EP3596466A1

EP3596466A1 - Biomarker for endocrine autoimmune-related dysfunction

Info

Publication number: EP3596466A1
Application number: EP18711317.0A
Authority: EP
Inventors: Lutz Schomburg; Joerg Johannes; Theresa PORST
Original assignee: Berysol GmbH
Current assignee: Berysol GmbH
Priority date: 2017-03-14
Filing date: 2018-03-14
Publication date: 2020-01-22
Also published as: WO2018167118A1; DE102017204268A1

Abstract

Instant invention is related to methods for diagnosing the presence or the risk of development, or for monitoring of a thyroid hormone-related endocrine autoimmune dysfunction (THREAD) of a subject, wherein the presence of autoantibodies against monocarboxylate transporter 8 (MCT8) is analyzed in a biological sample obtained from said subject, a kit for performing the methods of the invention and the use of MCT8 in the diagnosis of THREAD and the identification of a compound, which interferes in the complex formation of MCT8 and autoantibodies against MCT8.

Description

BIOMARKER FOR ENDOCRINE AUTOIMMUNE-RELATED

DYSFUNCTION

BACKGROUND OF THE INVENTION

Instant invention is related to methods for diagnosing the presence or the risk of development, or for monitoring of a thyroid hormone-related endocrine

autoimmune dysfunction (THREAD) of a subject, wherein the presence of autoantibodies against monocarboxylate transporter 8 (MCT8) is analyzed in a biological sample obtained from said subject, a kit for performing the methods of the invention and the use of MCT8 in the diagnosis of THREAD and the identification of a compound, which interferes in the complex formation of MCT8 and autoantibodies against MCT8. Most thyroid hormones (TH) that circulate in blood are bound to the TH binding proteins albumin, thyroxine binding globulin, and transthyretin, respectively. In order to exert their signaling via the nuclear TH receptors, TH need to pass the cell membrane. However, TH are unable to diffuse across the cell membranes and require transmembrane transporters. Amongst a growing number of well- characterized TH transporters, the family of monocarboxylate transporters (MCT) is of specific interest. MCT8 was the first TH transporter described to be associated with a genetic disease, i.e., the Allan-Herndon-Dudley syndrome (AHDS) (Friesema et al. 2004). MCT8 is located on the X-chromosome, and transport-impairing mutations in MCT8 are severely affecting the neuronal and intellectual development of the individual as well as causing problems with muscle control, coordination and movement. MCT10 is known to be highly related to MCT8, both in structure and transport characteristics (Roef et al., 2013).

TH are essential hormones implicated in growth and development, as well as in controlling metabolism and energy expenditure. Lack or excess of TH signaling is causing a number of diseases collectively associated with developmental or metabolic symptoms. Most of the known examples are related to mutations in central genes associated with TH signaling, such as the TH receptors, enzymes involved in TH biosynthesis, TH binding proteins or, more recently, TH transporters (e.g., Medici et al. 2015). Besides genetic causes, also autoimmune interactions can profoundly affect TH signaling, best known in the case of suppressing, neutral or activating (TRAK) autoantibodies to the TSH receptor, but also described for other autoantigens (McLachlan & Rapoport, 2015). Collectively, these diseases can be summarized as TH-related endocrine autoimmune dysfunction ("THREAD"). THREAD may relate to different organs involved in TH control, biosynthesis or action, including the hypothalamus, pituitary gland, thyroid gland or tissues responding to TH. THREAD of the pituitary gland is called autoimmune hypophysitis and constitutes a relatively rare disease with a prevalence of about 5/1 ,000,000. It was estimated that autoimmune destruction may take up to 40 years to present clinical symptoms (Stromberg et al, 1998). However, cases have been reported of more rapid severe attacks as a result of drug reactions (Weston et al, 2000), during or after pregnancy, or spontaneously without any identified causes. Due to present difficulties in detection and diagnosis it is, however, assumed that the prevalence of autoimmune

hypophysitis could be much higher.

At present the diagnosis of hypophysitis is essentially based on the fact that other diseases can be excluded. Since hormone level markers do not always provide satisfying results, patients may undergo surgery to exclude a pituitary tumor, whereby the biopsy of the pituitary gland is due to its location not easily and safely performed. The currently available most preferred diagnosis method may be Magnetic Resonance Imaging (MRI) to identify any visual abnormalities in the Sella Turcica region (Caturegli, 2007, Crock et al, 2006), with the disadvantage that only severe damages of the gland can be visualized and a differential diagnosis, e.g., of pituitary tumor versus hypophysitis remains problematic.

