Biomarkers in inflammatory diseases

The present invention relates to biomarkers in inflammatory diseases, namely CD1 B and CD1 D as biomarkers in certain disorders

CD1 (cluster of differentiation 1 ) is a family of glycoproteins expressed on the surface of various human antigen-presenting cells They are related to the class I MHC molecules, and are involved in the presentation of lipid antigens to T cells

CD1A, CD1 B and CD1 C (group 1 CD1 molecules) are expressed on cells specialized for antigen presentation, CD1 D (group 2 CD1 ) is expressed in a wider variety of cells Group 1 CD1 molecules have been shown to present foreign lipid antigens and specifically a number of mycobacterial cell wall components, to CD1 -specific T cells The natural antigens of group 2 CDIare not well characterized Group 2 CD1 molecules activate a group of T cells, known as Natural killer T cells because of their expression of NK surface markers such as CD161

We have now surprisingly found that both, CD1 B (NM_001764) and CD1 D (NM_001766) levels are increased in blood samples from patients which are suffering from Systemic Lupus Erythematosus (SLE), Lupus Anticoagulant (LA) Rheumatoid Arthritis (RA) and Idiopathic Thrombocytopenic Purpura (ITP) compared with levels in healthy control individuals Additionally it was found that CD1 D (NM_001766) is increased in blood samples from patients which are suffering from Multiple Sclerosis (MS) compared with levels in healthy control individuals

In several aspects the present invention provides 1 CD1 B and/or CD1 D for use, e g , or the use of CD1 B and/or CD1 D, e g CD1 B and/or CD1 D in human CD3 ⁺ cells,

1 1 as a target in a disorder selected from Systemic Lupus Erythematosus (SLE), Lupus Anticoagulant (LA) Rheumatoid Arthritis (RA) and Idiopathic Thrombocytopenic Purpura in humans, preferably in SLE,

1 2 for diagnosing a disorder selected from Systemic Lupus Erythematosus (SLE), Lupus Anticoagulant (LA) Rheumatoid Arthritis (RA) and Idiopathic Thrombocytopenic Purpura

(ITP), preferably SLE

CD1 B as used herein is known under the accession number NM 001764

CD1 D as used herein is known under the accession number NM_001766.

In one aspect of the present invention CD1 B may be used as indicated in 1.1 or 1.2 above..

In case of CD1 B a disorder is preferably SLE.

In another aspect of the present invention CD1 D may be used as indicated in 1.1 or 1.2 above..

In case of CD1 B a disorder is preferably MS, SLE, RA. LA or ITP, more preferably MS or SLE.

In another aspect the present invention provides CD1 D for use, e.g., or the use of CD1 D, e.g. CD1 D in human CD3 ⁺ cells,

1.3 as a target in Multiple Sclerosis

1.4 for diagnosing Multiple Sclerosis.

In several other aspects the present invention further provides

2. CD1 B and/or CD1 D, e.g. or the use of CD1 B and/or CD1 D, as a biomarker, for a use as indicated under 1.1 to 1.4 above, e.g. in a sample of an individual, e.g. in a sample of a body fluid or a tissue sample of an individual,

CD1 B and/or CD1 D as indicated under any of 1. or 2 above includes CD1 B and/or CD1 D in CD3 ⁺ cells.

According to the present invention the mRNA expression levels of both, CD1 B and CD1 D, in CD3 ⁺ cells isolated from SLE patients have been found to be surprisingly elevated (4 to 5- fold, p<0.05); e.g. and CD1 D levels in MS patients about 5-fold (p<0.001 ) in comparison with healthy control indiviuals. Elevated levels of CD1 B and CD1 D may also been found in RA, LA and ITP patients.

CD1 B or CD1 D as indicated herein includes CD1 B or CD1 D in any form, e.g. in the form of

- a nucleic acid encoding CD1 B, or CD1 D, respectively, e.g. including a nucleic acid encoding a derivative of CD1 B, or CD1 D, respectively,

- CD1 B or CD1 D protein, e.g. including protein which is a CD1 B derivative, or CD1 D derivative, respectively, or

- CD1 B or CD1 B derivative secreting cells, or CD1 D or CD1 D derivative secreting cells, respectively, preferably a nucleic acid encoding CD1 B or CD1 D.

"A derivative" of CD1 B or CD1 D nucleic acid or protein, e.g. in secreting cells, according to the present invention includes a fragment, a mutant, a variant, an homolog or a modification of a CD1 B or CD1 D protein, respectivly; or of a nucleic acid encoding CD1 B or CD1 D, respectively, which retains, e.g. essentially, the biological function of CD1 B or CD1 D, respectively, e.g. which retains, e.g. essentially, the biological function of CD1 B or CD1 D, respectively, e.g. in dendritic cells.

