RU2773344C2 - Composition - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the use of a composition including following peptides of a thyroid-stimulating hormone receptor (hereinafter – TSHR): (i) peptide with an amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence SEQ ID NO: 1; and (ii) peptide with an amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence SEQ ID NO: 2.

EFFECT: specified composition is used in the suppression or prevention of the production of auto-antibodies to TSHR in vivo, as well as in the treatment and/or prevention of Grave’s disease.

12 cl, 27 dwg, 1 tbl, 6 ex

Description

The field to which the invention relates

The present invention relates to a composition containing peptides derived from the thyroid stimulating hormone receptor (TSHR). The composition or peptides can be used to prevent and/or suppress the production of anti-TSHR autoantibodies, and therefore they can be used to treat and/or prevent Graves' disease.

Prior Art

Graves' disease is an autoimmune disease that affects the thyroid gland. This disease is characterized by an overactive thyroid gland, which leads to excessive production of thyroid hormone and enlargement of the thyroid gland (goiter). This state of 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 usually occurs in middle-aged people, but can also occur in children, adolescents, and the elderly. This disease affects up to 2% of women, and in women, it occurs 5-10 times more often than in men. The childhood form of Graves' disease affects approximately 6,000 children in the United States (USA) and 6,000 children in the European Union (EU). Graves' disease is also the most common cause of severe hyperthyroidism, which is associated with more severe clinical signs and symptoms, as well as laboratory-proven pathologies, compared with milder forms of hyperthyroidism.

The hereditary component plays a significant role in the development of Graves' disease. There has not been any recent population survey for the presence of Graves' disease, however, there have been several quasi-experimental population surveys for hyperthyroidism, and all estimates for the presence and incidence of Graves' disease are approximate. The incidence of hyperthyroidism varies from 26:100,000 to 93:100,000, and is estimated to have a worldwide prevalence of 1.3%, with 40% of cases being confirmed and 60% subclinical.

Approximately 30-50% of people with Graves' disease also have Graves' ophthalmopathy (also known as Graves' orbitopathy or "thyroid eye disease") (OH), which is a protrusion of one or both eyes. In many cases, OH occurs in a mild and self-limited form, however, in 20% of cases, such a disease manifests itself in a pronounced / moderate or severe form, and in at least half of the cases, such patients require the introduction of steroids, and in 3 - 5% of patients with OH have a vision-threatening disease, accompanied by pain in combination with dysthyroid optic neuropathy. Protrusion of the eyes can cause severe dryness of the cornea because the eyelids cannot close at night. Increased pressure on the optic nerve can cause visual field defects and lead to loss of vision. OH may also be associated with pretibial myxedema.

In fact, all symptoms and signs of Graves' disease are the result of the direct and indirect effects of hyperthyroidism, with the exception of its main symptoms, OH, goiter, and pretibial myxedema. Symptoms of hyperthyroidism can include insomnia, hand tremors, hyperactivity, hair loss, excessive sweating, heat intolerance, and weight loss despite increased appetite. The other most common signs are diffuse, painless enlargement (usually symmetrical) of the thyroid gland, non-closing of the eyelids, excessive watery eyes due to OH, cardiac arrhythmias, and hypertension. Patients with thyrotoxia may experience behavioral and personality changes such as psychosis, excitability, and depression. Patients with milder forms of hyperthyroidism may have less pronounced manifestations, such as anxiety, restlessness, irritability, and emotional instability.

Currently, there is no effective treatment for Graves' disease, and therefore the commonly used treatment is aimed at eliminating the symptoms. There are three treatments for Graves' disease, ie, oral administration of antithyroid drugs (ATD), radioactive iodine irradiation (RAI), and thyroidectomy. The last two methods increase the lifetime of thyroid hormones. RAI is the most common treatment in the US, and ATD is the first line treatment in Europe, Japan, and most other countries around the world.

ATD therapy is associated with some rare side effects and results in 50-60% remission. Currently, it is becoming increasingly clear that RAI can accelerate the development of OH or worsen the condition in active OH, and therefore, in the United States, the number of patients undergoing ATD treatment is increasing.

Since each of the chosen treatments is not always successful, patients are often subjected to more than one treatment if the first attempt was not entirely successful. The risk of recurrence or subsequent hypothyroidism is quite high, and the overall efficacy of available treatments for Graves' disease is poor.

The development of alternative therapies for Graves' disease is problematic due to the lack of relevant models, and in particular animal models, to assess the potential effectiveness of the proposed therapy. Known BALB/c mice with a model of Graves' disease have not been tested for the efficacy of drugs approved for the treatment of Graves' disease, such as methimazole and methylprednisolone.

Thus, it is necessary to develop alternative therapies for the treatment and prevention of a disease associated with the production of anti-TSHR autoantibodies, such as Graves' disease. There is also a need to develop alternative therapies that are effective in treating Graves' disease and reducing or alleviating the symptoms of the disease. In addition, alternative models need to be developed to evaluate the potential efficacy of proposed treatments for Graves' disease.

Brief description of aspects of the invention

The present inventors have found that a "cocktail" of two TSHR peptides is particularly effective in suppressing or preventing the production of anti-TSHR autoantibodies in vivo and in treating Graves' disease.

Thus, in its first aspect, the present invention relates to a composition containing the following TSHR peptides:

(i) all or part of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1), or part thereof, or a sequence that is at least 60% identical to the sequence of SEQ ID NO: 1; and

(ii) all or part of the amino acid sequence of GLKMFPDLTKVYSTD (SEQ ID NO: 2), or part thereof, or a sequence that is at least 60% identical to the sequence of SEQ ID NO: 2.

KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) is also referred to here as RNB-5D-K1. GLKMFPDLTKVYSTD (SEQ ID NO: 2) is also referred to here as RNB-9B.

The composition according to the invention can be used in the therapeutic aspects of the invention described here.

In its second aspect, the present invention provides a peptide of the invention as described herein for use in suppressing or preventing the production of anti-TSHR autoantibodies in vivo .

In its third aspect, the present invention provides a peptide of the invention as described herein for use in the treatment and/or prevention of Graves' disease in an individual.

In its fourth aspect, the present invention relates to the use of the peptide of the invention described herein in the irradiation of a drug for suppressing or preventing the production of anti-TSHR autoantibodies in vivo .

In its fifth aspect, the present invention relates to the use of the peptide of the invention described herein in the manufacture of a medicament for the treatment and/or prevention of Graves' disease.

In its sixth aspect, the present invention relates to a method for suppressing or preventing the production of autoantibodies to TSHR in an individual, comprising the step of administering to the individual all or part of the peptide of SEQ ID NO: 1, or a peptide whose sequence is at least 60% identical to the sequence of the peptide of SEQ ID NO: 1, or part thereof, and the entire peptide of SEQ ID NO: 2, or part thereof, or a peptide whose sequence is at least 60% identical to the sequence of the peptide of SEQ ID NO: 2, or part thereof.

In its seventh aspect, the present invention relates to a method of treating Graves' disease in an individual, comprising the step of administering to the individual all or part of the peptide of SEQ ID NO: 1, or a peptide whose sequence is at least 60% identical to the sequence of the peptide of SEQ ID NO: 1 , or part thereof, and the entire peptide of SEQ ID NO: 2, or part thereof, or a peptide whose sequence is at least 60% identical to the sequence of the peptide of SEQ ID NO: 2, or part thereof.

In one aspect of the invention, the composition does not include or does not contain the following peptide:

RNB-4K-GKK: KKGNLPNISRIYVSIDVTGKK

The peptide composition of the invention may include the amino acid sequences of the invention described herein. In one aspect of the invention, the peptide composition includes only the amino acid sequences described herein according to the invention.

The individual may have HLA-DR3. The individual may have HLA-DR4.

The peptides of the invention described herein or the composition of the invention may be administered in accordance with a dosage escalation schedule.

In its 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 part or part thereof, or a sequence that is at least 60% identical to the sequence of SEQ ID NO: 1;

and

(ii) all or part of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2), or part or part thereof, or a sequence that is at least 60% identical to the sequence of SEQ ID NO: 2;

for simultaneous, separate or sequential administration.

The kit can be used to treat or prevent a condition associated with the production of anti-TSHR autoantibodies, such as Graves' disease.

In a ninth aspect of the invention, the peptides or composition of the invention are combined with another therapeutic agent that is used to treat, prevent, or treat Graves' disease. For example, the peptides or composition may be combined with an antithyroid drug or a β-blocker.

In its ninth aspect, the present invention relates to an animal model of a disease associated with the production of anti-TSHR antibodies, wherein said animal is transgenic for human HLA-DR3, and wherein said animal has higher levels of TSHR than a corresponding control animal. In one aspect of the invention, the level of TSHR is increased by introducing a viral vector containing a nucleic acid molecule encoding a TSHR peptide.

The disease associated with the production of anti-TSHR antibodies may be Graves' disease.

The TSHR may be a human TSHR. Thus, for example, the TSHR may be the A subunit or the extracellular domain (ECD) of a human TSHR.

The viral vector may be an adenoviral vector or an adenoviral construct.

The adenovirus vector or construct may be administered at intervals of three weeks. The adenovirus vector or construct may be administered by two or three consecutive injections.

The adenovirus vector or construct is administered at a dose of 10 ⁹ -10 ¹¹ viral particles. The adenovirus vector or construct may be administered by intramuscular injection.

The animal may be a mouse. The mouse may be an HLA-BRD1*0301 transgenic mouse.

Description of drawings

Figure 1-5D-K1 - Treatment reduces TSHR-induced proliferation in DR3tg mice

DR3tg mice were pretreated with 5D-K1 (GLS) or control (HLA-DR3 binding peptide; GLS) (n=10/group) according to a dose escalation schedule up to a maximum dose of 100 μg. Animals were immunized with 50 μg of 5D (GLS) in PAF, and lymph nodes (LN) and spleen were taken 10 days later for evaluation of TSHR-specific proliferation. Data are the mean ± standard error of the mean (SEM) of stimulation index (SI) values for control-treated mice (red lines) and peptide-treated mice (blue lines). A two-way ANOVA was used to evaluate the overall effects of treatment on T cell proliferation and p-values are plotted. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). The mean percent reduction in T cell proliferation induced by peptide treatment is shown in the graph. SI - stimulation index; LN - lymph nodes.

Figure 2-9B-N-treatment reduces TSHR-induced proliferation in DR3tg mice

DR3tg mice were pretreated with 9B-N (PPL) or PBS (n=10/group) according to a dose escalation schedule up to a maximum dose of 33 μg. Animals were immunized with 50 μg of 9B (GLS) in CFA, and LN and spleen were taken 10 days later to assess TSHR-specific proliferation. Data are the mean ± standard error of the mean (SEM) of stimulation index (SI) values for control-treated mice (red lines) and peptide-treated mice (blue lines). A two-way ANOVA was used to evaluate the overall effects of treatment on T cell proliferation and p-values are plotted. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). The mean percent reduction in T cell proliferation induced by peptide treatment is shown in the graph. SI - stimulation index; LN - lymph nodes.

Figure 3 - ATX-GD-59 treatment reduces TSHR-induced proliferation in DR3tg mice

DR3tg mice were pre-treated with ATX-GD-59 (PPL) or HIP-15F-GKK (GLS) control peptide (n=10/group) according to a dose escalation schedule up to a maximum 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 LN and spleen were taken 10 days later to assess TSHR-specific proliferation. Data are the mean ± standard error of the mean (SEM) of stimulation index (SI) values for control-treated mice (red lines) and peptide-treated mice (blue lines). A two-way ANOVA was used to evaluate the overall effects of treatment on T cell proliferation and p-values are plotted. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). The mean percent reduction in T cell proliferation induced by peptide treatment is shown in the graph. SI - stimulation index; LN - lymph nodes. Data are representative of 2 independent experiments.

Figure 4 - ATX-GD-59 treatment reduces TSHR-specific splenocyte proliferation in Ad-TSHR-immunized DR3tg mice

DR3tg mice (n=11/group) were injected subcutaneously in the flank with 22.5 pmol, 225 pmol and 2250 pmol of ATX-GD-59 or control on days -15, -13 and -11, followed by 3 injections of 22.5 nmol/peptide ATX-GD-59 or control on days -8, -6, and -4 (dose escalation schedule). Mice were then injected 2 times intramuscularly with 10 ¹⁰ Ad-TSHR or Ad-LacZ at intervals of three weeks (day 0 and 21). The experiment was terminated 5 weeks after the first immunization and the spleen was taken for evaluation of TSHR-specific proliferation. Data are mean ± standard error of the mean (SEM) of absolute pulses per minute (ppm; left graph) or stimulation index (SI; right graph) for mice treated with control (red lines) and mice treated with peptide (blue lines). lines). A two-way ANOVA was used to evaluate the overall effects of treatment on T cell proliferation and p-values are plotted. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). The overall mean percent reduction in T cell proliferation induced by peptide treatment is shown in the graph. Data are representative of 2 independent experiments.

Figure 5 - ATX-GD-59 prophylactic treatment reduces anti-TSHR antibody levels

DR3tg mice were injected subcutaneously in the flank with 22.5 pmol, 225 pmol and 2250 pmol of ATX-GD-59 (n=11) or control (n=11) on days -15, -13 and -11, followed by 3 injections 22.5 nmol of each ATX-GD-59 peptide or control on days -8, -6 and -4 (dose escalation schedule). Mice were then injected 2 times intramuscularly with 10 ¹⁰ Ad-TSHR or Ad-LacZ (n=7) at three week intervals (day 0 and 21). Blood was taken before treatment and 2 and 5 weeks after the first immunization to assess the total levels of anti-TSHR IgG using ELISA. Each dot means data for one mouse and group mean ± SEM. One-way ANOVA was used to evaluate overall differences in anti-TSHR IgG levels. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). Data are representative of 2 independent experiments.