It is evident that diagnosing of hypophysitis is difficult, but early diagnosis is very important to prevent the further development of the disease and treatment of the symptoms. Due to the current limited understanding of the mechanism involved, treatment of hypophysitis is limited to the administration of immunosuppressive agents like corticosteroids, azatiorpine or methotrexate. A definitive reduction of the pituitary mass can be achieved by neurosurgical intervention, however long term side effects may be severe and may include panhypopituitarism with or without diabetes insipidus (Falorni et al., 2014). Further, bromocriptine may be administered to reduce symptoms of visual impairment and lowering prolactin levels. Thus, there is the need of an improved diagnostic assay for autoimmune pituitary disorder, which is safe and easy to perform. Furthermore, an improved

understanding of the mechanism involved in the development of hypophysitis could lead to novel therapeutic approaches. SUMMARY OF THE INVENTION

Surprisingly, it was found that autoantibodies against MCT8 polypeptide are biomarkers for the detection of the onset and/or presence a thyroid hormone- related endocrine autoimmune dysfunction ("THREAD") in a subject. As described hereinafter, the present invention teaches methods for the diagnosis of such autoimmune-related dysfunctions in a subject.

Therefore, the present invention relates to a method for diagnosing the presence or the risk of development, or for monitoring of a thyroid hormone-related endocrine autoimmune ("THREAD") dysfunction in a subject, preferably

autoimmune-mediated hypophysitis, the method comprising analyzing a biological sample obtained from the said subject for the presence of autoantibodies against monocarboxylate transporter 8 (MCT8). Hereby, the presence of said

autoantibodies is indicative of the presence and grade of the dysfunction or the risk of development, or as a marker for the therapy control of said dysfunction in said subject. In a preferred embodiment of the method of the invention the concentration, resp., the amount of said autoantibodies against MCT8, whereby the presence of autoantibodies at a value which is above a threshold, which is characteristic for said dysfunction or risk is indicative of the presence or grade of the dysfunction or the risk. In another preferred embodiment, a labeled MCT8 is employed in the method of the invention. In another preferred embodiment of the method of the invention said biological sample is contacted in an immunoassay with an MCT8, even more preferred with a labeled MCT8. Another embodiment of the invention is a method for diagnosing the presence or the risk of development, or for monitoring of an autoimmune-related dysfunction ("ARD"), preferably of an endocrine autoimmune-related dysfunction ("EARD") in a subject, the method comprising analyzing a biological sample obtained from the subject for the presence of autoantibodies against monocarboxylate transporter 8 (MCT8) , preferably whereby the presence of said autoantibodies indicates the presence or the risk of development of the said dysfunction in said subject.

In one embodiment of the invention said biological sample is contacted in an immunoassay comprising MCT8 polypeptide, preferably labeled MCT8.

In a particularly preferred embodiment the labeled MCT8 is derived from human.

In a preferred embodiment of the invention said biological sample is previously obtained from a body fluid of said subject, e.g., peripheral blood, serum, cerebrospinal fluid.

Another embodiment of the invention is a method for diagnosing ARD, preferably EARD, and more preferred THREAD in a subject by determining the presence of autoantibodies specific for MCT8 comprising the steps of: a) performing an immunoassay by contacting a biological sample obtained from the subject with MCT8; b) detecting the presence of autoantibodies in the sample which bind MCT8; whereby the presence of autoantibodies which bind MCT8 protein is indicative for the said dysfunction. The methods according to the present invention allow for the diagnosis of the presence or risk of development of THREAD or other types of an ARD involving MCT8 autoantibodies, as defined herein. Moreover, the method according to the present invention allows for the stratification of the therapeutic regimen of a subject afflicted with THREAD or other types of an ARD involving MCT8 autoantibodies, or being at risk of developing THREAD or other types of an ARD involving MCT8 autoantibodies as defined herein. Moreover, the methods according to the present invention allow to identify the stratification of the dysfunction, in particular, the activity of the dysfunction and the subject afflicted with THREAD or other types of an ARD involving MCT8 autoantibodies. Based on the results obtained, the medical practitioner will stratify the therapy, i.e. decide upon the appropriate type of therapy and will apply the type of therapy

accordingly. In another embodiment, the methods of the present invention allow for the stratification of the therapeutic regimen of a subject afflicted with THREAD or other types of an ARD involving MCT8 autoantibodies, or being at risk of developing THREAD or other types of an ARD involving MCT8 autoantibodies. That is, the present invention allows to identify the status of the dysfunction, in particular, the active state of the dysfunction in a subject afflicted with THREAD or other types of an ARD involving MCT8 autoantibodies, thereby allowing to decide for appropriate further therapeutic steps including the administration of suitable pharmaceutically effective compounds. In another embodiment of the present invention test kits or diagnostic kits are provided for performing a method according to the present invention. In particular, the use of immunological kits for detecting autoantibodies in biological samples allowing diagnosis of THREAD or other types of an ARD involving MCT8 autoantibodies. That is, the present invention provides the use of a test kit or diagnostic kit for use in a method according to the present invention for