CD1 B or CD1 D containing cells, e.g. including CD1 B or CD1 D producing cells, respectively, e.g. include CD3 ⁺ cells.

Thus, CD1 B or CD1 D, respectively, for use as provided by the present invention includes splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants, posttranslational modifications, such as glycosylation and phosphorylation variants, and modifications which are covalent derivatives of CD1 B, or CD1 D respectively, and which retain the biological function of CD1 B, or CD1 D respectively, e.g. in CD3 ⁺ cells. Exemplary CD1 B or CD1 D derivatives include modifications wherein the CD1 B, or CD1 D respectively, protein is covalently modified by substitution, e.g. substitution originating from appropriate means, e.g. chemical or enzymatic means, by a moiety in the CD1 B, or CD1 D respectively, protein. Such a moiety e.g. includes one or more amino acids, e.g. naturally occurring amino acids and other than naturally occurring amino acids, and/or a detectable moiety. A detectable moiety includes an enzyme, a radioisotope, tags, toxins and genes such as oncogenes and tumour suppressor genes. CD1 B, or CD1 D derivatives further include naturally occurring variants of CD1 B, or CD1 D respectively, e.g. provided within a particular species. Such a variant may be encoded by a related gene of the same gene family, by an allelic variant of a particular gene, or represent an alternative splicing variant of the CD1 B or CD1 D gene.

A CD1 B, or CD1 D derivative as used herein also includes fragments of a nucleic acid encoding CD1 B, or CD1 D respectively, or of the CD1 B, or CD1 D protein, and comprises individual CD1 B, or CD1 D respectively, domains and smaller polypeptides derived from CD1 B, or CD1 D respectively, domains. Preferably, smaller polypeptides derived from CD1 B,

or CD1 D, respectively, according to the present invention define a single functional activity which is characteristic of CD1 B, or CD1 D, respectively. Fragments may in theory be of almost any size, as long as they retain the biological characteristic of CD1 B, or CD1 D, respectively. Preferably, fragments will be between 12 and 210 nucleic acids in length or between 4 and 70 amino acids, respectively. Longer fragments are regarded as truncations of the full-length CD1 B, or CD1 D.

Derivatives of CD1 B, or CD1 D respectively, as used herein also comprise mutants thereof, which may contain amino acid deletions, additions or substitutions, subject to the requirement to retain the biological function of CD1 B, or CD1 D respectively, e.g. in CD3 ⁺ cells. Conservative amino acid substitutions may be made substantially without altering the nature of CD1 B, or CD1 D, respectively, e.g. by truncations from the 5' or 3' ends. Deletions and substitutions also include deletions and substitutions in fragments of CD1 B, or CD1 D respectively. CD1 B, or CD1 D, respectively, mutants may be produced from a DNA encoding CD1 B, or CD1 D respectively, which has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids in CD1 B, or CD1 D respectively. For example, substitutional, deletional or insertional variants of CD1 B or CD1 D, respectively, can be prepared by recombinant methods and screened for functional similarity to the native forms of CD1 B, or CD1 D respectively.

Derivatives of CD1 B or CD1 D as used herein also include CD1 B, or CD1 D respectively, homologs, preferably CD1 B or CD1 D homologs retain substantial homology with CD1 B, or CD1 D respectively. As used herein, "homology" means that CD1 B, or CD1 D, respectively and a CD1 B, or CD1 D, respectively, homolog share sufficient characteristics to retain the biological function of CD1 B, or CD1 D respectively, e.g. in CD3 ⁺ cells. Preferably, homology is used to refer to sequence identity. Thus, the derivatives of CD1 B, or CD1 D respectively, preferably retain substantial sequence identity with the nucleic acid sequence as indicated herein.

"Substantial homology", where homology indicates sequence identity, means more than 50% sequence identity, preferably more than 75% sequence identity and even more preferably a sequence identity of 80% and more, e.g. 90% and more, such as 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%..

Preferably CD1 B or CD1 D is originating from a mammal, e.g. a human. The nucleic acid encoding CD1 B, or CD1 D respectively, protein preferably has the nucleic acid sequence as disclosed in literature for CD1 B, or CD1 D respectively, such as

NM_001764 for CD1 B and NM_001766 for CD1 D.

