (cpm; left panel) or stimulation index (SI; right panel) values ​​for control mice (red line) and peptide-treated mice (blue). Two-way ANOVA was used to measure the overall effect of treatment on T cell proliferation; p-values ​​are shown in the figures. Bonferonni's post-hoc test was used, and significant differences are shown in the graphs (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). The overall mean percentage reduction in T cell proliferation induced by peptide treatment is shown in the graphs. Data represent two independent experiments.

[0039] Figure 5 --Prophylactic ATX-GD-59 treatment reduced anti-TSHR antibody levels. DR3tg mice were subcutaneously injected into the lateral region on days -15, -13, and -11 with 22.5 pmol, 225 pmol, and 2250 pmol ATX-GD-59 (n=11) or a control treatment (n=11), followed by three injections of 22.5 nmol of each peptide in the ATX-GD-59 peptide or the control treatment on days -8, -6, and -4 (dose escalation schedule). Mice were then intramuscularly injected with 1010 Ad-TSHR or Ad-LacZ (n=7) at two three-week intervals (day 0 and day 21). Blood was collected before treatment and at 2 and 5 weeks before and after the first immunization to measure total anti-TSHR IgG levels by ELISA. Each point represents data from one mouse and indicates group mean ± SEM. One-way ANOVA was used to measure overall variability in anti-TSHR IgG levels. Bonferonni's post-hoc test was used, and significant differences are shown in the graphs (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Data represent two independent experiments.

[0040] Figure 6 --Prophylactic ATX-GD-59 treatment reduced the levels of different anti-TSHR antibody isotypes. DR3tg mice were subcutaneously injected into the lateral region on days -15, -13, and -11 with 22.5 pmol, 225 pmol, and 2250 pmol ATX-GD-59 (n=11) or a control treatment (n=11), followed by three injections of 22.5 nmol of each peptide in the ATX-GD-59 peptide or the control treatment on days -8, -6, and -4 (dose escalation schedule). Mice were then intramuscularly injected with 1010 Ad-TSHR or Ad-LacZ (n=7) at two three-week intervals (days 0 and 3). Blood was collected before treatment and at weeks 2 and 5 after the first immunization; anti-TSHR isotype IgG levels were measured by ELISA at week 2. Each point represents data from one mouse and indicates mean ± SEM for each group. One-way ANOVA was used to measure overall variability in anti-TSHR IgG levels. Bonferonni's post-hoc test was used, and significant differences are shown in the graphs (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Data represent two independent experiments.

[0041] Figure 7 --Incidence of stimulating anti-TSHR antibodies in DR3tg mice immunized with Ad-TSHR DR3tg mice were subcutaneously injected into the lateral region on days -15, -13, and -11 with 22.5 pmol, 225 pmol, and 2250 pmol ATX-GD-59 (n=11) or a control treatment (n=11), followed by three injections of each peptide in the 22.5 nmol / ATX-GD-59 peptide or the control treatment on days -8, -6, and -4 (dose escalation schedule). Mice were then intramuscularly injected with 1010 Ad-TSHR or Ad-LacZ (n=7) at two three-week intervals (days 0 and 3). Serum was collected 5 weeks after the first immunization and analyzed in a CHO cell assay. Each point represents data from one mouse at week 5 and indicates mean ± SEM for each group. Differences in stimulating TSHR antibody levels were measured using the Mann-Whitney test, and significant differences are shown in the graphs (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Results represent more than 3 independent experiments. RLU, relative light units (light emission from stimulated cells / light emission from unstimulated cells).

[0042] Figure 8 --Illustrative overview of studies on the co-medication of MMI and ATX-GD-459 Mice were subcutaneously injected into the lateral parietal region on days -15, -13, and -11 with 15 pmol, 150 pmol, and 1500 pmol of each ATX-GD-59 peptide or control peptide, followed by three injections of 15 nmol of each ATX-GD-59 peptide or control peptide on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated with either a carrier or methimazole via a subcutaneously implanted osmotic pump. Mice were then intramuscularly injected with 109Ad-TSHR particles at two three-week intervals (week 0 and week 3). Blood was collected two weeks prior to treatment and immediately thereafter, and then at weeks 2, 4, and 5 following the first immunization. The experiment was terminated five weeks after the first immunization to obtain blood, thyroid, and spleen samples.

[0043] Figure 9 --Illustrative overview of studies on the combination therapy of propranolol and ATX-GD-459 Mice were subcutaneously injected into the lateral region of the body at 15 pmol, 150 pmol, and 1500 pmol of each ATX-GD-59 peptide or control peptide on days -15, -13, and -11, followed by three injections of 15 nmol of each ATX-GD-59 peptide or control peptide on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated with either the carrier or propranolol via daily intraperitoneal injection. Mice were then intramuscularly injected with 1010Ad-TSHR particles at two three-week intervals (week 0 and week 3). Blood was collected two weeks prior to treatment and immediately thereafter, and then at weeks 2, 4, and 5 following the first immunization. The experiment was terminated five weeks after the first immunization to obtain blood, thyroid, and spleen samples.

[0044] Figure 10 --TSHR-specific splenocyte proliferation in Ad-TSHR immunized DR3tg mice DR3tg mice (n = 7–10 / group) were subcutaneously injected into the lateral region on days -15, -13, and -11 with 15 pmol, 150 pmol, and 1500 pmol of ATX-GD-59 or a control treatment, followed by three injections of 15 nmol of ATX-GD-59 or a control treatment on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated with a carrier (solid symbol) or MMI (hollow symbol) via a subcutaneously implanted osmotic pump. Mice were then intramuscularly injected with 109Ad-TSHR particles at two three-week intervals (day 0 and day 3). The experiment was terminated 5 weeks after the first immunization, and spleens were harvested to assess TSHR-specific proliferation. Data represent the mean ± standard error of the mean (SEM) of absolute counts per minute (cpm; left panel), stimulation index value (SI; middle panel), or corrected count (Δcpm; right panel). Two-way ANOVA was used to measure the overall effect of treatment on T cell proliferation, and Bonferonni's post-hoc test was employed. Significance differences are described in the text.

[0045] Figure 11 --ATX-GD-459 treatment instead of MMI treatment reduces anti-TSHR IgG antibody levels DR3tg mice (n = 7–10 / group) were subcutaneously injected into the lateral region at days -15, -13, and -11 with 15 pmol, 150 pmol, and 1500 pmol of each ATX-GD-459 peptide or control peptide, followed by three injections of 15 nmol of each ATX-GD-459 peptide or control peptide at days -8, -6, and -4 (dose escalation schedule). Starting at day -15, mice also received either a carrier or MMI via a subcutaneously implanted osmotic pump. Mice were intramuscularly injected with Ad-TSHR at two three-week intervals (week 0 and week 3). The experiment was terminated 5 weeks after the first immunization. Blood samples were collected before treatment and at weeks 2, 4, and 5 before and after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse and indicates mean ± SEM for each group. One-way ANOVA was used to measure the overall variability in anti-TSHR IgG levels. Bonferroni post-hoc tests were used and statistical significance (if any) is shown in the graphs (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0046] Figure 12 --ATX-GD-459 treatment instead of MMI treatment reduces anti-TSHR IgG2b and IgG2c antibody levels DR3tg mice (n = 7–10 / group) were subcutaneously injected into the lateral region on days -15, -13, and -11 with 15 pmol, 150 pmol, and 1500 pmol of each ATX-GD-459 peptide or control peptide, followed by three injections of 15 nmol of each ATX-GD-459 peptide or control peptide on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated with a carrier or MMI via a subcutaneously implanted osmotic pump. Mice were then intramuscularly injected with Ad-TSHR at two three-week intervals (week 0 and week 3). The experiment was terminated 5 weeks after the first immunization. Blood samples were collected before treatment and at weeks 2, 4, and 5 before and after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse measured at week 2, and mean ± SEM is shown for each group. One-way ANOVA was used to measure the overall differences in anti-TSHR IgG1, IgG2b, and IgG2c levels. Bonferroni post-hoc tests were used and statistical significance is shown in the figures (if any) (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0047] Figure 13--MMI treatment significantly reduced serum T4 levels DR3tg mice (n = 7–10 / group) were subcutaneously injected with 15 pmol, 150 pmol, and 1500 pmol of each ATX-GD-459 peptide or control peptide into the lateral region on days -15, -13, and -11, followed by three injections of 15 nmol of each ATX-GD-459 peptide or control peptide on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated with a carrier or MMI via a subcutaneously implanted osmotic pump. Mice were then intramuscularly injected with Ad-TSHR at two three-week intervals (week 0 and week 3). The experiment was terminated 5 weeks after the first immunization. Blood samples were collected before treatment and at weeks 2, 4, and 5 before and after the first immunization, and T4 levels were analyzed by ELISA. Each point represents data from one mouse measured at week 5, and mean ± SEM is shown for each group. Overall differences in T4 levels were measured using one-way ANOVA. Bonferroni post-hoc tests were used and statistical significance (if any) is shown in the graphs (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0048] Figure 14 --TSHR-specific splenocyte proliferation in Ad-TSHR immunized DR3tg mice DR3tg mice (n=10 / group) were subcutaneously injected with 15 pmol, 150 pmol, and 1500 pmol ATX-GD-459 peptide or control peptide on days -15, -13, and -11, followed by three injections of 15 nmol ATX-GD-459 or control peptide on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated with daily intraperitoneal injections of the receiving carrier (solid symbol) or propranolol (hollow symbol). Mice were then intramuscularly injected with 1010Ad-TSHR twice at three-week intervals (days 0 and 3). The experiment was terminated 5 weeks after the first immunization, and spleens were harvested to assess TSHR-specific proliferation. Data represent the mean ± standard error of the mean (SEM) of absolute counts per minute (cpm; left panel), stimulation index value (SI; middle panel), or corrected count (Δcpm; right panel). Two-way ANOVA was used to measure the overall effect of treatment on T cell proliferation, and Bonferonni's post-hoc test was employed. Significance differences are described in the text.

[0049] Figure 15 Treatment with ATX-GD-459 instead of propranolol reduced anti-TSHR IgG antibody levels. DR3tg mice (n=10 / group) were subcutaneously injected into the lateral region on days -15, -13, and -11 with 15 pmol, 150 pmol, and 1500 pmol ATX-GD-459 or control treatment, followed by three injections of 15 nmol ATX-GD-459 or control treatment on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated daily with intraperitoneal injections of the receiving carrier (solid symbol) or propranolol (hollow symbol). Mice were then intramuscularly injected with 1010Ad-TSHR at two three-week intervals (day 0 and day 3). The experiment was terminated 5 weeks after the first immunization. Blood samples were collected before treatment and at weeks 2, 4, and 5 before and after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse and the mean ± SEM for each group is shown. One-way ANOVA was used to measure overall differences in anti-TSHR IgG levels. Bonferroni post-hoc tests were used and statistical significance is shown in the figures (if any) (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0050] Figure 16 --ATX-GD-459 treatment instead of propranolol reduced anti-TSHR IgG2b and IgG2c antibody levels. DR3tg mice (n=10 / group) were subcutaneously injected into the lateral region on days -15, -13, and -11 with 15 pmol, 150 pmol, and 1500 pmol ATX-GD-459 or control treatment, followed by three injections of 15 nmol ATX-GD-459 or control treatment on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated with daily intraperitoneal injections of the receiving carrier (solid symbol) or propranolol (hollow symbol). Then, mice were intramuscularly injected with 1010Ad-TSHR at two three-week intervals (day 0 and day 3). The experiment was terminated 5 weeks after the first immunization. Blood was collected before treatment and at weeks 2, 4, and 5 before and after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse and the mean ± SEM for each group is shown. One-way ANOVA was used to measure overall differences in anti-TSHR IgG1, IgG2b, and IgG2c levels. Bonferroni post-hoc tests were used and statistical significance is shown in the figures (if any) (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0051] Figure 17--Total T4 levels are unaffected by treatment with propranolol or ATX-GD-459. DR3tg mice (n=10 / group) were subcutaneously injected into the lateral region on days -15, -13, and -11 with 15 pmol, 150 pmol, and 1500 pmol ATX-GD-459 or a control treatment, followed by three injections of 15 nmol ATX-GD-459 or a control treatment on days -8, -6, and -4 (dose escalation schedule). Starting on day -15, mice were also treated daily with intraperitoneal injections of either the recipient carrier (solid symbol) or propranolol (hollow symbol). Mice were then intramuscularly injected twice with 1010Ad-TSHR at two three-week intervals (day 0 and day 3). The experiment was terminated 5 weeks after the first immunization. Blood samples were collected before treatment and at weeks 2, 4, and 5 before and after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse measured at week 5, and mean ± SEM is shown for each group. One-way ANOVA was used to measure the overall difference at the T4 level. Bonferroni post-hoc tests were used and statistical significance was shown in the figures (if any) (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0052] Figure 18 --Plasma levels of propranolol determine the pharmacological dosage Plasma propranolol levels were determined in control mice and mice that had been administered peptide and propranolol. Plasma levels were tested at weeks 0 and 5, and the results are shown.