diagnosing the presence or the risk of a development as well as for the therapy control of THREAD or other types of an ARD involving MCT8 autoantibodies in a subject comprising means for determining autoantibodies against MCT8 in a biological sample of a subject to be tested and instructions on how to use the test or diagnostic kit. In a preferred embodiment, said test kit is an

immunoprecipitation assay or a bridge assay involving the binding interaction of autoantibodies with MCT8 protein or fragments derived thereof.

Such kits can generally comprise one or more antigens, namely, full protein, oligo- or polypeptides of MCT8 that can immune react with the autoantibodies. Typically, the kits of the invention will comprise in suitable container(s), one or more immune reactive MCT8 polypeptide antigens for MCT8 autoantibodies, preferably one or more aliquots of one or more labeled MCT8 antigens, and optionally, MCT8 autoantibody, which allow for the determination of the concentration or the amount of MCT8 autoantibodies. Said antigens useful in the methods and test kits of the invention may be the full MCT8 proteins or immune reactive polypeptides derived therefrom, preferably labeled and more preferred from human and labeled.

In another embodiment of the invention the kit may comprise one or more detection agents and, optionally, further comprises substrate and other means for performing the reaction with to detect the said autoantibody-antigen-complex. The immunodetection agents of the kit can include detectable labels that are associated with or linked to the given detecting agent. Detectable labels that are associated with the antigen or attached to a secondary binding ligand are also contemplated. Detectable labels include dyes, chemiluminescent or fluorescent molecules, biotin, radiolabels or enzymes. Typical examples for suitable labels include commonly known chemiluminescent molecules like acridinium esters, fluorescent molecules, like rhodamine, fluorescein, green fluorescent protein, luciferase, or alkaline phosphatase and horseradish peroxidase as examples for suitable enzymes.

Optionally, the kits further comprise positive and negative controls for verifying the results obtained when using the kit as well as calibration standards. The components of the kits can be packaged either in aqueous medium or in lyophilized form and, in addition, the kits comprise one or more containers allowing to conduct the detection. In addition, the test kit comprises instructions for use of the kit. For example, the immune reactive peptide is at least one peptide selected from SEQ ID No. 1 and SEQ ID No. 2 or, optionally, a

polypeptide fragment thereof.

Another object of the invention is the use of an isolated MCT8 for the identification of a therapeutically effective compound in the treatment of a thyroid hormone- related endocrine autoimmune dysfunction ("THREAD") or other types of an ARD involving MCT8 autoantibodies, said compound inhibiting the binding of MCT8 autoantibodies to MCT8 protein. Moreover, the present invention relates to the use of an isolated MCT8 in the diagnosis, risk assessment or therapy control of a thyroid hormone-related endocrine autoimmune dysfunction ("THREAD") or other types of an ARD involving MCT8 autoantibodies.

Another object of the invention is the use of a method of the invention

for the identification of a therapeutically effective compound in the treatment of a thyroid hormone-related endocrine autoimmune dysfunction ("THREAD") or other types of an ARD involving MCT8 autoantibodies, said compound inhibiting the binding of MCT8 autoantibodies to MCT8 protein.

Another object of the invention is the use of an isolated MCT8 in an assay for the identification of a compound, which interferes in the complex formation of MCT8 and autoantibodies against MCT8, preferably of a labeled or human MCT8, especially of a labeled human MCT8.

Yet another object of the invention is the use of a kit of the invention for the identification of a therapeutically effective compound in the treatment of a thyroid hormone-related endocrine autoimmune dysfunction ("THREAD") or other types of an ARD involving MCT8 autoantibodies, said compound inhibiting the binding of MCT8 autoantibodies to MCT8 protein.

Yet another object of the invention is a composition for the performance of a diagnostic assay of the invention, especially for the identification of an

autoantibody against MCT8 comprising an MCT8 protein, or an immunogenic fragment thereof. In one preferred embodiment the composition of the invention comprises a labeled MCT8 protein or fragment thereof, more preferred said MCT8 protein or fragment thereof is derived from human. DEFINITIONS

The subsequent definitions shall provide the preferred understanding of each term in the specific context of instant invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of the ordinary skilled in the art to which the present disclosed subject matter belongs. Typically, all the methods according to the present invention are in vitro methods.

