Field of the invention

The present invention relates to methods for diagnosing the presence or the risk of development or the therapy control of sarcoidosis in human subjects. In addition, the present invention relates to test kits for use in the diagnosis of the presence or the risk of development, or for the therapy control of sarcoidosis in human subjects. In particular, the present invention relates to a method for diagnosing the presence or the risk of development, or for the therapy control of sarcoidosis, in a subject testing for the presence of antibodies against Negative elongation factor E (NELF-E)(RNA-binding protein RD) or a NELF-E analog protein, preferably bacterial NELF-E analog protein, or immune-reactive peptides thereof, in a subject. The presence of antibodies against NELF-E or immunoreactive peptides thereof, is indicative for the presence or the risk of development, or for the therapy control of sarcoidosis. That is, detection of the presence of antibodies against NELF-E or immunoreactive peptides thereof, allows early diagnosis of sarcoidosis.

Background of the invention

Sarcoidosis belongs to multisystemic inflammatory orphan diseases (lannuzzi M, Rybicki BA, Teirsten AS. Sacoidosis. Nejm 2007;375(21 ):2153-2165.). Sarcoidosis is characterized by noncaseeating granulomatous infiltrations of the soft tissue. It affects younger adults between 20 and 40 years (yrs) and older adults beyond the age of 50yrs. Its incidence varies between 10 and 50/100.000. In Germany, the prevalence is 40/100.00 and its incidence is 12/100.000.

Sarcoidosis can affect all organs, but the main organ involvement are the lymphe nodes, lung, skin, eyes and joints.

The diagnossis of sarkoidosis relies on the typical symptomatic trias, known as Loffgren trias, or positive biopsy in combination with radiologic findings or typical clinical findings, however exclusion of malignancies, infectious diseases and other inflammatory diseases is necessary.

So fare, no none invasive blood test for sarcoidosis has been established. Therefore, we wanted to investigate, weather our established screening using protein array technology could identify new autoantibodies for the diagnosis of sarcoidosis.

Brief description of the present invention

The present invention provides a diagnostic method for the presence or the risk of developing sarcoidosis, as well as for therapy control of sarcoidosis in a subject. That is, the present inventors recognized that subjects suffering from sarcoidosis have antibodies directed against NELF-E or immunoreactive peptides thereof, preferably human NELF-E or bacterial homologs, or

immunoreactive peptides thereof. Hence, early diagnosis of sarcoidosis possible before invasive and/or cost-intensive procedures have to be performed.

Thus, a first embodiment of the present invention relates to a method for diagnosing the presence or the risk of development, or for the therapy control of sarcoidosis in a subject comprising

a. obtaining from the subject a biological sample, and

b. analyzing the sample for the presence of antibodies against NELF-E or immunoreactive peptides thereof,

whereby the presence of antibodies against NELF-E or immunoreactive peptides thereof, is indicative of the presence or the risk of development, or for the therapy control of sarcoidosis.

Preferably, the biological sample is one from a human subject. Typically, the presence of antibodies against human NELF-E, i.e. autoantibodies, and/or bacterial NELF-E homologs, or immunoreactive peptides thereof, are detected.

Alternatively or in addition, it is preferred that antibodies against bacterial NELF- E homologs or immune reactive peptides thereof, are detected.

Preferably said detection of antibodies is conducted using immunoassays, like ELISA. Typically, the biological sample to be tested is obtained from blood, in particular serum.

Thus, the diagnostic method according to the present invention represents the first method allowing positive diagnosis of sarcoidosis in a subject via antibody markers. Diagnosis is possible at an early stage of the disease. Another embodiment of the present invention relates to a test kit for use in a method according to the present invention, namely for diagnosing the presence or the risk of a development as well as for therapy control of sarcoidosis in a subject comprising means of determining antibodies against NELF-E and

immunoreactive peptides thereof, in a biological sample of a subject to be tested and instruction on how to use said test kit. Preferably, the test kit is an ELISA assay.

Brief description of the drawings

Fig. 1 : The figure shows the titers of IgG-antibodies against the NELF-E (Seq.

ID. No.1 ) in different diseases. The titer is considered positive, if it is the same or higher than the cut off serum. From the left to the right the different test groups are listed: Sarcoidosis, small vessel vasculitis (SVV), systemic lupus erythematosus (SLE), febrile infectious diseases (FID), and blood donors (BD).

