Use of soluble cd46 concentration from peripheral blood as a parameter for the diagnosis of non-alcoholic fatty liver disease

Abstract

The present invention refers to a method of diagnosing a subject with non-alcoholic fatty liver disease (NAFLD), the method comprising determining a soluble CD46 (sCD46) concentration in a peripheral blood (PB) sample obtained from a subject, wherein said sCD46 concentration is indicative for whether or not said subject suffers from NAFLD. In addition, the present invention also relates to a data processing system comprising a processor configured to perform a method comprising the steps of obtaining a determined sCD46 concentration from a PB sample obtained from, a subject, comparing said sCD46 concentration with a cut-off value and then indicating whether or not said subject suffers from NAFLD.

Description

USE OF SOLUBLE CD46 CONCENTRATION FROM PERIPHERAL BLOODAS A PARAMETER FOR THE DIAGNOSIS OF NON-ALCOHOLIC FATTY LIVERDISEASE.CROSS-REFERENCE TO RELATED APPLICATIONSThe present application claims the benefit of priority of EP Patent Application No. 23183382.3 filed 04 July 2023, the content of which is hereby incorporated by reference in its entirety for all purposes.HELD OF THE INVENTION

[0001] The present invention refers to a method of diagnosing a subject with non-alcoholic fatty liver disease (NAFLD), the method comprising determining a soluble CD46 (sCD46) concentration in a peripheral blood (PB) sample obtained from a subject, wherein said sCD46 concentration is indicative for whether or not said subject suffers from NAFLD. In addition, the present invention also relates to a data processing system comprising a processor configured to perform a method comprising the steps of obtaining a determined sCD46 concentration from a PB sample obtained from a subject, comparing said sCD46 concentration with a cut-off value and then indicating whether or not said subject suffers from NAFLD.BACKGROUND OF THE INVENTION

[0002] Nonalcoholic fatty liver disease (NAFLD) has become one of the most common causes of liver diseases worldwide. Owing to the increasing prevalence of obesity and metabolic syndrome, NAFLD is becoming a greater clinical challenge because of no existing therapy and a lack of biomarkers.(003) Excessive deposition of fat as triglycerides in the liver forms lipid droplets within hepatocytes, a pathological condition known as hepatic steatosis which is an evidence of NAFLD (Wang, K. Expert Rev Mol Med 18, el4 (2016)). Hepatic steatosis often reflects a chronic imbalance between, on the one hand, hepatic fatty acid uptake and triglyceride synthesis, and on the other, triglyceride metabolism and excretion (Kawano, Y. & Cohen, D.E. J Gastroenterol 48, 434-441 (2013)). Steatosis itself is not harmful and can usually be reversed by treating the underlying cause; however, triglyceride deposition causes oxidative stress tohepatocytes, which ultimately leads to chronic inflammation of the liver (Ertunc, M.E. & Hotamisligil, G.S. J Lipid Res 57, 2099-2114 (2016)).

[0004] Such inflammatory changes of fat deposition can lead to a liver inflammation (short: steatohepatitis), which is determined as non-alcoholic steatohepatitis (short: NASH) within NAFLD. In some individuals, this NASH may even drive hepatic fibrosis, which can eventually progress to cirrhosis and hepatocellular carcinoma.

[0005] Currently, no pharmacological therapy is available to treat NAFLD. The reason might be that NAFLD is a multi-factorial disease with an incomplete understanding of the mechanisms involved, an absence of accurate and inexpensive imaging tools, and lack of adequate non- invasive or less invasive biomarker. Thus, at present a diagnosis of NAFLD is for example possible by applying an invasive liver biopsy. Mostly, such biopsies are performed these days if the patient simultaneously suffers from a liver tumor which will then also be removed surgically. Thus, NAFLD is only diagnosed in an advanced stage.

[0006] Accordingly, there is a need to provide a new method of diagnosing NAFLD. The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the figures and reflected in the claims.SUMMARY OF THE INVENTION

[0007] The present inventors have found a less invasive and less expensive, but still accurate method of diagnosing NAFLD based on a simple blood test whereby a soluble CD46 (sCD46) concentration in a peripheral blood (PB) sample from a subject is detected. With the method of the invention it is now possible to avoid invasive methods comprising obtaining liver tissue biopsies from a subject who is suffering from NAFLD and examining those as known by the skilled person or examining the liver with challenging imaging tools such as MRT and / or ultrasound.

[0008] Of immediate clinical relevance, sCD46 concentration in the PB, preferably in the plasma, performed extremely well as a predictive biomarker for steatosis grade, wherein a subject can be classified as non-steatotic or steatotic based on such steatosis grades as defined elsewhere herein which then allows to diagnose a subject with NAFLD or not. Thus the inventors found out a correlation of the steatosis grade as defined elsewhere herein and the sCD46 concentration in the PB. Therefore, it was concluded that there is great promise in sCD46 as biomarker in a less-invasive method for diagnosing NAFLD in a subject.

[0009] Accordingly, in a first aspect, the present invention relates to a method of diagnosing a subject with NAFLD, the method comprising determining a sCD46 concentration in a PB sample obtained from a subject, wherein said sCD46 concentration is indicative for whether or not said subject suffers from NAFLD.

[0010] In a second aspect, the present invention relates to a data processing system comprising a processor configured to perform a method comprising the steps of obtaining a determined sCD46 concentration from a PB sample obtained from a subject, comparing said sCD46 concentration with a cut-off value and then indicating whether or not said subject suffers from NAFLD.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings are included to further an understanding of the embodiments that are incorporated in and constitute a part of this specification. The drawings illustrate embodiments and together with the description serve to explain principles of embodiments. Other embodiments and many of the intended advantages of embodiments will be readily appreciated, as they become better understood by reference to the detailed description. The elements of the drawings are not necessarily to scale relative to each other.

[0012] Figure 1: (a) Frequency of intrahepatic iNKT cells relative to all intrahepatic CD3+ T cells of patients with a steatosis grade of 0-1 (n = 28) and 2-4 (n = 15) [two-tailed, unpaired Mann-Whitney test, unadjusted p-values]. (b) Frequency of intrahepatic IL-4+iNKT cells relative to all intrahepatic iNKT cells of patients with a steatosis grade of 0-1 (n = 24) and 2-4 (n = 14) [two-tailed, impaired Mann-Whitney test, unadjusted p-values]. (c) Frequency of IL-4+ iNKT cells after a 7-day expansion of iNKT cells in in coculture with unloaded (UL) or fat- loaded (FL) HepaRG cells with direct cell contact or transwell-separated. The frequency of IL4+ iNKT cells after 7 days of ex vivo expansion of iNKT cells alone are used as control [n = 8; paired one-way ANOVA with Tukey’s test for multiple comparisons], (d) Flow cytometric expression analysis of 361 surface markers on UL and FL-HepaRG cells [n = 6 each; two-tailed BH-coirected t-test. Red points represent selected markers significantly down-regulated after FL, including CD46]. (e) Concentration of soluble sCD46 in the supernatant of UL and FL-HepaRG cells measured with a competitive cell-based flow cytometry assay [n = 11; unpaired Mann- Whitney test], (f) Frequency of IL-4+ iNKT cells after a 7-day ex vivo expansion of iNKT cells untreated or treated with chimeric isotype IgG or CD46 IgG at 1 μg / ml or 10 μg / ml [n = 6, paired one-way ANOVA with Tukey’s test for multiple comparisons], (g) Frequency of IL-4+iNKT cells after a 7-day expansion of iNKT cells in coculture with UL-HepaRG cells under 2 μg / ml Isotype or 5 μg / ml antagonistically active antibody treatment directed against CD46 [n = 14; paired t-test]. (h) Frequency of IL-4+ iNKT cells after a 7-day expansion of iNKT cells in cocultures with UL-HepaRG cells stable transfected with random shRNA or CD46-knockdown shRNA. Cocultures with wild-type UL-HepaRG cells are used as controls, [n = 15, paired one- way ANOVA with Tukey’s test for multiple comparisons], (i) Frequency of IL-4+ iNKT cells after a 7-day expansion of iNKT cells in cocultures with UL- or FL-HepaRG cells untreated or treated with 10 μM MMP inhibitor (TAPI-1, GI254023X and batimastat). DMSO treated iNKT : HepaRG cocultures were used as solvent controls [n = 8, paired two-way ANOVA with Tukey’s test for multiple comparisons], (j) Relative increase of isotype-corrected MFI surface expression of CD46 on FL-HepaRG cells under dose-dependent treatment with the MMP inhibitors TAPI-1, GI254023X and batimastat. [n = 3; mean ± SD].