The term "ARD" according to instant invention preferably means an autoimmune- related dysfunction, preferably, an endocrine autoimmune-related dysfunction ("EARD"), more preferred a thyroid hormone-related endocrine autoimmune dysfunction ("THREAD"), whereby the term "dysfunction" preferably means any disorder, disease or abnormal function, which is associated with a clinical symptom in an individual. In one embodiment of instant invention the term

"THREAD" means a pituitary dysfunction, and especially autoimmune

hypophysitis.

According to instant invention, the term "autoantigen" is synonym with the term "MCT8". The term "monocarboxylate transporter 8" is synonym with "MCT8" and preferably means according to instant invention a polypeptide belonging to the monocarboxylate transporter family known in the art (cf. Roef et al. 2013), which is involved in the transport of thyroid hormones into or out of a responsive cell, preferably a thyroid hormone responsive cell, under physiological conditions, preferably MCT8, MCT10, and polypeptide fragments thereof, preferably MCT8 and MCT10, i.e., NCBI Ref: NP 006508.2 for MCT8 and NP 061063.2 for MCT10. An exemplary MCT8 polypeptide sequence is defined by SEQ ID NO. 1 . An exemplary MCT10 polypeptide sequence is defined by SEQ ID NO. 2.

The term "fragment of MCT8" according to instant invention preferably means a immunoreactive functional portion of the MCT8 polypeptide having at least about 75%, preferably 85%, and more preferred 95% amino acid sequence identity to NCBI Ref: NP 006508.2 (MCT8) or 061063.2 (MCT10) and contains, preferably, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the entire length of at least one of the said polypeptides. The term "identity" of amino acid sequences means according to the invention the identity between two amino acid sequences compared to each other within the corresponding sequence region having approximately the same amount of amino acids. For example, the identity of a full-length of the two amino acid sequences may be compared. The amino acid sequences of the two molecules to be compared may differ in one or more positions, which however does not alter the biological function or structure of the molecules. Such variation may occur naturally because of different but similar amino acids or mutations/polymorphisms in the gene defining the amino acid sequence or they may be achieved by specific mutagenesis. The variation may result from deletion, substitution, insertion, addition or combination of one or more positions in the amino acid sequence. The identity of the sequences is measured by using ClustalW alignment (e.g. in www.ebi.ac.uk/Tools/Clustalw). The matrix used is as follows: BLOSUM, Gap pen:10, Gap extension: 0.5.

The term "identical" in the context of two amino acid sequences means that the sequences, when optimally aligned, such as by the programs GAP or BEST-FIT using default gap weights, share at least about 50% sequence identity. Typically sequences that are identical according to instant invention will exhibit at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% percent sequence identity to at least one the said MCT polypeptides.

The percent identity between two amino acid sequences can be determined, e.g., using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. Polypeptide sequences can also be compared using FASTA, applying default or recommended parameters. A program in GCG Version 6.1 ., FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 1990;183: 63-98; Pearson, Methods Mol. Biol. 2000;132:185-219). The percent identity between two amino acid sequences can also be determined using the algorithm of Meyers & Miller (Comput. Appl. Biosci., 1988;1 1 -17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Another algorithm for comparing a sequence to other sequences contained in a database is the computer program BLAST, especially blastp, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 1990;215:403-410; Altschul et al., Nucleic Acids Res. 1997;25:3389-402 (1997). The protein sequences of MCT8 as defined according to the present invention can there be used as a "query sequence" to perform a search in public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. 1990 (supra). BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the MCT polypeptides. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997 (supra). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.

ncbi.nlm.nih.gov.

The term "autoantibody" means an antibody that is directed against an

autoantigen, i.e., an endogenous antigen in a subject. The term "autoantibody against a MCT8" according to instant invention preferably means an endogenous MCT8, most preferred of human origin.

The terms "diagnosing" or "diagnosis" as used herein refer to methods by which a skilled artisan can estimate and even determine whether or not a subject is suffering from a given dysfunction, disease, disorder or condition. The skilled artisan makes the diagnosis on the basis of one or more diagnostic indicators, namely the autoantibodies detectable by instant invention, the amount (including presence or absence) of which is indicative for the presence, severity, or absence of the condition, optionally together with alternative diagnostic markers or indicators.

Hence, "making a diagnosis" or "diagnosing", as used herein, may further include making a prognosis which can provide for predicting a clinical outcome, selecting an appropriate treatment, or monitoring a current treatment and potentially change in the treatment based on the measure of the diagnostic autoantibody. The term "monitoring" according to instant invention preferably means the accompanying diagnosis in order to monitor the success of the therapeutic measures ("therapy control") during the treatment of the dysfunction.