Fig.2: In figure 1 the positive patients for the human IgG anti-NELF-E antibodies and the positive patients for the human IgA anti-NELF-E (using Seq. ID. No.1 ) in sarcoidosis versus febrile infectious diseases (FID) are shown. In addition, a chi- squared distribution table shows the sensitivity and sensitivity.

Fig.3: In In figure 1 , the positive patients for the human IgG anti-NELF-E antibodies are shown, here the peptide of Seq. ID. No. 2, using different diseases: Sarcoidosis, systemic lupus erythematosus (SLE) and blood donors (BD). Fig.4: In figure 1 , the positive patients for the human IgG anti-NELF-E antibodies are shown, here the peptide of Seq. ID. No. 3, using different diseases:

Sarcoidosis, systemic lupus erythematosus (SLE), febrile infectious diseases (FID), lung diseases (including lung fibrosis, COPD, bronchial cancer) and blood donors (BD).

Fig.5: In figure 1 , the positive patients for the human lgG4 anti-NELF-E

antibodies are shown, here the peptide of Seq. ID. No. 1 , using different diseases: Sarcoidosis, systemic lupus erythematosus (SLE) and blood donors (BD).

Detailed description of the present invention

In a first aspect, the present invention relates to a method for diagnosing the presence or the risk of development, or for the therapy control of sarcoidosis in a subject comprising

a. obtaining from the subject a biological sample, and

b. analyzing the sample for the presence of antibodies against NELF-E,

preferably against NELF-E, or immunoreactive peptides thereof,

whereby the presence of antibodies against NELF-E or immunoreactive peptides thereof, is indicative for the presence or the risk of development, or for the therapy control of sarcoidosis.

That is, the present invention is based on the observation of the present inventor that subjects afflicted with sarcoidosis have antibodies against NELF-E, typically against the NELF-E, or immunoreactive peptides thereof.

The term NELF-E includes also fragments of NELF-E containing an immuno reactive peptide. The NELF-E peptide may be NELF-E derived from eukaryotes or prokaryotes. For example, the NELF-E may be human NELF-E, non-human eukaryotic or prokaryotic NELF-E which is a analog of the eukaryotic NELF-E, like bacterial NELF-E, or immunoreactive peptides derived from eukaryotic or prokaryotic NELF-E. Immunoreactive peptides are peptides of at least 7 amino acid residues, like 8 to 1 1 amino acid residues or longer. Immunoreactive peptides of NELF-E comprise an epitop of an anti-NELF-E antibody, like an autoantibody.

NELF-E is part of the NELF complex. The NELF complex negatively influences the elongation of transcription by RNA polymerase II. NELF-E shows the strongest RNA binding activity of the NELF complex and may start recruiting the NELF complex to RNA. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation.

References:

Yamaguchi Y., Takagi T., Wada T., Yano K., Furuya A., Sugimoto S., Hasegawa J., Handa H. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 1999 2;97(1 ):41 -51

Yamaguchi Y., Inukai N., Narita T., Wada T., Handa H. Evidence that negative elongation factor represses transcription elongation through binding to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA. Mol Cell Biol 2002 May;22(9):2918-2927

Narita T., Yamaguchi Y., Yano K., Sugimoto S., Chanarat S., Wada T., Kim D.-K., Hasegawa J., Omori M., Inukai N., Endoh M., Yamada T., Handa H., Human transcription elongation factor NELF: identification of novel subunits and

reconstitution of the functionally active complex. Mol Cell Biol 2003 Mar;23(6):1863- 1873

Vos S.M., Pollmann D., Caizzi L., Hofmann K.B., Rombaut P., Zimniak T., Herzog F., Cramer P. Architecture and RNA binding of the human negative elongation factor. Elife 2016 Jun 10;5.

The present invention demonstrates that NELF-E or immunoreactive peptides thereof, represents an entity to which antibodies can be found in subjects afflicted with sarcoidosis. Hence, determining the presence of antibodies against NELF-E (e.g. human NELF-E alone, bacterial NELF-E alone or a combination of both) is indicative of the presence or the risk of development, or for the therapy control of sarcoidosis in a subject. In particular, antibodies against NELF-E and/or the prokaryotic analog thereof, or immune reactive peptides thereof, e.g. against the protein sequence(s) according to Seq. ID No. 1 and/or Seq. ID No. 2 and/or Seq. ID No. 3 are detected.