[0053] Figure 19 Indicative T-cell tolerance regimen Mice were subcutaneously injected into the lateral region of the body at days -15, -13, and -11 with 0.1 μg, 1 μg, and 10 μg of peptide, followed by three injections of the top dose of peptide (ATX-GD-459 single peptide or mixture) at days -8, -6, and -4 (dose escalation schedule). The top dose and corresponding dose escalation doses may vary between experiments. On day 0, mice were subcutaneously immunized at the base of the tail with a peptide emulsified in complete Freund's adjuvant (CFA). The experiment was terminated 10 days post-immunization to determine the proliferation of lymph node (LN) cells and spleen cells after TSHR or peptide restimulation.

[0054] Figure 20 --ATX-GD-459 treatment effectively reduced TSHR-induced proliferation in HLA-DR3 mice. (A) HLA-DR4 mice (n = 10 / group) were pretreated with RNB-4K-GKK or PBS according to a dose-escalation schedule with a top dose of 100 μg; HLA-DR3 mice (n = 10 / group) were pretreated with RNB-5D-K1 or HIP-16E (HLA-DR3 binding control peptide) according to a dose-escalation schedule with a top dose of 100 μg; (C) HLA-DR3 mice (n = 10 / group) were pretreated with RNB-9B or PBS according to a dose-escalation schedule with a top dose of 33 μg. Animals were immunized with the parental peptide in CFA, and LN and spleen were harvested 10 days later to assess TSHR-specific proliferation. Data are presented as mean ± standard error (SEM) of the stimulation index (SI) values ​​for control-treated and peptide-treated mice. The overall treatment effect on T cell proliferation was determined using a two-way ANOVA, and p-values ​​are shown in the figure. Bonferonni post-hoc test was used and statistical significance is shown in the figures (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). The overall mean percentage reduction in T cell proliferation induced by peptide treatment is shown in the figures. SI, stimulation index; LN; lymph nodes. (D) HLA-DR3 mice (n=10 / group) were pretreated with ATX-GD-459 or PBS according to a dose escalation schedule of 75 μg top dose per peptide. Animals were immunized with 4K / 5D / 9B emulsified in CFA and LN and spleen were harvested 10 days later to assess TSHR-specific proliferation. Data represent the mean ± SEM of stimulation index (SI) values ​​for control-treated and peptide-treated mice. The overall effect of treatment on T cell proliferation was determined using two-way ANOVA and p-values ​​are shown in the figures. Bonferonni post-hoc test was used and statistical significance is shown in the figures (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). The overall mean percentage decrease in T cell proliferation induced by peptide treatment is shown in the figures. SI, stimulation index; LN; lymph node.

[0055] Figure 21 --A schematic overview of adenoviral Graves' disease models and validation (A) Mice were immunized intramuscularly with an adenovirus vector expressing the TSHR-A subunit (Ad-TSHR) or β-galactosidase (Ad-LacZ). 10¹⁰ adenovirus particles were injected twice at three-week intervals. Each experiment indicated deviation from the protocol. Blood was collected 2 and 4 weeks before and after the first immunization. The experiment was terminated 4 weeks after the first immunization, and blood, spleen, and thyroid tissue were collected for Graves' disease-like symptom studies. (B) Mice were immunized with Ad-TSHR at two three-week intervals (week 0 and week 3). Treatment began via subcutaneous pump insertion on the day of the first immunization and continued for 4 weeks until the end of the experiment. Blood was collected before and 2 and 4 weeks after the first immunization. Mice were euthanized 4 weeks after the first immunization to obtain blood and spleen samples. (C) Mice were subcutaneously injected into the lateral parietal region with 0.1 μg, 1 μg, and 10 μg peptides on days -15, -13, and -11, followed by three injections of 100 μg peptides on days -8, -6, and -4 (dose escalation schedule). Mice were then intramuscularly injected with Ad-TSHR or Ad-LacZ at two three-week intervals (week 0 and week 3). Blood was collected before treatment and before the first immunization, and at weeks 2 and 5 after the first immunization. Mice were euthanized 5 weeks after the first immunization to obtain blood and spleen samples.

[0056] Figure 22 --Evolution of T4 and anti-TSHR IgG levels over time in BALB / c mice immunized with Ad-TSHR BALB / c mice (n = 6–7 / group) were immunized intramuscularly with 10¹⁰ or 10¹¹ adenovirus vectors expressing the TSHR-A subunit (Ad-TSHR) or β-galactosidase (Ad-LacZ). The 10¹⁰ or 10¹¹ adenovirus particles were injected three times at three-week intervals (weeks 0, 3, and 6). Serum was collected before the first immunization and at 4 and 10 weeks after the first immunization. (A) T4 levels were analyzed by ELISA. Each point represents data from one mouse, and mean ± SEM is shown for each group. The number of hyperthyroid mice is shown in the figure. Consistent with published data, the cutoff for hyperthyroidism was defined as mean ± 2 standard deviations of T4 levels in normal-range mice among Ad-LacZ-immunized control mice. (B) Anti-TSHR IgG levels were analyzed by ELSA. Each point represents data from one mouse, and mean ± SEM is shown for each group. Overall variability in anti-TSHR IgG levels was measured using one-way ANOVA. Bonferroni post-hoc test was used, and the differences were indicated in the figure (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0057] Figure 23 --Evolution of T4, stimulating TSHR antibody, and anti-TSHR antibody titers over time in DR3tg mice immunized with Ad-TSHR (A) DR3tg mice were immunized intramuscularly with 109 adenovirus vectors expressing either the TSHR-A subunit (Ad-TSHR) (n=10) or β-galactosidase (Ad-LacZ) (n=12). Adenovirus particles were injected twice at three-week intervals (week 0 and week 3). Serum was collected before the first immunization and at 2 and 4 weeks after the first immunization, and T4 levels were analyzed by ELISA. Each point represents data from one mouse at week 4, and mean ± SEM is shown for each group. Differences in T4 levels were measured using the Mann-Whitney test, and statistically significant differences are indicated in the figure (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Results represent more than 3 independent experiments. (B) DR3tg mice were immunized intramuscularly with 109 adenovirus vectors expressing either the TSHR-A subunit (Ad-TSHR) or β-galactosidase (Ad-LacZ) (n = 10 / group). Adenovirus particles were injected twice at two three-week intervals (week 0 and week 3). Serum was collected before the first immunization and at 2 and 4 weeks after the first immunization, and T4 levels were analyzed by CHO luciferase reporter assay. Each point represents data from one mouse at week 4, and mean ± SEM is shown for each group. Differences in stimulating TSHR antibody levels were measured using the Mann-Whitney test, and statistical significance was indicated in the figure (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Results represent more than 3 independent experiments. RLU, relative light units (light emission from stimulated cells / light emission from unstimulated cells). (C) BALB / c mice (n=10; left panel) and DR3tg mice (n=7; right panel) were immunized intramuscularly with 10¹⁰ adenovirus vectors expressing the TSHR-A subunit (Ad-TSHR). Adenovirus particles were injected three times at three-week intervals (weeks 0, 3, and 6). Serum was collected before the first immunization and at 4, 7, and 10 weeks after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse, and mean ± SEM is shown for each group. One-way ANOVA was used to measure overall differences in anti-TSHR IgG levels. Bonferroni post-hoc tests were used, and statistical significance was indicated in the figures (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0058] (D)DR3tg mice were immunized intramuscularly with either 10¹⁰ or 10¹¹ adenovirus vectors expressing the TSHR-A subunit (Ad-TSHR) or 10¹⁰ adenovirus vectors expressing β-galactosidase (Ad-LacZ). Adenovirus particles were injected three times at three-week intervals (weeks 0, 3, and 6). After 10 weeks, spleens were collected, and TSHR-induced splenocyte proliferation was measured by incorporation of tritylthymidine. Results are shown as mean ± SEM per group. Cpm, counts per minute; SI, stimulation index.

[0059] Figure 24 --Effects of methimazole treatment on T4 and anti-TSHR levels in DR3tg mice immunized with Ad-TSHR DR3tg mice (n = 7-8 / group) were immunized with Ad-TSHR at weeks 0 and 3. Treatment with methimazole (50 or 500 μg / mouse / day) or the carrier was initiated via subcutaneous pump insertion on the day of the first immunization and continued for 4 weeks until the end of the experiment. Serum was collected before the first immunization and at 2 and 4 weeks after the first immunization. (A) T4 levels were analyzed by ELISA. Each point represents data from one mouse, and mean ± SEM is shown for each group. One-way ANOVA was used to measure the overall difference in T4 levels. Bonferroni post-hoc test was used, and statistical significance was indicated in the figure (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (B) Anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse, and mean ± SEM is shown for each group. One-way ANOVA was used to measure the overall difference in anti-TSHR IgG levels. Bonferroni post-hoc test was used and the differences were indicated in the figure (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0060] Figure 25 --Treatment with methylprednisolone reduced anti-TSHR IgG levels DR3tg mice (n=10 / group) were immunized with 1010Ad-TSHR at weeks 0 and 3. Treatment with methylprednisolone (Mpred) (7 mg / kg / day) or a carrier was initiated 3 days prior to the first immunization and continued for 4 weeks until the end of the experiment. Serum was collected before the first immunization and at 2 and 4 weeks after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Each point represents data from one mouse, and mean ± SEM is shown for each group. Differences in anti-TSHR IgG levels were measured using the Mann-Whitney test, and statistical significance was indicated in the figure (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0061] Figure 26 --ATX-GD-459 treatment reduced anti-TSHR antibody levels DR3tg mice (n=10 / group) were subcutaneously injected into the lateral region on days -15, -13, and -11 with 0.1 μg, 1 μg, and 10 μg of ATX-GD-459 or the control treatment, followed by three injections of 100 μg of ATX-GD-459 or the control treatment on days -8, -6, and -4 (dose escalation schedule). Mice were then intramuscularly injected with Ad-TSHR or Ad-LacZ at two three-week intervals (days 0 and 3). Blood samples were collected before treatment and at weeks 2 and 5 after the first immunization to measure total anti-TSHR IgG levels by ELISA. Each point represents data for one mouse, and group mean ± SEM is shown. One-way ANOVA was used to measure overall variability in anti-TSHR IgG levels. Bonferroni post-hoc test was used and the differences were indicated in the figure (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0062] Figure 27 --APIPS assay showing responses in hybridoma clones to RNB-5D-K1 and RNB-4K-GKK Five x 10⁴ T-cell hybridoma clones specifically targeting RNB-5 (clones 50+35) or RNB-4 (clones 164+455) and TSHR were cultured in the presence of TSHR and RNB-5D-K1 or RNB-4K-GKK with five x 10⁴ individual DR3-expressing antigen-presenting B cells (VAVY) (clone 50) or with DR4-expressing antigen-presenting B cells (BM14) (clones 35, 164, 455). Antigen-presenting cells (APCs) were either viable or previously fixed with 0.5% paraformaldehyde before being added to the culture wells. After 48 h of co-culture, the supernatant was collected and analyzed by ELISA to assess IL-2 secretion levels. Results from representative clones are presented. Invention Details This invention provides a novel and alternative treatment option for preventing or inhibiting the production of TSHR autoantibodies, which is useful in the treatment and / or prevention of Graves' disease. As demonstrated in Example 1 of this application, the combination of peptides of SEQ ID NO: 1 and 2 results in tolerance to TSHR in a Graves' disease model.

[0064] Therefore, a first aspect of the present invention relates to a composition comprising a plurality of peptides from TSHR, namely the peptides of the present invention as defined herein, preferably peptides of SEQ ID NOs:1 and 2.

[0065] Thyroid-stimulating hormone receptor Graves' disease is an autoimmune disease caused by autoreactive T and B lymphocytes that target the primary autoantigen, the thyroid-stimulating hormone receptor (TSHR).

[0066] TSHR is a G protein-coupled receptor on thyroid follicular cells in the thyroid gland. Upon binding to its ligand (thyroid-stimulating hormone (TSH)), it stimulates the production of thyroxine (T4) and triiodothyronine (T3) via a cAMP signaling cascade. After internalization, degradation, and presentation of TSHR by APCs, T cells are activated and interact with autoreactive B cells, leading to the production of stimulatory autoantibodies against TSHR. Thyroid-stimulating immunoglobulins bind to the same receptor pocket as TSH, thereby activating TSHR-mediated signal transduction and resulting in excessive production of thyroid hormones and thyroid growth.

[0067] TSHR, also known as thyroid-stimulating hormone receptor, is mainly expressed on thyroid epithelial cells.

[0068] The TSHR holoreceptor has 764 residues and includes an N-terminal extracellular domain for TSH binding, a serpentine (or transmembrane domain), and a C-terminal intracellular domain.