[0013] Figure 2: (a) Distribution of sCD46 concentration in plasma samples from patients with varying degrees of steatosis (Grade 0, n=48; Grade 1, n=31; Grade ≥2, n=12; one-way ANOVA with Dunnett’s test for multiple comparisons). Diagnostic cut-off values are given in blue, (b) Receiving Operator Characteristic (ROC) curve illustrating the capacity of sCD46 concentrations as a discriminator of Grade 0 versus Grade ≥1 steatosis in training set patients (n=45). An optimal cut-off value of 26.19 ng / ml sCD46 was set, AUC = area under curve, (c) ROC curve discriminating Grade 0 and Grade ≥1 steatosis in validation set patients (n=46). (d) ROC curve discriminating Grade ≤1 and Grade ≥2 steatosis in training set patients (n=45). An optimal cut- off value of 45.55 ng / ml sCD46 was set (e) ROC curve discriminating Grade ≤1 and Grade ≥2 steatosis in validation set patients (n=46). (f) A diagnostic tree to predict steatosis grade in individual patients based upon plasma sCD46 concentrations, (g) Cross-tabulation of predicted steatosis grade in validation set patients (Fisher’s exact test, n=46). (h) Cross-tabulation of predicted steatosis grade in validation set patients considering only the most clinically relevant distinction between Grade ≤1 and Grade ≥2 steatosis (Fisher’s exact test, n=46). (i) Distribution of sCD46 concentration in plasma samples of an independent test cohort comprising patients evaluated for living liver transplant donation, who exhibited varying degrees of steatosis determined sonographically (none steatosis, n=112; moderate steatosis, n=34; severe steatosis, n=12; one-way ANOVA with Dunnett’s test for multiple comparisons). Diagnostic cut-off values from the training and validation sets are given in blue, (j) ROC (Receiving Operator Characteristic) curve illustrating the capacity of the sCD46 concentration to discriminate between none steatosis and moderate steatosis in patients. AUC = area under curve, (k) ROC curve discriminating moderate steatosis and severe steatosis. (1) ROC curve discriminating noneand existing steatosis, (m) Cross-tabulation of predicted steatosis level (Fisher’s exact test), (n) Cross-tabulation of predicted steatosis level considering only the most clinically relevant distinction between none and existing steatosis (Fisher’s exact test), (o) A diagnostic tree to predict steatosis level (none, moderate, severe) in individual patients evaluated for living liver transplant donation based upon plasma sCD46 concentrations.

[0014] Figure 3: Recruitment of patients undergoing liver surgery for a prospective, non- randomised observational clinical study.

[0015] Figure 4: Competitive cell-based flow cytometry assay to measure the concentration of soluble sCD46. (a) 2-colour FACS panel, (b) Representative standard curve, (c) Mathematical formula for calculating sCD46 concentrations.DETAILED DESCRIPTION

[0016] The following language and descriptions of certain preferred embodiments of the present invention are provided in order for further understanding of the principles of the present invention. However, it will be understood that no limitations of the present invention are intended, and that further alterations, modifications, and applications of the principles of the present invention are also included.

[0017] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments described throughout the specification should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all elements described herein should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0018] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members,integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. When used herein the term “comprising” can be substituted with the term “containing" or “including” or sometimes when used herein with the term “having”. When used herein “consisting of excludes any element, step, or ingredient not specified.

[0019] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Additionally, the singular of a term also comprises the plural of a term and vice versa. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0020] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0021] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. The term “at least one” refers to one or more such as two, three, four, five, six, seven, eight, nine, ten and more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0022] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0023] When used herein “consisting of excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0024] The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0025] The term “about” means plus or minus 10%, more preferably plus or minus 5%, most preferably plus or minus 1%.

[0026] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0027] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0028] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0029] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0030] A better understanding of the present invention and of its advantages will be gained from the examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.Method

[0031] As used herein, the method of the present invention as defined in more detail herein is applied to diagnose a subject with NAFLD.

[0032] The term “Nonalcoholic fatty liver disease (NAFLD)” as used herein and which can also be called “nonalcoholic hepatic steatosis” refers to a condition caused by a build-up of fat in the liver (a fat deposition in the liver). Generally for defining NAFLD, there must be (1) evidence of hepatic steatosis (HS; fat deposition), either by imaging or histology, and (2) lack of secondary causes of hepatic fat accumulation such as significant alcohol consumption, long-term use of a steatogenic medication, or monogenic hereditary disorders. Hepatic steatosis occurs when intrahepatic fat is ≥5% of liver weight. NAFLD can be categorized histologically into nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) as the most severe form of NAFLD including inflammatory components. NAFLD is defined as the presence of ≥5% HS without evidence of hepatocellular injury in the form of hepatocyte ballooning as inflammatory components. NASH is defined as the presence of ≥5% HS and additionally inflammation with hepatocyte injury (e.g., ballooning), with or without any fibrosis (Chalasani, N. et al. Hepatology, Volume 67, Issue 1, 328-357 (2018)). With the method of the invention it cannot be distinguished explicitly whether said subject suffers from NAFL or from NASH, but whether said subject suffers from NAFLD or not.

[0033] A subject with < 5% HS may be Classified as non-steatotic (steatosis grade 0 or 1, meaning a steatosis grade ≤1), whereas a subject with ≥ 5% HS may be classified as steatotic (steatosis grades 2 - 4, meaning a steatosis grade ≥2) (Chalasani, N. et al. Hepatology, Volume 67, Issue 1, 328-357 (2018). Having a steatosis grade of 0 as it is defined by the clinical guidelines published by the American Association for the Study of Liver Diseases (AASLD, see again Chalasani, N. et al. Hepatology, Volume 67, Issue 1, 328-357 (2018)), histopathologically there is no evidence of any fat deposition in the liver. Having a steatosis grade of 1 as it is defined by the clinical guidelines published by the AASLD, histopathologically the evidence of fat deposition in the liver is < 5%. Having a steatosis grade of ≥2 as it is defined by the clinical guidelines published by the AASLD, histopathologically the evidence of fat deposition in the liver is ≥ 5%. Steatosis grades can be determined by histopathological determination including liver biopsy and microscopic investigations as known to a person skilled in the art.

[0034] The term “diagnose / diagnosing” as used herein and through the entire description refers to confirming a diagnosis that a subject suffers indeed from NAFLD, which has been suspected to suffer from said disease, before said method of the present invention has been applied to said subject. In that sense, a diagnosis means that it is determined that a subject being examined suffers indeed from said disease.

[0035] The term “to have / having NAFLD” can be used interchangeably with the term “to suffer from / suffering from NAFLD”. In general, when a subject suffers from a disease, said subject shows specific symptoms of the disease, whereas when a subject has a disease, said subject does not always have to show certain symptoms of the disease, but still is diagnosed with said disease. However, this general concept does not apply to NAFLD. When the subject of the invention suffers from said disease, such subject may or may not (being asymptomatic) show the specific symptoms of NAFLD as known to the person skilled.

[0036] The term “subject” as used herein and throughout the entire description, also addressed as an individual, refers to a human or non-human animal, generally a mammal. A subject may be a mammalian species such as a rabbit, a mouse, a rat, a Guinea pig, a hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an ape or a human. Preferably, the subject being used in the present invention is a human. More preferably, said subject is an adult. Thus, the present invention may comprise the method of the invention, wherein the subject is a human, preferably an adult. Where the subject is a human, said subject can also refer to a “patient”.