The terms "determining" or "analyzing" as used herein refer to the assessment of the presence, absence, quantity, level or amount of the respective autoantibodies within the subject derived sample, including qualitative or quantitative

concentration levels of therapeutically effective substances. In particular, said terms include the physical detection or analysis, respectively. According to instant invention it has been determined that autoantibodies against an MCT8 gene product can be correlated with the presence or the risk of developing an ARD, in particular an EARD, and more specific a THREAD.

The skilled person is well aware of useful immunodetection methods allowing to analyze the sample for the presence or absence of autoantibodies in general. For example, the biological sample obtained from the subject is contacted with an antigen representing the autoantibodies immunoreactive antigenic epitopes, thus, allowing binding of the autoantibodies to said polypeptide. The knowledge of the skilled person about such techniques and methods in immunodetection can be easily transferred to the antigen MCT8 in order to practice the present invention. In this connection, the terms "polypeptide" and "protein" which are used

interchangeably herein, refer to a polymer of amino acids having a length of at least 50 amino acids. The term "oligopeptide" refers to a polypeptide having a length of from 5 to 49 amino acids.

Preferably, an autoantibody can be detected in a method of the invention by an immunoassay, which can, e.g., be selected from immunoprecipitation, bridge assay, enzyme-linked immunosorbent assay (ELISA), fluorescent immunosorbent assay (FIA), fluoresecence resonance energy transfer (FRET) assay, chemical linked immunosorbent assay (CLIA), radioimmunoassay (RIA), immunoblotting, immunometric assay, flow cytometry, western blot, and immunohistochemistry or any other method known to the skilled person. The autoantibodies to be detected in the method of the invention may be of the IgG or IgM type, which immunoglobulin subtypes additionally allow the use of specific detection reagents known to the skilled person.

Contacting the chosen biological sample comprising the antigen under suitable conditions and period of time sufficient to allow the formation of immune complexes, is generally a matter of adding the antigen (i.e., according to the present invention MCT8 or any of its immunoactive fragments) to the sample and incubating the mixture for a sufficient time period form immune complexes. Said antigen-antibody complexes can be detected by known means and methods as mentioned above. That is, the detection of immunocomplex formation of antigen- autoantibody can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker directly or indirectly to the autoantigen, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. Additional advantages may be employed through the use of a secondary binding ligand such as a second antibody or a biotine/avidine (streptavidine) ligand binding

arrangement as known in the art.

In some embodiments, the primary immune complex can be detected by a second binding ligand that has binding affinity for the antigen or the autoantibody presented in the sample, for example reactivity to the Fc region of the

autoantibodies or having reactivity to a region of the antigen different to the binding region of the autoantibody. In these cases, the second binding ligand can be linked to a detectable label or marker molecule. The second binding ligand is itself often an antibody which may thus be termed a secondary antibody.

Typically, the primary immune complexes are contacted with the labeled, secondary binding ligand or antibody, under suitable conditions and sufficient period of time. The secondary immune complexes are then generally washed to remove of any unbound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complex is then detected. Alternatively, comparative immunodetection methods may be used of which the skilled person is well aware of. According to instant invention the terms "individual", "patient" and "subject" are used interchangeably and preferably mean a mammalian individual, more preferred a human. In one embodiment of the invention the term "subject" means an individual, which is supposed to suffer from THREAD, especially hypophysitis. However, it will be understood that "patient" does not imply that overt symptoms are present.

The term "biological sample" according to instant invention preferably means a sample obtained before from the subject that may be extracted, untreated, treated, isolated, diluted or concentrated therefrom. Of note, the method according to the present invention does not include the step of taking the sample from the subject. Rather, the sample is an isolated sample which may be further processed. The biological sample is selected from any part of the patient's body, including, but not limited to hair, skin, nails, tissues or body fluids, such as saliva, synovia and blood as well as cerebrospinal fluid (liquor), preferably body fluids, more preferred serum, blood, plasma or liquor and most preferred serum. The term "labeled" according to instant invention preferably means directly or indirectly labeled, optionally, via a carrier, which comprises the antigen in an immobilized form. In other words, the immobilizing carrier may comprise the label to allow detection of the autoantibody-antigen complex according to the method of the invention. In another embodiment of the invention, the provided antigen is bound to a solid support, such as for example the material of a column matrix or the surface of a well of a microtiter plate, a membrane, beads, dip sticks or the like. Alternatively, such solid support can be provided as an additional element in the kit of instant invention. In a yet alternative embodiment of the invention, both ways of presenting the antigen may be combined, i.e. an immobilized antigen and one in solution, allowing the detection and quantification of the autoantibodies in a bridge assay format. BRIEF DESCRIPTION OF THE FIGURES

Figure 1A shows the presence of an MCT8 autoantibody interaction related signal in a healthy cohort of adult humans (n=200).