[0069] TSHR contains a large extracellular domain (418 amino acids) with highly conserved Cys residues that promote the formation of the extracellular domain's tertiary structure, which is likely important in both ligand-binding and inactive receptor conformations. The extracellular domain comprises more than half the total protein length and is sufficient for high-affinity ligand binding. Upon transport to the cell surface, the receptor molecule undergoes intramolecular cleavage, resulting in the removal of a 50-amino acid sequence between residues 316 and 366. As a result, the receptor contains two subunits: an α subunit containing the extracellular ligand-binding domain and a β subunit containing the transmembrane domain and a short C-terminal sequence, which are bound together by disulfide bonds. In subsequent steps, the α subunit is masked, leading to excessive stripping of the β subunit of the ligand-binding domain from the cell membrane.

[0070] Upon binding of circulating TSH to TSHR, the G protein signaling cascade activates adenylate cyclase and increases intercellular cAMP levels. cAMP activates all functional aspects of thyroid cells, including iodine pumping, thyroglobulin synthesis, iodination, endocytosis and proteolysis, thyroid peroxidase activity, and hormone release.

[0071] The amino acid sequence of mature TSHR is given below (SEQ ID No. 3). Tolerance T-cell epitopes play a central role in adaptive immune responses to any antigen, whether self-generated or exogenous. Their central role in hypersensitivity disorders, including allergic reactions, autoimmune diseases, and transplant rejection, has been demonstrated using experimental models. Inflammatory or allergic diseases may be induced by injecting synthetic peptides (based on the structure of T-cell epitopes) in combination with adjuvants.

[0072] In contrast, the administration of soluble peptide epitopes has been shown to potentially induce immune tolerance against specific antigens. The administration of soluble peptides has been demonstrated as an effective means of suppressing disease in experimental autoimmune encephalomyelitis (EAE–MS) models (Metzler and Wraith (1993) Int. Immunol. 5:1159-1165; Liu and Wraith (1995) Int. Immunol. 7:1255-1263; Anderton and Wraith (1998) Eur. J. Immunol. 28:1251-1261) and experimental models of arthritis, diabetes, and uveoretinitis (reviewed in Anderton and Wraith (1998) above). This has also been shown as a means of treating disease progression in EAE (Anderton and Wraith (1998) above).

[0073] Tolerance is the failure of the immune system to respond to an antigen. Tolerance to self-antigens is a fundamental characteristic of the immune system; its loss can lead to autoimmune diseases. The adaptive immune system must maintain the ability to respond to a large number of infectious agents while avoiding autoimmune attacks from self-antigens contained within its own tissues. This is largely controlled by the sensitivity of immature T lymphocytes in the thymus to apoptotic cell death (central tolerance). However, not all self-antigens are detected in the thymus, so the death of self-reactive thymocytes is not yet complete. Therefore, there are also mechanisms by which tolerance can be acquired through mature self-reactive T lymphocytes in peripheral tissues (peripheral tolerance). A review of the mechanisms of central and peripheral tolerance is given in Anderton et al. (1999) (Immunological Reviews 169:123-137).

[0074] Graves' disease is currently believed to be caused by TSHR-stimulating autoantibodies, which bind to and activate TSHR, thereby stimulating thyroid hormone synthesis and secretion, as well as thyroid growth. The compositions of the present invention can induce tolerance to TSHR, such that when administered to subjects, they can regain tolerance to TSHR autoproteins and reduce pathogenic immune responses.

[0075] APITOPES In the adaptive immune response, T lymphocytes can recognize internal epitopes of protein antigens. APCs take up protein antigens and degrade them into short peptide fragments. Peptides can bind to major histocompatibility molecules within the cell and be carried to the cell surface. When presented on the cell surface by binding to MHC molecules, peptides can be recognized by T cells (via T cell receptors (TCRs)), in which case the peptide is a T cell epitope.

[0076] Therefore, an epitope is a peptide that can be derived from an antigen and is able to bind to the peptide-binding groove of an MHC molecule and be recognized by T cells.

[0077] A minimal epitope is the shortest fragment of an epitope that can bind to the peptide-binding groove of an MHC molecule and be recognized by T cells. For a given immunogenic region, it is typically likely to produce a “nested set” of overlapping peptides that act as epitopes, with all epitopes containing a minimal epitope but differing in their flanking regions.

[0078] Similarly, it may be possible to identify minimal epitopes targeting specific MHC molecules:T cell combinations by measuring responses to truncated peptides. For example, if a response is obtained to a peptide containing residues 1-15 in an overlapping library, minimal epitopes can be identified using truncated sets (i.e., 1-14, 1-13, 1-12, etc., and 2-15, 3-15, 4-15, etc.).

[0079] The inventors have previously established a link between the ability of a peptide to bind to an MHC molecule and be presented to T cells without further processing and the peptide's ability to induce tolerance in vivo (WO 02 / 16410). If a peptide is too long to bind to the peptide-binding groove of an MHC molecule without further processing (e.g., trimming), or binds in an inappropriate conformation, it will not be tolerogenic in vivo. On the other hand, if a peptide has an appropriate size and conformation to bind directly to the MHC peptide-binding groove and be presented to T cells, the peptide can be predicted to be useful for inducing tolerance.

[0080] Therefore, it is possible to study the tolerogenic capacity of peptides by investigating whether they can bind to MHC molecules and be presented to T cells without further in vitro antigen processing.

[0081] TSHR apitopes (antigen-independent epiTOPEs) can bind to MHC molecules and stimulate responses from TSHR-specific T cells without further antigen processing. According to the rule-based approach described in WO 02 / 16410, the resulting tolerance to TSHR can be predicted.

[0082] Peptides that bind to MHC class I molecules are typically 7 to 13 amino acids long, but more commonly 8 to 10. The binding of a peptide is stable at its two ends through interatomic contact between atoms in the peptide backbone and atoms at invariant sites in the peptide-binding groove of all MHC class I molecules. Invariant sites exist at both ends of the groove at the amino and carboxyl terms of the binding peptide. Variations in peptide length are accommodated by kinking in the peptide backbone, often at flexible proline or glycine residues.

[0083] Peptides that bind to MHC class II molecules are typically between 8 and 20 amino acids in length, more commonly between 10 and 17 amino acids, and can be longer (e.g., up to 40 amino acids). These peptides reside within a conformation extending along the MHC class II peptide-binding groove, which (unlike the MHC class I peptide-binding groove) is open at both ends. The peptide is held in place primarily by contact with the backbone atoms of conserved residues that line the peptide-binding groove.

[0084] In a preferred embodiment, the peptide is able to bind to MHC class II molecules without further processing.

[0085] peptides The term "peptide" in its general sense refers to a series of residues (usually L-amino acids) that are typically linked together by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The term includes modified peptides and synthetic peptide analogs.

[0086] Peptides can be prepared using chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin). For example, peptides can be synthesized using solid-phase techniques (Roberge JY et al. (1995) Science 269:202-204), cleaved from resin, and purified by preparative high-performance liquid chromatography (e.g., Creighton (1983) Proteins Structures and Molecular Principles, WH Freeman and Co., New York, NY). Automated synthesis can also be achieved, for example, using an ABI 43 1A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0087] Peptides can be prepared alternatively through recombinant methods or by cleaving from longer peptides. For example, peptides can be obtained by cleaving from the thyroid-stimulating hormone receptor protein, which may then be modified at one or both ends. The composition of the peptide can be determined by amino acid analysis or sequencing (such as the Edman degradation procedure).

[0088] The peptides used in this invention are as follows: (i) All or part of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO:1), or a sequence having at least 60% sequence identity with SEQ ID NO:1; and (ii) All or part of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO:2), or a sequence having at least 60% sequence identity with SEQ ID NO:2; The peptides according to the invention may include or consist of amino acid sequences having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 70%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the peptides of SEQ ID NOs:1 or 2. In one aspect, the peptide has at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the peptides of SEQ ID NOs:1 or 2.

[0089] In a preferred embodiment, the peptide comprises SEQ ID NO:1 and 2. In a further preferred embodiment, the peptide consists of SEQ ID NO:1 and 2.

[0090] Sequence identity can be assessed using any conventional method. However, computer programs for performing multiple sequence alignments are useful for determining the degree of sequence identity between sequences, such as Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22:4673-4680). Programs for comparing and aligning sequence pairs, such as ALIGN (Myers et al., (1988) CABIOS, 4:1-17), FASTA (Pearson et al., (1988) PNAS, 85:2444-2448; Pearson (1990), Methods Enzymol., 183:63-98), and BLAST (Altschul et al., (1997) Nucleic Acids Res., 25:3389-3402), can also be used for this purpose. In addition, the Dali server of the European Institute of Bioinformatics provides structure-based alignment of protein sequences (Holm (1993) J. Mol. Biol., 233: 123-38; Holm (1995) Trends Biochem. Sci., 20: 478-480; Holm (1998) Nucleic Acid Res., 26: 316-9).

[0091] Using standard BLAST parameters (using sequences from all available organisms, matrix Blosum 62, gap cost: presence 11, extension 1), multiple sequence alignment and percentage identity calculations can be determined.

[0092] Alternatively, you can use the following program and parameters: Align Plus 4, version 4.10 (Sci Ed CentralClone Manager Professional Suite). DNA alignment: global alignment, standard linear scoring matrix, mismatch penalty = 2, open gap penalty = 4, extended gap penalty = 1. Amino acid alignment: global alignment, BLOSUM 62 scoring matrix.

[0093] Therefore, the scope of this invention includes variants of the stated or given sequence, provided that the variant retains the parental functional activity, i.e., the variants are functionally equivalent, in other words, they have or exhibit the activity of the parental peptide as defined herein. Such variants may comprise amino acid substitutions, additions, or deletions (including truncation at one or both ends) of the parental sequence (e.g., one or more, e.g., 1 to 14 amino acids).

[0094] It also includes functionally equivalent derivatives, in which one or more amino acids are chemically derived, for example, by substitution with chemical groups.

[0095] Therefore, the peptides of the present invention may comprise portions or fragments of SEQ ID NO:1-3, provided that the peptide retains the desired activity. Fragments or portions of SEQ ID NO:1-3 may, for example, range in length from 6 to 14 residues, such as 6, 7, 8, 9, 10, 11, 12, or 13 residues.

[0096] The peptides of the present invention may contain between 8 and 30 amino acids, for example, between 8 and 25 amino acids, between 8 and 20 amino acids, between 8 and 15 amino acids, or between 8 and 20 amino acids. Therefore, in one aspect, the peptides of the present invention may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.

[0097] TSHR peptides can be in the form of a composition, preferably in the form of a pharmaceutical composition.

[0098] TSHR peptides can be formulated into neutral or salt compositions. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the peptide), which form with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and maleic acid. Salts formed with the free carboxyl group can also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium, or iron, or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0099] Composition The inventors have surprisingly discovered that the peptides and / or compositions according to the invention can be administered in combination with other therapies for the treatment or management of Graves' disease while maintaining the therapeutic effects of both the peptides / compositions of the invention and other therapeutic agents.

[0100] Therefore, on the one hand, the peptides or compositions of the present invention can be combined with other therapeutic agents for the treatment or prevention of Graves' disease.

[0101] For example, peptides or compositions can be combined with antithyroid agents or beta-blockers.

[0102] As demonstrated in Example 2, administration of the peptides SEQ ID NO:1 and SEQ ID NO:2 in combination with an antithyroid agent does not affect the reduction in anti-TSHR antibody production due to the TSHR peptide, nor does it reduce the antithyroid effect (measured by T4 production); that is, both therapeutic effects are maintained. Similar results have been achieved with combinations of the compositions according to the invention with a β-blocker. Therefore, the inventors have demonstrated that the peptides according to the invention can be used in combination with existing therapies for the treatment and management of Graves' disease.

[0103] The composition according to the invention is advantageous because it promotes a reduction in the autoimmune response (i.e., the underlying mechanism) in Graves' disease, while simultaneously allowing for the treatment or management of the symptoms of Graves' disease.

[0104] "Antithyroid agents" are hormone antagonists that act on thyroid hormones. Antithyroid agents are used to treat Graves' disease. Administering antithyroid agents to patients with Graves' disease achieves their inhibitory effect on thyroid hormone synthesis, thereby directly lowering their thyroxine (T4) levels.

[0105] In one embodiment, the antithyroid agent is selected from carbimazole, methimazole (MMI), propylthiouracil (PTU), and potassium perchlorate. In a preferred embodiment, the antithyroid agent is methimazole (MMI).

[0106] Administering beta-blockers to patients with Graves' disease to combat the beta-adrenergic consequences of hyperthyroidism (without treating the disease's immune compounds).

[0107] Beta-blockers are a class of drugs used to manage cardiac arrhythmias. Beta-blockers block the effects of endogenous catecholamines adrenaline (epinephrine) and noradrenaline (noradrenaline) in the sympathetic nervous system, particularly on adrenergic beta receptors.

[0108] The β-blockers according to the present invention include: propranolol, buspirolol, carteolol, carvedolol, labetalol, naldolol, oxenolol, pentbuprofenolol, pindolol, sotalol, and timolol. In a preferred embodiment, the β-blocker is propranolol.