[0037] In a preferred embodiment, the present invention comprises the method of the invention, wherein said subject is suspected to suffer from NAFLD. Based on the symptoms the subject may indicate, it can already be suspected before the method of the invention is applied to said subject that the subject suffers from NAFLD. The method of the invention may then refer as confirmation that such subject indeed suffers from said disease without applying histopathological procedures including a liver biopsy to determine the steatosis grade. The term “suspected to suffer” thus refers to assuming that said subject who will be examined by using the method of the present invention might suffer from NAFLD based on the symptoms indicated before and / or based on general diagnosing test(s) available in the prior art (e.g., ultrasound, MRT).

[0038] According to the present invention, it may also be comprised that a subject not having any symptoms at all and / or not being diagnosed with NAFLD based on the general diagnosing test(s) available in the prior art (e.g., ultrasound, MRT), which has / have been applied to said subject, may then be diagnosed with NAFLD by applying the method of the present invention. Thus, the method of the present invention may also diagnose a specific patient group, which has not been diagnosed with NAFLD or which failed to be diagnosed with NAFLD by applying classical diagnosing procedures known to a person skilled in the art.

[0039] In another preferred embodiment, the present invention comprises the method of the invention, wherein said subject is suspected to be a liver transplant donor. Normally, as assessment of the liver steatosis of the potential donor as part of the evaluation process sonographical determination (e.g. ultrasound) may be applied for classifying a non-steatotic or steatotic liver. In sonography, a steatotic liver appears hyperechogenic (= "whiter") compared to a non-steatotic liver due to the increased fat deposits. The parenchymal structure of the kidney is usually used as a comparison for this subjective assessment. Such assessment rather refers to a descriptive description of the liver (less accurate) also using expensive imaging tools such as ultrasound.

[0040] If a subject may be suspected to be a liver transplant donor, the method of the invention can be performed on said subject which then gives certainty whether said subject suffers from NAFLD or not without applying the less accurate assessment as defined above and ususally known to the person skilled using expensive tools such as ultrasound. If not (meaning the subject does not suffer from NAFLD), s / he could be used as a liver transplant donor. Said subject being a liver transplant donor may be an adult, particularly a parent, who gives his / her child part of the liver, preferably part of the left liver lobe, which is then transplanted into the child who has a failed or diseased liver.

[0041] In more detail, the present invention demonstrates a new method of diagnosing NAFLD in a subject by determining a sCD46 concentration in a PB sample obtained from a subject. CD46 (also known as membrane cofactor protein) is a member of the membrane-bound complement regulatory protein family and a key costimulatory molecule on human CD4+T cells, specifically regulating Th1 responses (Le Friec, G., et al. Nat Immunol 13, 1213-1221 (2012)), Particularly, in humans, CD46 is ubiquitously expressed on all nucleated cells in four distinct isoforms that arise through alternative splicing of a single gene (West, E.E., Kolev, M. & Kemper, C. Annu. Rev. Immunol. 36, 309-338 (2018)).

[0042] CD46 is an immune receptor which is shed from fat-loaded hepatocytes. Loss of surface CD46 expression in fat loaded hepatocytes such as the hepatocyte-like hepatocellular carcinoma cell line HepaRG appeared to be a post-secretional effect that is consistent with shedding of immune receptors from the surface of HepaRG cells, e.g. through enzymatic cleavage by Matrix metalloproteinases (MMPs) as it was discovered by the present invention. MMPs are zinc- dependent endopeptidases that are involved in remodeling of the extracellular matrix. MMPsparticipate in the process of liver regeneration along with many liver diseases including NAFLD. MMPs are responsible for turnover of matrix proteins, as well as non-matrix substrates like growth factors, chemokines and adhesion molecules. MMPs are secreted as inactive forms and are subsequently activated by proteolytic cleavage. Their expression and activation are regulated at different levels, including gene transcription, enzyme secretion and endogenous inhibition by tissue inhibitor of metalloproteinases (TIMPs).

[0043] Based on the discovery of the inventors, fat deposition results in increased levels of MMPs that remove the outside domain from the CD46 immune receptor, so that HepaRG cells expressing CD46 can no longer inhibit iNKT cells, resulting in the activation of iNKT cells, thereby having a IL-4 increase. In sum, such shedding of immune receptors such as CD46 from fat-loaded hepatocytes contributes to dysregulated activation of iNKT cells. Thereby, CD46 is not attached on said hepatocytes anymore, but freely floating in the blood of the subject which may refer inter alia to the meaning of “soluble” CD46 as used throughout in the specification. Such sCD46 concentration in the PB as defined elsewhere herein thus increases with increasing degree of fat deposition in the liver. This means that by determing sCD46 concentration in the PB sample from a subject, the degree of fat deposition in the liver may be determined which allows the assignment of a steatosis grade so that NAFLD can be diagnosed.

[0044] The liver is densely populated by innate-like lymphocytes, including natural killer (NK) cells, natural killer T (NKT) cells and mucosal-associated invariant T (MAIT) cells (Heymann, F. & Tacke, F. Nature reviews. Gastroenterology & hepatology 13, 88-110 (2016)). Of special interest, NKT cells are a minor subpopulation of TCRαβ-expressing T cells that respond to certain glycolipids presented by CD1d (Gapin, L. D. Curr Opin Immunol 39, 68-74 (2016)). NKT cells are classified as Type I (invariant) and Type II NKT cells. Human invariant Natural Killer T cells (iNKT) are characterised by co-expression of classic NK cell markers and the invariant TCR-Vα24-Jα18 chain, often in conjunction with TCR-Vβ11, which allows them to recognise α-galactosylceramide (α-GalCer) in the context of CD1d. Human iNKT cells can be further divided into CD4+, CD8+and double-negative (DN) subsets, which appear to have distinct functional characteristics. Similar to conventional CD4+T cells, CD4+iNKT cells produce Th1, Th2 and Th17 cytokines depending upon the conditions under which they become activated. An essential feature of iNKT cells is their ability to respond rapidly by secreting high levels of cytokines such as interleukin-4 (IL-4) which is produced by intrahepatic iNKT cells, in particular when said iNKT cells are activated as mentioned above and as discovered by the inventors.

[0045] In sum, it was found by the inventors that a dysregulated proteolytic cleavage of surface receptors such as CD46 following fat loading is a key mechanism leading to inflammatory activation of human iNKT cells in the presence of fat-loaded hepatocytes. It was further discovered by the inventors that shedding of immune regulatory molecules (such as CD46) from fat-loaded hepatocytes represents a mechanism of innate-like lymphocyte activation in NAFLD patients, which allows diagnosing of NAFLD with a simple blood test for sCD46 as biomarker which is not invasive compared to a liver biopsy and also less expensive in comparison to using imaging tools such as ultrasound.

[0046] The term “detect” or “detecting”, as well as the term “determine” or “determining” or “measure” or “measuring” when used herein in combination with the word “concentration”, the words “detect”, “detecting”, “determine”, “determining”, “measure” or “measuring” are understood to generally refer to a quantitative or a qualitative concentration. For example, when used in the context of detecting the concentration of sCD46, “detect”, “detecting”, “determine” “determining” “measure” or “measuring” are understood to generally refer to a quantitative concentration. Such concentration of sCD46 is understood as the abundance of a constituent (here sCD46) divided by the total volume of a mixture. In more detail, such concentration refers to a mass concentration defined as the mass of a constituent (such as sCD46) divided by the volume of the mixture, expressed in units normally in g / L. In the present invention such concentration of sCD46 can be measured using any detection method known to a person skilled in the art. Hereby, the level, i.e. number or (relative) amount of sCD46 in said sample of the invention which is defined in more detail as PB sample may be detected as defined herein before the concentration is determined as mentioned above (see also Figure 4). The level of sCD46 may be expressed by the amount of CD46 being soluble within the sample under investigation. Such detection method (e.g. flow cytometry) in principle relies upon competition between sCD46 and membrane-expresed CD46. Based on this data and an external calibration curve one can infer how much sCD46 was present in the test PB sample.