Figure 6 - ATX-GD-59 prophylactic treatment reduces levels of anti-TSHR antibodies of various isotypes

DR3tg mice were injected subcutaneously in the flank with 22.5 pmol, 225 pmol and 2250 pmol of ATX-GD-59 (n=11) or control (n=11) on days -15, -13 and -11, followed by 3 injections 22.5 nmol of each ATX-GD-59 peptide or control on days -8, -6 and -4 (dose escalation schedule). Mice were then injected intramuscularly with 10 ¹⁰ Ad-TSHR or Ad-LacZ (n=7) 2 times intramuscularly at intervals of three weeks (day 0 and 3). Blood was taken prior to treatment and 2 and 5 weeks after the first immunization, and at week 2, isotype specific anti-TSHR IgG levels were assessed by ELISA. Each dot means data for one mouse and group mean ± SEM. One-way ANOVA was used to evaluate overall differences in anti-TSHR IgG levels. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). Data are representative of 2 independent experiments.

Figure 7 - Stimulating anti-TSHR antibody levels in Ad-TSHR-immunized DR3tg mice

DR3tg mice were injected subcutaneously in the flank with 22.5 pmol, 225 pmol and 2250 pmol of ATX-GD-59 (n=11) or control (n=11) on days -15, -13 and -11, followed by 3 injections 22.5 nmol of each ATX-GD-59 peptide or control on days -8, -6 and -4 (dose escalation schedule). Mice were then injected intramuscularly with 10 ¹⁰ Ad-TSHR or Ad-LacZ (n=7) 2 times intramuscularly at intervals of three weeks (day 0 and 3). Serum was taken 5 weeks after the first immunization and evaluated in a CHO cell assay. Each point represents data for one mouse at week 5, and the mean ± SEM is presented for each group. The Mann-Whitney test was used to assess differences in levels of stimulating anti-TSHR antibodies, and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001). The results are representative of more than 3 independent experiments. RLU - relative units of radiation (radiation emitted by stimulated cells / radiation emitted by unstimulated cells).

Figure 8 - General design of the study on combination therapy with MMI and ATX-GD-459

Mice were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol of each ATX-GD-459 peptide or control peptide on days -15, -13 and -11, and then received 3 injections of 15 pmol of each ATX-GD-459 peptide or control peptide on days -8, -6 and -4 (according to the dose increase scheme). Starting at day -15, mice were also injected with vehicle or methimazole using a subcutaneously implanted osmotic pump. Mice were then injected intramuscularly with 10 ⁹ Ad-TSHR particles 2 times at three week intervals (weeks 0 and 3). Serum was taken 2 weeks later and immediately prior to treatment, and then 2, 4 and 5 weeks after the first immunization. The experiment was terminated 5 weeks after the first immunization and blood and thyroid and spleen samples were taken.

Figure 9 - General design of the study on combination therapy with propranolol and ATX-GD-459

Mice were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol of each ATX-GD-459 peptide or control peptide on days -15, -13 and -11, and then received 3 injections of 15 pmol of each ATX-GD-459 peptide or control peptide on days -8, -6 and -4 (according to the dose increase scheme). Starting at day -15, mice were also given vehicle or propranolol by daily intraperitoneal injections. Mice were then injected intramuscularly with 10 ¹⁰ Ad-TSHR particles 2 times at three week intervals (weeks 0 and 3). Blood was taken 2 weeks later and just prior to treatment, and then 2, 4 and 5 weeks after the first immunization. The experiment was terminated 5 weeks after the first immunization and blood and thyroid and spleen samples were taken.

Figure 10 - Proliferation of TSHR-specific splenocytes in Ad-TSHR-immunized DR3tg mice

DR3tg mice (n=7-10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol ATX-GD-459 or control on days -15, -13 and -11, followed by 3 injections of 15 nmol ATX -GD-459 or control on days -8, -6 and -4 (dose escalation schedule). Starting at day -15, mice were also injected with vehicle (shaded symbols) or MMI (open symbols) using subcutaneously implanted osmotic pumps. Mice were then injected intramuscularly with 10 ⁹ Ad-TSHR 2 times at three week intervals (days 0 and 3). The experiment was terminated 5 weeks after the first immunization and the spleen was taken for evaluation of TSHR-specific proliferation. Data are the mean ± standard error of the mean (SEM) of absolute pulses per minute (ppm; left plot), stimulation index (SI; middle plot), or corrected pulses ((Δppm; right plot). of overall treatment effects on T cell proliferation, two-way ANOVA and post-hoc test with Bonferroni correction were used.Significant differences are indicated in the text.

Figure 11 - Treatment with ATX-GD-459 but not MMI reduces levels of anti-TSHR IgG antibody

DR3tg mice (n=7-10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol of each ATX-GD-459 peptide or control peptide on days -15, -13 and -11, followed by 3 injections 15 nmol of each ATX-GD-459 peptide or control peptide on days -8, -6 and -4 (in dosing escalation schedule). Starting at day -15, mice were also injected with vehicle or MMI using a subcutaneously implanted osmotic pump. Mice were then injected intramuscularly with Ad-TSHR 2 times at three week intervals (weeks 0 and 3). The experiment was terminated 5 weeks after the first immunization. Blood was taken before treatment and 2, 4 and 5 weeks after the first immunization and then anti-TSHR IgG levels were assessed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. One-way ANOVA was used to evaluate overall differences in anti-TSHR IgG levels. Bonferroni's post-hoc test was applied and significant differences, if any, are plotted (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001).

Figure 12 - Treatment with ATX-GD-459 but not MMI reduces anti-TSHR levels of IgG2b and IgG2c antibodies

DR3tg mice (n=7-10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol of each ATX-GD-459 peptide or control peptide on days -15, -13 and -11, followed by 3 injections 15 nmol of each ATX-GD-459 peptide or control peptide on days -8, -6 and -4 (in dosing escalation schedule). Starting at day -15, mice were also injected with vehicle or MMI using a subcutaneously implanted osmotic pump. Mice were then injected intramuscularly with Ad-TSHR 2 times at three week intervals (weeks 0 and 3). The experiment was terminated 5 weeks after the first immunization. Blood was taken before treatment and 2, 4 and 5 weeks after the first immunization and then anti-TSHR IgG levels were assessed by ELISA. Each point represents data for one mouse at week 2, and the mean ± SEM is presented for each group. One-way ANOVA was used to assess overall differences in anti-TSHR IgG1, IgG2b, and IgG2c levels. Bonferroni's post-hoc test was applied and significant differences, if any, are plotted (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001).

Figure 13 - MMI treatment significantly reduces serum T4 levels

DR3tg mice (n=7-10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol of each ATX-GD-459 peptide or control peptide on days -15, -13 and -11, followed by 3 injections 15 nmol of each ATX-GD-459 peptide or control peptide on days -8, -6 and -4 (in dosing escalation schedule). Starting at day -15, mice were also injected with vehicle or MMI using a subcutaneously implanted osmotic pump. Mice were then injected intramuscularly with Ad-TSHR 2 times at three week intervals (weeks 0 and 3). The experiment was terminated 5 weeks after the first immunization. Blood was taken before treatment and 2, 4 and 5 weeks after the first immunization, and then assessed the levels of T4 using ELISA. Each point represents data for one mouse at week 2, and the mean ± SEM is presented for each group. A one-way ANOVA was used to assess overall differences in T4 levels. Bonferroni's post-hoc test was applied and significant differences, if any, are plotted (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001).

Figure 14 - Proliferation of TSHR-specific splenocytes in Ad-TSHR-immunized DR3tg mice

DR3tg mice (n=10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol of ATXGD-459 or control on days -15, -13 and -11, followed by 3 injections of 15 nmol/ATX-GD- 459 or controls on days -8, -6, and -4 (dose escalation schedule). Starting at day -15, mice were also treated with vehicle (shaded symbols) or propranolol (open symbols) via daily intraperitoneal injections. Mice were then injected intramuscularly with 10 ¹⁰ Ad-TSHR 2 times at three week intervals (days 0 and 3). The experiment was terminated 5 weeks after the first immunization and the spleen was taken for evaluation of TSHR-specific proliferation. Data are the mean ± standard error of the mean (SEM) of absolute pulses per minute (ppm; left plot), stimulation index (SI; middle plot), or corrected pulses (Δppm; right plot). A two-way ANOVA and Bonferroni-adjusted post-hoc test were used to evaluate the overall effects of treatment on T cell proliferation. Significant differences are indicated in the text.

Figure 15 - Treatment with ATX-GD-459 but not propranolol reduces levels of anti-TSHR IgG antibody

DR3tg mice (n=10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol ATX-GD-459 or control on days -15, -13 and -11, followed by 3 injections of 15 nmol ATX-GD -459 or controls on days -8, -6, and -4 (dose escalation schedule). Starting at day -15, mice were also treated with vehicle (shaded symbols) or propranolol (open symbols) via daily intraperitoneal injections. Mice were then injected intramuscularly with 10 ¹⁰ Ad-TSHR 2 times at three week intervals (days 0 and 3). The experiment was terminated 5 weeks after the first immunization. Blood was taken before treatment and 2, 4 and 5 weeks after the first immunization and then anti-TSHR IgG levels were assessed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. One-way ANOVA was used to evaluate overall differences in anti-TSHR IgG levels. Bonferroni's post-hoc test was applied and significant differences, if any, are plotted (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001).

Figure 16 - Treatment with ATX-GD-459 but not propranolol reduces anti-TSHR levels of IgG2b and IgG2c antibodies

DR3tg mice (n=10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol ATX-GD-459 or control on days -15, -13 and -11, followed by 3 injections of 15 nmol ATX-GD -459 or controls on days -8, -6, and -4 (dose escalation schedule). Starting at day -15, mice were also treated with vehicle (shaded symbols) or propranolol (open symbols) via daily intraperitoneal injections. Mice were then injected intramuscularly with 10 ¹⁰ Ad-TSHR 2 times at three week intervals (days 0 and 3). The experiment was terminated 5 weeks after the first immunization. Blood was taken before treatment and 2, 4 and 5 weeks after the first immunization and then anti-TSHR IgG levels were assessed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. One-way ANOVA was used to assess overall differences in anti-TSHR IgG1, IgG2b, and IgG2c levels. Bonferroni's post-hoc test was applied and significant differences, if any, are plotted (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001).

Figure 17 - Administration of propranolol or ATX-GD-459 had no effect on total T4 levels

DR3tg mice (n=10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol ATX-GD-459 or control on days -15, -13 and -11, followed by 3 injections of 15 nmol ATX-GD -459 or controls on days -8, -6, and -4 (dose escalation schedule). Starting at day -15, mice were also treated with vehicle (shaded symbols) or propranolol (open symbols) via daily intraperitoneal injections. Mice were then injected intramuscularly with 10 ¹⁰ Ad-TSHR 2 times at three week intervals (days 0 and 3). The experiment was terminated 5 weeks after the first immunization. Blood was taken before treatment and 2, 4 and 5 weeks after the first immunization and then anti-TSHR IgG levels were assessed by ELISA. Each point represents data for one mouse at week 5, and the mean ± SEM is presented for each group. A one-way ANOVA was used to assess overall differences in T4 levels. Bonferroni's post-hoc test was applied and significant differences, if any, are plotted (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001).

Figure 18 - Plasma propranolol levels confirmed the pharmacologic dose

Plasma propranolol levels were assessed in control mice and in mice treated with peptides and propranolol. Plasma was analyzed at weeks 0 and 5 and the results were recorded.

Figure 19 - Schematic protocol for T cell tolerization

Mice were injected subcutaneously in the flank with 0.1 μg, 1 μg and 10 μg of the peptide on days -15, -13 and -11, and then received 3 injections of the maximum dose of the peptide (one ATX-GD-459 peptide or cocktail) on days -8 , -6 and -4 (according to the scheme of increasing doses). Maximum doses and correspondingly higher doses may differ in different experiments. On day 0, mice were immunized subcutaneously at the base of the tail with a peptide emulsified in complete Freund's adjuvant (CFA). The experiment was terminated 10 days after immunization to assess the proliferation of lymph node (LN) cells and splenocytes after re-stimulation with TSHR or peptide.

Figure 20 - ATX-GD-459 treatment effectively reduces TSHR-induced proliferation in HLA-DR3 mice

(A) HLA-DR4 mice were pretreated with RNB-4K-GKK or PBS (n=10/group) in a dose escalation schedule up to a maximum dose of 100 μg; (B) HLA-DR3 mice were pretreated with RNB-5D-K1 or HIP-16E (HLA-DR3 binding control peptide) (n=10/group) in a dose escalation schedule up to a maximum dose of 100 μg; (C) HLA-DR3 mice were pretreated with RNB-9B or PBS (n=10/group) in a dose escalation scheme up to a maximum dose of 33 μg. Animals were immunized with the parent peptide in CFA, and after 10 days LN and spleen were taken to assess TSHR-specific proliferation. Data are the mean ± standard error of the mean (SEM) of stimulation index (SI) values for mice treated with control and mice treated with peptide. A two-way ANOVA was used to evaluate the overall effects of treatment on T cell proliferation and p-values are plotted. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). The mean percent reduction in T cell proliferation induced by peptide treatment is shown in the graph. SI - stimulation index; LN - lymph nodes.

(D) HLA-DR3 mice were pretreated with ATX-GD-459 or PBS (n=10/group) in a dose escalation scheme up to a maximum dose of 75 μg for each peptide. Animals were immunized with 4K/5D/9B emulsified in CAF and LN and spleen were taken 10 days later to assess TSHR-specific proliferation. Data are the mean ± standard error of the mean (SEM) of stimulation index (SI) values for mice treated with control and mice treated with peptide. A two-way ANOVA was used to evaluate the overall effects of treatment on T cell proliferation and p-values are plotted. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). The mean percent reduction in T cell proliferation induced by peptide treatment is shown in the graph. SI - stimulation index; LN - lymph nodes.