[0109] A pharmaceutically acceptable salt of a β-blocker can be used. In a preferred embodiment, the β-blocker is propranolol hydrochloride.

[0110] The appropriate dosage of the antithyroid agent and the beta-blocker can be determined by those skilled in the art. For example, the antithyroid agent can be administered at a dose between 10 and 1000 μg, such as between 20 and 900 μg, 30 and 800 μg, 40 and 700 μg, 50 and 600 μg, 60 and 500 μg, 70 and 400 μg, 80 and 300 μg, or 90 and 200 μg. In a preferred embodiment, a dose between 300 and 700 μg is administered, for example, about 500 μg, and preferably 500 μg.

[0111] In a preferred embodiment, the antithyroid agent may be administered daily, for example every 20-28 hours, preferably every 24 hours.

[0112] Antithyroid agents can be administered via any suitable route, as will be known to those skilled in the art. In a preferred embodiment, the antithyroid agent is administered subcutaneously.

[0113] The β-blocker can be administered at doses, for example, between 1 and 100 mg / kg, such as between 5 and 90 mg / kg, between 10 and 80 mg / kg, between 15 and 75 mg / kg, between 20 and 70 mg / kg, between 25 and 60 mg / kg, between 30 and 55 mg / kg, between 35 and 50 mg / kg, and between 40 and 45 mg / kg. In a preferred embodiment, the β-blocker is administered at a dose of 5-20 mg / kg, preferably about 10 mg / kg, more preferably 10 mg / kg.

[0114] In a preferred embodiment, the β-blocker can be administered daily, for example every 20-28 hours, preferably every 24 hours.

[0115] The β-blocker can be administered via any suitable route, as will be known to those skilled in the art. In a preferred embodiment, the β-blocker is administered intraperitoneally.

[0116] In one embodiment, the peptide or composition according to the invention is administered simultaneously with an antithyroid agent and / or a β-blocker. In an alternative embodiment, the peptide or composition according to the invention is administered with the antithyroid agent and / or a β-blocker at different times.

[0117] In one aspect of the invention, the subject receiving (or intending to receive) the peptide / composition has already taken an antithyroid agent and / or a beta-blocker. Therefore, the invention covers uses where the subject is currently or has previously taken an antithyroid agent and / or a beta-blocker.

[0118] In another aspect of the invention, the subject is not taking and / or has not yet taken any treatment for Graves' disease.

[0119] As a first step, one aspect of the invention discussed herein is the identification of subjects who have or are at risk of developing Graves' disease.

[0120] The peptides or compositions according to the present invention may be used for preventive or therapeutic purposes.

[0121] When administered for prophylactic purposes, the peptide or composition may reduce or prevent the production of an immune response to TSHR. The level of the immune response is lower than that obtained in patients who have not been treated with the composition. The term "reduction" refers to a partial reduction in the immune response, such as a reduction of 50%, 70%, 80%, or 90% compared to the response observed in patients who have not been treated with the composition (or the response observed in untreated patients over the same time period). The term "prophylaxis" refers to the absence of a significant immune response against TSHR.

[0122] When administered for therapeutic purposes, the peptide or composition may suppress an ongoing immune response against TSHR. The term "suppression" refers to a reduction in the level of an ongoing immune response compared to pre-treatment levels of the peptide, or compared to levels observed at the same time point before treatment.

[0123] Treatment with the peptides or compositions of the present invention can cause a reduction in any or all of the following levels: i) TSHR autoantibodies ii) CD4+ T cells specifically targeting TSHR iii) B cells that secrete TSHR autoantibodies.

[0124] All factors can be detected using techniques known in the art, such as ELISA and flow cytometry.

[0125] Furthermore, or additionally, treatment with the peptides or compositions of the present invention can induce anergy in CD4+ T cells specific to TSHR. For example, anergy can be detected by subsequent TSHR challenge in vitro.

[0126] Preparations Peptides or compositions can be prepared as injectable solutions or suspensions; they can also be prepared in solid forms suitable for use as solutions or suspensions in liquids prior to injection. Preparations can also be emulsified or peptides encapsulated in liposomes. The active ingredient can be mixed with pharmaceutically acceptable and compatible excipients. Suitable excipients include, for example, water, saline (e.g., phosphate-buffered saline), dextran, glycerol, ethanol, and combinations thereof.

[0127] In addition, the composition may contain small amounts of auxiliary substances such as wetting agents or emulsifiers and / or pH buffers, if desired. Buffer salts include phosphates, citrates, and acetates. Hydrochloric acid and / or sodium hydroxide may be used for pH adjustment. For stabilization, disaccharides such as sucrose or trehalose may be used.

[0128] In the composition, the relative ratio of peptides (RNB-5D-K1 and RNB-9B) can be approximately 1:1. Alternatively, the relative ratio of each peptide can be variable, for example, to focus the tolerogenic response on a specific subset of autoreactive T cells or if one peptide is found to work better than others in a particular HLA type.

[0129] After preparation, the peptide or composition can be incorporated into a sterile container, then sealed and stored at a low temperature (e.g., 4°C), or it can be freeze-dried.

[0130] Conveniently, peptides or compositions can be prepared as lyophilized (freeze-dried) powders. Freeze-drying allows for long-term storage in a stable form. Freeze-drying procedures are known in the art; see, for example, http: / / www.devicelink.com / ivdt / archive / 97 / 01 / 006.html. Fillers commonly used prior to freeze-drying include mannitol, dextran, or glycine, or sugars such as trehalose.

[0131] Peptides or compositions can be administered in convenient ways, such as via oral, intravenous (in the case of water solubility), intramuscular, subcutaneous, sublingual, intranasal, intradermal, or suppository routes, or implantation (e.g., using sustained-release molecules).

[0132] Advantageously, the peptide or composition can be administered via intranasal, subcutaneous or intradermal routes.

[0133] The methods, peptides, and compositions of this invention can be used to treat human subjects. Typically, a physician will determine the most appropriate actual dose for an individual subject, and this will vary depending on the specific patient's age, weight, and response.

[0134] The peptides or compositions of the present invention can be used to treat human subjects. Subjects may have Graves' disease. Subjects may have TSHR autoantibodies.

[0135] Subjects may be of HLA haplotypes associated with the formation of inhibitory THSR autoantibodies. Subjects may express HLA-DR3 or HLA-DR4. Methods for determining an individual's HLA haplotype are known in the art.

[0136] In a preferred embodiment, a dose-escalation protocol can be followed, in which multiple doses are administered to the patient at escalating concentrations. Such a protocol has been used, for example, in the immunotherapy of bee venom allergies to phospholipase A2 peptide (Mülleret et al. (1998) J. Allergy Clin Immunol. 101:747-754 and Akdis et al. (1998) J. Clin. Invest. 102:98-106).

[0137] Reagent test kit Conveniently, the two TSHR peptides can be administered together as a mixed composition or cocktail. However, it may be preferable to provide the peptides separately in the form of a kit for simultaneous, separate, sequential, or combined administration.

[0138] For example, the kit may contain two peptides in separate containers. The components of the containers may or may not be combined before application.

[0139] The kit may also include mixing and / or administration means (e.g., a vaporizer for intranasal administration; or a syringe or needle for subcutaneous / intradermal administration). Instructions for use may also be included.

[0140] The pharmaceutical compositions or kits of the present invention can be used to treat and / or prevent diseases.

[0141] Specifically, the composition / kit can be used to inhibit or prevent the production of TSHR autoantibodies in vivo. The composition / kit can be used to treat and / or prevent Graves' disease in subjects.

[0142] animal models Current animal models of Graves' disease are associated with various drawbacks. For example, currently used animal models do not develop the symptoms or characteristics associated with Graves' disease. Furthermore, available animal models—such as those developed in BALB / c mice—have not been tested for response to approved therapies for Graves' disease.

[0143] Therefore, animal models of Graves' disease are not currently optimal for researching potential treatments.

[0144] The inventors have surprisingly discovered that human HLA-DR3 transgenic animals provide a suitable context for studying potential therapies. Therefore, in another aspect, the present invention provides an animal model of a disease associated with the production of TSHR antibodies, wherein said animals are human HLA-DR3 transgenic and contain elevated TSHR levels compared to control animals.

[0145] The term “elevated TSHR level” is intended to cover elevated levels of any full-length TSHR sequence (e.g., TSHR peptide).

[0146] The TSHR level is elevated relative to a suitable control animal (e.g., an animal that is a human HLA-DR3 transgenic animal but does not have elevated TSHR). The TSHR level can be elevated by any suitable means. For example, by administering a vector containing nucleic acid encoding a TSHR peptide. In a preferred embodiment, the vector is a viral vector.

[0147] In a further embodiment, the present invention provides a method for producing an animal model of a disease associated with the production of TSHR antibodies, the method comprising increasing the level of TSHR in animals transgenic with human HLA-DR3.

[0148] In a preferred embodiment, the method includes increasing TSHR levels by introducing a vector containing a nucleic acid encoding the TSHR peptide. In a more preferred embodiment, the vector is a viral vector.

[0149] Animal models exhibit characteristics associated with a disease related to the production of TSHR antibodies. For example, TSHR antibody levels are elevated in animal models compared to equivalent control animals (where TSHR levels are not increased). TSHR antibody levels can be at least 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than in equivalent control animals (where TSHR antibody levels are not increased).

[0150] TSHR antibody levels can be determined using standard methods known in the art, such as ELISA assays. The level of TSHR antibodies in a sample can be determined in vitro. For example, the sample can be a serum sample.

[0151] "Human HLA-DR3 transgenic" means that the animal expresses the human MHC class II cell surface molecule HLA-DR3. Transgenic animals can be produced using appropriate methods known in the art.

[0152] "Associated with the production of TSHR antibodies" means that TSHR antibodies contribute to the cause of the disease. For example, TSHR antibody levels can be elevated compared to levels not present in the disease. A disease associated with TSHR antibodies is Graves' disease.

[0153] TSHR can be a human TSHR. For example, TSHR can be a human TSHR A subunit or a portion thereof.

[0154] Nucleic acids encoding TSHR can be provided using methods known in the art, such as those based on the TSHR sequences described herein.

[0155] Nucleic acids can be natural, synthetic, or recombinant. They can be double-stranded or single-stranded, and can be DNA or RNA or a combination thereof. For example, they can be cDNA, PCR products, genomic sequences, or mRNA.

[0156] The nucleotide sequence can be a codon used for optimization in a selected host / host cell.

[0157] Delivery of the nucleotide sequence encoding TSHR can be mediated by viral infection. Suitable viral vectors are well known in the art. For example, viral vectors may be adenoviruses, retroviruses, or lentiviruses.

[0158] In particular, the viral vector may be an adenovirus.

[0159] The creation of animal models can involve multiple administrations of a vector.

[0160] The vector (e.g., adenovirus) can be administered at 3-week intervals. For example, the vector can be administered at intervals of 18-25, 18-23, 19-23, or 20-22 days. The vector can also be administered at intervals of 20, 21, or 22 days.

[0161] Animal models may include administration of the adenovirus vector at at least two points. For example, the adenovirus vector may be administered at two or three points. In particular, animal models may include administration of the adenovirus vector at two points.

[0162] Animal models may include administration of the vector at two different times, with a three-week interval between administrations.

[0163] The vector (e.g., adenovirus) can be administered at a dose of 10⁸ to 10¹¹ viral particles per application. Specifically, adenovirus can be administered at a dose of 10⁹ to 10¹¹ viral particles per application. Adenovirus can be administered at a dose of 10⁹ viral particles per application.

[0164] The vector can be administered by any suitable means. For example, in the case of an adenovirus vector, the vector can be administered via intramuscular injection.

[0165] The animal can be a mammal. For example, the animal can be a mouse, rat, rabbit, guinea pig, or primate. Preferably, the animal is a mouse.

[0166] In one embodiment, the mice are transgenic mice with a mixed genetic background of HLA-DRA1*01:01 and HLA-DRB1*03:01. Methods for producing such mice are known to those skilled in the art.

[0167] For example, the mouse model described in the Examples section was generated using the following method: a 6-kb NdeI fragment cloned from the HLA-DRA genome in pUC and a 24-kb ClaIxSalI fragment containing the B gene at cos 4.1 were co-injected into fertilized eggs from a (C57BL / 6xDBA / 2) F1 donor mated with a C57BL / 6 male. The offspring were then bred into an IA-beta knockout C57BL / 6 genetic background (AB0 mice) lacking mouse MHC-II molecule expression. These DR3tg mice express HLA-DR3 molecules but not mouse MHC-II molecules. This method is not exhaustive, and alternative methods for generating HLA-DR3 transgenic mice (e.g., on a C57BL / 10 background) are known in the art.

[0168] The present invention will now be further described by way of embodiments intended to help those skilled in the art to implement the invention, and not intended to limit the scope of the invention in any way.