Figure 1 B shows the presence of an MCT10 autoantibody interaction related signal in a healthy cohort of adult humans (n=200).

Figure 2A shows the presence of an MCT8 autoantibody interaction related signal in a cohort of adult humans suffering from hypophysitis (n=18).

Figure 2B shows the presence of an MCT10 autoantibody interaction related signal in a cohort of adult humans suffering from hypophysitis (n=18).

Binding Index means the value of the quotient of the signals of sample/negative control (buffer).

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987);

"Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991 ). Generally, these techniques are applicable to the production of the polynucleotides and polypeptides used in the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the example sections.

SEQUENCES:

Seq. ID No. 1 amino acid sequence (539 aa) from hMCT8

Seq. ID No. 2 amino acid sequence (515 aa) from hMCT10

Seq. ID No. 3 Primer P1 (27mer)

Seq. ID No. 4 Primer P2 (27mer)

Seq. ID No. 5 DNA (1656 bp) encoding firefly luciferase

Seq. ID No. 6 amino acid sequence (550 aa) from firefly luciferase Seq. ID No. 7 DNA (1620 bp) encoding hMCT8

Seq. ID No. 8 Primer P3 (32mer)

Seq. ID No. 9 Primer P4 (33mer)

Seq. ID No. 10 DNA (3276bp) encoding a fusion protein of hMCT8-luciferase

Seq. ID No. 1 1 amino acid sequence (1091 aa) of the fusion protein hMCT8-luc

Seq. ID No. 12 DNA (1548 bp) encoding hMCTI O

Seq. ID No. 13 Primer P5 (32mer)

Seq. ID No. 14 Primer P6 (43mer)

Seq. ID No. 15 DNA (3204bp) encoding a fusion protein of hMCTI O-luciferase

Seq. ID No. 16 amino acid sequence (1067 aa) of the fusion protein hMCT10-luc

The following examples illustrate the invention and provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods of the invention. Those of skill in the art will appreciate that the following examples are intended to be exemplary only as numerous changes, alterations and variations can be employed without departing from the scope of the present invention.

EXAMPLES DNA primers were obtained from BioTeZ Berlin Buch GmbH (Berlin, Germany); pSP-luc+NF vector was obtained from Promega GmbH (Mannheim, Germany); pIRESneo vector was obtained from Clontech (Palo Alto, CA, USA); Goat anti human IgG (SIGMA) was labeled with Acridinium NHS Ester (Cayman Chemical Company, Ann Arbor, Ml, USA); CellTiter-Glo luminescent cell viability assay kit was obtained from Promega GmbH.

If not otherwise stated, all other reagents and chemicals were obtained from Sigma- Aldrich Chemie GmbH (Munich, Germany) or Merck KGaA (Darmstadt, Germany); enzymes were obtained from Promega or New England Biolabs (Ipswich, MA, USA). Hereinafter "PBS" means 10 mM PO ^3", 137 mM NaCI and 2.7mM KCI, pH 7.4; "room temperature" means a temperature in the range of 20°C to 24°C. Example 1 : Construction of fusion proteins

Example 1A: Construction of an MCT8-luciferase fusion protein

DNA (Seq. ID No. 5) encoding firefly luciferase (Seq. ID No. 6) on pSP-luc+NF was amplified by PCR using primers P1 (Seq. ID No. 3) and P2 (Seq. ID No. 4) containing EcoRI and BamHI restriction sites, respectively. pIRESneo was digested with EcoRI and BamHI restriction endonucleases; the obtained fragment was replaced with the DNA encoding firefly luciferase obtained from the aforementioned PCR resulting in plasmid pIRESneo-Luc. The DNA (Seq. ID No. 7) encoding human MCT8 (Seq. ID No. 1 ) was amplified by PCR using primers P3 (Seq. ID No. 8) and P4 (Seq. ID No. 9) containing Notl and EcoRI restriction sites, respectively. pIRESneo-Luc was digested with Notl and EcoRI restriction endonucleases and the obtained fragment was replaced with the DNA sequence encoding hMCT8 obtained from the aforementioned PCR resulting in vector plRESneo-hMCT8-Luc containing Seq. ID No. 10 encoding the hMCT8-Luc fusion polypeptide of Seq. ID No. 1 1 .

Example 1 B: Construction of an MCT10-luciferase fusion protein

Plasmid pIRESneo-Luc was obtained as described in Example 1A.