Figure 21 - Schematic description of the model of adenoviral Graves' disease and its confirmation

(A) Mice were immunized intramuscularly with adenovirus vectors expressing the TSHR-A subunit (Ad-TSHR) or β-galactosidase (Ad-LacZ). 10 ¹⁰ adenoviral particles were injected twice with an interval of 3 weeks. Protocol deviations for this experiment are also indicated. Blood was taken before immunization and 2 and 4 weeks after the first immunization. The experiment was terminated 4 weeks after the first immunization and blood, spleen and thyroid were taken for examination for the presence of a symptom similar to Graves' disease. (B) Mice were immunized twice with Ad-TSHR 3 weeks apart (weeks 0 and 3). Treatment was started by subcutaneous injection of the pump on the day of the first immunization and continued for 4 weeks until the end of the experiment. Blood was taken before immunization and 2 and 4 weeks after the first immunization. Mice were euthanized 4 weeks after the first immunization and blood and spleen samples were taken. (C) Mice were injected subcutaneously in the flank with 0.1 μg, 1 μg and 10 μg of the peptide on days -15, -13 and -11, and then received 3 injections of 100 μg of the peptide on days -8, -6 and -4 (by dose escalation schedule). Mice were then injected intramuscularly with Ad-TSHR or Ad-LacZ twice 3 weeks apart (weeks 0 and 3). Blood was taken before immunization and 2 and 5 weeks after the first immunization. Mice were euthanized 5 weeks after the first immunization and blood and spleen samples were taken.

Figure 22 - Increase in T4 and anti-TSHR IgG levels over time in BALB/c mice after Ad-TSHR immunization

BALB/c mice (n=6-7/group) were intramuscularly immunized with 10 ¹⁰ or 10 ¹¹ adenoviral vectors expressing the TSHR-A subunit (Ad-TSHR) or β-galactosidase (Ad-LacZ). 10 ¹⁰ or 10 ¹¹ adenoviral particles were injected three times with an interval of 3 weeks (weeks 0, 3 and 6). Serum was taken before immunization and 4 and 10 weeks after the first immunization. (A) T4 levels were analyzed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. The number of hyperthyroid mice is indicated in the graphs. In accordance with published data, the threshold for hyperthyroidism is defined as the mean ± 2 standard deviations from normal for mouse T4 levels in Ad-LacZ-immunized control mice. (B) Anti-TSHR IgG levels were analyzed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. One-way ANOVA was used to evaluate overall differences in anti-TSHR IgG levels. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001).

Figure 23 - Increase in T4 titers, stimulatory anti-TSHR antibody titers and anti-TSHR antibody titers over time in DR3tg mice after Ad-TSHR immunization

(A) DR3tg mice were intramuscularly immunized with 10 ⁹ adenoviral vectors expressing the TSHR-A subunit (Ad-TSHR) (n=10) or β-galactosidase (Ad-LacZ) (n=12). Adenovirus particles were injected 2 times with an interval of 3 weeks (weeks 0 and 3). Serum was taken before immunization and 2 and 4 weeks after the first immunization and T4 levels were assessed by ELISA. Each point means data for one mouse at week 4 and group mean ± SEM. The Mann-Whitney test was used to assess differences in T4 levels, and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001) . The results are representative of more than 3 independent experiments.

(B) DR3tg mice (n=10/group) were intramuscularly immunized with 10 ⁹ adenoviral vectors expressing the TSHR-A subunit (Ad-TSHR) or β-galactosidase (Ad-LacZ). Adenovirus particles were injected twice with an interval of 3 weeks (weeks 0 and 3). Serum was taken before immunization and 2 and 4 weeks after the first immunization and T4 levels were assessed in the CHO cell luciferase reporter gene assay. Each point means data for one mouse at week 4 and group mean ± SEM. The Mann-Whitney test was used to evaluate differences in levels of stimulating anti-TSHR antibodies, and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p< 0.0001). The results are representative of more than 3 independent experiments. RLU - relative units of radiation (radiation emitted by stimulated cells / radiation emitted by unstimulated cells).

(C) BALB/c mice (n=10; left panel) and DR3tg mice (n=7; right panel) were intramuscularly immunized with 10 ¹⁰ adenoviral vectors expressing the TSHR-A subunit (Ad-TSHR). Adenovirus particles were injected twice with an interval of 3 weeks (weeks 0, 3 and 6). Serum was taken before immunization and 4, 7 and 10 weeks after the first immunization and anti-TSHR IgG levels were assessed by ELISA. Each dot means data for one mouse and group mean ± SEM. One-way ANOVA was used to assess overall differences in stimulatory anti-TSHR IgG levels, and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0 ,0001).

(D) DR3tg mice were intramuscularly immunized with 10 ¹⁰ or 10 ¹¹ adenoviral vectors expressing the TSHR-A subunit (Ad-TSHR) or 10 ¹⁰ adenoviral vectors expressing β-galactosidase (Ad-LacZ). Adenovirus particles were injected three times with an interval of 3 weeks (weeks 0, 3 and 6). After 10 weeks, the spleen was taken and TSHR-induced splenocyte proliferation was assessed by incorporation of tritium-treated thymidine. Results are presented as mean ± SEM per group. Cpm - number of pulses per minute; SI - stimulation index.

Figure 24 - Effect of methimazole treatment on T4 and anti-TSHR antibody levels in DR3tg mice after Ad-TSHR immunization

DR3tg mice (n=7-8/group) were immunized with Ad-TSHR at week 0 and week 3. Treatment with methimazole (50 or 500 μg/mouse/day) or vehicle treatment was performed by insertion of a subcutaneous pump starting from the day of the first immunization, and continued for 4 weeks until the end of the experiment. Serum was taken before immunization and 2 and 4 weeks after the first immunization. (A) T4 levels were analyzed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. A one-way ANOVA was used to assess overall differences in T4 levels. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001). (B) Anti-TSHR IgG levels were analyzed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. One-way ANOVA was used to evaluate overall differences in anti-TSHR IgG levels. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001).

Figure 25 - Anti-TSHR IgG levels decrease after treatment with methylprednisolone

DR3tg mice (n=10/group) were immunized with 10 ¹⁰ Ad-TSHR at week 0 and week 3. Treatment with methylprednisolone (Mpred) (7 mg/kg/day) or vehicle treatment was performed by insertion of a subcutaneous pump starting from day 3 to first immunization, and continued for 4 weeks until the end of the experiment. Serum was taken before immunization and 2 and 4 weeks after the first immunization and anti-TSHR IgG levels were assessed by ELISA. Each dot means data for one mouse, and the mean ± SEM is presented for each group. The Mann-Whitney test was used to assess differences in anti-TSHR IgG levels, and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0 ,0001).

Figure 26 - ATX-GD-459 treatment reduced levels of anti-TSHR antibodies

DR3tg mice (n=10/group) were injected subcutaneously in the flank with 0.1 μg, 1 μg and 10 μg of ATX-GD-459 or control on days -15, -13 and -11, followed by 3 injections of 100 μg of ATX -GD-459 or control on days -8, -6 and -4 (dose escalation schedule). Mice were then injected intramuscularly with Ad-TSHR or Ad-LacZ 2 times at intervals of three weeks (days 0 and 3). Blood was taken before treatment and 2 and 5 weeks after the first immunization to assess the total levels of anti-TSHR IgG using ELISA. Each point represents data for one mouse at week 2, and the mean ± SEM is presented for each group. One-way ANOVA was used to evaluate overall differences in anti-TSHR IgG levels. Bonferroni's post-hoc test was applied and significant differences are shown in the graphs (*p<0.05;**p<0.01;***p<0.001:****p<0.0001).

Figure 27 - APIPS analysis showed that hybridoma clones respond to RNB-5D-K1 and RNB-4K-GKK

5×10 ⁴ T-cell hybridoma clones specific for RNB-5 (clone 50+35) or RNB-4 (clone 164+455) and TSHR were cultured in the presence of TSHR and RNB-5D-K1 or RNB-4K-GKK in combination with 5×10 ⁴ human DR3 expressing antigen presenting B cells (VAVY) (clone 50) or DR4 expressing antigen presenting B cells (BM14) (clone 35, 164, 455). Antigen presenting cells (APCs) were viable or pre-fixed with 0.5% paraformaldehyde before being added to the culture wells. After co-culture for 48 hours, supernatants were collected and analyzed by ELISA to assess levels of IL-2 secretion. The results are presented for representative clones.

Detailed description of the invention

The present invention relates to a new and alternative therapeutic agent for preventing or suppressing the production of anti-TSHR autoantibodies, which can be used to treat and/or prevent Graves' disease. As demonstrated in Example 1 of the present invention, the combination of peptides of SEQ ID NO: 1 and 2 conferred TSHR tolerance in a Graves' disease model.

Thus, in its first aspect, the present invention provides a composition comprising a plurality of peptides derived from TSHR, namely the peptides of the invention as defined herein, preferably the peptides of SEQ ID NOs: 1 and 2.

thyroid stimulating hormone receptor

Graves' disease is an autoimmune disease caused by autoreactive T and B lymphocytes targeting a primary autoantigen, thyroid stimulating hormone receptor (TSHR).

TSHR is a G protein-coupled receptor on thyroid follicular cells in the thyroid gland that stimulates the production of thyroxine (T4) and triiodothyronine (T3) through the cAMP signal transduction cascade after its binding to a ligand, i.e., thyroid stimulating hormone ( TSH). After internalization, degradation, and presentation of TSHR by APC, T cells become activated and interact with autoreactive B cells, which in turn produce excitatory agonist autoantibodies against TSHR. Thyroid-stimulating immunoglobulins bind to the same receptor pocket as TSH, activating TSHR-mediated signaling, resulting in excess thyroid hormone production from the thyroid gland and enlargement of the thyroid gland.

TSHR, also known as thyrotropin receptor, is expressed primarily on thyroid epithelial cells.

The TSHR holoreceptor has 764 residues and contains an N-terminal extracellular domain to which TSH binds; serpentine (or transmembrane domain) and C-terminal intracellular domain.

TSHR includes a large extracellular domain (418 amino acids) with highly conserved Cys residues that facilitate the formation of a tertiary extracellular domain structure that may play an important role in ligand binding and in the formation of the inactive receptor conformation. The extracellular domain comprises more than half the length of the total protein and is sufficient for high affinity binding to the ligand. The receptor molecule, after its transport to the cell surface, undergoes intramolecular cleavage, which leads to the removal of the 50 amino acid sequence located between residues 316 and 366. As a result, the receptor contains two subunits, an α-subunit, including an extracellular ligand-binding domain, and a β-subunit comprising a transmembrane domain and a short C-terminal sequence which, when linked together, form disulfide bonds. In subsequent steps, the α-subunit is removed, resulting in the formation of an excess of the β-subunit-free ligand-binding domain on the cell membrane.

After TSH binds to TSHR in the circulation, the G-protein signaling cascade activates adenylyl cyclase, resulting in increased levels of intercellular cAMP. cAMP activates all functional properties of thyroid cells, including iodine pumping, thyroglobulin synthesis, iodination, endocytosis and proteolysis, thyroid peroxidase activity, and hormone release.

The amino acid sequence of mature TSHR is shown below (SEQ ID No. 3).

1 mrpadllqlv llldlprdlg gmgcssppce chqeedfrvt ckdiqripsl ppstqtlkli

61 ethlrtipsh afsnlpnisr iyvsidvtlq qleshsfynl skvthieirn trnltyidpd

121 alkelpllkf lgifntglkm fpdltkvyst diffileitd npymtsipvn afqglcnetl

181 tlklynngft svqgyafngt kldavylnkn kyltvidkda fggvysgpsl ldvsqtsvta

241 lpskglehlk eliarntwtl kklplslsfl hltradlsyp shccafknqk kirgileslm

301 cnessmqslr qrksvnalns plhqeyeenl gdsivgykek skfqdthnna hyyvffeeqe

361 deiigfgqel knpqeetlqa fdshydytic gdsedmvctp ksdefnpced imgykflriv

421 vwfvsllall gnvfvllill tshyklnvpr flmcnlafad fcmgmyllli asvdlythse

481 yynhaidwqt gpgcntagff tvfaselsvy tltvitlerw yaitfamrld rkirlrhaca

541 imvggwvccf llallplvgi ssyakvsicl pmdtetplal ayivfvltln ivafvivccc

601 yvkiyitvrn pqynpgdkdt kiakrmavli ftdficmapi sfyalsailn kplitvsnsk

661 illvlfypln scanpflyai ftkafqrdvf illskfgick rqaqayrgqr vppknstdiq

721 vqkvthdmrq glhnmedvye lienshltpk kqgqiseeym qtvl

Tolerance

T cell epitopes play a central role in generating an adaptive immune response to any antigen, whether self or foreign. The central role played by T-cell epitopes in the development of hypersensitivity reactions (including allergies, autoimmune diseases and transplant rejection) has been demonstrated in experimental models. Inflammatory or allergic reactions can be induced by injection of synthetic peptides (derived from the structure of T cell epitopes) in combination with an adjuvant.

In contrast, it has been shown that immunogenic tolerance to specific antigens can be induced by administering peptide epitopes in soluble form. The administration of a soluble peptide has been shown to be effective in inhibiting the disease in an experimental autoimmune encephalomyelitis (EAE - multiple sclerosis (MS) model) model (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 in experimental models with arthritis, diabetes, and uveoretinitis (see Anderton and Wraith (1998) supra). This agent has also been demonstrated as a treatment for EAE-associated disease (see Anderton and Wraith (1998) supra).

Tolerance is the inability of the immune system to respond to an antigen. Tolerance to self antigens is an important feature of the immune system; and when it is lost, an autoimmune disease can occur. The adaptive immune system must maintain the body's ability to respond to a vast array of infectious agents, but must not carry out an autoimmune attack on its own antigens contained in its own tissues. This is highly regulated by the sensitivity of immature T-lymphocytes to apoptotic cell death in the thymus (central tolerance). However, not all self antigens can be detected in the thymus, and therefore not all autoreactive thymocytes die. Thus, there are also mechanisms by which tolerance can be acquired by mature autoreactive T-lymphocytes in peripheral tissues (peripheral tolerance). For a description of the mechanisms of central and peripheral tolerance, see Anderton et al. (1999) (Immunological Reviews 169:123-137).

It is now known that Graves' disease is caused by stimulatory anti-TSHR autoantibodies that bind to and activate TSHR, resulting in stimulation of thyroid hormone synthesis and secretion and enlargement of the thyroid gland. The composition according to the invention has the ability to induce tolerance to TSHR, so that when administered to an individual, it can restore tolerance to the TSHR autoprotein and reduce the pathogenic immune response.