[0169] Example Example 1 – ATX-GD-59 induces TSHR-specific T cells in an HLA-DR3 transgenic mouse model Tolerance and reduced anti-TSHR antibody titer Materials, methods and procedures mice DR3tg mice were bred externally at Charles River, UK, or InnoSer, NL under specific pathogen-free conditions. The DR3tg breed was originally created by Straub et al. Briefly, the genomic construct used was a 6kb Nde I fragment from the HLA-DRA genome clone in pUC 13 and a 24kb CLaI x SalI fragment from cos 4.1 (a viscous granule containing the DRB1*0301 B gene (pTCF)). Solutions containing 1–2 μg / mL of each construct were co-injected into zygotes from (C57Bl / 6x DBA / 2) F1 donors mated with C57Bl / 6 males. The offspring were cultured into an IA-beta knockout C57BL / 6 genetic background (AB0 mice) lacking mouse MHC class II molecule expression. These DR3tg mice expressed human MHC class II, HLA-DR3 molecules, but not mouse MHC class II molecules. Transgenic mice were identified by Southern blotting analysis of tail DNA digested with Eco RI, and the detection was performed using a 1.35 kb Bam HI fragment of DRA cDNA and a 1.25 kb Bam HI fragment of DRB1*0301 cDNA. DR3tg mice were used for these experiments because this MHC class II molecule has been proposed to be associated with increased susceptibility to Graves' disease.

[0170] Animal research was conducted in batches by the Ethical Committee for Animal Experiments at Hasselt University and in a pathogen-free facility with care standards.

[0171] antigen Peptides were synthesized by PolyPeptide Laboratories (PPL; Strasbourg, France) and stock solutions of 8 mg / ml in PBS were stored at -20°C. The preparation process was based on solid-phase peptide synthesis using N-α-Fmoc-protected amino acids as building blocks in peptide assembly. C-terminal residues were coupled to MBHA resin as the part of the Fmoc-lys(Boc)-MPPA-linker. Other amino acids were incorporated through a continuous cycle of Fmoc deprotection and amino acid coupling. The figure caption indicates when PPL peptides were used.

[0172] Alternatively, peptides were synthesized via GL Biochemistry Ltd (GLS; Shanghai, China) or Severn Biotech Ltd (Severn, Kidderminster Worcs., UK) and stored in stock solutions of 20 mg / ml in DMSO (Sigma-Aldrich) at -80°C. Peptides were synthesized using F-moc chemistry with N-terminal free amine and C-terminal amide. Different peptide suppliers were indicated for each experiment.

[0173] The peptides are used for treatment as a single peptide (9B-N, 5D-K1) or as a mixture of peptides, ATX-GD-59. ATX-GD-59 is an equimolar mixture of 9B-N and 5D-K1, and a specified dose of ATX-GD-59 refers to the individual peptide content, i.e., the total amount of peptides applied is twice the specified treatment dose.

[0174] The recombinant human extracellular domain of TSHR (TSHR-ECD, AA19-417) was generated in the Trichoplusia ni larval expression system using the Chesapeake PERLXpress technology from Chesapeake PERL (Savage, USA). Protein mass of each batch of TSHR-ECD was assessed by SDS-PAGE gel and Western blot analysis.

[0175] Adenovirus vectors were purchased from Viraquest (North Liberty, IA, USA). The construction and purification of adenovirus containing amino acid residues 1-289 of TSHR (Ad-TSHR) have been previously described. Briefly, adenovirus Ad-TSHR and a control vector expressing β-galactosidase (Ad-LacZ) were amplified in HEK293 cells and purified by CsCL density gradient centrifugation. Viral particle concentration was determined by measuring absorbance at 260 nm.

[0176] In vitro tolerance experiment DR3tg mice were subcutaneously injected with 0.1 μg, 1 μg, and 10 μg peptides into the lateral region on days -15, -13, and -11, respectively, followed by three injections of 100 μg (top dose / peptide) of soluble peptides (5D-K1 or 9B-N) on days -8, -6, and -4. ATX-GD-59 is an equimolar mixture of peptides 5D-K1 (SEQ ID NO:1) and 9B-N (SEQ ID NO:2), and the mentioned top dose (nmol) refers to each peptide in the mixture. A 100 μg peptide dose corresponds to approximately 45 nmol, as both systems have been used in different experiments. The peptides were administered subcutaneously in PBS. Changes in the top dose / peptide ratio and corresponding dose escalation were indicated in each experiment. On day 0, mice were subcutaneously immunized at the base of the tail with 50 μg of parental peptide (unmodified 5D and / or 9B) emulsified in complete Freund's adjuvant (CFA; 2 mg / ml Mycobacterium tuberculosis H37RA (Difco Laboratories, Michigan, USA) or in incomplete Freund's adjuvant (Difco)). Ten days after immunization, drained lymph nodes (LNs) and spleens were harvested. LN cells and spleen cells were isolated and cultured in 96-well plates in X-vivo 15 medium supplemented with 2 mM L-glutamine, 50 U / mL penicillin, and 50 U / mL streptomycin (all from Lonza, Verviers, Belgium). To investigate antigen-induced cell proliferation, 0.5 x 10⁶ cells / well were cultured for 72 hours (200 μL / well) with TSHR-ECD at concentrations ranging from 0 to 25 μg / mL or with a purified protein derivative (PPD; immune control; Statens serum institut, Copenhagen, Denmark) at a concentration of 12.5 μg / mL. After 72 hours of incubation at 37°C with 5% CO₂, 60 μL of the supernatant was collected and frozen. Tritized thymidine (PerkinElmer, Zaventem, Belgium) was then added to the cells at 20 μL / well to obtain a final concentration of 1 μCi / well. Cells were incubated at 37°C with 5% CO₂ and plated after 16 hours. Thawed plates were harvested and read on a β-counter (Wallac 1450 Microbeta Trilux Liquid Scintillation Counter) to assess cell proliferation.

[0177] Adenovirus animal model for GD DR3tg mice were subcutaneously injected into the lateral region on days -15, -13, and -11 with 22.5 pmol, 225 pmol, and 2250 pmol ATX-GD-59 or a control, respectively, followed by three injections of 22.5 nmol ATX-GD-59 (top dose / peptide) or a control on days -8, -6, and -4 (dose escalation schedule). Changes in the top dose and corresponding dose escalation doses were indicated in each experiment.

[0178] On day 0, mice were injected intramuscularly into the thigh muscle with either Ad-TSHR or Ad-LacZ (1010 viral particles). All mice were simultaneously immunized with the same batch of adenovirus for each experiment. Mice were injected at two three-week intervals (week 0 and week 3), and according to the instructions shown. Figure 2 The protocol involved collecting blood at different time points. Five weeks after the first immunization, mice were euthanized to obtain blood, spleen cells, and thyroid glands. Spleen cells were isolated and cultured in 96-well plates in X-vivo 15 medium supplemented with 2 mM glutamine, 50 U / mL penicillin, and 50 U / mL streptomycin (all from Lonza, Verviers, Belgium). To investigate antigen-induced cell proliferation, 0.5 x 10⁶ cells / well were cultured with different concentrations of TSHR-ECD (0–25 μg / mL) (200 μl / well) for 72 hours. After 72 hours of incubation at 37°C with 5% CO₂, 60 μL of cell supernatant was collected and frozen. Tritated thymidine (PerkinElmer, Zaventem, Belgium) was then added to the cells at 20 μL / well to obtain a final concentration of 1 μCi / well. Cells were incubated at 37°C and plated after 16 hours. The melted plates were harvested and read on a β-counter (Wallac 1450 Microbeta Trilux Liquid Scintillation Counter) to assess cell proliferation.

[0179] Detection of anti-TSHR antibodies Anti-TSHR antibodies (IgG class) against purified recombinant TSHR-ECD (Chesapeake-Perl) were measured using an ELISA. 96-well plates (half-region 96-well, Fisher Scientific) were coated overnight at room temperature (RT) with 50 μl / well of TSHR-ECD protein (0.5 μg / ml) in PBS. After washing with PBS-0.05% Tween 20, the wells were blocked with 1% BSA (w / v) in PBS at RT for 1 hour and incubated with test serum (1:50 or 1:500 dilution). Mouse anti-TSHR antibody (A9, Abcam, Cambridge, UK) was used as a positive control. Antibody binding was then detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam). To detect anti-TSHR antibodies of IgG1, IgG2a, IgG2b, and IgG2c isotypes, HRP-conjugated rat anti-mouse IgG1 (Southern Biotech, Alabama, USA), goat anti-mouse IgG2a (Southern Biotech), goat anti-mouse IgG2b (Abcam), and goat anti-mouse IgG2c (Abcam) antibodies were used. The signals were visualized using tetramethylbenzidine (TMB). Optical density (OD) was measured at 450 nm using a plate reader (Tecan Benelux).

[0180] Detection of irritant anti-TSHR antibodies (TSAbs) Lulu* cells were kindly provided by Professor M. Ludgate (Cardiff University, UK) and were Chinese hamster ovary K1 (CHO-K1) cells stably transfected with pA3Luc (cAMP-responsive luciferase construct), pcDNA3-TSHR, human TSHR, and G418 resistance. Cells were maintained at 37°C in 5% CO2 at Ham's 12 medium (Lonza) supplemented with 2 mM L-glutamine, 50 U / mL penicillin, 50 U / mL streptomycin (all Lonza), 10% fetal bovine serum (FBS) (Fisher Scientific, Aalst, Belgium), and 0.2 mg / mL genimycin (Fisher). For TSAbs measurements, cells were seeded at 2 x 10⁴ cells / well in 96-well plates containing Ham's F12 medium with 10% charcoal-exfoliated serum (Sigma-Aldrich, Bornem, Belgium). The following day, the culture medium was removed and the cells were incubated for 4 hours at 37°C in fresh Ham's medium containing 5% PEG and 10% test serum. Cells were then lysed using luciferase cell culture lysis buffer (Promega, Leiden, Netherlands). cAMP-responsive luciferase production was measured using a luciferase reporter assay (Promega) according to the manufacturer's instructions, and light emission was measured using a FLUOstar omega spectrophotometer (BMG Labtech, Ortenberg, Germany). Results are expressed as relative light units (RLU; light emission of stimulated cells / light emission of unstimulated cells).

[0181] Testing for hyperthyroidism Total thyroxine (T4) was measured in undiluted mouse serum (10 μl) using the CBI Mouse / Rat Thyroxine ELISA Kit (Calbiotech, Spring Valley, CA, USA) according to the manufacturer's instructions. T4 values ​​were calculated from the standards in the kit and expressed in μg / dl.

[0182] result Single peptide treatment induced TSHR-specific T cell tolerance. To determine the tolerogenic effects of the individual peptides 5D-K1 and 9B-N, transgenic mice were first treated with one of the peptides individually according to a dose-escalation schedule. In the first experiment, DR3tg mice received either 5D-K1 or control treatment, as described in the Methods section. Pretreatment with the modified peptide reduced TSHR-induced T cell proliferation by 43% in spleen samples and by 60% in LN samples compared to control-treated animals (see [link to relevant documentation]). Figure 1 and2 ).

[0183] To investigate the tolerogenic capacity of peptide 9B-N, DR3tg mice were treated with either 9B-N or PBS. The study showed that pretreatment with 9B-N significantly reduced T cell proliferation against TSHR by 41% and 33% in spleen and LN samples, respectively.

[0184] Combined peptide treatment induces TSHR-specific T cell tolerance The finding that peptide treatment with individual peptides 5D-K1 or 9B-N reduced TSHR-specific lymphocyte proliferation in mice led to an investigation into whether combined treatments (administered as a mixture of peptides) could also reduce TSHR-specific responses. DR3tg mice were treated with ATX-GD-59 according to a dose-escalation schedule and immunized with emulsions containing two parental unmodified peptides from the CFA. Potentially different tolerogenic capabilities were tested at three top doses of ATX-GD-59 (15; 22.5 and 45 nmol / peptide), but no differences were observed between treatment doses (data not shown). Representative results are presented in... Figure 3 As shown in the figure, it demonstrates the significant level at which ATX-GD-59 treatment induced TSHR-specific tolerance. TSHR-induced proliferation was reduced by 58% and 54% in splenocytes and LN cells, respectively.

[0185] Combined peptide treatment reduced the production of anti-TSHR antibodies in an adenovirus-based animal model of Graves' disease. To demonstrate the efficacy of ATX-GD-59 treatment, it was investigated whether treatment with the peptide of ATX-GD-59 could inhibit the presence of Graves' disease parameters, such as TSHR-specific splenocyte proliferation and anti-TSHR antibody formation. DR3tg mice were treated with ATX-GD-59 or a control according to a dose-escalation schedule, followed by two immunizations with Ad-LacZ or Ad-TSHR viral particles. First, the effect of ATX-GD-59 treatment on TSHR-specific splenocyte proliferation was investigated. Splenocytes from Ad-LacZ-immunized mice did not proliferate in vitro after TSHR stimulation (data not shown). In control-treated, Ad-TSHR-immunized DR3tg mice, the absolute proliferation rate of TSHR-specific splenocytes was low, but increased in a dose-dependent manner. Figure 4 As shown, treatment with ATX-GD-59 at a top dose of 22.5 nmol / peptide significantly reduced TSHR-specific splenocyte proliferation by more than 40%. Treatment with ATX-GD-59 at 15 nmol per peptide showed similar results (data not shown). Similar tolerance-inducing effects of ATX-GD-59 treatment were observed in replicate experiments (data not shown), thus confirming the reproducibility of these results.