Biomarker as used herein means that determination of CD1 B, or CD1 D, respectively, in elevated levels compared to healthy control indiviuals in a sample of an individual has been found to be an indicator for disorders selected from SLE, LA, RA and ITP, preferably SLE; e.g. and in case of CD1 D additionally for MS, as such and/or is useful for monitoring the status of disorders selected from SLE, LA, RA and ITP, preferably SLE; e.g. and in case of CD1 D additionally of MS.

In another aspect the present invention provides a method for diagnosing a disorders selected from SLE, LA, RA and/or ITP, preferably SLE; e.g. and MS, comprising a) providing a sample of an individual, b) determining the level of CD1 B, or CD1 D respectively, in said sample, e.g. the level of mRNA of CD1 B, or CD1 D respectively, in CD3 ⁺ cells, c) comparing the level of CD1 B, or CD1 D respectively, as determined in step b) with a reference level from a sample of a healthy control individual, and d) diagnosing in case of CD1 B, or CD1 D respectively, SLE, LA, RA and/or ITP, and in case of CD1 D additionally MS, if the level of of CD1 B, or CD1 D respectively, is elevated compared with said reference level.

Elevated level as used herein includes a level which is 2-fold up to 20-fold or more increased compared with the level of a healthy control (donor) individual, such as 3-fold to 20-fold, e.g. 3-fold to 15-fold, e.g. 3-fold to 10-fold.

In another aspect the present invention provides a method for monitoring the therapeutic efficacy in the treatment of an individual with a substance which is expected to have an effect on reducing or curing SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS, which method comprises determining the level of CD1 B, or CD1 D respectively, in cells, e.g. CD3 ⁺ cells, in a sample of said individual and comparing that level with the level of CD1 B, or CD1 D respectively, prior to administration of said substance.

A sample of an individual according to a use or a method of the present invention includes a sample of a body fluid or a tissue sample. A body fluid may be derived e.g. from blood, e.g.

including isolated mononuclear cells, or from a blood fraction, e g including plasma or serum, preferably serum A tissue sample may be a biopsy, e g such as a skin biopsy

In another aspect the present invention provides the a use or a method of the present invention wherein a sample is a body fluid or a tissue sample of an individual, e g a body fluid may be derived from blood, e g isolated cells, such as CD3 ⁺ cells, or from a blood fraction, e g plasma or serum, e g serum, e g the tissue sample may be a biopsy, e g such as a skin biopsy

Cells, e g CD3 ⁺ cells, from a sample of an individual may be isolated as appropriate, e g according, e g analogously, to a a method as conventional

Detection means in cells for determining the level of CD1 B, or CD1 D respectively, include means as conventional, e g immunoassays, such as an immunodiagnostic method, an enzyme linked immunoassay (ELISAs), a fluorescence based assay, such as dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), an radiometric assay or by carrying out a CD1 B, or CD1 D, respectively, specific Polymerase Chain Reaction (PCR), specifically detection means include a molecule which specifically recognizes CD1 B, or CD1 D respectively, e g a molecule which is directly or indirectly detectable, preferably comprising an antibody, including antibody derivatives or fragments thereof, e g an antibody which recognizes CD1 B, or CD1 D respectively, e g a label bearing CD1 B, or CD1 D, respectively, recognizing antibody

Such label may be a conventional label, e g biotin or an enzyme such as alkaline phosphatase (AP), horse radish peroxidase (HRP) or peroxidase (POD) or a fluorescent molecule, e g a fluorescent dye, such as e g fluorescein isothiocyanate Preferably the label is biotin The label bearing molecule, e g the label bearing antibody, may be detected according to methods as conventional, e g via fluorescence measurement or enzyme detection methods

An antibody fragment or antibody derivative includes a fragment or a derivative, e g chemically or enzymatically modified, of an antibody which still is capable of recognising CD1 B, or CD1 D, respectively

CD1 B, or CD1 D, respectιvely,-secretιng cells in a sample of a body fluid of an individual, e g blood, may be determined by a method as conventional, e g by the following method

CeIIs, e.g. dendritic cells may be purified, e.g. separated by a density gradient, from the sample, e.g. blood, and the purified cells obtained are stained. Anti- CD1 B, or CD1 D respectively, antibodies, e.g. fluorescence labeled anti-CD1 B, or CD1 D, antibodies, respectively, are added to the stained cell preparation, optionally after stimulation of the cells, e.g. with interleukin-4, and the level of CD1 B, or CD1 D, respectively, secreting cells is determined.