Apitopes

In an adaptive immune response, T-lymphocytes have the ability to recognize internal epitopes of a protein antigen. APCs absorb protein antigens and decompose them into short peptide fragments. The peptide can bind to a major histocompatibility complex molecule present inside cells and on the cell surface. When presented on the cell surface in combination with the MHC model, the peptide can be recognized by the T cell (via the T cell receptor (TCR), in which case, the peptide is a T cell epitope.

Thus, an epitope is a peptide derived from an antigen that binds to the peptide-binding groove of the MHC molecule and is recognized by the T cell.

A minimal epitope is the shortest fragment derived from an epitope that binds to the peptide-binding groove of an MHC molecule and is recognized by a T cell. For a given immunogenic region, a "nested series" of overlapping peptides is usually prepared that act as epitopes, and all of these epitopes contain a minimal epitope but differ in their flanking regions.

The same trait can identify the minimum epitope for a particular MHC molecule: T cell combination by evaluating the response to truncated peptides. So, for example, if a response is observed to a peptide containing residues 1-15 in an overlapping library, then a series truncated at both ends (i.e., 1-14, 1-13, 1-12, etc.) can be used to identify the minimum epitope. .p and 2-15, 3-15, 4-15, etc.).

The present inventors have already determined that there is a relationship between the ability of a peptide to bind to an MHC molecule and its presentation to a T cell without additional processing, and the ability of this peptide to induce tolerance in vivo (WO 02/16410). If a peptide is too long to bind to the peptide-binding groove of the MHC molecule without additional processing (eg, truncation), or binds in an inappropriate conformation, then it may not be tolerogenic in vivo . On the other hand, if the peptide is of the appropriate size and conformation to directly bind to the MHC peptide-binding groove and is presented to a T cell, then that peptide can be used to induce tolerance.

Thus, the tolerogenic ability of a peptide can be assessed independently of its ability to bind to an MHC molecule and present it to a T cell without additional in vitro antigen processing.

TSHR apitopes (antigen processing independent epitopes) are able to bind to the MHC molecule and stimulate a TSHR-specific cell response without any additional antigen processing. Such apitopes that confer TSHR tolerance can be predicted by a method according to the rules described in WO 02/16410.

Peptides that bind to MHC class I molecules are typically 7-13, and most commonly 8-10 amino acids in length. Peptide binding is stabilized at both ends through contacts between atoms in the peptide main chain and in invariant sites of the peptide-binding groove of all MHC class I molecules. At both ends of the groove, there are invariant sites that bind to the amino and carboxy ends of the peptide. Changes in peptide length can be made by creating a loop in the peptide backbone, often in proline or glycine residues, to provide flexibility.

Peptides that bind to class II MHC molecules are typically 8 to 20 amino acids in length, and most commonly 10 to 17 amino acids or more (eg, up to 40 amino acids). These peptides have an extended conformation throughout the groove that binds to the MHC II peptide (as opposed to the groove that binds to the MHC I peptide) and are "open" at both ends. The peptide is held in a certain position, mainly due to the contacts of the main chain atom with conservative residues that make up the peptide-binding groove.

In a preferred embodiment of the invention, the peptide has the ability to bind to an MHC class II molecule without additional processing.

Peptide

The term "peptide", used in the conventional sense, means a series of usually L-amino acid residues connected to each other by peptide bonds, often located between the α-amino and carboxyl groups of adjacent amino acids. This term includes modified peptides and synthetic peptide analogs.

Peptides can be obtained by chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin). For example, peptides can be synthesized by solid phase methods (Roberge J.Y. et al. (1995) Science 269: 202-204), cleaved from the resin and purified using preparative high performance liquid chromatography (e.g. Creighton (1983) Proteins Structures And Molecular Principles , W. H. Freeman and Co, New York, NY). Automatic synthesis can be carried out, for example, on an ABI 431 A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

Alternatively, the peptide may be obtained by recombinant methods or by cleavage from a longer polypeptide. For example, a peptide can be obtained by cleaving a thyrotropin receptor protein from a protein, followed by modification at one or both ends. The composition of the peptide can be confirmed by amino acid analysis or sequencing (eg, according to the Edman degradation procedure).

The following peptides are used in the present invention:

(i) all or part of the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or part thereof, or a sequence that is at least 60% identical to the sequence of SEQ ID NO: 1; and

(ii) all or part of the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or part thereof, or a sequence that is at least 60% identical to the sequence of SEQ ID NO: 2.

The peptide of the invention may comprise or consist of an amino acid sequence that is 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% identical to the peptide of SEQ ID NN: 1 or 2. B in one aspect of the invention, the peptide has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the sequence of SEQ ID NN:1 or 2.

In a preferred aspect of the invention, the peptides comprise SEQ ID NN: 1 and 2. In another preferred aspect of the invention, the peptides consist of SEQ ID NN: 1 and 2.

Sequence identity can be assessed by any standard method. However, to determine the degree of sequence identity, there are suitable computer programs for matching multiple sequences, for example, Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22: 4673-4680). For this purpose, programs for comparing and aligning pairs of sequences can be used, such as ALIGN (Myers ef al., (1988) CABIOS, 4: 1-17), FASTA (Pearson er al., (1988) PNAS, 85:2444 -2448; Pearson (1990), Methods Enzymol., 183: 63-98) and BLAST with gaps (Altschul er al., (1997) Nucleic Acids Res., 25: 3389-3402). In addition, the European Bioinformatics Institute has a Dali server to align protein sequences based on their structure (Holm (1993) J. Mol. Biol., 233: 123-38; Holm (1995) Trends Biochem. Sci., 20: 478- 480; Holm (1998) Nucleic Acid Res. 26: 316-9).

Aligning multiple sequences and calculating their percent identity can be done using standard BLAST parameters (using sequences derived from all available organisms; Blosum 62 matrices, gap costs: presence 11, extension 1).

Alternatively, the following program and options can be used: Program: Align Plus 4, version 4.10 (Sci Ed Central Clone Manager Professional Suite). DNA Comparison: General Comparison, Standard Linear Score Matrix, Mismatch Penalty=2, Gap Penalty=4, Extension Penalty=1. Amino Acid Comparison: General Comparison, BLOSUM 62 Scoring Matrix.

Variants with or with these sequences are within the scope of the present invention, provided that these variants will retain the functional activity of the parent molecule, that is, these variants are functionally equivalent or, in other words, have or exhibit the activity of the parent peptide as defined in this application. Such variants may have amino acid substitutions, additions, or deletions (including truncations at one or both ends) of the parent sequence, eg, one or more amino acids, eg, 1 to 14 amino acids.

Also included are functionally equivalent derivatives in which one or more amino acids are chemically derivatized, eg, substituted with a chemical group.

Thus, the peptides according to the invention may include parts or fragments of SEQ ID NN:1-3, provided that they will retain the desired activity. Fragments or portions of SEQ ID NN:1-3 may, for example, be 6 to 14 residues in length, such as 6, 7, 8, 9, 10, 11, 12 or 13 residues.

The peptide according to the invention may contain from 8 to 30 amino acids, for example 8-25 amino acids, 8-20 amino acids, 8-15 amino acids or 8-12 amino acids. In one aspect of the invention, the peptide according to the invention may have a length of 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.

The TSHR peptides may be in the form of a composition, and preferably a pharmaceutical composition.

The TSHR peptides may be in the form of a composition prepared in neutral or salt form. Pharmaceutically acceptable salts are acid addition salts (formed by the free amino groups of the peptide) and such salts are formed by inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric and maleic acids. Salts formed by free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and procaine.

Combination

The inventors of the present invention have surprisingly found that the peptides and/or composition of the invention can be administered in combination with other therapeutic agents used in the treatment or therapy of Graves' disease while maintaining the therapeutic effects of the peptides/composition of the invention and the other therapeutic agent.

Thus, for example, in one aspect of the invention, the peptides or composition of the invention may be combined with another therapeutic agent used to treat or prevent Graves' disease.

For example, the peptides or composition may be combined with an antithyroid agent or with a β-blocker.

As shown in Example 2, the administration of the peptide of SEQ ID NO:1 and SEQ ID NO:2 in combination with an antithyroid agent does not reduce the level of anti-TSHR antibody production by the TSHR peptides and does not reduce the antithyroid effect (determined by T4 production), that is, both of these therapeutic effects are preserved. Similar results have also been achieved for the combination of the composition of the invention together with a β-blocker. For example, the authors of the present invention have demonstrated that the peptides according to the invention can be used in combination with existing agents for the treatment and therapy of Graves' disease.

The advantage of the combination according to the invention is that it facilitates the reduction of the autoimmune response in Graves' disease, i.e., the mechanism underlying this disease, and therefore such a combination can be used simultaneously to treat or alleviate the symptoms of Graves' disease.

An "antithyroid agent" is a hormone antagonist acting after thyroid hormones. Antithyroid agents are used to treat Graves' disease. Antithyroid agents are administered to patients with Graves' disease to inhibit the synthesis of thyroid hormones and thereby directly reduce thyroxine (T4) levels.

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

β-blockers are administered to patients with Graves' disease to suppress β-adrenergic responses in hyperthyroidism (without resorting to treatment of the disease with immunological compounds).

β-blockers belong to a class of drugs used to treat cardiac arrhythmias. Beta-blockers block the action of endogenous catecholamines such as epinephrine (adrenaline) and norepinephrine (noradrenaline) - and in particular, the action of adrenergic beta receptors of the sympathetic nervous system.

The β-blockers according to the invention are: propranolol, bucindolol, carteolol, carvediol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol and timolol.

In a preferred embodiment of the invention, the β-blocker is propranolol.

Pharmaceutically acceptable salts of β-blockers may be used. In a preferred embodiment of the invention, the β-blocker is propranolol hydrochloride.

Suitable doses of the antithyroid agent and β-blocker can be determined by the skilled person. Thus, for example, the antithyroid agent may be administered at a dose of 10 to 1000 micrograms, such as 20 to 900, 30 to 800, 40 to 700, 50 to 600, 60 to 500, 70 to 400, 80 to 300 or 90 to 200 mcg. In a preferred embodiment of the invention, the dose administered is from 300 to 700 micrograms, for example about 500 micrograms, and preferably 500 micrograms.

In a preferred embodiment of the invention, the antithyroid agent is administered daily, for example every 20-28 hours, and preferably every 24 hours.

The antithyroid agent may be administered by any suitable route known to those skilled in the art. In a preferred embodiment of the invention, the antithyroid agent is administered subcutaneously.

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

In a preferred embodiment of the invention, the β-blocker is administered daily, for example every 20-28 hours, and preferably every 24 hours.

The β-blocker may be administered by any suitable route known to those skilled in the art. In a preferred embodiment of the invention, the β-blocker is administered intraperitoneally.

In one of the embodiments of the invention, the peptides or composition according to the invention is administered simultaneously with an antithyroid agent and/or β-blocker. In an alternative embodiment of the invention, the peptides or composition according to the invention and the antithyroid agent and/or β-blocker are administered at different times.

In one aspect of the invention, the individual to whom the peptides/composition are administered (or the individual to whom they are to be administered) has already been administered an antithyroid agent and/or a β-blocker. Thus, for example, the present invention covers the use according to the invention, where such an individual is an individual who is administered or who has previously been administered an antithyroid agent and/or a β-blocker.

In another aspect of the invention, such an individual is an individual who is not administered or who has not previously administered any therapeutic agents for the treatment of Graves' disease.

In one of the aspects of the invention discussed here, the first step includes identifying an individual suffering from Graves' disease or at risk of developing Graves' disease.

The peptides or composition according to the invention may be used prophylactically or therapeutically.

The peptides or composition, when administered prophylactically, may reduce or prevent the development of an immune response to TSHR. The level of the immune response is lower than the response if the patient was not administered the composition. The term "reduction" means a partial reduction in the immune response, for example, 50%, 70%, 80%, or 90%, than the response that would be observed if the composition was not administered to the patient (or than the response that would be observed if if a patient who was not treated in a given period of time). The term "warning" means the absence of any overt immune response to TSHR.

The peptides or composition, when administered prophylactically, can suppress an existing immune response to TSHR. The term "suppression" means a decrease in the level of the present immune response compared to the level before treatment with the peptide or the levels that were observed in the same period of time in an untreated patient.

Treatment with the peptides or composition of the invention may result in a reduction in any or all of the following:

i) anti-TSHR autoantibodies

ii) TSHR-specific CD4 ⁺ T cells

iii) B cells secreting anti-TSHR autoantibodies.

All of these factors can be detected by known methods such as ELISA, flow cytometry, and the like.

Treatment with the peptides or composition according to the invention can also or alternatively induce anergy of TSHR-specific CD4 ⁺ T cells. Anergy can be detected, for example, by subsequent in vitro TSHR stimulation.

Compound

The peptides or composition may be provided as an injectable liquid solution or suspension, while a solid form may also be prepared which, prior to injection, may be dissolved or suspended in a liquid. The drug may also be emulsified, or the peptides may be encapsulated in liposomes. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline (eg, phosphate buffered saline), dextrose, glycerol, ethanol, or the like. and their combinations.

In addition, if necessary, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents and/or pH buffering agents. The buffering salts are phosphate, citrate, acetate. Hydrochloric acid and/or sodium hydroxide can be used to adjust the pH. For stabilization, disaccharides such as sucrose or trehalose can be used.

In the composition, the ratio of peptides (RNB-5D-K1 and RNB-9B) may be approximately 1:1. Alternatively, the relative content of each peptide can be altered, for example, to target a tolerogenic response to a particular subset of autoreactive T cells, or if one peptide has been found to function better than other peptides in particular HLA types.

The peptides or composition, once prepared, can be introduced into a sterile container, which is then sealed and stored at low temperature, for example at 4°C, or they can be lyophilized.

Typically, the peptides or composition are prepared as a lyophilized (freeze-dried) powder. Freeze-drying allows long-term storage in a stabilized form. Lyophilization procedures are well known to those skilled in the art, see eg http://www.devicelink.com/ivdt/archive/97/01/006.html. Before lyophilization, bulking agents such as mannitol, dextran or glycine, or sugars such as trehalose are usually added.

The peptides or composition can be administered in a standard way, for example, orally, intravenously (if they are water soluble), intramuscularly, subcutaneously, sublingually, intranasally, intradermally, or in the form of suppositories or implants (for example, using sustained release molecules).