[0047] Detecting said sCD46 concentration may comprise any one of an enzyme-linked immunosorbent assay (ELISA), a bead-based sandwich assay, a flow cytometry-based competitive binding assay or an immunoturbidimetric assay or the like. Thus, the present invention may also comprise the method as defined elsewhere herein, wherein said sCD46 concentration is detected using any one of an enzyme-linked immunosorbent assay (ELISA), abead-based sandwich assay, a flow cytometry-based competitive binding assay or an immunoturbidimetric assay.

[0048] Generally, the invention envisages that the detection of sCD46 can be carried out in a single step or can be carried out in more than one step, e.g. two steps, three steps or four steps. In preferred embodiments, the detection is carried out in a single step. A preferred method for the detection of said sCD46 concentration as defined above is flow cytometry, preferably FACS. Thus, it can also be expressed by the strength of a signal measured when immunofluorescence may be used, which may be combined with flow cytometry. Immunofluorescence being used in flow cytometry is generally achieved using a binding partner as defined herein, which is linked to, or includes, a fluorophore as a detectable marker. Typically a binding partner of sCD46 may be used in combination with a detectable marker or the binding partner is functionally linked to a detectable marker.

[0049] Flow cytometry is a technique for counting, examining, and sorting microscopic particles such as biological cells or shedded surface receptors such as sCD46 (e.g. by cleaving said receptor by MMPs) suspended in a stream of fluid. It allows a simultaneous multi-parametric analysis of the physical and chemical characteristics of single cells flowing through an optical or electronic detection device. An illustrative example of a well-established flow cytometry based analysis in the art is FACS. FACS allows sorting a heterogeneous mixture of particles into a plurality of containers, one particle at a time, based upon the specific light scattering and fluorescent characteristics of each cell. FACS is often used in combination with monoclonal immunoglobulins as a reagent to detect particles such as sCD46.

[0050] This technique allows the concurrent fast, objective and quantitative recording of fluorescent signals from individual particles and the physical separation of respective particles according to particular interest. Fluorescent signals used in flow cytometry, for instance when quantifying and / or sorting particles by any marker present, or being on or in the cell, are typically fluorescently-tagged antibody preparations or fluorescently-tagged ligands for binding to antibodies or other antigen-, epitope- or ligand-specific agent, such as with biotin / avidin binding systems or fluorescently-labeled and optionally addressable beads (e.g. LUMINEX® microspheres). Depending of the equipment used, any desired detectable marker or combination of detectable markers can be detected by the optics and / or electronics of a flow cytometer.

[0051] The present invention may also comprise the method as defined herein, wherein a binding partner for sCD46 is used when determining the sCD46 concentration as defined herein. Thus, the present inevtnion may also comprise the method as defined herein, wherein a binding partner for sCD46 is used. A respective binding partner of sCD46 may be an immunoglobulin, a fragment thereof or a proteinaceous binding molecule with immunoglobulin-like functions, or a recombinant receptor dependent on the detection method used as mentioned elsewhere herein, preferably an immunoglobulin as defined herein. Thus, the present invention may further comprise the method as definded herein, wherein said binding partner is any one of an immunoglobulin or a fragment thereof, a proteinaceous binding molecule with immunoglobulin- like functions, or a recombinant receptor. For example, when an ELISA, flow cytometry or an immunoturbidimetric assay is applied as detection method, as binding agent an immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions may be chosen. When for example a bead-based sandwich assay is used, then again an immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions may be used as binding agent, further applying beads (such as LUMINEX® microspheres) coated on a plate capturing such binding agent as defined herein. In this context, the term “bead" refers to a small spherical object / particle (also called microsphere), e.g., made of glass, plastic, metal, agarose, latex, metallic nano- or microparticle, metal oxide nano- or microparticle or magnetic material.

[0052] An immunoglobulin (antibody) fragment generally contains an antigen binding or variable region. Examples of (recombinant) antibody fragments are immunoglobulin fragments such as Fab fragments, Fab’ fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies or domain antibodies (Holt, L.J., et al., Trends Biotechnol. (2003), 21, 11, 484-490). Such antibody fragment has the same functional activity as the antibody used as binding agent for sCD46 within an exemplified detection method as mentioned herein.

[0053] An example of a proteinaceous binding molecule with immunoglobulin-like functions is a mutein based on a polypeptide of the lipocalin family (WO 03 / 029462, Beste et al., Proc Nat. Acad Sci 1999; 96:1898-1903). Lipocalins, such as the bilin binding protein, the human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D or glycodelin, possess natural ligand-binding sites that can be modified so that they bind to selected small protein regions known as haptens. Examples of other proteinaceous binding molecules are the so-called glubodies (see e.g. international patent application WO 96 / 23879 or Napolitano et al., Chemistry & Biology 1996; 3(5):359-367), proteins based on the ankyrin scaffold (Mosavi et al., Protein Science 2004; 13(6): 1435- 1448) or crystalline scaffold (e.g. intemation patent application WO01 / 04144), the proteins described in Skerra, J. Mol. Recognit. 2000; 13:167-187, AdNectins, tetranectins and avimers. Avimers contain so called A-domains that occur as strings of multiple domains in several cell surface receptors (Silverman et al., Nature Biotechnology 2005; 23:1556- 1561). Adnectins, derived from a domain of human fibronectin, contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill & Damle, Current Opinion in Biotechnology 2006; 17:653-658). Tetranectins, derived from the respective human homotrimeric protein, likewise contain loop regions in a C-type lectin domain that can be engineered for desired binding. A suitable antibody may in some embodiments also be a multispecific antibody that includes several immunoglobulin fragments.

[0054] An immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions may be PEGylated or hyperglycosylated if desired. In some embodiments a proteinaceous binding molecule with immunoglobulin-like functions is a fusion protein of one of the exemplary proteinaceous binding molecules above and an albumin-binding domain, for instance an albumin-binding domain of streptococcal protein G. In some embodiments, a proteinaceous binding molecule with immunoglobulin-like functions is a fusion protein of an immunoglobulin fragment, such as a single-chain diabody, and an immunoglobulin binding domain, for instance a bacterial immunoglobulin binding domain. As an illustrative example, a single-chain diabody may be fused to domain B of staphylococcal protein A as described by Unverdorben et al., Protein Engineering, Design & Selection 2012; 25:81-88.

[0055] An immunoglobulin may be monoclonal or polyclonal. The term “polyclonal” refers to immunoglobulins that are heterogenous populations of immunoglobulin molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal immunoglobulins, one or more of various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species. “Monoclonal immunoglobulins”, also called “monoclonal antibodies”, are substantially homogenous populations of immunoglobulins to a particular antigen. They may be obtained by any technique which provides for the production of immunoglobulin molecules by continuous cell lines in culture. Monoclonal immunoglobulins may be obtained by methods well known to those skilled in the art (see for example, Kohler et al., Nature (1975) 256, 495-497, and U.S. Patent No. 4,376,110). An immunoglobulin or immunoglobulin fragment with specific binding affinity only for e.g. sCD46 can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of both immunoglobulins orimmunoglobulin fragments and proteinaceous binding molecules with immunoglobulin-like functions, in both prokaryotic and eukaryotic organisms.

[0056] In more detail, an immunoglobulin may be isolated by comparing its binding affinity to a protein of interest, e.g. sCD46, with its binding affinity to other polypeptides. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting. In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art. Any animal such as a goat, a mouse or a rabbit that is known to produce antibodies can be immunized with the selected polypeptide, e.g. sCD46. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization and the immunization regimen will vary based on the animal which is immunized, including the species of mammal immunized, its immune status and the body weight of the mammal, as well as the antigenicity of the polypeptide and the site of injection.