As demonstrated in the examples, the determination of antibodies against at least one of these molecules allows to identify individuals suffering from sarcoidosis. Particular preferred, the immunoreactive peptide of NELF-E is derived from the NELF-E, preferably of human NELF-E, or its analogs in prokaryotes. For example, the NELF-E peptide is any one of Seq. ID No. 1 .

In particular, it is demonstrated herein that determining the presence of antibodies against NELF-E allows a specific diagnosis of sarcoidosis, in particular, at early stages. Until today, said diseases can be diagnosed by extensive and cost-intensive diagnosis or exclusion of other diseases, disorders or conditions, only. Thus, it is possible for the first time to diagnose the presence or the risk of development of sarcoidosis in a subject with a simple test system or test kit based on the method described herein. Further, it is possible to allow therapy control of subjects afflicted with sarcoidosis.

For example, the present invention allows to identify the therapy regimen of an individual in need thereof. That is, identifying the presence of antibodies against NELF-E or immune reactive peptides thereof, suggests a therapy comprising 13- cell depletion, inducing tolerance in T-cells or to any other therapy in order to eliminate NELF-E specific antibodies. In a preferred embodiment, the presence of antibodies against both, human NELF-E and bacterial protein is determined. That is, by determining the presence of antibodies against both, human NELF-E, and bacterial homologs, or immunoreactive peptides thereof, the specificity of diagnosing sarcoidosis.

The terms“patient” and“subject” are used interchangeably and refer to patients and subjects of humans or other mammals and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that“patient” does not imply that symptoms are present.

The term“biological sample” as used herein refers to a sample that may be extracted, untreated, treated, isolated, concentrated from a patient. Suitably, 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. It is preferred that the samples are from the blood of said individual, like from the sera. Throughout this specification, unless the content requires otherwise, the word “comprise” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

With the term“obtained from” is meant that a sample such as, for example serum is isolated from or derived from a particular source of the subject. For example, the extract can be obtained from tissue or a body fluid isolated directly from the subject.

As used herein the terms“a”,“an” and“the” mean“one or more” when used in this application, including the claims. 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 matters belongs.

The terms“diagnosing” and“diagnosis” as used herein refers to methods by which a skilled artisan can estimate and even determine whether or not a subject is suffering from a given disease, disorder or condition. The skilled artisan makes the diagnosis on the basis of one or more diagnostic indicators, namely antibodies, the amount (including presence or absence) of which is indicator for the presence, severity, or absence of the condition.

Along with diagnosis, therapy control and clinical prognosis is also an area of great concern and interest. It is important to know the severity of the disease as well as the activity of the disease in order to design the most effective therapy.

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 current treatment and

potentially change the treatment based on the measure of a diagnostic antibody.

The term“determining” or“analyzing” as used herein refers to assessing the presence, absence, quantity, level or amount of the respective antibodies within the subject derived sample, including qualitative or quantitative concentration levels of said substances otherwise evaluating the values or categorisation of a subject clinical parameter.

It has been determined by the inventor that antibody production against NELF-E or immune reactive peptides thereof, can be correlated with the presence of sarcoidosis.

In addition, in some embodiments of the presently disclosed subject matter, multiple determinations of the antibodies over time can be made to facilitate stratification, diagnosis and/or prognosis.

In some embodiments of the methods disclosed herein, detecting, determining or analyzing the presence of the antibodies in the sample can include binding the antibodies to antigen and then detecting either the binding event or the presence of the antibody isolated from the biological sample. Exemplary techniques for detecting the antibodies include, but are not limited to, enzyme-linked

immunosorbent assay (ELISA), radio immunoassay (RIA), multiplex

immunoassay, immunoprecipitation and immunoblotting (including for example western blotting and dot blotting).