The peptides or composition may advantageously be administered intranasally, subcutaneously or intradermally.

The method, peptides and composition according to the invention can be used to treat humans. Typically, the physician will determine the actual dose that is most appropriate for a particular individual, and such dose may vary depending on the age, weight and response of the particular patient.

The peptides and composition according to the invention can be used for human treatment. The individual may be suffering from Graves' disease. This individual may have anti-TSHR autoantibodies.

An individual may have an HLA haplotype that is associated with a predisposition to produce inhibitory anti-THSR autoantibodies. This individual may express HLA-DR3 or HLA-DR4. Methods for determining the HLA haplotype in an individual are known to those skilled in the art.

In a preferred embodiment of the invention, a "dose escalation" protocol may be implemented, where multiple doses are administered to a patient in increasing concentrations. Such a method has been used, for example, to administer phospholipase A2 peptides for immunotherapeutic purposes in the treatment of bee venom allergy (Müller et al (1998) J. Allergy Clin Immunol. 101:747-754 and Akdis et al (1998) J. Clin. Invest. 102:98-106).

Kit

Typically, the two TSHR peptides may be administered together in the form of a mixed formulation or cocktail. However, in some cases, preferably, the peptides are obtained separately in the form of a kit for simultaneous, separate, sequential or combined administration.

For example, the kit may include two peptides contained in separate containers. The contents of these containers may or may not be combined prior to administration.

The kit may also include a means for mixing and/or administration (eg, a steam inhaler for intranasal administration; or a syringe and needle for subcutaneous/intradermal dosing). The kit may also include instructions for its use.

The pharmaceutical composition or kit according to the invention can be used for the treatment and/or prevention of a disease.

In particular, the composition/kit can be used to suppress or prevent the production of anti-TSHR autoantibodies in vivo. The composition/kit may be used to treat and/or prevent Graves' disease in an individual.

animal model

Current animal models of Graves' disease have a number of disadvantages. For example, currently used animal models do not develop the symptoms or signs of Graves' disease. In addition, suitable animal models, such as BALB/c mouse disease models, have not been tested for susceptibility to the approved therapy for Graves' disease.

Therefore, current animal models of Graves' disease are not optimal for investigating potential therapies.

The present inventors have unexpectedly found that an animal that is transgenic for human HLA-DR3 is a suitable basis for research into potential therapies. Thus, in another aspect, the present invention relates to an animal model of a disease associated with the production of antibodies to TSHR, where the specified animal is transgenic for human HLA-DR3 and has elevated levels of HLA-DR3 compared to a control animal.

The term "elevated levels of TSHR" includes an increased level of any full-length TSHR sequence, such as TSHR peptides.

The level of TSHR was increased compared to that of a corresponding control animal (eg, an animal that is transgenic for human HLA-DR3 but does not show elevated levels of TSHR). The level of TSHR can be increased by any suitable means, for example, by introducing a vector comprising a nucleic acid encoding a TSHR peptide. In a preferred embodiment of the invention, said vector is a viral vector.

In another embodiment, the present invention relates to a method of obtaining an animal model of a disease associated with the production of antibodies to TSHR, which method includes increasing the level of TSHR in an animal that is transgenic for human HLA-DR3.

In a preferred embodiment of the invention, said method comprises increasing the level of TSHR by introducing a vector comprising a nucleic acid encoding a TSHR peptide. In a more preferred embodiment of the invention, the vector is a viral vector.

Animal model has features inherent in the disease associated with the production of antibodies to TSHR. Thus, for example, in this animal model, TSHR antibody levels were elevated compared to equivalent control animals in which TSHR levels were not elevated. Anti-TSHR antibody levels can be increased by at least 2, 3, 5, 10, 100, or 1000 times that of an equivalent control animal in which TSHR antibody levels have not been increased.

The levels of antibodies to TSHR can be determined by standard methods known in the art, for example, using an ELISA assay. The level of antibodies to TSHR can be determined in the sample in vitro. For example, the sample may be a serum sample.

The term "transgenic for human HLA-DR3" means that the HLA-DR3 molecule of human MHC class II is expressed on the surface of animal cells. Such a transgenic animal can be obtained by suitable methods well known to those skilled in the art.

The term "associated with the production of antibodies to TSHR" means that anti-TSHR antibodies are involved in the development of the disease. Thus, for example, in the case of a disease, the levels of antibodies to TSHR may be elevated compared to the levels observed in the absence of the disease. The disease associated with anti-TSHR antibodies may be Graves' disease.

The TSHR may be a human TSHR. Thus, for example, the TSHR may be the A subunit of human TSHR or a portion thereof.

Nucleic acid encoding TSHR can be obtained by methods well known in the art, for example, based on the TSHR sequence described here.

The nucleic acid may be natural, synthetic or recombinant. It may be double stranded or single stranded, and may be DNA or RNA, or a combination thereof. The nucleic acid may be, for example, cDNA, PCR product, genomic sequence or mRNA.

The nucleotide sequence can be codon optimized for production in the selected host/host cell.

Delivery of the nucleotide sequence encoding the TSHR can be accomplished through a viral infection. Suitable viral vectors are well known to those skilled in the art. Thus, for example, the viral vector may be an adenovirus, a retrovirus, or a lentivirus.

In particular, the viral vector may be an adenovirus.

Obtaining an animal model may include several introductions of the vector.

A vector, eg adenovirus, may be administered at 3 week intervals. Thus, for example, the vector may be administered at 18-25, 18-23, 19-23, or 20-22 day intervals. The vector may be administered at 20, 21 or 22 day intervals.

The production of an animal model may include the introduction of an adenoviral vector at least twice. Thus, for example, the adenoviral vector may be administered two or three times. In particular, obtaining an animal model may include the introduction of the adenoviral vector twice.

Obtaining an animal model may include the introduction of the vector twice with intervals of three weeks between injections.

The vector, eg adenovirus, may be administered at a dose of 10 ⁸ -10 ¹¹ viral particles per administration. In particular, adenovirus may be administered at a dose of 10 ⁹ -10 ¹¹ viral particles per administration. Adenovirus can be administered at a dose of 10 ⁹ viral particles per injection.

The vector may be entered in any suitable manner. Thus, for example, in the case of an adenoviral vector, the vector may be administered by intramuscular injection.

The animal may be a mammal. Thus, for example, the animal may be a mouse, a rat, a rabbit, a guinea pig, or a primate. The preferred animal is a mouse.

In one embodiment, the mouse is an HLA-DRA1*01:01- and HLA-DRB1*03:01- transgenic mouse from a mixed genetic background. The method of obtaining such a mouse is known to those skilled in the art.

For example, the mouse model described in the Examples section was generated as follows: 6-kb. Nde I fragment of HLA-DRA genomic clone in pUC and 24 kb. The Cla Ix Sal I cos 4.1 fragment containing the B gene was co-injected into fertilized eggs of F1 donors (C57BL/6xDBA/2) crossed with C57BL/6 males. The progeny were then bred to produce IA-beta C57BL/6 knockout mice with a genetic background (AB0 mice) that did not express the mouse MHC class II molecule. These DR3tg mice expressed the HLA-DR3 molecule but did not express the mouse MHC-II molecule. This method is not the only one and there are known alternative methods for obtaining HLA-DR3 transgenic mice, for example, mice with a background of C57BL/10.

The present invention is described in more detail in the examples, which are provided for a better understanding of the invention and are not construed as limiting the scope of the invention.

Examples

Example 1 - ATX-GD-59 induces tolerance in TSHR-specific T cells and reduces anti-TSHR antibody titers in animal models, i.e., HLA-DR3 transgenic mice

Materials, methods and procedures

Mice

DR3tg mice were bred under specific non-pathogenic conditions at other laboratories, Charles River, UK or InnoSer, NL. DR3tg mice were first created by Strauβ et al. Briefly, the genomic constructs used were a 6 kb-Nde I fragment of the HLA-DRA genomic clone at pUC 13 and a 24 kb-CLa I × Sal I fragment cos 4.1, i.e., cosmids (pTCF) containing the B DRB1*0301 gene. A solution containing 1-2 μg/ml of each construct was used for co-injection into fertilized eggs of F1 donors (C57BL/6xDBA/2) crossed with C57BL/6 males. The progeny were then bred to produce IA-beta C57BL/6 knockout mice with a genetic background (AB ⁰ mice) that did not express the mouse MHC class II molecule. These DR3tg mice expressed the human HLA-DR3 MHC class II molecule but did not express the mouse MHC class II molecule. Transgenic mice were identified by Southern blot analysis of DNA tail digested with EcoRI and probed with 1.35 kb-Bam HI DRA cDNA and 1.25 kb-Bam HI- cDNA fragment DRB1*0301. DR3tg mice were used in these experiments because it has been suggested that this MHC class II molecule is associated with increased susceptibility to Graves' disease.

Animal studies were approved by the "Committee on the Ethics of Animal Experimentation" at Hasselt University and were performed according to standard treatment methods in a pathogen-free laboratory.

Antigens

Peptides were synthesized at PolyPeptide Laboratories (PPL; Strasbourg, France) and stored as a stock solution at 8 mg/ml in PBS at -20°C. The method for obtaining peptides is based on solid-phase synthesis of peptides, carried out by adding N-α-Fmoc-protected amino acids in the form of structural elements used to assemble the peptide. The C-terminal residue was attached to the MBHA resin as part of the Fmoc-lys(Boc)-MPPA linker. Other amino acids were introduced by successive removal of the Fmoc protecting group and amino acid addition cycles. If PPL peptides were used, this is indicated in the inscriptions on the drawing.

Alternatively, peptides were synthesized at GL Biochemistry Ltd (GLS; Shanghai, China) or at Severn Biotech Ltd (Severn, Kidderminster Worcs., UK) and stored as a stock solution at 20 mg/mL in DMSO (Sigma-Aldrich) at -80°C. Peptides were synthesized by chemical addition of F-moc at the N-terminal free amine and C-terminal amide. For each experiment, suppliers of different peptides are indicated.

Treatment peptides were used as a single peptide (9B-N, 5D-K1) or as an ATX-GD-59 peptide cocktail. ATX-GD-59 is an equimolar mixture of 9B-N and 5D-K1, and the indicated doses of ATX-GD-59 represent the content of a single peptide, that is, the total amount of peptides administered is twice the dose used for treatment.

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

Adenovirus vectors were purchased from Viraquest (North Liberty, IA, USA). Construction and purification of adenovirus containing amino acid residues TSHR 1-289 (Ad-TSHR) have been described previously. Briefly, the Ad-TSHR adenoviral construct and a control construct (Ad-LacZ) expressing β-galactosidase were propagated in HEK293 cells and purified by CsCL density gradient centrifugation. The concentration of viral particles was determined by measuring the optical density at 260 nm.

Ex vivo tolerization experiment

DR3tg mice were injected subcutaneously in the flank with 0.1 μg, 1 μg and 10 μg of peptides on days -15, -13 and -11, respectively, and then received 3 injections of 100 μg (maximum dose/peptide) of soluble peptide (5D-K1 or 9B-N) on days -8, -6 and -4 (dose escalation schedule). ATX-GD-59 is an equimolar mixture of peptides 5D-K1 (SEQ ID NO:1) and 9B-N (SEQ ID NO:2) and the maximum dose (nmol) mentioned is for the peptides in the cocktail. Since both systems are used in various experiments, a peptide dose of 100 μg corresponds to ~45 nmol. The peptides were injected subcutaneously in PBS. Changes in maximum doses/peptide and, accordingly, higher doses are indicated for each experiment. On day 0, mice were immunized subcutaneously at the base of the tail at a dose of 50 μg, corresponding to the dose of the parent peptide (unmodified 5D and/or 9B) emulsified in Freund's complete adjuvant (CFA; 2 mg/ml Mycobacterium tuberculosis H37RA (Difco Laboratories, Michigan, US ) and in incomplete Freund's adjuvant (Difco)) (50 μg peptide in 100 μl CAF (100 μg H37RA)/injection). Ten days after immunization, LN and spleen were removed. LN cells and splenocytes were isolated and cultured 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) in 96-well flat-bottomed tablets. To study antigen-induced cell proliferation, 0.5×10 ⁶ cells per well were cultured (200 μl/well) for 72 hours with TSHR-ECD at a concentration of 0-25 μg/ml or with 12.5 μg/ml of purified protein derivative (PPD; immunization controls; Statens serum institut, Copenhagen, Denmark). After a 72 hour incubation at 37° C. in a 5% CO ₂ incubator, 60 μl of cell supernatant was collected and frozen. Then, 20 μl/well of tritiated thymidine (PerkinElmer, Zaventem, Belgium) was added to the cells to a final concentration of 1 μCi/well. Cells were incubated at 37°C and 5% CO ₂ and after 16 hours the plates were frozen. Thawed plates were harvested and counted on a β-counter (Wallac 1450 Microbeta Trilux liquid scintillation counter) to assess cell proliferation.

Animal with adenoviral HD model

DR3tg mice were injected subcutaneously in the flank with 22.5 pmol, 225 pmol and 2250 pmol ATX-GD-590 or control on days -15, -13 and -11, respectively, and then received 3 injections of 22.5 pmol ATX-GD- 59 (maximum dose/peptide) or controls on days -8, -6 and -4 (dose escalation schedule). Changes in maximum doses, and correspondingly higher doses, are indicated for each experiment.

On day 0, mice were intramuscularly injected into the thigh with Ad-TSHR or Ad-LacZ (10 ¹⁰ viral particles). All mice were simultaneously immunized with the same batch of adenovirus per experiment. Mice were given two injections three weeks apart (weeks 0 and 3) and bled at different times according to the protocol shown in Figure 2. Five weeks after the first immunization, mice were euthanized and blood, spleen cells and thyroid gland. Splenocytes were isolated and cultured 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) in 96 well flat bottom plates. To study antigen-induced cell proliferation, 0.5×10 ⁶ cells per well were cultured (200 μl/well) for 72 hours with various concentrations of TSHR-ECD (0-25 μg/ml). After a 72 hour incubation at 37° C. in a 5% CO ₂ incubator, 60 μl of cell supernatant was collected and frozen. Then, 20 μl/well of tritiated thymidine (PerkinElmer, Zaventem, Belgium) was added to the cells to a final concentration of 1 μCi/well. Cells were incubated at 37°C, and after 16 hours the plates were frozen. Thawed plates were harvested and counted on a β-counter (Wallac 1450 Microbeta Trilux liquid scintillation counter) to assess cell proliferation.