The DNA (Seq. ID No. 12) encoding human MCT10 (Seq. ID No. 2) was amplified by PCR using primers P5 (Seq. ID No. 13) and P6 (Seq. ID No. 14) containing Notl and EcoRI restriction sites, respectively. pIRESneo-Luc was digested with Notl and EcoRI restriction endonucleases and the obtained fragment was replaced with the DNA sequence encoding hMCTI O obtained from the

aforementioned PCR resulting in vector plRESneo-hMCT10-Luc containing Seq. ID No. 15 encoding the hMCT10-Luc fusion polypeptide of Seq. ID No. 16.

Example 2: Generation of hMCT-Luc fusion protein producing cells

Example 2A: Generation of HEK293 cells producing hMCT8-Luc or hMCT10-Luc HEK 293 cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum. Cells were cultivated in a 5% CO2 atmosphere at 37°C. HEK 293 cells were transfected with plRESneo-hMCT8-Luc vector and plRESneo-hMCT10-Luc vector, respectively, using FuGENE6 transfection reagent (obtained from Roche Deutschland Holding GmbH, Grenzach-Wyhlen, Germany) according to the manufacturer's instruction. 48 hours after transfection, selection was started with 0.8 mg/ml G418 (Gibco™ BRL, Invitrogen). Stable clones expressing high levels of fusion protein were selected.

Example 3: Production of MCT-Luciferase fusion protein

Example 3A: Confluent HEK 293 cells (producing either hMCT8-Luc or hMCTI O- Luc) grown until confluent in a 75 cm² plate were resuspended by scraping into PBS buffer and were washed in the same buffer by centrifugation at 2,000g at 4°C for 5 minutes. The resulting cell pellets were lysed in 0.5 ml lysis buffer containing 50 mM Tris-HCI, 100mM NaCI, 2% (v/v) Triton X-100, 10% (v/v) glycerol pH 7.5. The suspension was centrifuged at 2,000g at 4°C for 15min and the supernatant was collected and stored at -80°C until used. Example 4: Isolation of human IgG

0.3 ml of human serum was diluted with 0.3 ml PBS and mixed with 0.3 ml of 10% (v/v) POROS protein A suspension. The mixture was incubated overnight at 4°C under constant movement. The beads were pelleted at 2,000g and washed six times with PBS. Bound IgG was eluted with 25mM citric acid and the pH was adjusted to 7.0 using 1 M Hepes-NaOH (pH 8.0). Eluted IgG was concentrated to 300 μΙ using Centricon Filters at 4°C and 1 ,000g.

Example 5: Immunoprecipitation assay for hMCT-8 autoantibodies

The hMCT8-Luc cell extract was diluted with dilution buffer containing 50 mM KH2PO4/ K2HPO4 (pH 7.5), 100 mM NaCI, 10% (v/v) Glycerol, 1 % (v/v) Triton X- 100 and 5 mg/ml BSA. For the immunoprecipitation 100 μΙ of the diluted extract (about 1 .0e07 RLU) was mixed with 10 μΙ sample and incubated overnight at 4°C under constant movement. Immune complexes were precipitated by addition of 100μΙ of 10% (v/v) POROS Protein A suspension for 1 .5 h at room temperature under constant movement. POROS Protein A was pelleted at 2,000g, 5 min. at 4°C and washed 3 times with 1 ml of washing buffer (50 mM KH2PO4/ K2HPO4 (pH 7.5), 100 mM NaCI and 0.1 % Triton X-100. Luciferase activity was measured in a luminometer Berthold LB 953 for 5 seconds and results are expressed as RLU bound. Example 6: MCT8 autoantigen is a biomarker for THREAD

Patient sera was analyzed for the presence of autoantibodies against MCT8 and MCT10. The results are shown in the Figures and Tables below, wherein "SD" means Standard deviation, "IQR" means inter quartile range, "PT" means positive threshold, i.e., the threshold value for positive samples indicating the presence or onset of a THREAD. The values in tables 1 to 4 are shown in relative units.

Table 1 : hMCT8 autoantibody level in the serum of a healthy cohort of male and female adults (each n=100) (cf. Fig. 1A)

Number of values 100 female 100 male

Minimum 0.710 0.560

25% Percentile P25 1 .030 0.990

Median 1 .150 1 .105

75% Percentile P75 1 .268 1 .273

Maximum 2.120 2.020

Mean 1 .198 1 .157

Standard Deviation 0.2531 0.2621

PT > mean + 2^*SD 1 .70 1 .70

PT > P75 + 1 .5^*IQR 1 .63 1 .69

Table 2: hMCTI 0 autoantibody level in the serum of a healthy cohort of male and female adults (each n=100) (cf. Fig. 1 B)