[0057] As indicated above, a detectable marker may be coupled to a binding partner of sCD46, as the case may be, or a molecule that forms a complex with the binding partner of sCD46. In some embodiments, a detectable marker being coupled to a binding partner may refer to a “fluorescently labelled binding partner”. A respective detectable marker, which may be coupled to a binding partner of sCD46, or a molecule that forms a complex therewith, may be an optically detectable label, a fluorophore, or a chromophore. Examples of suitable labels include, but are not limited to, an organic molecule, an enzyme, a radioactive, fluorescent, and / or chromogenic moiety, a luminescent moiety, a hapten, digoxigenin, biotin, a metal complex, a metal and colloidal gold. Accordingly an excitable fluorescent dye, a radioactive amino acid, a fluorescent protein or an enzyme may for instance be used to detect e.g. the concentration of sCD46. Examples of suitable fluorescent dyes include, but are not limited to, Krome Orange (KrO), fluorescein (FITC), fluorescein isothiocyanate, 5,6-carboxymethyl fluorescein, Cascade Blue®, Oregon Green®, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl, coumarin, dansyl chloride, rhodamine, amino-methyl coumarin, DAPI, Eosin, Erythrosin, BODIPY®, pyrene, lissamine, xanthene, acridine, a fluorescent brightener (PB), an oxazine, phycoerythrin, a Cy dye such as Cy3, Cy3.5, Cy5, Cy5PE, Cy5.5, Cy7, Cy7PE or Cy7APC, an Alexa dye such as Alexa 647, Alexa 750 or Alexa 700, and NBD (Naphthol basic dye). Examples of suitable fluorescent proteins include, but are not limited to, EGFP, emerald, EYFP, a phycobiliprotein such asphycoerythrin (PE) or allophycocyanin (APC), Monomeric Red Fluorescent Protein (mRFP), mOrange, mPlum and mCherry. In some embodiments a reversibly photoswitchable fluorescent protein such as Dronpa, bsDronpa and Padron may be employed (Andresen, M., et al., Nature Biotechnology (2008) 26, 9, 1035). Regarding suitable enzymes, alkaline phosphatase, soybean peroxidase, or horseradish peroxidase may serve as a few illustrative examples. In a further embodiment, tandem conjugates such as PE-Cy5.5 or PE-Cy7 may also be used. In some embodiments other methods of detection may include electrophoresis, HPLC, fluorescence correlation spectroscopy or a modified form of these techniques. Some or all of these steps may be part of an automated separation / detection system.

[0058] The sample used herein within the method of the invention is a PB sample. The PB is the flowing, circulating blood of the body. It is composed of erythrocytes, leukocytes and thrombocytes. These blood cells are suspended in blood plasma, through which the blood cells are circulated through the body.

[0059] Preferably, the invention comprises a method as defined elsewhere herein, wherein the PB sample is a plasma or a serum sample, preferably a plasma sample. A plasma sample corresponds to the untreated cell-free part of the blood. A serum sample on the other hand is obtained after clotting and centrifugation, which allows the removal of fibrin clots, blood cells, and related coagulation factors, whereas a plasma sample is obtained by adding anticoagulants (i.e., EDTA, citrate, heparin) before removal of blood cells by centrifugation.

[0060] The respective method according to the present invention may also involve analysis of one or more PB samples from the subject in vitro. Typically the sample is, essentially consists of, or includes PB from the subject. The term “essentially consists of’ is understood to allow the presence of additional components in said PB sample or a composition that do not affect the properties of the sample or a composition. Examples of additional components may include, but are not limited to certain types of media, protease inhibitors which may help to stabilize a stored sample or any type of buffer.

[0061] The methods of the invention may include providing a sample from the subject or obtaining said sample from the subject. The sample may be obtained by venous puncture following typical procedures known to a person skilled in the art and then collected in particular tubes. The sample may have been taken at any desired point in time before carrying out the method of the invention. It is envisaged by the invention that the sample may have been taken onthe same or on the previous day, such as about 72 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 4 hours, about 2 hours or less before the method of the invention is being carried out. Preferably, it is envisaged by the invention that the sample is taken about 2 hours before the method of the invention is carried out.

[0062] The invention also contemplates that the sample from the individual may be a fresh sample (and then stored on ice) or may be a frozen sample particularly if analyzed after 24h. A frozen sample may be formed by freezing an obtained sample after adding a cryoprotective agent such as DMSO, glycerol and / or hydroxyethyl starch. In general, steps taken to prevent possible sCD46 degradation may improve test accuracy of the method of the invention. These steps may include (1) minimizing preanalytical storage time, (2) faster processing and measurement - avoiding multiple freeze-thaw cycles as this tends to degrade sCD46, (3) storage at 4°C for hours or -80°C for longer periods before analysis, (4) addition of stabilizers.

[0063] Further, the method of the invention comprises that said determined sCD46 concentration in PB sample obtained from a subject as will be defined elsewhere herein is indicative for whether or not said subject suffers from NAFLD. Thus, by determining the sCD46 concentration in the PB obtained from said subject as defined elsewhere herein it can be determined whether the subject being examined and the PB sample is obtained from suffers indeed from said disease NAFLD which is based on the fact as outlined above that such sCD46 concentration in the PB increases with increasing degree of fat deposition in the liver of said subject being examined, which allows the assignment of a steatosis grade so that NAFLD can be diagnosed. Thus, the method of the invention may also comprise after the step of determining a sCD46 concentration in a PB sample from a subject, a further step of determining a steatosis grade as defined herein based on said determined sCD46 concentration in the PB sample, wherein said sCD46 concentration which allows a subject to be classified as non-steatotic or steatotic based on the particular steatosis grade is indicative for whether or not said subject suffers from NAFLD. The higher the degree of fat deposition (e.g. above ≥5% HS), the more increased the sCD46 concentration is which then allows a subject to be classified into a steatosis grade indicating the subject to be steatotic which then allows the diagnosis of NAFLD. In sum, the higher the sCD46 concentration, the higher the probability that a patient has a high-grade steatosis and suffers from NAFLD.

[0064] In a preferred embodiment, the method may further comprise comparing said sCD46 concentration determined in said PB sample with a discriminatory cut-off value. The term “cut-off value” is understood to generally refer to a qualitative value. Thus, if the cut-off value is understood to refer to a qualitative value, said sCD46 concentration may be equal or above said cut-off value. The term “cut-off” may be used interchangeably with the term “cut-off value”. An optimal discriminatory cut-off value is a value that most accurately divides a training dataset into two classes (e.g. non-steatotic vs. steatotic based on the steatosis grades as mentioned elsewhere herein, see e.g. Figures 2b to f - particularly for non-steatotic vs. steatotic grade see Figures 2d, e, f; or e.g. none vs. moderate / moderate vs. severe steatotic level, see e.g. Figures 2j to 1 - particularly for moderate vs. severe level see Figure 2o). Therefore, it depends upon the distribution of cases in the two classes being investigated so that the cut-off may vary depending on the initial situation. Selecting a diagnostic cut-off involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test cut-offs, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. Suitable cut-offs may be determined in a variety of ways such as Receiver Operating Characteristic ("ROC”) which is able to best distinguish a well responding subpopulation from a poorly responding subpopulation.

[0065] A false positive in this case occurs when a person tests positive (poor responder or diagnosed with NAFLD, grade ≥2), but actually is a good responder and thus does not suffer from NAFLD (grade 0 or ≤1). A false negative, on the other hand, occurs when the person tests negative (good responder or not being diagnosed with NAFLD - grade 0 or ≤1), when it actually suffers from NAFLD (grade ≥2). To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the cut-off value is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1 - specificity, the ROC graph is sometimes called the sensitivity vs (1 - specificity) plot. A perfect test will have an area under the ROC curve (AUC) of 1.0; a random test will have an area of 0.5. Preferably, the tests described herein provide a ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95. A cut- off value is selected to provide an acceptable level of specificity and sensitivity. A cut-off value that can provide an acceptable level of specificity and sensitivity in separating a population of subjects into “bins” such as a “first” subpopulation (e.g., which is diagnosed with NAFLD, grade ≥2) and a “second” subpopulation which is not diagnosed with NAFLD (grade 0 or ≤1). A cut- off value is selected to separate this first and second population by one or more of the following measures of test accuracy:an odds ratio greater than 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a specificity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95; a sensitivity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding specificity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95; at least about 75% sensitivity, combined with at least about 75% specificity; a positive likelihood ratio (calculated as sensitivity / (l-specificity)) of greater than 1, at least about 2, more preferably at least about 3, still more preferably at least about 5, and most preferably at least about 10; or a negative likelihood ratio (calculated as (l-sensitivity) / specificity) of less than 1, less than or equal to about 0.5, more preferably less than or equal to about 0,3, and most preferably less than or equal to about 0.1.