Optionally, CD1 B, or CD1 D respectively, comprised in the sample or the CD1 B, or CD1 D, respectively, recognizing, e.g. detectable, molecule comprised in the detection means is immobilized on a solid phase. An appropriate solid phase includes e.g. conventional solid phases used for immobilization, e.g. a plastic plate like a polystyrene or polyvinyl plate, especially a microtiter plate. Also microbeads can be used as a solid phase, e.g. coated microbeads. The solid phase can be coated with a coating material the nature of which depends e.g. on the label comprised in the detection means. The coating material should be able to bind to the label, e.g. if the label is biotin a coating material includes streptavidin, e.g. covalently bound to the solid phase.

Preferably determination of CD1 B, or CD1 D, respectively, in cells, e.g. CD3 ⁺ cells, is carried out by PCR

In another aspect the present invention provides a method for diagnosing a disorder or disease which is mediated, e.g. associated with, e.g. driven, by eleated levels of CD1 B, or CD1 D, resόpectively, or CD1 B, or CD1 D, resόpectively, activity according to the present invention wherein the level of CD1 B, or CD1 D respectively, in cells, e.g. in CD3 ⁺ cells, is determined by use of PCR.

In another aspect the present invention provides a method for the preparation of a kit comprising providing a) a molecule which recognizes CD1 B, or CD1 D respectively, optionally in a labeled form, b) instructions how to use said kit, e.g. in CD3 ⁺ cell isolates, c) optionally detection means, d) optionally a solid phase. in case of CD1 B, or CD1 D respectively, for use in the diagnosis of SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS, e.g. in a sample of an individual.

Such kit may further comprise a substantial component, e.g. including an appropriate environment of a sample to be tested and, e.g. appropriate means to determine CD1 B, or CD1 D, respectively, in a sample to be tested.

In a further aspect the present invention provides an assay for identifying an agent that modulates, e.g. mediates, e.g. is associated with a disorder selected from SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS, comprising a) determining the level of CD1 B, or CD1 D respectively, in a sample, e.g. in CD3 ⁺ cells of a sample, of an individual, in the absence and in the presence of a candidate compound which may be expected to modulate the level of CD1 B, or CD1 D, respectively, b) identifying a candidate compound which modulates the level of CD1 B, or CD1 D respectively, as determined in step a) as an agent, e.g. and optionally c) using such agent as a pharmaceutical in case of CD1 B, or CD1 D respectively, in the treatment of a disorder selected from SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS..

In another aspect the present invention provides an assay for identifying an agent that is modulated, e.g. mediated, e.g. is associated with, elevated levels of CD1 B, or CD1 D, respectively, e.g. or CD1 B, or CD1 D, respectively, activity, comprising a) determining the level of CD1 B, or CD1 D, respectively, in a sample of an individual, in the absence and in the presence of a candidate compound which is expected to modulate the level of CD1 B, or CD1 D, respectively, b) identifying a candidate compound which modulates the level of CD1 B, or CD1 D, respectively, as determined in step a) as an agent, e.g. and, optionally c) using such agent as a pharmaceutical in the treatment of disorders mediated by elevated levels of CD1 B, or CD1 D, respectively, or CD1 B, or CD1 D, respectively, activity, e.g. wherein such disorder is selected from SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS.

Preferably a candidate compound identified decreases the level of CD1 B, or CD1 D respectively..

The level of CD1 B, or CD1 D respectively, may be determined as appropriate, e.g. as described herein.

A candidate compound as described herein is a compound which may be expected to modulate the level of CD1 B, or CD1 D respectively, e.g. or CD1 B or CD1 D activity, or CD1 B or CD1 D secreting cells, and includes compound(s)(libraries) from which its influence on CD1 B, or CD1 D respectively, can be determined. Compound (libraries) include for example oligopeptides, polypeptides, proteins, antibodies, mimetics, small molecules, e.g. low molecular weight compounds (LMWs).

An agent is a candidate compound which modulates the level of the level of CD1 B, or CD1 D respectively, or CD1 B or CD1 D activity, or CD1 B, or CD1 D secreting cells, e.g. in cells, such as CD3 ⁺ cells in a sample form a patient, e.g. a blood sample, such as serum, e.g. or a skin biopsy. An agent includes oligopeptides, polypeptides, proteins, antibodies, mimetics, small molecules, e.g. low molecular weight compounds (LMW ^' s).

In another aspect the present invention provides an agent identified by an assay or a method of the present invention.

An agent of the present invention may exhibit pharmacological activity and is therefore useful as a pharmaceutical. An agent of the present invention may show therapeutic activity, e.g. in disorders selected from SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS, e.g. which disorders are mediated, e.g. associated with, e.g. driven by elevated CD1 B, or CD1 D , respectively, levels, e.g. or CD1 B, or CD1D, respectively, activity.