[0186] The effect of ATX-GD-59 treatment on the formation of anti-TSHR antibodies was then investigated. Serum samples were collected to measure anti-THHR IgG levels by ELISA. No anti-TSHR IgG antibodies were present before ATX-GD-59 peptide pretreatment (W-2, data not shown) and after pretreatment (W0). Figure 5 Corresponding to the proliferation data, no anti-TSHR antibodies were detected in mice immunized with Ad-LacZ control. Two weeks after the first Ad-TSHR immunization, high levels of anti-TSHR IgG were observed in peptide-controlled mice. At week 2, ATX-GD-59 treatment reduced the increase in anti-TSHR antibodies seen in peptide-controlled mice after Ad-TSHR immunization by up to 95%. Furthermore, by week 5, anti-TSHR IgG levels were still reduced by 93% with ATX-GD-59 peptide treatment.

[0187] To further investigate the reduction in anti-TSHR antibody levels, the effect of ATX-GD-59 treatment on different anti-TSHR isotype antibodies was studied. Immunization with 1010 viral particles using Ad-TSHR induced high levels of isotype IgG2b and IgG2c anti-TSHR antibodies. Figure 6 The results showed that ATX-GD-59 significantly reduced IgG1, IgG2b, and IgG2c isoforms of anti-TSHR antibodies in mice immunized with Ad-TSHR, thereby achieving the same effect as shown in the study. Figure 5 The pattern of total IgG antibody levels was correlated. In two independent experiments, treatment regimens of 15 nmol and 22.5 nmol ATX-GD-59 were tested, and both significantly reduced anti-TSHR total IgG and IgG isotype levels (data for the 15 nmol dose are not shown), demonstrating that both doses are effective in the adenovirus-based GD model.

[0188] In summary, these results demonstrate that prophylactic ATX-GD-59 treatment is effective in inducing TSHR-specific T cell tolerance in an in vitro tolerance model and in reducing anti-TSHR antibody levels in an adenovirus Graves' disease model.

[0189] Example 2 – Combination therapy of ATX-GD-459 for Graves' disease and clinically used drugs Materials and methods mice DR3tg mice were bred externally at Charles River, UK, under specific pathogen-free conditions. The DR3tg breed was originally created by Strauss et al. (Strauss et al., 1994, Immunogenetics 3, 104-108). Briefly, the genomic construct used was a 6kb NdeI fragment from the HLA-DRA genome clone in pUC 13 and a 24kb ClaIxSalI fragment from the cos 4.1 viscera (pTCF) containing the DRB1*0301 B gene. Solutions containing 1–2 μg / mL of each construct were co-injected into zygotes from (C57BL / 6x DBA / 2) F1 donors mated with C57BL / 6 males. The offspring were then bred into IA-beta knockout C57BL / 6 genetic backgrounds lacking mouse MHC type II molecule expression (AB0 mice). These DR3tg mice expressed the HLA-DRB1*0301 molecule but not the mouse MHC class II molecule. Mice were maintained by backcrossing with C57BL / 6 and B10.Q. Transgenic mice were identified by Southern blot analysis of tail DNA digested with EcoRI, and probed with a 1.35 kb BamHI fragment of DRACDNA and a 1.25 kb BamHI fragment of DRB1*0301 cDNA. DR3tg mice were used for these experiments because this MHC class II molecule has been proposed to be associated with an elevated risk of developing Graves' disease in individuals.

[0190] The DR4 mouse breed was initially created by Lars Fugger et al. (PNAS 1994, volume 91:6151-55) through co-microinjection of HLA-DRA*0101 / HLA-DRB1*0401 and mCD3-huCD4c / g constructs into embryos from a (DBA / 1xA / CA) F1 mating, and the viable embryos were transferred to pseudopregnant females (BALB / cx 129) F1 for development. The offspring were then bred into IA-b knockout genetic backgrounds lacking expression of mouse MHC class II molecules (AB0 mice). Therefore, the only MHC class II molecule expressed in these DR4 mice is the human HLA DR4 molecule.

[0191] BALB / cJOlaHsd mice were obtained from Harlan's laboratory (Venray, Netherlands).

[0192] The animal research was approved by Haselt University’s Animal Experimentation Ethics Committee and conducted in a pathogen-free facility.

[0193] antigen All individual peptides were synthesized by GL Biochem Ltd (Shanghai, China) and stored at -80°C in a stock solution of 20 mg / ml in DMSO (Sigma-Aldrich). Peptides with N-terminal free amine and C-terminal amide were synthesized.

[0194] The ATX-GD-459 peptide (see Example 3 for discussion of these peptides) was synthesized by PolyPeptide Laboratories (Strasbourg, France) and a stock solution of 8 mg / ml in PBS was stored at -20°C. The preparation process was based on solid-phase peptide synthesis using N-α-Fmoc-protected amino acids as building blocks in peptide assembly. C-terminal residues were coupled to MBHA resin as the part of the Fmoc-lys(Boc)-MPPA-linker. Other amino acids were incorporated through a continuous cycle of Fmoc deprotection and amino acid coupling.

[0195] The recombinant human extracellular domain of TSHR (TSHR-ECD, AA19-417) was generated in the Trichoplusia ni larval expression system using the Chesapeake PERL (Savage, USA) Chesapeake PERL technology PERLXpress. Protein mass of each batch of TSHR-ECD was assessed by SDS-PAGE gel and Western blot analysis.

[0196] Adenovirus vectors were purchased from Viraquest (North Liberty, IA, USA). The construction and purification of adenovirus containing amino acid residues 1-289 of TSHR (Ad-TSHR) have been previously described (Chen et al. 1999 J Clin Endocrinol Metab 84:3182-3186). In summary, adenovirus Ad-TSHR and a control vector expressing β-galactosidase (Ad-LacZ) were amplified in HEK293 cells and purified by CsCL density gradient centrifugation. Viral particle concentration was determined by measuring absorbance at 260 nm.

[0197] Combination therapy of ATX-GD-459 and clinically used GD drugs in adenovirus-based GD animal model. DR3tg mice (n = 9–10 / group) were subcutaneously injected with 15 pmol, 150 pmol, and 1500 pmol of each ATX-GD-459 peptide or control peptide on days -15, -13, and -11, respectively, into the lateral region. This was followed by three injections of 15 nmol of each ATX-GD-459 peptide (top dose) or control peptide (dose escalation schedule) on days -8, -6, and -4. Changes in the top dose and corresponding variations in the dose escalation doses are indicated for each experiment. Mice were treated with MMI or propranolol from the day of the first peptide treatment until the end of the experiment. MMI (Sigma-Aldrich, Bornem, Belgium) was dissolved in sterile water at the desired concentration and administered subcutaneously to mice at a daily dose of 500 μg via an ALZET osmotic pump (model 1004, 0.11 μL / h; Charles River, France). The novel osmotic pump was implanted after 4 weeks. Propranolol hydrochloride (Sigma-Aldrich) was freshly dissolved in 0.9% saline and administered daily via intraperitoneal (IP) injection at a dose of 10 mg / kg / day. Mice were immunized on day 0 by intramuscular injection of 109 or 1010Ad-TSHR virus particles, with a repeat immunization three weeks later. Mouse weight was recorded weekly to assess health status. Five weeks after the first immunization, mice were euthanized to obtain blood, thyroid, and spleen samples. TSHR-induced spleen cell proliferation, cytokine secretion, and anti-TSHR antibody levels were assessed as described below.

[0198] Detection of anti-TSHR antibodies Anti-TSHR antibody (IgG grade) against purified recombinant TSHR-ECD (Chesapeake-Perl) was measured using an ELISA. 96-well plates (half-region 96-well, Fisher Scientific) were coated overnight at room temperature (RT) with 50 μl / well of TSHR-ECD protein in PBS (0.5 μg / ml). After washing with PBS-0.05% Tween 20, the wells were blocked with 1% BSA (w / v) in PBS at RT for 1 hour and incubated with test serum (1:50 or 1:500 dilution). Mouse anti-TSHR antibody (A9, Abcam, Cambridge, UK) was used as a positive control. Antibody binding was then detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam). To detect anti-TSHR antibodies of IgG1, IgG2a, IgG2b, and IgG2c isotypes, HRP-conjugated rat anti-mouse IgG1 (Southern Biotech, Alabama, USA), goat anti-mouse IgG2a (Southern Biotech), goat anti-mouse IgG2b (Abcam), and goat anti-mouse IgG2c (Abcam) antibodies were used. Signals were visualized using tetramethylbenzidine (TMB). Optical density (OD) was measured at 450 nm using a plate reader (Tecan Benelux).

[0199] Detection of irritant anti-TSHR antibodies (TSAbs) Lulu* cells were kindly provided by Professor M. Ludgate (Cardiff University, UK) and were Chinese hamster ovary K1 (CHO-K1) cells stably transfected with pA3Luc (cAMP-responsive luciferase construct), pcDNA3-TSHR, human TSHR, and G418 resistance. Cells were maintained at 37°C in 5% CO2 at Ham's 12 medium (Lonza) supplemented with 2 mM L-glutamine, 50 U / mL penicillin, 50 U / mL streptomycin (all Lonza), 10% fetal bovine serum (FBS) (Fisher Scientific, Aalst, Belgium), and 0.2 mg / mL genimycin (Fisher). For TSAbs measurements, cells were seeded at 2 x 10⁴ cells / well in 96-well plates containing Ham's F12 medium with 10% charcoal-exfoliated serum (Sigma-Aldrich, Bornem, Belgium). The following day, the culture medium was removed and the cells were incubated for 4 hours at 37°C in fresh Ham's medium containing 5% PEG and 10% test serum. Cells were then lysed using luciferase cell culture lysis buffer (Promega, Leiden, Netherlands). cAMP-responsive luciferase production was measured using a luciferase reporter assay (Promega) according to the manufacturer's instructions, and light emission was measured using a FLUOstar omega spectrophotometer (BMG Labtech, Ortenberg, Germany). Results are expressed as relative light units (RLU; light emission from stimulated cells / light emission from unstimulated cells).

[0200] Testing for hyperthyroidism Total thyroxine (T4) was measured in undiluted mouse serum (10 μl) using the CBI Mouse / Rat Thyroxine ELISA Kit (Calbiotech, Spring Valley, CA, USA) according to the manufacturer's instructions. T4 values ​​were calculated from the standards in the kit and expressed in μg / dl.

[0201] Spleen cell proliferation assay Spleen cells were isolated and cultured in 96-well flat-bottom plates in X-vivo 15 medium supplemented with 2 mM glutamine, 50 U / mL penicillin, and 50 U / mL streptomycin (all from Lonza, Verviers, Belgium). To investigate antigen-induced cell proliferation, 0.5 x 10⁶ cells / well were cultured with different concentrations of TSHR-ECD (0–25 μg / mL) (200 μL / well) for 72 h. After culturing for 72 h in a 37°C incubator with 5% CO₂, 60 μL of the supernatant was collected and frozen. Tritized thymidine (PerkinElmer, Zaventem, Belgium) was then added to the cells at 20 μL / well to obtain a final concentration of 1 μCi / well. Cells were incubated at 37°C and the plates were frozen after 16 h. The thawed plates were harvested and read on a β-counter (Wallac 1450 Microbeta Trilux Liquid Scintillation Counter) to assess cell proliferation.

[0202] result Combination therapy of MMI and ATX-GD-459 in an adenovirus-based GD model To verify whether ATX-GD-459 treatment could be combined with MMI to treat GD symptoms, the effects of combined MMI and ATX-GD-459 administration were investigated in an adenovirus-based GD animal model (see Examples 4 and 5 for further discussion of the model). In this experiment, DR3tg mice were treated with the following combinations of MMI and ATX-GD-459: vector + control peptide, MMI + control peptide, vector + ATX-GD-459, or MMI + ATXGD-459. The dosage was increased according to a dose escalation schedule (…). Figure 8 During the first two weeks of the experiment, participants were treated with either ATX-GD-459 or the control peptide. Intradermal or MMI treatment was initiated simultaneously but continued until the end of the experiment. This experimental design allowed for investigation of the interaction between MMI and ATX-GD-459 treatments on GD-like disease parameters.

[0203] First, the effects of MMI and ATX-GD-459 treatments on TSHR-specific splenocyte proliferation were investigated. In control mice, the absolute TSHR-specific splenocyte proliferation value was relatively low ( Figure 10 However, these values ​​in this experiment were significantly higher than in all other control groups in previous experiments (data not shown) or propranolol experiments. Therefore, as Figure 10 As shown, the significant decrease in TSHR-specific spleen cell proliferation induced by MMI or ATX-GD-459 treatment should be interpreted with caution.