[0066] In addition to cut-off comparisons, other methods for correlating assay results to a patient selection or classification (e.g. likelihood of being a good responder or a poor responder) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications.

[0067] Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (“AUC”) of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann- Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

[0068] The comparison to a cut-off value may be carried out manually, semi-automatically or in a fully automated manner. In some embodiments, the comparison may be computer assisted. A computer assisted comparison may employ values stored in a database as a reference for comparing an obtained value or a determined amount, for example via a computer implemented algorithm. Likewise, the comparison to a cut-off measurement may be carried out manually, semi-automatically or in a fully automated manner, including in a computer assisted manner.

[0069] In a further preferred embodiment, the present invention comprises the method as defined elsewhere herein, wherein if said sCD46 concentration as defined elsewhere herein is equal or above said cut-off value, it is indicative for whether or not said subject suffers from NAFLD. The term “above” means that said sCD46 concentration being determined in said PB sample is bigger than the exact value of the cut-off, e.g. if the cut-off value is for example 4.5, the sCD46 concentration is 4.51 (e.g. in ng / ml) or above. For the term “equal” it applies that for example if the cut-off value is 4.5, the sCD46 concentration is also 4.5 (e.g. in ng / ml).

[0070] If said sCD46 concentration is equal or above said cut-off value, it is possible to determine whether the subject being examined suffers from NAFLD or not. This is due to the fact that by equal or above said cut-off value, the subject can be clearly assigned to a steatosis grade ≥2 according to Chalasani, N. et al. Hepatology, Volume 67, Issue 1, 328-357 (2018), thus being diagnosed as steatotic with ≥ 5% HS (fat deposition) which is an evidence for NAFLD so that said subject can be diagnosed with NAFLD.

[0071] Additionally or alternatively, the subject can be assigned to suffering from “severe” steatosis, if the sCD46 concentration is equal or above the cut-off value. Classifying hepatic steatosis into different levels such as none (which refers to non-steatotic, steatosis grade 0), moderate (which refers to non-steatotic, steatosis grade 1) and severe (which refers to steatotic,steatosis grade ≥2) may normally be based upon imaging by ultrasound or radiological studies as known to the person skilled and thus refers to a less accurate approach compared to classifying a subject into different steatosis grades which may be achieved by liver biopsy and further microscopic investigation as mentioned elsewhere herein. Even though classifying a subject with different steatosis levels is as mentioned herein less accurate compared to the grading as defined herein which is however invasive, by applying the method of the invention if said sCD46 concentration may be equal or above the cut-off value, it may be indicative for severe steatosis (which is based on the characteristics - comprising a high degree of steatosis (fat deposition) - considered as steatotic (steatosis grade ≥2) with regard to the grading), thus the subject being examined can be diagnosed with NAFLD.

[0072] There are different ways of determining discriminatory cut-off values known to a person skilled in the art. Said cut-off value may be established as explained by WJ Youden, Index for rating diagnostic test, from the National Bureau of Standards, Washington D.C. (1949)).

[0073] The present invention may also comprise the method as defined elsewhere herein, wherein said cut-off value as defined above is in the range between about 43 ng / ml and about 47 ng / ml, such as about 43, 44, 45, 46 and 47ng / ml, preferably between about 44 ng / ml and about 46 ng / ml, even more preferably about 45 ng / ml, most preferably about 45.55 ng / ml (see Figures 2a and f which refer to data based on subjects being examined and having different steatosis grades; as well as see Figures 2i and o which refer to data based on subjects evaluated for living liver transplant donation and having different steatosis levels). For both scenarios, the same most preferred cut-off value has been established (about 45 ng / ml, in particular 45.55 ng / ml) which explains stable definitions of sCD46 as predictive biomarker and its relation to fat deposition in the liver. The concentration of sCD46 determined in the PB sample of the subject as defined herein is expressed preferably in ng / ml.

[0074] The present invention may also comprise the method of the invention as defined elsewhere herein, wherein said cut-off value as defined above is about 45 ng / ml - such value being the optimal discriminatory cutoff value that most accurately divides the training dataset used in the Examples into the two classes (non-steatotic, grade ≤1 vs. steatotic, grade ≥2 (see Figure 2f) and e.g. moderate vs. severe level (see Figure 2o)).

[0075] The present invention may also comprise the method as defined elsewhere herein, wherein if said sCD46 concentration is below said cut-off value as defined above generally andparticularly, it can be determined that said subject does not suffer from NAFLD - thus classifying the subject into grade ≤1 or 0 according to Chalasani, N. et al. Hepatology, Volume 67, Issue 1, 328-357 (2018), non-steatotic with < 5% HS (fat deposition) which is an evidence of not having NAFLD. Additionally or alternatively, the subject can be assigned to suffering from “moderate” or “none” steatosis, if the determined sCD46 concentration is below the cut-off value. Even though classifying a subject with different steatosis levels is as mentioned elsewhere herein less accurate compared to the grading as defined herein which is however invasive, by applying the method of the invention if said sCD46 concentration may be below the cut-off value, it may be indicative for moderate steatosis (which is based on the characteristics - comprising a small degree of steatosis (fat deposition) - still considered as non-steatotic with regard to the grading), thus the subject being examined cannot be diagnosed with NAFLD. The term “below” means that said sCD46 concentration being determined in said PB sample is smaller than the exact value of the cut-off, e.g. if the cut-off value is for example 4.5, the sCD46 concentration is 4.49 (e.g. in ng / ml) or below. In a particular context, said cut-off value can be in the range between about 43 ng / ml and about 47 ng / ml, such as about 43, 44, 45, 46 and 47ng / ml, preferably between about 44 ng / ml and about 46 ng / ml, even more preferably about 45 ng / ml, most preferably about 45.55 ng / ml.(0076] The present invention may also comprise the method as defined elsewhere herein, wherein if said sCD46 concentration is below said cut-off value and equal or above another cut- off value, it can also be determined that said subject does not suffer from NAFLD - classifying the subject into just grade 1 according to Chalasani, N. et al. Hepatology, Volume 67, Issue 1, 328-357 (2018), non-steatotic with < 5% HS (fat deposition) which is an evidence of not having NAFLD. Additionally or alternatively, the subject can be assigned to suffering from “moderate” steatosis as defined herein, if the sCD46 concentration is below said cut-off value and equal or above another cut-off value. The “another cut-off value” can be established as the “cut-off value” mentioned herein. All definitions regarding the cut-off value mentioned herein may be applicable - where appropriate - to the “another cut-off value”. In a preferred embodiment, said “another cut-off value” is in the range between about 24 ng / ml and about 28 ng / ml, such as about 24, 25, 26, 27 and 28 ng / ml, preferably between about 25 ng / ml and about 27 ng / ml, even more preferably about 26 ng / ml, most preferably about 26.19 ng / ml (see Figures 2a and f as well as see Figures 2i and o). For both scenarios, the same most preferred another cut-off value has been established (about 26 ng / ml, in particular 26.19 ng / ml). In a particular context, the “cut-off value” as used herein can be in the range between about 43 ng / ml and about 47 ng / ml, such as about 43, 44, 45, 46 and 47ng / ml, preferably between about 44 ng / ml and about 46 ng / ml, evenmore preferably about 45 ng / ml, most preferably about 45.55 ng / ml.Computer-implemented features

[0077] The subject-matter of the defined method steps of the present invention may also fully be carried out by computer program instructions running on means which, in the context of the invention, provide generic data processing functions. Such means may, for example, be embedded in a personal computer, smartphone, printer. A computer-implemented invention may therefore be one which involves the use of a computer, computer network or other programmable apparatus, where one or more features are realised wholly or partly by means of a computer program. Any definition made with regard to the method of the invention may also be applicable to the computer-implemented features as well.