In another aspect the present invention provides the use of an agent of the present invention as a pharmaceutical in case of CD1 B, or CD1 D , respectively, for the treatment of disorders selected from SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS, e.g. which disorders are mediated, e.g. associated with, e.g. driven by elevated levels of CD1 B or CD1 D, respectively, or CD1 B or CD1 D activity.

It may reasonably be expected that elevated levels of CD1 B or CD1 D, respectively, are connected with of CD1 B or CD1D, respectively, activity.

For pharmaceutical use an agent of the present invention for treatment includes one or more, preferably one, agent of the present invention, e.g. a combination of two or more agents of the present invention.

In another aspect the present invention provides the use of an agent of the present invention for the manufacture of a medicament for the treatment of disorders selected from SLE, LA, RA and/or ITP ₁ preferably SLE; e.g. and in case of CD1 D additionally MS, e.g. which disorders are mediated, e.g. associated with, e.g. driven by elevated levels of CD1 B or CD1 D, respectively, or CD1 B or CD1 D activity.

In another aspect the present invention provides a pharmaceutical composition comprising an agent of the present invention beside at least one pharmaceutical excipient, e.g. appropriate carrier and/or diluent, e.g. including fillers, binders, disintegrators, flow conditioners, lubricants, sugars and sweeteners, fragrances, preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers.

In another aspect the present invention provides a method for the treatment disorders of in case of CD1 B or CD1 D, respectively, selected from SLE, LA, RA and/or ITP, preferably SLE; e.g. and in case of CD1 D additionally MS, e.g. which disorders are mediated, e.g. associated with, e.g. driven by elevated levels of CD1 B or CD1 D, respectively, or CD1 B or CD1 D activity, comprising administering an effective amount of an agent of the present invention to a subject in need of such treatment.

For such treatment, the appropriate dosage will, of course, vary depending upon, for example, the chemical nature and the pharmakokinetic data of a compound of the present invention used, the individual host, the mode of administration and the nature and severity of the conditions being treated. However, in general, for satisfactory results in larger mammals, for example humans, an indicated daily dosage includes a range

- from about 0.001 g to about 1.5 g, such as 0.001 g to 1.5 g;

- from about 0.01 mg/kg body weight to about 20 mg/kg body weight, such as 0.01 mg/kg body weight to 20 mg/kg body weight, for example administered in divided doses up to four times a day.

An agent of the present invention may be administered by any conventional route, for example enterally, e.g. including nasal, buccal, rectal, oral, administration; parenterally, e.g. including intravenous, intramuscular, subcutanous administration; or topically; e.g. including epicutaneous, intranasal, intratracheal administration; via medical devices for local delivery,

e g stents, e g in form of coated or uncoated tablets, capsules, (injectable) solutions, solid solutions, suspensions, dispersions, solid dispersions, e g in the form of ampoules, vials, in the form of creams, gels, pastes, inhaler powder, foams, tinctures, lip sticks, drops, sprays, or in the form of suppositories

For topical use, e g including administration to the eye, satisfactory results may be obtained with local administration of a 0 5-10 %, such as 1-3% concentration of active substance several times daily, e g 2 to 5 times daily An agent of the present invention may be administered in the form of a pharmaceutically acceptable salt, e g an acid addition salt or metal salt, or in free form, optionally in the form of a solvate An agent of the present invention in the form of a salt may exhibit the same order of activity as an agent of the present invention in free form, optionally in the form of a solvate An agent of the present invention may be used for pharmaceutical treatment according to the present invention alone, or in combination with one or more other pharmaceutically active agents

Combinations include fixed combinations, in which two or more pharmaceutically active agents are in the same formulation, kits, in which two or more pharmaceutically active agents in separate formulations are sold in the same package, e g with instruction for co- administration, and free combinations in which the pharmaceutically active agents are packaged separately, but instruction for simultaneous or sequential administration are given

Description of the Figures Figure 1 Shows the relative expression of CD1 B mRNA in CD3 ⁺ cells of patients with autoimmune diseases compared with healthy control individuals (ND)

From Figure 1 it is evident that in patients with autoimmune disease the expression of CD1 B mRNA in CD3 ⁺ cells is increased compared with healthy control individuals (ND)

Figure 2

Shows the relative expression of CD1 D mRNA in CD3 ⁺ cells of patients with autoimmune diseases compared with healthy control individuals (ND)

From Figure 2 it is evident that in patients with autoimmune disease the expression of CD1 D mRNA in CD3 ⁺ cells is increased compared with healthy control individuals (ND)

In sum, according to the present invention mRNA expression of CD1 B and/or CD1 D in CD3 ⁺ cells has been found to be associated with autoimmune disorders as indicated herein.