Detection of anti-TSHR antibodies

Anti-TSHR antibodies (IgG class) against purified recombinant TSHR-ECD (Chesapeake-Perl) were evaluated by ELISA. 96-well plates (half the area of a 96-well plate, Fisher Scientific) were sensitized overnight at room temperature (RT) with 50 microliters/well of TSHR-ECD protein in PBS (0.5 μg/ml). After washing with PBS-0.05% Tween 20, wells were blocked with 1% BSA (w/v) in PBS for 1 hour at RT and incubated with test serum (1:50 or 1:500 dilutions). A 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). Anti-TSHR antibodies of IgG1, IgG2a, IgG2b, and IgG2c isotypes were detected using 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), respectively. The signal was developed using tetramethylbenzidine (TMB). Optical density (OD) was measured with a 450 nm plate reader (Tecan Benelux).

Detection of stimulatory anti-TSHR antibodies (TSAbs)

Lulu*, Chinese Hamster Ovary-K1 (CHO-K1) cells stably transfected with pA3Luc, cAMP-responsive luciferase construct and pcDNA3-TSHR, and human TSHR, and possessing G418 resistance, were kindly provided by Prof. M. Ludgate (Cardiff University, UK)9. Cells were maintained in Hams 12 medium (Lonza) supplemented with 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin (all from Lonza), 10% fetal bovine serum (FBS) (Fisher Scientific , Aalst, Belgium) and 0.2 mg/ml geneticin (Fisher) at 37° C. in 5% CO ₂ . For TSAb evaluation, cells were seeded at a density of 2×10 ⁴ cells/well in 96-well plates in Hams' medium 12 with 10% charcoal-treated serum (Sigma-Aldrich, Bornem, Belgium). The next day, the culture medium was removed and the cells were incubated with fresh Hams medium containing 5% PEG and 10% test serum for 4 hours at 37°C. The cells were then lysed using luciferase cell culture lysis buffer (Promega, Leiden, The Netherlands). The production of cAMP-responsive luciferase was assessed in a luciferase reporter assay (Promega) according to the manufacturer's instructions and light emission was measured on a FLUOstar omega luminometer (BMG labtech, Ortenberg, Germany). The results are expressed in relative units of radiation (RLU, radiation emitted by stimulated cells/radiation emitted by unstimulated cells).

Detection of 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 manufacturers instructions. T4 values were calculated from the standards provided in the kit and expressed in µg/dl.

results

Single Peptide Treatment Induced TSHR-Specific T Cell Tolerance

To determine the tolerogenic effect of individual 5D-K1 and 9B-N peptides, HLA-DR transgenic mice were treated with a dose escalation scheme, starting with a single dose of one of the peptides. In the first experiment, DR3tg mice were treated with 5D-K1 or control as described in the Method section. Pretreatment with this modified apitope resulted in a 43% reduction in TSHR-induced T cell proliferation in spleen samples and 60% in LN samples compared to control treated animals (see Figures 1 and 2).

To test the tolerogenic capacity of the 9B-N peptide, DR3tg mice were treated with 9B-N or PBS. This study showed that 9B-N pretreatment significantly reduced TSHR-induced T cell proliferation by 41% and 33% in spleen and LN samples, respectively.

Combined Peptide Treatment Induces TSHR-Specific T Cell Tolerance

The finding that treatment with single 5D-K1 or 9B-N peptides reduced TSHR-specific lymphocyte proliferation in mice prompted a study to determine whether combined peptide treatment in a cocktail could lead to a reduction in TSHR-specific response. . DR3tg mice were treated with ATX-GD-59 on a dose escalation schedule and immunized with an emulsion containing the parental unmodified peptides in CAF. Three maximum doses (15, 22.5 and 45 nmol/peptide) of ATX-GD-59 were tested for their potentially different tolerization capacity, but no differences were found between the administered doses (data not shown). The representative results presented in Figure 3 showed that ATX-GD-59 treatment induced significant levels of TSHR-specific tolerance. TSHR-induced proliferation was reduced by 58% and 54% in splenocytes and LN cells, respectively.

Combined Peptide Treatment Decreases Anti-TSHR Antibody Production in Animals with an Adenovirus Model of Graves' Disease

To illustrate the therapeutic efficacy of ATX-GD-59, a study was conducted to determine if treatment with the ATX-GD-59 peptide could result in inhibition of Graves' disease parameters such as TSHR-specific splenocyte proliferation and anti-TSHR antibody production. DR3tg mice were treated with ATX-GD-59 or control in a dose escalation schedule followed by two immunizations with Ad-LacZ or Ad-TSHR virus 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 after re-stimulation with TSHR in vitro (data not shown). The absolute values of TSHR-specific splenocyte proliferation in control-treated and Ad-LacZ-immunized DR3tg mice were low but increased in a dose-dependent manner. As shown in Figure 4, ATX-GD-59 treatment at a maximum dose of 22.5 nmol/peptide resulted in a significant reduction in TSHR-specific splenocyte proliferation by more than 40%. ATX-GD-59 treatment with 15 nmol of each peptide gave similar results (data not shown). A similar tolerizing effect of ATX-GD-59 treatment was observed in a repeat experiment (data not shown), confirming the reproducibility of these results.

The effect of ATX-GD-59 treatment on anti-TSHR antibody production was then evaluated. Serum samples were taken to assess anti-TSHR IgG levels using ELISA. Anti-TSHR IgG antibodies were not observed either before (W-2, data not shown) or after (W0) pretreatment with the ATX-GD-59 peptide (Figure 5). Consistent with proliferation data, no anti-TSHR antibodies were detected in the Ad-LacZ immunized control mice. High levels of anti-TSHR IgG were observed in control peptide treated mice 2 weeks after the first Ad-TSHR immunization. ATX-GD-59 treatment slowed the increase in anti-TSHR antibody levels observed in control peptide-treated mice after Ad-TSHR immunization by 95% at week 2. In addition, anti-TSHR IgG levels decreased by 93% at week 2. 5 after treatment with ATX-GD-59 peptide.

To further investigate the reduction in anti-TSHR antibody levels, the effect of ATX-GD-59 treatment on anti-TSHR antibody levels of various isotypes was evaluated. Ad-TSHR immunization at a viral particle density of 10 ¹⁰ induced high levels of anti-TSHR antibodies of the IgG2b and IgG2c isotypes. Figure 6 shows that ATX-GD-59 significantly reduces levels of anti-TSHR antibodies of the IgG1, IgG2b and IgG2c isotypes in Ad-TSHR-immunized mice, thus correlating with the pattern of total IgG antibody levels as shown in Figure 5. Treatment peptide doses of 15 nmol and 22.5 nmol ATX-GD-59 were analyzed twice in two independent experiments, resulting in a significant decrease in the levels of total anti-TSHR IgG and IgG of various isotypes (data for a dose of 15 nmol are not shown), which indicates that both doses are effective in the adenoviral HD model.

Overall, the results indicate that ATX-GD-59 prophylactic treatment is effective in inducing TSHR-specific T cell tolerance in an ex vivo tolerance model and in reducing anti-TSHR antibody levels in an adenoviral Graves' disease model.

Example 2 Combined Treatment of Graves' Disease by Administering ATX-GD-459 and Clinically Used Drugs for the Treatment of Graves' Disease

Materials and methods

Mice

DR3tg mice were bred under specific non-pathogenic conditions at another laboratory, Charles River, UK or InnoSer, NL. DR3tg mice were first created by Strauβ et al. (Strauss et al, 1994, Immunogenetics 3, 104-108). Briefly, the genomic constructs used were a 6 kb Nde I fragment of the HLA-DRA genomic clone at pUC 13 and a 24 kb CLaI × SalI fragment cos 4.1, i.e., cosmids (pTCF ) containing the B DRB1*0301 gene. A solution containing 1-2 μg/ml of each construct was used for co-injection into fertilized eggs of F1 donors (C57BL/6xDBA/2) crossed with C57BL/6 males. The progeny were then bred to produce IA-beta C57BL/6 knockout mice with a genetic background (AB0 mice) that did not express the mouse MHC class II molecule. These DR3tg mice expressed the HLA-DR3 molecule but did not express the mouse MHC class II molecule. Mice were obtained by backcrossing with C57BL/6 and with B10.Q. Transgenic mice were identified by Southern blot analysis of DNA tail digested with EcoRI and probed with 1.35 kb BamHI DRA cDNA and 1.25 kb BamHI cDNA DRB1*0301. DR3tg mice were used in both experiments because it has been suggested that this MHC class II molecule is associated with an increased risk of individuals developing Graves' disease.

DR4 mice were first created by Lars Fugger et al. (PNAS 1994 volume 91: 6151-55), i.e., the HLA-DRA*0101/HLA-DRB1*0401 and mCD3-huCD4c/g constructs were co-microinjected into F1 progeny (DBA/1xA/CA) embryos, and viable embryos were transferred to F1 pseudopregnant females (BALB/c × 129) to develop pregnancy. The progeny were then bred to produce IA-beta C57BL/6 knockout mice with a genetic background (AB0 mice) that did not express the mouse MHC class II molecule. These DR4 mice expressed only the MHC class II molecule, namely the human HLA DR4 molecule.

BALB/cJOlaHsd mice were obtained from the Harlan laboratory (Venray, The Netherlands).

Animal studies were approved by the "Committee on the Ethics of Animal Experimentation" (ECD) at Hasselt University and were performed according to standard practices in a pathogen-free laboratory.

Antigens

All individual peptides were synthesized at GL Biochem Ltd (Shanghai, China) and stored as a stock solution at 20 mg/ml in DMSO (Sigma-Aldrich) at -80°C. Peptides were synthesized by the addition method at the N-terminal free amine and C-terminal amide.

ATX-GD-459 peptides (see Example 3 for a discussion of these peptides) were synthesized at PolyPeptide Laboratories (PPL; Strasbourg, France) and stored as a stock solution at 8 mg/ml in PBS at -20°C. The method for obtaining peptides is based on solid-phase synthesis of peptides, carried out by adding N-α-Fmoc-protected amino acids in the form of structural elements used to assemble the peptide. The C-terminal residue was attached to the MBHA resin as part of the Fmoc-lys(Boc)-MPPA linker. Other amino acids were introduced by successive removal of the Fmoc protecting group and amino acid addition cycles.

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

Adenovirus vectors were purchased from Viraquest (North Liberty, IA, USA). The construction and purification of an adenovirus containing amino acid residues TSHR 1-289 (Ad-TSHR) has been described previously (Chen et al. 1999 J Clin Endocrinol Metab 84:3182-3186). Briefly, adenoviruses Ad-TSHR and control adenovirus (Ad-LacZ) expressing β-galactosidase were propagated in HEK293 cells and purified by CsCL density gradient centrifugation. The concentration of viral particles was determined by measuring the optical density at 260 nm.

Co-administration of ATX-GD-459 and drugs used in clinical practice for the treatment of Graves' disease in animals with an adenoviral model of HD

DR3tg mice (n=9-10/group) were injected subcutaneously in the flank with 15 pmol, 150 pmol and 1500 pmol of each ATX-GD-459 peptide or control peptide on days -15, -13 and -11, respectively, and then injected 3 injections of 15 pmol of each ATX-GD-459 peptide (maximum dose) or control peptide on days -8, -6 and -4 (dose escalation schedule). Changes in maximum doses and correspondingly higher doses are indicated for each experiment. From the day of the first peptide treatment until the end of the experiment, mice were treated with either MMI or propranolol. MMI (Sigma-Aldrich, Bornem, Belgium) was dissolved in sterile water at the desired concentration and administered to mice at a daily dose of 500 μg by subcutaneous injection using ALZET osmotic pumps (model 1004, 0.11 μl/hour; Charles River, France). After 4 weeks, new osmotic pumps were implanted. Fresh propranolol hydrochloride (Sigma-Aldrich) was dissolved in 0.9% saline and administered daily by intraperitoneal (IP) injection at a dose of 10 mg/kg/day. On day 0, mice were immunized by intramuscular injection of 10 ⁹ or 10 ¹⁰ Ad-TSHR virus particles and the immunization was repeated three weeks later. The body weight of the mice was recorded every week to assess their condition. Five weeks after the first immunization, mice were euthanized and blood, thyroid and spleen samples were taken. TSHR-induced splenocyte proliferation, cytokine secretion, and anti-TSHR antibody levels were assessed as described below.

Detection of anti-TSHR antibodies

Anti-TSHR antibodies (IgG class) against purified recombinant TSHR-ECD (Chesapeake-Perl) were evaluated by ELISA. 96-well plates (half the area of a 96-well plate, Fisher Scientific) were sensitized overnight at room temperature (RT) with 50 microliters/well of TSHR-ECD protein in PBS (0.5 μg/ml). After washing with PBS-0.05% Tween 20, wells were blocked with 1% BSA (w/v) in PBS for 1 hour at RT and incubated with test serum (1:50 or 1:500 dilutions). A 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). Anti-TSHR antibodies of IgG1, IgG2a, IgG2b, and IgG2c isotypes were detected using 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), respectively. The signal was developed using tetramethylbenzidine. Optical density (OD) was measured with a 450 nm plate reader (Tecan Benelux).

Detection of stimulating anti-TSHR antibodies (TSAbs)

Lulu* cells, Chinese hamster ovary-K1 (CHO-K1) cells stably transfected with pA3Luc, cAMP-responsive luciferase construct and pcDNA3-TSHR, and human TSHR, and possessing G418 resistance, were kindly provided by Prof. M. Ludgate (Cardiff University, UK) ⁶ . Cells were maintained in Hams 12 medium (Lonza) supplemented with 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin (all from Lonza), 10% fetal bovine serum (FBS) (Fisher Scientific , Aalst, Belgium) and 0.2 mg/ml geneticin (Fisher) at 37° C. in 5% CO ₂ . For TSAb evaluation, cells were seeded at a density of 2×10 ⁴ cells/well in 96-well plates in Hams' medium 12 with 10% charcoal-treated serum (Sigma-Aldrich, Bornem, Belgium). The next day, the culture medium was removed and the cells were incubated with fresh Hams medium containing 5% PEG and 10% test serum for 4 hours at 37°C. The cells were then lysed using luciferase cell culture lysis buffer (Promega, Leiden, The Netherlands). The production of cAMP-responsive luciferase was assessed in a luciferase reporter assay (Promega) according to the manufacturer's instructions and light emission was measured on a FLUOstar omega luminometer (BMG labtech, Ortenberg, Germany). The results are expressed in relative units of radiation (RLU, radiation emitted by stimulated cells/radiation emitted by unstimulated cells).