The skilled person is well aware of useful immunodetection methods allowing analyzing the sample for the presence or absence of antibodies against N ELF-E, in particular heavy chain N ELF-E, or immunoreactive peptides thereof. For example, the biological sample obtained from the subject is contacted with an antigen, namely with N ELF-E or oligo-, polypeptide or protein containing the antibody immunoreactive peptide including a NELF-E epitope, thus, allowing binding of the antibody to said peptide. In this connection, the term“polypeptide” or“protein” which are used interchangeably herein, refer to a polymer of amino acids heaving a length of at least 50 aa. The term“oligopeptide” refers to a polymer of amino acids heaving a length of from 5 to 49 aa. The term“peptide” include polypeptides, oligopeptides as well as proteins. Contacting the chosen biological sample of the antigen under conditions effective and for a period of time sufficient to allow the formation of immune complexes, is generally a matter of adding the composition to the sample and incubating the mixture for a period of time along enough for the antibodies to form immune complexes with the antigens presented. Said antigen antibody mixture can be detected by known means and methods. That is, detection of immune complex 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, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotine/avidine (streptavidine) ligand binding arrangement as it is 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 antibody presented in the sample, for example reactivity to the Fc region of the antibodies or having reactivity to a region of the antigen different to the binding region of the antibody. 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 labelled, secondary binding ligand or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove off any unbound labelled secondary antibodies or ligands, and the remaining label in the secondary immune complex is then detected.

The second binding ligand, such as an antibody, having binding activity for either the antigen or antibody, may also be used to bind to the primary immune complexes. The second binding ligand contains an enzyme capable of

processing a substrate detectable to a product and, hence, amplify a signal over time. After washing, the secondary immune complexes are contacted with substrate, permitting detection. Alternatively, comparative immunodetection may be used. The skilled person is well aware of suitable methods.

It is particularly preferred that the biological sample is a body fluid, preferably blood. In particular, the biological sample is serum of the subject to be

diagnosed.

Further, in another embodiment, the methods according to the present invention allows for the stratification of the therapeutic regimen of a subject afflicted with sarcoidosis or being at risk of developing sarcoidosis.

That is, the present invention allows to identify the status of disease, in particular, the active state of the disease in a subject afflicted with sarcoidosis.

The antibodies to be detected may be of the IgA, IgM, IgE, IgD and/or IgG type or IgG 1 , lgG2, lgG3, lgG4 subtype. That is, it is possible to determine the presence of the IgA, IgM, IgE, IgD and/or IgG type or lgG1 , lgG2, lgG3, lgG4 antibodies in the biological sample obtained from the subject. Although it is sufficient to detect only one type of antibodies, either IgA, IgM, IgE, IgD and/or IgG type or IgG 1 , lgG2, lgG3, lgG4 antibodies, it is preferred that IgA and IgG and lgG4 antibodies against NELF-E are detected. In a particular preferred embodiment, the subject is a human and the antibodies are human

autoantibodies.

In a further embodiment, the presently disclosed subject matter provides test kits or diagnostic kits for the use in a method according to the present invention. In particular, immunological kits for use in detecting antibodies in biological samples allowing diagnosis of sarcoidosis. That is, the present invention provides a test kit for use in a method according to the present invention for diagnosing the presence of the risk of a development as well as for the therapy control of sarcoidosis, in a subject comprising means or determining antibodies against NELF-E or immunoreactive peptides derived from NELF-E in a biological sample of a subject to be tested and instructions on how to use the test kit. In preferred embodiment said test kit is an ELISA. Such kits can generally comprise or more antigens, namely oligo- or polypeptides of NELF-E that can immunoreact with the antibodies. Typically, the immunodetection kits will comprise in suitable container(s), one or more antibody immunoreactive peptide antigens derived from NELF-E. Said antigens useful in the presently claimed methods and test kits may be the full NELF-E or

immunoreactive peptides derived therefrom.

In certain embodiments, the antigen can be provided bound to a solid support, such as for example a column matrix or a well of a microtiter plate, a membrane, beads, dip sticks or the like. Alternatively, the support can be provided as a separate element of the kit.

That is, the test kit according to the present invention for diagnosing sarcoidosis includes beside the antigen a detection agent for the antibodies which may be an antibody, antibody fragment etc. In addition, the kit may comprise more than one detection agent. If required, the kit further comprises substrate and further means for allowing reaction with an enzyme used as label for the detecting agent which may be an antibody.

The immunodetection agents of the kit can include detectable labels that are associated with or linked to the given detecting agent, in particular, the detecting antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Detectable labels include dyes,

illuminescent or fluorescent molecules, biotin, radiolabels or enzymes. Typical examples for suitable labels include commonly known fluorescent molecules, like rhodamine, fluorescein, green fluorescent protein or 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. The components of the kits can be packaged either in aqueous medium or lyophilised 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. Moreover, the present invention relates to the use of NELF-E, immune reactive sequences or analogs thereof in the diagnosis, risk assessment or therapy control of sarcoidosis.