Number of values 100 female 100 male

Minimum 0.620 0.480

25% Percentile P25 1 .060 0.975

Median 1 .170 1 .175

75% Percentile P75 1 .490 1 .420

Maximum 1 1 .55 7.490

Mean 1 .527 1 .369

Standard Deviation SD 1 .343 0.883

PT > mean + 2^*SD 4.21 3.13

PT > P75 + 1 .5^*IQR 2.78 2.75 Table 3: hMCT8 autoantibody level in the serum of a cohort of patients suffering from hypophysitis (n=18) vs a negative control of healthy individuals (n=10) (cf. Fig. 2A)

Number of values 18

Minimum 2.140

25% Percentile P25 3.408

Median 4.345

75% Percentile P75 6.593

Maximum 1 1 .98

Mean 5.019

Standard Deviation SD 2.324

PT > mean + 2^*SD 9.67

PT > P75 + 1 .5^*IQR 1 1 .37

Table 4: hMCTI O autoantibody level in the serum of a cohort of patients suffering from hypophysitis (n=18) vs. a negative control of healthy individuals (n=10) (cf. Fig. 2B)

Number of values 18

Minimum 1 .240

25% Percentile P25 2.040

Median 3.005

75% Percentile P75 4.640

Maximum 15.95

Mean 4.277

Standard Deviation SD 3.693

PT > mean + 2^*SD 1 1 .66

PT > P75 + 1.5^*IQR 8.54

The results indicate that the presence of autoantibodies against hMCT8 or hMCT10 in the serum of an individual in an amount greater than the defined threshold indicates the presence or the onset of a THREAD. LITERATURE

Caturegli, P Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s). J. Clin. Endocrinol. Metab. 2007; 92(6):2038-2040.

Crock, PA., et al., Pituitary autoantibodies, Neuroendocrinology,

Current Opinion in Endocrinology & Diabetes 2006; 13(4):344-350.

Falorni A et al, Diagnosis and classification of autoimmune hypophysitis, Autoimm reviews 2014; 13(4-5):412-6.

Friesema, ECH et al. Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. Lancet 2004; 364:1435- 1437.

McLachlan SM & Rapoport B Breaking tolerance to thyroid antigens: changing concepts in thyroid autoimmunity. Endocr. Rev. 2014;35(1 ):59-105.

Medici M et al. Genetic determination of the hypothalamic-pituitary-thyroid axis: where do we stand? Endocr. Rev. 2015; 36(2):214-244.

Roef GL et al. Associations between single nucleotide polymorphisms in thyroid hormone transporter genes (MCT8, MCT10 and OATP1 C1 ) and circulating thyroid hormones Clinica Chimica Acta 2013; 425, 227-232.

Stromberg S et al. Pituitary autoantibodies in patients with hypopituitarism and their relatives. J. Endocrinol. 1998; 157(3):475-480.

Weston SN & Weston CF The mysterious case of the lost pituitary: amiodarone- induced hypothyroidism. Hosp Med. 2000; 61 (1 ):64-65.

Claims

1 . An in vitro method for diagnosing the presence or the risk of development, or for monitoring of a thyroid hormone-related endocrine autoimmune dysfunction (THREAD) in a subject, the method comprising analyzing a biological sample obtained from said subject for the presence of autoantibodies against monocarboxylate transporter 8 (MCT8).

2. The method according to claim 1 , wherein the concentration of an

autoantibody against MCT8 is determined.

3. The method according to claim 1 or 2, wherein the MCT8 is a labeled MCT8.

4. The method according to any of claims 1 -3, wherein said autoantibody

concentration in the sample is compared to one or more reference values, whereby a concentration of the autoantibody against MCT8 greater than a threshold indicates the presence and grade of a dysfunction.

5. The method according to any of claims 1 -4, wherein said MCT8 is human.

6. A kit for the diagnosis and/or monitoring of a thyroid hormone-related

endocrine autoimmune dysfunction (THREAD) by determining in a sample of a subject the presence or concentration of a MCT8 autoantibody comprising one or more aliquots of one or more labeled MCT8.

7. The kit according to claim 6, wherein said MCT8 is derived from a human

MCT8.

8. The use of a MCT8 derived from human in a method for the diagnosis, risk assessment or therapy control of a thyroid hormone-related endocrine autoimmune dysfunction.

9. The use of an isolated MCT8 in an assay for the identification of a compound, which interferes in the complex formation of MCT8 and autoantibodies against MCT8.

10. Connposition for the performance of a diagnostic assay for the identification of an autoantibody against MCT8 comprising MCT8 protein, or an immunogenic fragment thereof.

1 1 . The composition according to claim 10, wherein said MCT8 protein or

fragment thereof is labeled.

12. The composition according to claim 10 or 1 1 , wherein said MCT8 protein or fragment thereof is derived from human.