[0078] Thus, the present invention comprises a data processing system comprising a processor configured to perform a method comprising the steps of a) obtaining a determined sCD46 concentration from a PB sample obtained from a subject; b) comparing said sCD46 concentration obtained in step a) with a cut-off value; and then c) indicating whether or not said subject suffers from NAFLD. Additionally, the present invention may comprise the data processing system as defined elsewhere herein, the method further comprising indicating whether or not said subject suffers from NAFLD, if said sCD46 concentration is equal or above said cut-off value.

[0079] According to the present invention, said sCD46 concentration as defined above may be determined in a PB sample obtained from a subject according to the present invention using any one of the detection methods as defined herein such as any one of an enzyme-linked immunosorbent assay (ELISA), a bead-based sandwich assay, a flow cytometry-based competitive binding assay or an immunoturbidimetric assay, preferably using a flow cytometry, in particular using a flow cytometry device. The detected concentration of sCD46 may be entered into said data processing system comprising a processor which is configured to perform the abovementioned steps. In some embodiments, the step of obtaining said determined concentration as defined above may be an optional step. As used herein, by using the term “obtaining / obtain the determined CD46 concentration” in this context means that the data for the concentration of sCD46 being detected in e.g. a flow cytometry device as defined elsewhere herein is entered into the data processing system by any way known to a person skilled in the art.

[0080] Then, within the data processing system the obtained sCD46 concentration is comparedwith a cut-off value as defined herein. Such cut-off value may be established as defined herein and then also entered before, simultaneously or after entering the detected concentration of sCD46 into said data processing system for the comparison step. By comparing said sCD46 concentration with said established cut-off value, it is then possible to indicate with the data processing system whether or not said subject suffers from NAFLD. For the step of comparing said sCD46 concentration with said cut-off value it may be of importance that said sCD46 concentration is equal or above said cut-off value as it is defined elsewhere herein. Any definition regarding the “cut-off value” mentioned for the method of the invention may be applicable - where appropriate - for the data processing system and vice versa.

[0081] A more meaningful characterization of modem digital data processing systems is the functional classification as either computational or input / output (“I / O") oriented. As the classifying labels imply, computational oriented data processing systems are designed primarily for performing long, complicated calculations. I / O oriented data processing systems are designed to handle large quantities of digital data, thereby requiring extensive I / O operations. Both data processing systems may be comprised by the present invention. The data processing system of the present invention may also be utilized as a peripheral subsystem of a larger computational computer. The structural design of a data processing system may necessarily directly be related to the functional use to which the data processing system is put. A data processing system of the present invention may refer to a programmable or programmed device / apparatus, where one or more features are realised wholly or partly by means of a computer program. A data processing system may include, but are not limited to, a computer, smartphone, tablet, chip.

[0082] As used herein, the term “processor” may refer to one functional element being a physical unit of a data processing system, which is configured by a computer program as defined elsewhere herein to perform the specified steps mentioned above.

[0083] The present invention further comprises the interaction between the data processing steps and other technical means such as a flow cytometry device.

[0084] Thus, the present invention comprises a device capable of detecting a sCD46 concentration in a PB sample obtained from a subject, comprising the data processing system as defined elsewhere herein.

[0085] As defined above, said sCD46 concentration may be detected by using any device, preferably using a flow cytometry, even more preferably using a flow cytometry analysis being combined with immunofluorescence as described elsewhere herein. In a preferred embodiment said flow cytometry device as defined above may be a FACS. In some embodiments said FACS may be connected to a computer, tablet, smartphone or any other technical device / apparatus being used as a further means for performing said FACS analysis. In another embodiment said FACS may not be connected to a computer, tablet, smartphone or any other technical device / apparatus being used as a further means for performing said FACS analysis.

[0086] The present invention also envisages a computer program comprising instructions to cause the data processing system as defined above or the flow cytometry device as defined above to execute the steps of a) obtaining a determined sCD46 concentration from a PB sample obtained from a subject; b) comparing said sCD46 concentration obtained in step a) with a cut- off value; and then c) indicating whether or not said subject suffers from NAFLD. The present invention may also comprise the computer program comprising instructions to cause the data processing system as defined above or the flow cytometry device as defined above to execute the steps of further comprising indicating whether or not said subject suffers from NAFLD, if said sCD46 concentration is equal or above said cut-off value.

[0087] Again, any definition regarding the “cut-off value” mentioned for the method of the invention may be applicable - where appropriate - for the computer program and vice versa. Again, in some embodiments, the step of obtaining said determined sCD46 concentration as defined above may be an optional step. Everything being defined above for said data processing system may apply mutatis mutandis to the corresponding computer program as defined above.

[0088] According to the present invention a computer program as mentioned above may refer to a program listing written in a programming language to implement an algorithm, and to binary code loaded in a computer-based apparatus, encompassing the accompanying documentation. A computer program as defined above may include, but is not limited to any software or any downloadable internet link known to a person skilled in the art comprising instructions to cause the data processing system as defined elsewhere herein to execute the defined steps.

[0089] Also comprised is a computer-readable medium having stored thereon the computer program as defined above. A computer-readable medium having stored thereon all of the computer programs as defined above may also be comprised herein. As used herein, a computer-readable medium having stored thereon the computer program may include, but is not limited to, a USB stick, a disc, DVD, CD, CD-ROM.

[0090] EXAMPLESMaterial and Methods.

[0091] Study approval and patient cohort.

[0092] This study involving human research participants was performed in accordance with the Declaration of Helsinki, as well as all applicable German and European laws and ethical standards. Samples for the first cohort (Fig. 3) were obtained from 105 patients undergoing liver surgery for primary liver malignancies (hepatocellular or cholangiocellular carcinoma), liver metastases or benign liver lesions at the Department of Surgery, University Hospital Regensburg. No study patients received systemic chemotherapy before surgery. This single- centre observational study was authorised by the Ethics Committee of the University of Regensburg (approval number 13-257-101 and 13-257-5-101) and registered with clinicaltrials.gov (NCT04943978). All participants gave full, informed written consent. The first reported patient was recruited in August 2014 and the last reported patient was recruited in September 2019. Patients received standard-of-care treatment according to local guidelines. Analysis of collected plasma samples from the second cohort (living liver donors) was also authorised by the Ethics Committee of the University of Regensburg (approval number 13-257- 5-101) without the need of a written consent.

[0093] Histopathological examination of human liver specimens.

[0094] All liver tissue specimens were fixed in neutral buffered formalin then embedded in paraffin. Histological sections with a thickness of 4 pm were prepared, deparaffinized with ethanol and xylene, and then stained according to standard protocols with Haematoxylin and Eosin (HE) and Elastica van Gieson (EvG) or Sirius Red. Staining with EvG or Sirius Red was used to evaluate liver fibrosis according to the Ishak scoring system (Ishak, K., et al. Histological grading and staging of chronic hepatitis. J Hepatol 22, 696-699 (1995). Steatosis of the liver was reported as percentage of hepatocytes containing fat droplets. By convention, patients with <5% steatosis were considered to be non-steatotic (Rinella, M.E., Tacke, F., Sanyal, A.J. & Anstee, Q.M. Report on the AASLD / EASL Joint Workshop on Clinical Trial Endpoints in NAFLD. Hepatology 70, 1424-1436 (2019).

[0095] Processing of PBMC and IHL from clinical material.