[0204] Following TSHR-induced spleen cell proliferation, serum levels of anti-TSHR IgG were measured in all mice. Prior to the start of the experiment, anti-TSHR IgG antibodies were not detected in any mice (data not shown). As detected at week 0, dose-escalation treatment with ATX-GD-459 did not induce any production of anti-TSHR antibodies. Figure 11 Anti-TSHR immunization induced the production of anti-TSHR antibodies in mice treated with the control peptide but not in mice treated with ATX-GD-459. Although there was no significant difference due to substantial changes between control-treated mice, ATX-GD-459 pretreatment reduced mean anti-TSHR antibody production by more than 90%. Conversely, MMI treatment had no effect on anti-TSHR antibody levels. Furthermore, MMI treatment did not affect the antibody-reducing ability of ATX-GD-459 peptide treatment.

[0205] The anti-TSHR antibody profile was further investigated by measuring specific IgG isotypes. Figure 12 As shown, none of the mice immunized with Ad-TSHR produced anti-TSHR antibodies of the IgG1 isotype. Ad-TSHR immunization induced anti-TSHR IgG2b and IgG2c antibodies in mice treated with the control peptide but not in mice treated with ATX-GD-459. ATX-GD-459 pretreatment reduced the production of both anti-TSHR IgG2b and IgG2c antibodies by more than 90%. Conversely, MMI treatment had no effect on any anti-TSHR IgG isotype and did not affect the antibody-reducing ability of ATX-GD-459 treatment. These data correspond perfectly to the observed profile of total anti-TSHR IgG antibodies.

[0206] In addition to allotype assays, the bioactivity of anti-TSHR antibodies and the potential role of MMI or ATX-GD-459 were investigated. Since none of the mice immunized with Ad-TSHR exceeded the threshold for irritant antibodies (data not shown), no conclusions were drawn regarding the effect of MMI or ATX-GD-459 treatment on the incidence of irritant antibodies. The low incidence of irritant antibodies is consistent with previously described experiments (reported as ATX-GD-15-003).

[0207] Finally, the effects of MMI and ATX-GD-459 treatments on T4 levels were examined. Since Ad-LacZ immunization was not used as an immune control in this study, the incidence of hyperthyroidism could not be determined. Although no information on hyperthyroidism levels was available, the results clearly showed that MMI treatment significantly reduced T4 levels (…). Figure 13 However, ATX-GD-459 pretreatment had no effect on serum T4 levels. Furthermore, ATX-GD-459 pretreatment had no effect on the T4-reducing ability of MMI.

[0208] In summary, these data demonstrate that the MMI and ATX-GD-459 processes do not interfere with each other's functionality and can be safely combined in adenovirus-based GD models.

[0209] Combination therapy of propranolol and ATX-GD-459 in adenovirus-based GD models Patients with Graves' Disease (GD) often use beta-blockers to combat the adrenergic symptoms of hyperthyroidism. Therefore, this study investigated the effects of combination therapy with ATX-GD-459 and the beta-blocker propranolol in an adenovirus-based GD model. First, TSHR-specific splenocyte proliferation was investigated. (For example...) Figure 14 As shown, neither propranolol nor ATX-GD-459 treatments had a significant effect on the proliferation of TSHR-specific splenocytes. Combined treatment with propranolol and ATX-GD-459 also had no effect on the proliferation response.

[0210] The effects of propranolol and ATX-GD-459 treatments on the formation of anti-TSHR antibodies were then investigated. Serum samples were collected to measure anti-TSHR IgG levels by ELISA. No anti-TSHR IgG antibodies were present before (W-2) and after (W0) ATX-GD-459 peptide pretreatment. Figure 15 Ad-TSHR immunization induced strong production of anti-TSHR antibodies in mice treated with the control peptide, but to a lesser extent in mice treated with ATX-GD-459. Significantly, Ad-TSHR immunization using 10¹⁰ viral particles, as used in this experiment, resulted in higher anti-TSHR antibody titers than immunization using 10⁹ Ad-TSHR particles used in previous experiments (data not shown). When measured at W2 and W5 post-immunization, ATX-GD-459 treatment reduced anti-TSHR antibody production by more than 90%. In contrast, propranolol treatment had no effect on anti-TSHR antibody levels. Furthermore, propranolol treatment did not affect the antibody-reducing ability of ATX-GD-459 peptide treatment.

[0211] The anti-TSHR IgG isotype profile was then determined to further investigate the antibody profile. Anti-TSHR IgG1 antibodies were significantly induced by immunization with Ad TSHR, although at lower levels than IgG2b and IgG2c. These data contradict previous experiments where anti-TSHR IgG1 antibodies were absent (data not shown), but can be explained by the increased dose of adenovirus particles (10⁹–10¹⁰) used in the immunization. Figure 16As shown, ATX-GD-459 treatment significantly reduced anti-TSHR IgG2b and IgG2c antibody levels. Anti-TSHR IgG1 antibody titers were also reduced by peptide treatment, but not significantly. In contrast to peptide treatment, propranolol treatment had no effect on any anti-TSHR IgG isoform. Furthermore, propranolol treatment did not affect the antibody-reducing ability of ATX-GD-459 treatment, thus perfectly corresponding to the spectrum observed against total anti-TSHR IgG antibodies.

[0212] In summary, ATX-GD-459 treatment significantly reduced anti-TSHR antibody titers in this animal model. However, no effect of propranolol treatment on any GD-like disease parameters was observed. To verify whether routine IP administration induces pharmacological levels of propranolol in mouse blood, plasma propranolol levels were measured by LC-MSMS (Anacura). Blood samples were collected 60–90 minutes after administration, and individual plasma levels were compared between W0 and W5. Results are as follows. Figure 18 As shown.

[0213] Example 3 – In vitro T cell tolerance using ATX-GD-459 Hybridoma clones specific to RNB-4K and RNB-5D were selected to determine whether the RNB-4K-GKK and RNB-5D-K1 peptides were apitopes. Antigen treatment-independent presentation (APIPS) assays were performed and confirmed that the RNB-4K-GKK and RNB-5D-K1 peptides were apitopes. Figure 27 ).

[0214] Compositions containing the peptide shown in Table 1 (referred to as ATX-GD-459), or individual peptides administered alone, were identified as having the ability to induce tolerance to TSHR. Figure 19 An in vitro tolerance regimen is shown.

[0215] Table 1 HLA-DR3 mice were treated with ATX-GD-459 according to a dose-escalation schedule and then immunized with the parental peptides 4K / 5D / 9B in CFA. Results showed that ATX-GD-459 treatment induced significant levels of TSHR-specific tolerance in both the spleen and LN cells. Figure 20 Tolerance induced by ATX-GD-459 treatment was greater than tolerance induced by administration of individual peptides. Figure 20 ).

[0216] Example 4 - Establishment of an adenovirus-based Graves' disease model To verify the efficacy of ATX-GD-459 peptide treatment, an adenovirus-based animal model was first established in wild-type BALB / c mice. TSHR-induced splenocyte proliferation, anti-TSHR IgG antibody levels, serum T4 levels, and thyroid histopathological changes were investigated as parameters of disease symptoms. In the first experiment, BALB / c mice were immunized with 108 Ad-TSHR viral particles. Although this viral dose was described as inducing hyperthyroidism and anti-TSHR antibody production (Chen et al, 2004, Endocrinology 145(11):4927-33), only 2 / 10 mice were considered to have (marginal) hyperthyroidism, and none of the mice produced anti-TSHR IgG antibodies (data not shown).

[0217] During the second experiment using elevated viral particle doses, approximately 30% of the Ad-TSHR-immunized mice showed elevated serum T4 levels measured 4 weeks after the first immunization. Figure 22 However, the incidence was observed to be only 15% at week 10. The decline in elevated T4 levels in wild-type BALB / c mice over time, following an initial rise several weeks post-immunization, has been previously described (McLachlan et al, 2012, Thyroid 8:1-7). However, the apparent hyperthyroidism in isolated mice did not fully correspond to histopathological findings in the thyroid tissue (such as thyroid epithelial cell hypertrophy or lymphocytic infiltration) (data not shown).

[0218] Although the literature provides evidence that mice with a C57BL / 6 genetic background or DR3tg mice retain resistance to the formation of Graves' disease-like symptoms, the inventors tested the ability of Ad-TSHR to induce Graves' disease-like symptoms in DR3tg mice with a mixed C57BL / 10, DBA / 2, and C57BL / 6 non-MHC class II genetic background.

[0219] Figure 23 A shows serum T4 levels in DR3tg mice immunized with Ad-LacZ and Ad-TSHR. Although the four Ad-TSHR immunized mice showed higher T4 levels than the Ad-LacZ immunized mice (mean +2 SD), they were only borderline hyperthyroidism and the increased T4 levels were considered too low to be used as a disease parameter when studying the efficacy of peptide treatment.

[0220] Ad-TSHR immunization induced high anti-TSHR IgG titers in the serum of DR3tg mice. Figure 11(See right figure). Anti-TSHR IgG levels reached their highest at week 4, then declined at weeks 7 and 10, in contrast to the stable anti-TSHR IgG levels observed in BALB / c mice. Figure 23 (C, left figure). The finding that anti-TSHR IgG levels in DR3tg mice were even higher at week 4 than at week 4 after the first immunization (data not shown) led to the conclusion that the adenovirus Graves' disease model was terminated at week 4 instead of week 10.

[0221] Example 5 – Validation of the adenovirus Graves' disease model To validate the DR3tg adenovirus Graves' disease model, the effects of different immunomodulatory drugs on the production of anti-TSHR antibodies were investigated in the adenovirus Graves' disease model. The antithyroid drug methimazole (MMI) is currently used to directly reduce thyroid hormone levels in Graves' disease patients. Therefore, the effect of MMI treatment in DR3tg mice immunized with Ad-TSHR was determined. Based on literature describing the hyperthyroidism-inducing effect of MMI in mice (Jeong et al, 2012, Endocrinology 153:683-9; Mozes et al, 1998, J. Clin Immunology 18(2):106-13), two different MMI doses (50-500 μg / mouse / day) were tested. At 2 and 4 weeks after the first immunization, the daily dose of 500 μg MMI significantly reduced T4 levels, while the 50 μg dose only showed a trend towards reducing T4 levels. Figure 24 A). Although Ad-TSHR immunization did not induce hyperthyroidism in this experiment (data from Ad-LacZ-immunized mice are not shown) and previous experiments, the decrease in T4 levels induced by MMI treatment suggests that it can affect hormones produced by the thyroid gland of DR3tg mice.

[0222] Besides its antithyroid function, MMI also plays an immunomodulatory role (Mozes et al., 1998, ibid.; Wang et al., 2003, J. Leukoc. Biol. 73:57-64). Therefore, it was determined whether MMI treatment could reduce anti-TSHR IgG production in Ad-TSHR immunized mice. Although MMI treatment significantly reduced T4 levels, no change in anti-TSHR IgG levels was observed ( Figure 24 B).

[0223] Patients with Graves' disease (especially those with GO) often require immunosuppressive treatment with glucocorticoids. The effect of methylprednisolone (a synthetic glucocorticoid) on anti-TSHR antibody production in Ad-TSHR immunized mice was tested. Treatment with methylprednisolone at 7 mg / kg / day significantly reduced anti-TSHR IgG levels in Ad-TSHR immunized DR4tg mice, while a dose of 1 mg / kg / day was insufficient to reduce antibody titers (data not shown). When measured 2 weeks after the first immunization, methylprednisolone treatment at 7 mg / kg / day in Ad-immunized DR3tg mice significantly reduced anti-TSHR IgG levels, but this reduction was not observed at week 4, as anti-TSHR antibody levels in untreated mice naturally decreased over time. Methylprednisolone treatment also reduced thymus and spleen cell counts, indicating that adequate pharmacological dose levels are necessary in an adenovirus Graves' disease model. Figure 25 ).

[0224] These data demonstrate that the efficacy of apitopes in diseased DR3tg mice can be measured by measuring the reduction of anti-TSHR IgG antibodies.

[0225] Example 6—Demonstrating the efficacy of ATX-GD-459 in an adenovirus Graves' disease model To determine whether the combination of RNB-4K-GKK, RNB-5D-K1, and RNB-9B peptides (ATX-GD-459) could inhibit the formation of anti-TSHR antibodies, DR3tg mice were treated with ATX-GD-459 or a control according to a dose-escalation schedule, followed by immunization with Ad-LacZ or Ad-TSHR. Serum samples were collected to measure anti-TSHR IgG levels by ELISA. Anti-TSHR IgG antibodies were absent before ATX-GD-459 peptide treatment (W-2, data not shown) and after treatment (W0). Figure 25 Two weeks after the first Ad-TSHR immunization, high levels of anti-TSHR IgG were observed in mice treated with PBS. At weeks 2 and 5 post-Ad-TSHR immunization, ATX-GD-459 treatment reduced the increase in anti-TSHR IgG antibodies by 72% and 65%, respectively. These results indicate that ATX-GD-459 is effective in this adenovirus Graves' disease model.