Detection of 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 manufacturers instructions. T4 values were calculated from the standards provided in the kit and expressed in µg/dL.

Splenocyte proliferation assay

Splenocytes were isolated and cultured 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) in 96 well flat bottom plates. To study antigen-induced cell proliferation, 0.5×10 ⁶ cells per well were cultured (200 μl/well) for 72 hours with various concentrations of TSHR-ECD (0-25 μg/ml). After a 72 hour incubation at 37° C. in a 5% CO ₂ incubator, 60 μl of cell supernatant was collected and frozen. Then, 20 μl/well of tritiated thymidine (PerkinElmer, Zaventem, Belgium) was added to the cells to a final concentration of 1 μCi/well. Cells were incubated at 37°C and 5% CO ₂ and after 16 hours the plates were frozen. Thawed plates were harvested and counted on a β-counter (Wallac 1450 Microbeta Trilux liquid scintillation counter) to assess cell proliferation.

results

Combined injection of MMI and ATX-GD-459 in an adenoviral HD model

In order to confirm whether the combined ATX-GD-459 treatment and MMI treatment can be used to treat the symptoms of HD, the effect of co-administration of MMI and ATX-GD-459 in an animal model of adenoviral HD was evaluated (for further discussion of this model, see . in Examples 4 and 5). In this experiment, DR3tg mice were treated with the following combinations of MMI and ATX-GD-459: vehicle+control peptide, MMI+control peptide, vehicle+ATX-GD-459, or MMI+ATXGD-459. Treatment with ATX-GD-459 or control peptide was performed in the first two weeks of the experiment on a dose escalation schedule (Figure 8). Subcutaneous administration of vehicle or MMI was started at the same time, but ended before the completion of the experiment. This experimental design allows investigation of the reciprocal effect of MMI and ATX-GD-459 treatment on HD-like disease parameters.

First, the effect of MMI and ATX-GD-459 treatment on TSHR-specific splenocyte proliferation was investigated. The absolute values of TSHR-specific splenocyte proliferation were relatively low in control treated mice (FIG. 10). However, in this experiment, these values were much higher than in all other control groups in previous experiments (data not shown) or in the propranolol administration experiment. Therefore, the significant reduction in TSHR-specific splenocyte proliferation due to MMI or ATX-GD-459 treatment, as shown in Figure 10, must be interpreted with some caution.

After TSHR-specific splenocyte proliferation, serum levels of anti-TSHR IgG were determined in all mice. Prior to the start of the experiment, no anti-TSHR IgG antibodies were detected in any mice (data not shown). ATX-GD-459 dose escalation treatment did not induce any production of anti-TSHR antibodies as determined at week 0 (Figure 11). Ad-TSHR immunization stimulated the production of anti-TSHR antibodies in mice treated with the control peptide but not in mice treated with ATX-GD-459. Although no significant difference was observed in mice treated with different control doses, however, pre-treatment with ATX-GD-459 reduced the production of anti-TSHR antibodies by more than 90% on average. In contrast, MMI treatment did not affect anti-TSHR antibody levels. In addition, MMI treatment did not affect the antibody-lowering effect of ATX-GD-459 peptide treatment.

The anti-TSHR antibody profile was further analyzed by determining specific IgG isotypes. As shown in Figure 12, none of the Ad-TSHR-immunized mice produced anti-TSHR antibodies of the IgG1 isotype. Ad-TSHR immunization induced the production of anti-TSHR IgG2b and IgG2c antibodies in control peptide treated mice but not in ATX-GD-459 treated mice. ATX-GD-459 pre-treatment reduced the production of anti-TSHR IgG2b and IgG2c antibodies by more than 90%. In contrast, MMI treatment did not affect anti-TSHR IgG isotype antibody levels and the antibody-lowering effect of ATX-GD-459 peptide treatment. These data are fully consistent with the profile observed for all anti-TSHR IgG antibodies.

In addition to isotype determination, the biological activity of anti-TSHR antibodies and the potential effect of MMI or ATX-GD-459 treatment were evaluated. Since the threshold for stimulatory antibodies was not exceeded in all Ad-TSHR immunized mice (data not shown), no data were obtained on the effect of MMI or ATX-GD-459 treatment on the production of stimulatory antibodies. The low level of stimulatory antibodies is consistent with previously described experiments (report ATX-GD-15-003).

Finally, the effect of MMI or ATX-GD-459 processing on T4 levels was evaluated. Since Ad-LacZ immunization was not used as a control immunization in this experiment, the incidence of hyperthyroidism could not be determined. Despite the lack of information on the level of hyperthyroidism, the results obtained clearly indicated that MMI treatment led to a significant decrease in T4 levels (figure 13). However, ATX-GD-459 pre-treatment did not affect serum T4 levels. In addition, ATX-GD-459 pre-treatment did not affect the T4-reducing efficiency of MMI.

Overall, these data indicated that combined MMI or ATX-GD-459 treatment did not adversely affect all other functions and could be safely used in the adenoviral HD model.

Combined administration of propranolol and ATX-GD-459 in an adenoviral HD model

To control the adrenergic symptoms of hyperthyroidism, patients with HD often take β-blockers. Therefore, in this experiment, the effect of the combined administration of ATX-GD-459 and a β-blocker, namely propranolol, was investigated in an adenoviral HD model. First, TSHR-specific splenocyte proliferation was assessed. As shown in Figure 14, neither propranolol treatment nor ATX-GD-459 treatment had a significant effect on TSHR-specific splenocyte proliferation. Combined treatment with propranolol and ATX-GD-459 also did not affect the proliferative response.

The effect of propranolol treatment and ATX-GD-459 treatment on anti-TSHR antibody production was then evaluated. Serum samples were taken to assess anti-TSHR IgG levels using ELISA. Anti-TSHR IgG antibodies were not observed either before (W-2) or after (W0) pretreatment with the ATX-GD-459 peptide (FIG. 15). Ad-TSHR immunization induced high levels of anti-TSHR antibody production in control peptide treated mice and to a lesser extent in ATX-GD-459 treated mice. Notably, Ad-TSHR immunization with ¹⁰¹⁰ viral particles in this experiment resulted in increased anti-TSHR antibody titers, in contrast to ¹⁰⁹ Ad-TSHR immunizations with previous experiments (data not shown) . ATX-GD-459 treatment resulted in a decrease in the level of production of anti-TSHR antibodies by more than 90%, as determined on W2 and W5 after immunization. In contrast, propranolol treatment did not affect anti-TSHR antibody levels. In addition, propranolol treatment did not affect the antibody-lowering efficacy of ATX-GD-459 peptide treatment.

To further study the antibody profile, anti-TSHR antibody profiles of the IgG isotype were determined. Although the levels of these antibodies were lower than those of IgG2b and IgG2c, however, anti-TSHR IgG antibodies were clearly induced by Ad-TSHR immunization. These data contrasted with those obtained in the absence of anti-TSHR IgG1 antibodies in previous experiments (data not shown), but this can be explained by increasing the dose of adenovirus particles from 10 ⁹ to 10 ¹⁰ used in immunization. As shown in Figure 16, ATX-GD-459 treatment significantly reduced levels of anti-TSHR IgG2b and IgG2c antibodies. The titers of anti-TSHR IgG1 antibodies also decreased after treatment with the peptide, but not significantly. In contrast to peptide treatment, propranolol treatment did not affect anti-TSHR antibody levels of any IgG isotypes. In addition, propranolol treatment did not affect the antibody-lowering efficacy of ATX-GD-459 peptide treatment, which was consistent with the profile observed for all IgG anti-TSHR antibodies. Overall, ATX-GD-459 treatment significantly reduced anti-TSHR antibody titers in this model animal. However, no effect of propranolol treatment on any disease parameters similar to HD was observed. In order to confirm whether daily IP administration could induce pharmacological blood levels of propranolol in mice, mouse plasma propranolol levels were assessed by LC-MS-MS (Anacura). Blood samples were taken 60-90 minutes post-dose and the plasma levels of propranolol in the individuals were compared for W0 and W5. The results are shown in figure 18.

Example 3 - ex vivo tolerization of T cells by ATX-GD-459

In order to determine whether the RNB-4K and RNB-5D peptides are apitopes, hybridoma clones specific for RNB-4K-GKK and RNB-5D-K1 were selected. Antigen processing-independent presentation (APIPS) assays were performed and the peptides RNB-4K-GKK and RNB-5D-K1 were confirmed to be apitopes (FIG. 27).

Was determined by the ability of the composition containing the peptides presented in table 1 (designated ATX-GD-459), or separately administered peptides, to induce tolerance to TSHR. The ex vivo tolerization protocol is shown in Figure 19.

Table 1

Peptide Subsequence SEQID NO: RNB-5D-K1 KKKKYVSIDVTLQQLESHKKK SEQ ID NO: 1 RNB-4K-GKK KKGNLPNISRIYVSIDVTGKK SEQ ID NO: 4 RNB-9B GLKMFPDLTKVYSTD SEQ ID NO: 2

HLA-DR3 mice were treated with ATX-GD-459 on a dose escalation schedule and immunized with the combination of parental 4K/5D/9B peptides in CFA. The results showed that ATX-GD-459 treatment induced significant levels of TSHR-specific tolerance in spleen and LN cells (Figure 20). Tolerance induced by ATX-GD-459 treatment was higher than that induced by administration of the individual peptides (Figure 20).

Example 4 - Building a Model for Adenovirus Graves' Disease

To confirm the therapeutic effect of treatment with the ATX-GD-459 peptide, an animal model of adenovirus disease was first created, namely wild-type BALB/c mice. TSHR-induced splenocyte proliferation, anti-TSHR IgG antibody levels, serum T4 levels, and pathological changes in thyroid tissue were examined as parameters of disease symptoms. In the first experiment, BALB/c mice were immunized with 10^eight viral particles Ad-TSHR. Although this dose of virus was intended to induce hyperthyroidism and produce anti-TSHR antibodies (Chen et al., 2004, Endocrinology 145 (11): 4927-33), however, only 2 out of 10 mice were considered hyperthyroid (borderline) and none of these mice produced anti-TSHR IgG antibodies (data not shown).

In a second experiment using an increased dose of viral particles, approximately 30% of Ad-TSHR-immunized mice showed an increase in serum T4 levels, as determined 4 weeks after the first immunization (Figure 22). However, at week 10, the incidence was only 15%. This decrease in elevated T4 levels in wild-type BALB/c mice over a period of time after the initial increase weeks after immunization has been described previously (McLachlan et al, 2012, Thyroid 8:1-7). However, the marked hyperthyroidism in individual mice was clearly consistent with the histopathological findings obtained for thyroid tissue, such as hypertrophy of thyroid epithelial cells or lymphocyte infiltration (data not shown).

Although there is evidence in the literature that mice with a genetic background of C57BL/6 or DR3tg mice will remain resistant to the development of symptoms similar to Graves' disease, however, the authors of the present invention conducted an analysis in order to determine whether Ad- TSHR immunization induced the development of Graves' disease-like symptoms in DR3tg mice with a mixed genetic background of C57BL/10, DBA/2, C57BL/6, non-MHC class II.

Figure 23A shows serum T4 levels in Ad-LacZ and Ad-TSHR immunized DR3tg mice. Although the 4 Ad-TSHR-immunized mice had T4 levels higher than the mean+2SD of the Ad-LacZ-immunized mice, they only had borderline hyperthyroid syndrome and the increase in T4 levels was considered too low to be useful. as a disease parameter when investigating the effectiveness of peptide treatment.

Ad-TSHR immunization induced high titers of anti-TSHR IgG antibodies in the sera of DR3tg mice (FIG. 11, right panel). The highest levels of anti-TSHR IgG were reached at 4 weeks followed by a decrease at weeks 7 and 10, which contrasted with the stable levels of anti-TSHR IgG observed in BALB/c mice (Figure 23C, left panel). The fact that levels of anti-TSHR IgG in DR3tg mice were higher not only after 4 weeks, but even 2 weeks after the first immunization (data not shown), led the authors to the decision to terminate the study in a model of adenoviral Graves' disease not after 10 weeks, and already in 4 weeks.

Example 5 - Model Validation with Adenovirus Graves' Disease

In order to validate the DR3tg model of adenoviral Graves' disease, the effect of various immunomodulatory drugs on the production of anti-TSHR antibodies in an adenoviral Graves' disease model was evaluated. The antithyroid drug methimazole (MMI) is currently used to directly lower thyroid hormone levels in patients with Graves' disease. Therefore, the effects of MMI treatment in Ad-TSHR-immunized DR3tg mice were determined. Based on literature descriptions of the hyperthyroidism-inducing effect of MMI in mice, two different doses of MMI (50-500 µg/mouse/day) were tested (Jeong et al, 2012, Endocrinology 153: 683-9; Mozes et al., 1998, J. Clin Immunology 18 (2): 106-13). A daily dose of 500 μg MMI significantly reduced T4 levels at 2 and 4 weeks after the first immunization, while the 50 μg dose only showed a trend towards lower T4 levels (Figure 24A). Although Ad-TSHR immunization did not induce hyperthyroidism in this experiment (data for Ad-LacZ-immunized mice not shown) and in previous experiments, however, the decrease in T4 levels induced by MMI treatment indicated that in DR3tg mice, The production of hormones can be influenced by the thyroid gland.

MMI, in addition to its antithyroid function, also has immunomodulatory effects (Mozes et al, 1998, as described above; Wang et al, 2003, J. Leukoc. Biol. 73: 57-64). Therefore, it was determined whether MMI treatment could reduce levels of anti-TSHR IgG production in Ad-TSHR immunized mice. Although MMI treatment significantly reduced T4 levels, however, no change in anti-TSHR IgG levels was observed (Figure 24B).