Finally, the present invention relates to the use of peptides derived from NELF-E including immune reactive peptides in prophylaxis and/or treatment of

sarcoidosis. That is, the use of NELF-E including the immune reactive peptides of said molecules allow to induce tolerance, thus, being useful in prophylaxis and therapy of sarcoidosis. For example, NELF-E, immunoreactive peptides thereof, may be used for systemic or local therapy of diseases, disorders or conditions which are associated with an immunoreaction against said

compounds. The administration thereof may be effected orally, parenterally or via mucosal membranes.

Hence, the present invention provides pharmaceutically compositions containing NELF-E molecules including immunoreactive peptides for use in the prophylaxis and treatment of the disease identified herein as well as in diseases, disorders or conditions associated with an immune reaction against these compounds. The pharmaceutical composition may be provided in a suitable form. The skilled person is well aware of useful forms, dosages etc.

Examples

The following examples have been included to illustrate modes of the present disclosed subject matter. In light of the present disclosure and the general level of the skilled in the art, those of skilled will appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the scope of the present disclosed subject matter.

Example 1

Sera of patients with different inflammatory and rheumatic diseases were screened via protein array technology. New markers of sarcoidosis have been identified which have been evaluated further for frequency and association by different ELISA.

The evaluation was performed with sera of patients with sarcoidosis and with sera of controls (blood donors, patients with systemic lupus erythematosus, malignant diseases, different lung diseases, infectious diseases, HIV infected persons and small vessel vasculitis). The patients had been well characterized with regard to the disease activity, demographic data and treatment. The sera of the patients and of the controls had been stored in a -20°C freezer until use.

For performing the ELISA tests, 96 well plates (Nunc Maxisorb) were coated with 10 pg recombinant NELF-E (amino acids, purchased from Abeam, Cambridge, UK)(Seq. ID. No. 1 ), or with 100 pg of peptide consisting of the NELF-E peptid I (synthesized by Biomatik, U.S.A.)(Seq. ID. No.2) or with 100 pg of the NELF-E peptid II (synthesized by Biomatik, U.S.A.)(Seq. ID. No. 3). The plates were incubated with sera in a 1 :100 dilution in PBS for 30 to 60 minutes at room temperature. As a standard, a serum of a patient with active sarcoidosis was used. The concentration of antibodies in this serum was defined as 1 AU for IgG- type antibodies against NELF-E and the NELF-E peptides as well as for IgA-type NELF-E antibodies. After 30 to 60 minutes incubation, the plates were washed 3 times with PBS. Next, 100 pi of a secondary peroxidase-goat anti-human IgG or IgA antibody labelled with horse radish peroxidase (HRP) (Jackson

ImmunoResearch Europe Ltd.) was added in a dilution of 2:10000 or 2 pi in 8 ml PBS and 2 ml 5% BSA or 100 pi of a secondary peroxidase-mouse anti-human lgG4 labelled with HRP (Abeam, Cambridge, UK). The plates were incubated for 30 minutes at room temperature and washed 3 times with PBS. The color reaction was performed with tetramethylbenzidine (TMB) (Therma Scientific) for up to 15 minutes according to the manufacturer’s instructions and the ODs were read at 450 nm in an ELISA reader.

The test for IgG-type antibodies against NELF-E protein and against the NELF-E peptide I and mycobacterial homolog sequence NELF-E II (“NELF-E analog protein”) had a sensitivity for sarcoidosis of 48%, 56% and 50%, respectively, and a specificity (considering only the non-autoimmune controls) of 90%, 97% and 96%. IgG- type antibodies against NELF-E peptide II showed a specificity of 94% considering the infectious diseases and of 95% considering non-sarcoidosis lung diseases. IgG- type antibodies against NELF-E peptide II showed a specificity considering only the systemic lupus erythematosus patients of 89%. The test for lgG4-subtype antibodies against NELF-E had a sensitivity for sarcoidosis of 31 % and a specificity (considering only the non-autoimmune controls) of 96% and (considering only the infectious diseases) 96%, respectively.

By using the combination of Anti-lgG NELF-E peptide I as positive biomarker of sarcoidosis and Anti-IgA NELF-E as negative biomarker of sarcoidosis sensitivity and specificity changes from 61 % and 72% to 61 % and 86% considering FID.