[0226] High levels of anti-TSHR IgG were observed after Ad-TSHR immunization. Therefore, this disease parameter was used to investigate the effects of immunomodulatory drugs or ATX-GD-459 peptide treatment in an adenovirus Graves' disease model. Methylprednisolone treatment successfully reduced anti-TSHR IgG levels in Ad-TSHR immunized DR3tg mice. Furthermore, ATX-GD-459 peptide treatment according to a dose-escalation schedule reduced the increase in anti-TSHR IgG antibody formation by up to 70%.

[0227] Materials and methods mice Mice were used as described in Example 2.

[0228] antigen The antigen is as described in Example 2.

[0229] Antigen treatment-independent presentation system (APIPS) assay The responsiveness of antigen-specific T-cell hybridomas to peptides presented by fixed or unfixed VAVY or BM14 cells (=APCs) was tested. 5 x 10⁴ cells from individual clones were cultured with 25 μg / ml peptide and 5 x 10⁴ fixed or fresh APCs. To fix the APCs, cells were incubated with 0.5% paraformaldehyde (Merck, Darmstadt, Germany) (pH 7) at room temperature (RT) for 5 min. Fixation was stopped by adding 0.4 M glycine (Sigma-Aldrich) and the cells were washed in RPMI-10% FCS. Additionally, responsiveness to human TSHR-ECD protein (Chesapeake-PERL, Savage, Maryland, USA) was measured to identify epitopes. After 48 hours, antigen-induced IL-2 production was measured by ELISA.

[0230] In vitro tolerance experiment Mice were subcutaneously injected with 0.1 μg, 1 μg, and 10 μg peptide into the lateral region of DR3tg or DR4tg mice on days -15, -13, and -11, respectively, followed by three injections of 33, 75, or 100 μg peptide on days -8, -6, and -4 (dose escalation schedule) (depending on the top dose, see figure caption). Changes in the top dose and the corresponding changes in the dose escalation doses are indicated in each experiment. On day 0, mice were subcutaneously immunized at the base of the tail with 50 μg of antigen (unmodified parental 15-mer peptide) emulsified in CFA (peptide / CFA). Ten days post-immunization, draining lymph nodes (LNs) and spleens were collected. LN cells and spleen cells were isolated and cultured in 96-well flat-bottom plates in X-vivo 15 medium (supplemented with 2 mM L-glutamine, 50 U / mL penicillin, and 50 U / mL streptomycin; Lonza). To investigate antigen-induced cell proliferation, 0.5 x 10⁶ cells / well were cultured for 72 h with different antigen concentrations (0–25 μg / ml) or with 12.5 μg / ml of purified protein derivatives (PPD; initiation control; Statens serum institut, Copenhagen, Denmark). After 72 h, 60 μL of cell supernatant was collected and frozen. Then, 20 μL / well of tritated thymidine (PerkinElmer, Zaventem, Belgium) was added to the cells to obtain a final concentration of 1 μCi / well. Cells were incubated at 37 °C and plated after 16 h. The thawed plates were harvested and read using a β-counter (Wallac 1450 Microbeta Trilux liquid scintillation counter) to assess cell proliferation.

[0231] Adenovirus animal model of Graves' disease Adenovirus expressing the human TSHR A subunit (amino acid residues 1-289; Ad-TSHR) and control adenovirus expressing β-galactosidase (Ad-LacZ) were purchased from Viraquest (North Liberty, IA, USA). Six-week-old female BALB / cJOlaHsd mice (Harlan Laboratories, Venray, Netherlands) or DR3tg mice were injected intramuscularly into the thigh muscle with Ad-TSHR or Ad-LacZ (109, 1010, or 1011 viral particles). All mice were simultaneously immunized with the same batch of adenovirus for each experiment. Mice were injected at two or three times every three weeks (week 0, week 3, and week 4), and blood was collected one week before the first immunization and one week after the second immunization. Mice were euthanized 4, 5, or 10 weeks after the first immunization to obtain blood, spleen cells, and thyroid cells. Spleen cells were analyzed and cultured in 96-well plates in X-vivo 15 medium (supplemented with glutamine, penicillin, and streptomycin; Lonza). To investigate antigen-induced cell proliferation, 0.5 x 10⁶ cells / well were cultured for 72 h with different antigen concentrations (0–25 μg / ml). After 72 h, 60 μL of cell supernatant was collected and frozen. Then, 20 μL / well of tritated thymidine (PerkinElmer, Zaventem, Belgium) was added to the cells to obtain a final concentration of 1 μCi / well. Cells were incubated at 37 °C and plated after 16 h. The thawed plates were harvested and read using a β-counter (Wallac 1450 Microbeta Trilux liquid scintillation counter) to assess cell proliferation.

[0232] Detection of anti-TSHR antibodies Anti-TSHR antibodies against purified recombinant TSHR-ECD (Chesapeake-Perl) were measured using an ELISA. 96-well plates (half-domain 96-well, Fisher Scientific) were coated overnight at room temperature (RT) with 50 μl / well of TSHR-ECD protein (0.5 μg / ml) in PBS. After washing with PBS-0.05% Tween, the wells were blocked with 1% bovine serum albumin (BSA) (w / v) in PBS at RT for 1 hour and incubated with test serum (repeated aliquots, 1:50 dilution). Mouse anti-TSHR antibody (A9, Abcam, Cambridge, UK) was used as a positive control. Antibody binding was then detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam). To detect anti-TSHR antibodies of IgG1, IgG2a, IgG2b, and IgG2c isotypes, HRP-conjugated rat anti-mouse IgG1 (Southern Biotech, Alabama, USA), goat anti-mouse IgG2a (Southern Biotech), goat anti-mouse IgG2b (Abcam), and goat anti-mouse IgG2c (Abcam) antibodies were used. Signals were visualized using tetramethylbenzidine (TMB). Optical density (OD) was measured at 450 nm using a plate reader (Tecan Benelux).

[0233] Detection of irritant anti-TSHR antibodies (TSAbs) Lulu* cells were kindly provided by Professor M. Ludgate (Cardiff University, UK). These cells were Chinese hamster ovary K1 (CHO-K1) cells stably transfected with pA3Luc (cAMP-responsive luciferase construct), pcDNA3-TSHR, human TSHR, and G418 resistance. Cells were maintained at 37°C in 5% CO2 at Ham's 12 medium (Lonza) supplemented with 2 mM L-glutamine, 50 U / mL penicillin, 50 U / mL streptomycin (Lonza), 10% fetal bovine serum (FBS) (Fisher Scientific, Aalst, Belgium), and 0.2 mg / mL genimycin (Fisher). For TSAbs measurements, cells were seeded at 2 x 10⁴ cells / well in 96-well plates containing Ham's F12 medium with 10% charcoal-exfoliated serum (Sigma-Aldrich, Bornem, Belgium). The following day, the culture medium was removed and the cells were incubated for 4 hours at 37°C in fresh Ham's medium containing 5% PEG and 10% test serum. Cells were then lysed with luciferase cell culture lysis buffer (Promega, Leiden, Finland). cAMP-responsive luciferase production was measured using a luciferase reporter assay (Promega) according to the manufacturer's instructions, and light emission was measured using a FLUOstar omega spectrophotometer (BMG Labtech, Ortenberg, Germany). Results are expressed as relative light units (RLU; light emission from stimulated cells / light emission from unstimulated cells).

[0234] Testing for hyperthyroidism Total thyroxine (T4) was measured in undiluted mouse serum (10 μl) using the CBI Mouse / Rat Thyroxine ELISA Kit (Calbiotech, Spring Valley, CA, USA) according to the manufacturer's instructions. T4 values ​​were calculated from standards in the kit and expressed in μg / dl. In addition to serum T4 measurement, thyroid histology was used as a parameter for hyperthyroidism. Thyroid glands were fixed in 10% neutral buffered formalin (pH 7.5), sectioned, and stained with hematoxylin and eosin. Pathological changes (hypertrophy, epithelial supercellular and lymphocytic infiltration) were observed and scored (KWS Biotest, Bristol, UK).

[0235] Validation of an animal model of Graves' disease DR3tg mice were intramuscularly immunized with Ad-TSHR at two three-week intervals (day 0 and day 21). Mice were treated with either MMI or 6α-methylprednisolone 21-hemisuccinate (methylprednisolone) from the day of the first immunization until the end of the experiment. All compounds were administered subcutaneously via an ALZET osmotic pump (model 1004, 0.11 μL / h, Charles River, France). MMI (Sigma) was dissolved in sterile water at the desired concentration to administer to mice at daily doses of 50 or 500 μg. Methylprednisolone (Sigma) was dissolved in sterile water and administered to mice at doses of 1 mg / kg / day or 7 mg / kg / day. Mouse weight was recorded weekly to assess health status. Mice were euthanized four weeks after the first immunization to obtain blood and spleen samples. TSHR-specific spleen cell proliferation and anti-TSHR antibody levels were evaluated as described above.

[0236] Animal model of preventive Graves' disease DR3tg mice were subcutaneously injected into the lateral region on days -15, -13, and -11 with 0.1 μg, 1 μg, and 10 μg of ATX-GD-459 or a control, respectively. This was followed by three injections of 100 μg of ATX-GD-459 (top dose) or a control on days -8, -6, and -4 (dose escalation schedule). Changes in the top dose and corresponding variations in the dose escalation doses are indicated for each experiment. Mice were immunized intramuscularly with 109Ad-TSHR or Ad-LacZ virus particles on day 0, and this immunization was repeated three weeks later. Blood and spleens were collected five weeks after the first immunization. TSHR-specific splenocyte proliferation and anti-TSHR antibody levels were evaluated as above.

[0237] Various modifications and variations of the invention will be apparent to those skilled in the art without departing from its scope and spirit. While the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, the invention is intended to cover various modifications of the descriptive paradigm for carrying out the invention that will be obvious to those skilled in the art of chemistry, biology, or related fields. All publications mentioned in the foregoing specification are incorporated herein by reference.

Claims

1. A composition comprising the following thyroid-stimulating hormone receptor (TSHR) peptide: (i) All or part of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO:1), or a sequence having at least 60% sequence identity with SEQ ID NO:1; and (ii) All or part of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO:2), or a sequence having at least 60% sequence identity with SEQ ID NO:2; And optionally antithyroid agents and / or beta-blockers.

2. The use of the peptide as defined in claim 1, and optionally a thyroid agent and / or a beta-blocker, or the composition of claim 1, in a therapeutic manner.

3. The peptide or composition for use according to claim 1 or claim 2, for use in inhibiting or preventing the production of TSHR autoantibodies in vivo.

4. The peptide or composition for use according to claim 1 or claim 2, for use in treating and / or preventing Graves' disease in a subject.

5. Use of the peptide or composition according to claim 1 or claim 2 in the preparation of a medicament for inhibiting or preventing the production of TSHR autoantibodies in vivo.

6. Use of the peptide or composition according to claim 1 or claim 2 in the preparation of a medicament for the treatment and / or prevention of Graves' disease.

7. A method for inhibiting or preventing the production of TSHR autoantibodies in a subject, comprising administering to the subject a peptide or composition according to claim 1 or claim 2.

8. A method for treating Graves' disease in a subject, comprising administering to the subject a peptide or composition according to claim 1 or claim 2.

9. The use or method according to any one of claims 3 to 8, wherein the subject is HLA-DR3.

10. The use or method according to any one of claims 3 to 8, wherein the subject is HLA-DR4.

11. The use or method according to any one of claims 3 to 10, wherein the composition is administered according to a dose escalation regimen.

12. A kit containing the following TSHR peptides; (i) All or part of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO:1), or a sequence having at least 60% sequence identity with SEQ ID NO:1; and (ii) All or part of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO:2), or a sequence having at least 60% sequence identity with SEQ ID NO:2; And optionally antithyroid agents and / or beta-blockers, which may be administered simultaneously, separately or sequentially.

13. The kit of claim 12, for use in the prevention or treatment of Graves' disease, administered simultaneously, separately, or sequentially.

14. An animal model for a disease associated with the production of TSHR antibodies, wherein the animals are transgenic for human HLA-DR3 and wherein the level of TSHR is elevated in the animals compared with a control animal.

15. The animal model of claim 14, wherein the disease associated with the production of TSHR antibodies is Graves' disease.

16. The animal model of claim 14 or 15, wherein the TSHR is a human TSHR.

17. The animal model according to any one of claims 14 to 16, wherein TSHR is the human TSHR A subunit.

18. The animal model according to any one of claims 14 to 17, wherein the viral vector is an adenovirus vector.

19. The animal model of claim 18, wherein the adenovirus is administered by intramuscular injection.

20. The animal model of claim 18 or 19, wherein the animal is a mouse.

21. The animal model of claim 20, wherein the mouse is an HLA-BRD1*0301 transgenic mouse.

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