Patients with Graves' disease, and in particular patients with OH, often require immunosuppressive therapy with glucocorticoids. The effect of methylprednisolone, a synthetic glucocorticoid drug, on the production of anti-TSHR antibodies in Ad-TSHR-immunized mice was tested. Treatment with methylprednisolone at a dose of 7 mg/kg/day resulted in a significant decrease in 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). Treatment of Ad-immunized DR3tg mice with methylprednisolone at a dose of 7 mg/kg/day resulted in a significant decrease in anti-TSHR IgG levels not after 4 weeks, but already 2 weeks after the first immunization, which is due to a natural decrease in anti-TSHR antibody levels in untreated mice for a certain period of time. Treatment with methylprednisolone also resulted in a decrease in thymus and spleen cells, indicating a pharmacological dose level sufficient to administer the adenoviral Graves' disease model (FIG. 25).

These data demonstrated that the efficacy of apitopes in diseased DR3tg mice can be determined by assessing the reduction in anti-TSHR IgG antibodies.

Example 6 Illustrating the Therapeutic Efficacy of ATX-GD-459 in an Adenovirus Graves' Disease Model

To determine whether combined treatment with peptides RNB-4K-GKK, RNB-5D-K1 and RNB-9B (ATX-GD-459) could suppress the production of anti-TSHR antibodies, DR3tg mice were treated with ATX-GD-459 or controlled dose escalation scheme and then subjected to Ad-LacZ or Ad-TSHR immunization. Serum samples were taken to assess anti-TSHR IgG levels using ELISA. Anti-TSHR IgG antibodies were not observed either before (W-2, data not shown) or after (W0) pretreatment with the ATX-GD-459 peptide (Figure 25). High levels of anti-TSHR IgG were observed in PBS-treated mice 2 weeks after the first Ad-TSHR immunization. ATX-GD-459 treatment resulted in a delay in the increase in anti-TSHR IgG antibody levels after Ad-TSHR immunization by 72% and 65% at weeks 2 and 5, respectively. These results indicate that ATX-GD-459 is effective in this model of adenoviral Graves' disease.

High levels of anti-TSHR IgG were observed after Ad-TSHR immunization. Therefore, this disease parameter was applied to investigate the effect of treatment with immunomodulatory drugs or the ATX-GD-459 peptide in a model of adenoviral Graves' disease. Methylprednisolone treatment was shown to successfully reduce anti-TSHR IgG levels in Ad-TSHR-immunized DR3tg mice. In addition, treatment with the ATX-GD-459 peptide in a dose escalation schedule resulted in a 70% decrease in the increase in anti-TSHR IgG antibody production.

Materials and methods

Mice

Mice were described in Example 2.

Antigens

Antigens have been described in Example 2.

Analysis per presentation system independent of antigen processing (APIPS)

Antigen-specific T-cell hybridomas were tested for their reactivity with peptides presented by fixed or non-fixed VAVY or BM14 (=APC) cells. 5x10 ⁴ cells from individual clones were cultured with 25 μg/ml peptide and 5x10 ⁴ fixed or fresh APCs. For APC fixation, cells were incubated with 0.5% paraformaldehyde (Merck, Darmstadt, Germany) (pH7) for 5 minutes at room temperature (RT). The fixation reaction was terminated by adding 0.4M glycine (Sigma-Aldrich) and washing the cells in RPMI-10%FCS. In addition, reactivity with human TSHR-ECD protein (Chesapeake-PERL, Savage, Maryland, USA) was assessed to identify epitopes. After 48 hours, antigen-induced IL-2 production was assessed by ELISA.

Ex vivo tolerization experiment

DR3tg or DR4tg mice were injected subcutaneously in the flank with 0.1 μg, 1 μg and 10 μg of peptides on days -15, -13 and -11, respectively, and then received 3 injections of 33, 75 or 100 μg of the peptide (depending on the maximum dose, see the inscriptions on the drawing) on days -8, -6 and -4 (according to the dose increase scheme). Changes in the maximum doses and, accordingly, higher doses are indicated for each experiment. On day 0, mice were immunized subcutaneously at the base of the tail with 50 μg of antigen (unmodified parental 15-mer peptide) emulsified in CAF (peptide/CAF). Ten days after immunization, the draining lymph nodes (LN) and spleen were removed. LN cells and splenocytes were isolated and cultured in X-vivo 15 medium (supplemented with 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin; Lonza) in 96 well flat bottom plates. To study antigen-induced cell proliferation, 0.5×10 ⁶ cells per well were cultured (200 μl/well) for 72 hours with various concentrations of antigen (0-25 μg/ml) or with 12.5 μg/ml of purified protein derivative (PPD; priming control; Statens serum institute, Copenhagen, Denmark). After 72 hours, 60 μl of cell supernatant was collected and frozen. Then, 20 μl/well of tritiated thymidine (PerkinElmer, Zaventem, Belgium) was added to the cells to a final concentration of 1 μCi/well. Cells were incubated at 37°C and after 16 hours the plates were frozen. Thawed plates were harvested and counted on a β-counter (Wallac 1450 Microbeta Trilux liquid scintillation counter) to assess cell proliferation.

Animals with a model of adenovirus Graves' disease

Adenovirus expressing the A subunit of human TSHR (amino acid residues 1-289; Ad-TSHR) and control adenovirus (Ad-LacZ) expressing β-galactosidase were purchased from Viraquest (North Liberty, IA, USA). 6-week-old female BALB/cJOlaHsd mice (Harlan Laboratories, Venray, The Netherlands) or DR3tg mice were intramuscularly injected with Ad-TSHR or Ad-LacZ (10 ⁹ , 10 ¹⁰ or 10 ¹¹ virus particles) in the thigh muscle. All mice were simultaneously immunized with the same batch of adenovirus per experiment. Mice were given two or three injections three weeks apart (weeks 0, 3 and 6) and bled prior to the first immunization and one week after the second immunization. At 4, 5 or 10 weeks after the first immunization, mice were euthanized and blood, spleen and thyroid cells were taken. Splenocytes were isolated and cultured in X-vivo 15 medium (to which glutamine, penicillin and streptomycin were added; Lonza) in 96 well flat bottom plates. To study antigen-induced cell proliferation, 0.5×10 ⁶ cells per well were cultured (200 μl/well) for 72 hours with various concentrations of antigen (0-25 μg/ml). After 72 hours, 60 μl of cell supernatant was collected and frozen. Then, 20 μl/well of tritiated thymidine (PerkinElmer, Zaventem, Belgium) was added to the cells to a final concentration of 1 μCi/well. Cells were incubated at 37°C and after 16 hours the plates were frozen. Thawed plates were harvested and counted on a β-counter (Wallac 1450 Microbeta Trilux liquid scintillation counter) to assess cell proliferation.

Detection of anti-TSHR antibodies

Anti-TSHR antibodies (IgG class) against purified TSHR-ECD protein (Chesapeake-Perl) were evaluated by ELISA. 96-well plates (half the area of a 96-well plate, Fisher Scientific) were sensitized overnight with 50 μl/well of TSHR-ECD protein in PBS (0.5 μg/ml) at RT. After washing with PBS-0.05% Tween, the wells were blocked with 1% bovine serum albumin (BSA) (w/v) in PBS for 1 hour at RT and incubated with test serum (aliquots in duplicate, 1:50 dilution). A 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). Anti-TSHR antibodies of IgG1, IgG2a, IgG2b, and IgG2c isotypes were detected using 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), respectively. The signal was developed using tetramethylbenzidine (TMB). Optical density (OD) was measured with a 450 nm plate reader (Tecan Benelux).

Detection of stimulating anti-TSHR antibodies (TSAbs)

Lulu* cells, Chinese Hamster Ovary-K1 (CHO-K1), stably transfected with pA3Luc, cAMP-responsive luciferase construct and pcDNA3-TSHR, and human TSHR, and possessing G418 resistance, were kindly provided by Prof. M. Ludgate (Cardiff University, UK). Cells were maintained in Hams 12 medium (Lonza) supplemented with 2 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 U/ml streptomycin (Lonza), 10% fetal bovine serum (FBS) (Fisher Scientific, Aalst, Belgium) and 0.2 mg/ml geneticin (Fisher) at 37° C. in 5% CO ₂ . For TSAb evaluation, cells were seeded at a density of 2×10 ⁴ cells/well in 96-well plates in Hams' medium 12 with 10% charcoal-treated serum (Sigma-Aldrich, Bornem, Belgium). The next day, the culture medium was removed and the cells were incubated with fresh Hams medium containing 5% PEG and 10% test serum for 4 hours at 37°C. The cells were then lysed using luciferase cell culture lysis buffer (Promega, Leiden, The Netherlands). The production of cAMP-responsive luciferase was assessed in a luciferase reporter assay (Promega) according to the manufacturer's instructions and light emission was measured on a FLUOstar omega luminometer (BMG labtech, Ortenberg, Germany). The results are expressed in relative units of radiation (RLU, radiation emitted by stimulated cells/radiation emitted by unstimulated cells).

Detection of 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 manufacturers instructions. T4 values were calculated from the standards provided in the kit and expressed in µg/dl. In addition to determining the level of T4 in serum, a histological analysis of the thyroid gland was performed to assess the parameter of hyperthyroidism. The thyroid gland was fixed in 10% neutral buffered formalin (pH 7.5), sectioned and stained with hematoxylin and eosin. Sections were examined for pathological changes (hypertrophy, increased epithelial cells and lymphocyte infiltration) and evaluated (KWS Biotest, Bristol, UK).

Confirmation of the Animal Model of Graves' Disease

DR3tg mice were immunized twice intramuscularly with Ad-TSHR three weeks apart (day 0 and 21). From the day of the first immunization until the end of the experiment, mice were treated with either MMI or sodium salt of 6α-methylprednisolone hemisuccinate 21 (methylprednisolone). All compounds were injected subcutaneously with ALZET osmotic pumps (model 1004, 0.11 μl/h; Charles River, France). MMI (Sigma) was dissolved in sterile water at the desired concentration and administered to mice at a daily dose of 50 or 500 μg. Methylprednisolone (Sigma) was dissolved in sterile water and administered to mice at a dose of 1 mg/kg/day or 7 mg/kg/day. The body weight of the mice was recorded every week to assess their condition. Four weeks after the first immunization, mice were euthanized and blood and spleen samples were taken. TSHR-specific splenocyte proliferation and anti-TSHR antibody levels were assessed as described above.

Animal with a prophylactic model of Graves' disease

DR3tg mice were injected subcutaneously in the flank with 0.1 µg, 1 µg, and 10 µg of ATX-GD-459 or control on days -15, -13, and -11, respectively, followed by 3 injections of 100 µg of ATX-GD-459 ( maximum dose) or control on days -8, -6 and -4 (according to the dose increase scheme). Changes in the maximum doses and, accordingly, higher doses are indicated for each experiment. On day 0, mice were immunized by intramuscular injection of 10 ⁹ Ad-TSHR or Ad-LacZ virus particles and immunized again three weeks later. Five weeks after the first immunization, blood and spleen were taken. TSHR-specific splenocyte proliferation and anti-TSHR antibody levels were assessed as described above.

Various modifications and changes will be obvious to a person skilled in the art and should not go beyond the essence and scope of the invention. While the present invention has been described in terms of specific preferred embodiments, it should be noted that the claimed invention should not be unreasonably limited to such specific embodiments. Indeed, various modifications can be made to the described methods for carrying out the invention, which are obvious to specialists in the field of chemistry, biology or related fields and are included in the scope of the present invention. All of the above publications are introduced into the present description by reference.

Claims (38)

1. Use of a composition comprising the following thyroid stimulating hormone receptor (TSHR) peptides: (i) a peptide having the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1; and (ii) a peptide having the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2, in suppressing or preventing the production of anti-TSHR autoantibodies in vivo . 2. Use of a composition comprising the following thyroid stimulating hormone receptor (TSHR) peptides: (i) a peptide having the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1; and (ii) a peptide having the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2, in the treatment and/or prevention of Graves' disease in an individual. 3. Use of a composition comprising the following thyroid stimulating hormone receptor (TSHR) peptides: (i) a peptide having the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1; and (ii) a peptide having the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2, in the preparation of a medicament for inhibiting or preventing the production of anti-TSHR autoantibodies in vivo . 4. Use of a composition comprising the following thyroid stimulating hormone receptor (TSHR) peptides: (i) a peptide having the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1; and (ii) a peptide having the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2, in obtaining a medicinal product for the treatment and/or prevention of Graves' disease. 5. A method of suppressing or preventing the production of TSHR autoantibodies in an individual, comprising the step of administering to the individual a composition comprising the following thyroid stimulating hormone receptor (TSHR) peptides: (i) a peptide having the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1; and (ii) a peptide having the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2. 6. A method of treating Graves' disease in an individual, comprising the step of administering to the individual a composition comprising the following thyroid stimulating hormone receptor (TSHR) peptides: (i) a peptide having the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1; and (ii) a peptide having the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2. 7. Application according to any one of paragraphs. 1-4 or the method of claim 5 or 6, wherein the composition comprises an antithyroid agent and/or a β-blocker. 8. Application according to any one of paragraphs. 1-4 or 7 or the method according to any one of paragraphs. 5-7, where the individual has HLA-DR3. 9. Application according to any one of paragraphs. 1-4 or 7 or the method according to any one of paragraphs. 5-7, where the individual has HLA-DR4. 10. Application according to any one of paragraphs. 1-4 or paragraphs. 7-9 or the method according to any one of paragraphs. 5-9, where the composition is administered in accordance with the dose escalation protocol. 11. A kit for the treatment and/or prevention of Graves' disease, the kit containing the following TSHR peptides: (i) a peptide having the amino acid sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 1) or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1; and (ii) a peptide having the amino acid sequence GLKMFPDLTKVYSTD (SEQ ID NO: 2) or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2, and optionally an antithyroid agent and/or a β-blocker used for simultaneous, separate or sequential administration. 12. The kit according to claim 11 for simultaneous, separate or sequential administration for the prevention or treatment of Graves' disease.

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