[0096] Human peripheral blood mononuclear cells (PBMC) were isolated from CPDA tubes by Ficoll gradient centrifugation. Intrahepatic lymphocytes (IHL) were isolated as previously described (Morsy, M.A., et al. Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique. Laboratory Investigation 85, 285-296 (2005)). In brief, surgically resected liver material was verified to be tumour-free by a pathologist and then washed with HBBS medium before dissection into small fragments. After digestion with collagenase type IV and DNase I, hepatocytes were removed by filtration through a 40 μm mesh. IHL were then collected by Ficoll gradient centrifugation. iNKT cells and iNKT- depleted PBMC were prepared from leucocyte apheresates obtained as a by-product of thrombocyte collection. Isolated cells were frozen in RPMI containing 10% DMSO and stored at -160°C in liquid nitrogen. For thawing, cryovials were placed in a 37°C waterbath, then the cell suspension was transferred to prewarmed RPMI containing lOOμg / ml DNase I (Applichem Cat. A3778,0100) and slowly diluted.

[0097] Isolation, expansion and treatment of iNKT cells.

[0098] Vα24-Jα18+iNKT cells were magnetically isolated from PBMCs using anti-iNKT microbeads (Miltenyi - Cat. 130-094-842) with the program POSSEL_S on an AutoMACS Pro device. iNKT expansion medium comprised RPMI 1640 GlutaMAX™, 10% HyClone FetalClone II serum, 1% sodium pyruvate, 1% MEM non-essential amino acids, 7.5% NaHCO3, 1% penicillin / streptomycin and 50 mM 2-mercaptoethanol. Enriched iNKT cells were seeded at 5 x 104cells / cm2in iNKT medium supplemented with 50 ng / ml of animal-free rhIL-2 and 100 ng / ml a-Gal-Cer before expansion for 7 days. The medium was replaced on day 2 with iNKT medium supplemented with rhIL-2 and a-Gal. On day 5, the medium was replaced with iNKT medium supplemented with recombinant human (rh)IL-2 only. Treatment of ex v / vo-expanded iNKT cells with chimeric CD46-Fcy (R&D - Cat. 10257-CD) or the corresponding chimeric isotype (R&D - Cat. 110-HG) was renewed with each medium change.

[0099] Differentiation, fat loading (FL) and manipulation of HepaRG cells.

[0100] The HepaRG cell line was obtained from Biopredic International, France. For two weeks, HepaRG cells were cultured in 12-well plates (3.9 cm2- TPP) or in T75 flasks (TPP) at 2.5 x 104cells / cm2in HepaRG growth medium (William’s E medium supplemented with 10% HyClone FetalClone II serum, 1% penicillin / streptomycin, 1% L-glutamine, 0.023 lE / ml insulin, 4.7 μg / ml hydrocortisone and 80 μg / ml gentamycin). HepaRG cells were then cultured for a further 2 weeks in HepaRG growth medium supplemented with 1.8% DMSO to aid differentiation into hepatocyte-like cells. Differentiated HepaRG cells were cultured for 24 hours in serum-free HepaRG growth medium and then treated with bovine serum albumin (BSA)conjugated-palmitic acid and oleic acid (1:2 - 0.5 mM) for a further 24 hours to induce fat loading (FL) of HepaRG cells (FL-HepaRG). The inventors used unloaded (UL)-HepaRG as control cells that were generated using only isopropanol, which was used as a solvent for fatty acids. Fat loading of HepaRG cells was confirmed by Oil Red O staining (ScienCell™ - Cat. 0843) according to the manufacturer's instructions. For flow cytometry analysis, adherent HepaRG cells were detached from plastic culture surfaces with Cell Staining Buffer (Biolegend - Cat. 420201) and mechanical scraping. For hypoxia experiments, HepaRG cells were cultured for 12 h in an atmosphere of 0% O2, 5% CO2 and 95% N2. Metalloprotease inhibitors (TAPI-1 [Cat. 18505]; GI 254023X [Cat. 28284]; Batimastat [Cat. 14742]) were applied to HepaRG cells for 24 h in parallel to the FL protocol.

[0101] iNKT cell and HepaRG cell cocultures.

[0102] To investigate the interaction of iNKT cells and HepaRG cells in vitro, coculture experiments were performed. 2 x 105freshly sorted iNKT cells were added to differentiated UL- or FL-HepaRG cells in 12-well plates with iNKT expansion medium, which was changed after 4 days. Indirect coculture experiments were performed with Transwell inserts (Coming). After 7 days’ coculture, the non-adherent iNKT cells were harvested and further analyzed. In treated iNKT cell and HepaRG cell coculture experiments, the respective treatment (CD46 [R&D - Cat. AF2005], goat Ig control [R&D - Cat. AB-108-C]) started with the addition of the freshly isolated iNKT cells. In the MMP-I treated cocultures, however, the inhibitors were already added in parallel to the FL.

[0103] Flow cytometry.

[0104] Briefly, surface staining was performed at 4°C in Cell Staining Buffer (Biolegend - Cat. 420201) and 10% FcR-block (Miltenyi - Cat. 130-059-901) for 30 to 60 min. FoxP3 Fixation- and-Permeabilization buffers (eBioscience - Cat. 00-5523-00) were used for intracellular staining. Dead cells were excluded with Fixable Viability Dye eFluor506 (Invitrogen - 65-0866- 14) or ViaKr-808 (Beckman Coulter - Cat. C36628). For intracellular detection of cytokines, cultured iNKT cells were harvested, washed and then stimulated with 50 ng / ml PMA [Cat. Pl 585] and 1 μg / ml ionomycin [Cat. 10634] in the presence of brefeldin A [BD - Cat. 555029] and monensin [BD - Cat. 554724] for 5 hours prior to staining. Data were collected with a Navios cytometer or a CytoFlex LX instrument (Beckman Coulter, Krefeld, Germany) as previously described (Hutchinson, J. A., et al. Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis. Nat Commun 12, 1439 (2021)). Analyses were performed with Kaluza 2.1 (Beckman Coulter). Marker expression levels were estimated bycalculating background (isotype)-subtracted geometric mean fluorescence intensities.Background-subtracted MFI values ≤ 0 were set to 1.

[0105] Stable knockdown of CD46 in HepaRG cells.

[0106] Stable CD46 knockdown of HepaRG cells was performed using Lipofectamine 3000 (ThermoFisher - Cat. L300000) according to the manufacturer's instructions. The shRNA plasmids used (CD46knockdownand random with a scrambled sequence) were obtained from SantaCruz (Cat. sc-35004-SH). After 1.5 μg / ml puromycin selection, low CD46-expressing transfectants were sorted using a BD FACSAria™ IL

[0107] Statistics.

[0108] Significance tests, curve-fitting and predictive modelling were performed with GraphPad or SPSS® software. As indicated, t-tests, Mann-Whitney, one-way and two-way ANOVA were used for tests of significance. P-values were adjusted for multiple comparison as indicated in the figure legends.Results.

[0109] Example 1: Steatosis is associated with intrahepatic enrichment of IL-4+iNKT cells.

[0110] To confinn activation of invariant NKT cells in patients with hepatic steatosis, the inventors first investigated peripheral blood (PBMC) and intrahepatic lymphocytes (IHL) in patients with varying degrees of steatosis undergoing liver resection due to primary liver cancer. By convention, patients with < 5 % liver fat were classified as non-steatotic (Grade 0 or 1; n=55), whereas patients with ≥ 5 % liver fat were classified as steatotic (Grades 2 -4; n=50; Fig. 3). Analysis of flow cytometry data showed an increase of iNKT cells in the liver of steatotic patients (Fig. 1). Furthermore, intracellular staining for effector cytokines revealed higher IL-4+iNKT cell frequencies (Fig. lb) in steatotic livers whereas no difference was detected in IL-17+iNKT cells (data not shown).

[0111] To study the interaction of fat-loaded hepatocytes with human iNKT cells, the inventors established an in vitro coculture system using HepaRG cells and primary human iNKT cells (data not shown). Isolated iNKT cells were expanded for 7 days with rhIL-2 and α-GalCer in direct coculture with fat-loaded (FL)- or unloaded (UL)-HepaRG cells. A higher proportion of IL-4+iNKT cells was obtained after coculture with FL-HepaRG compared to control UL- HepaRG cells (Fig. 1c). No differences in IFN-γ+or JL-17A+iNKT cells were observed (data notshown). Comparing iNKT cells expanded alone or in coculture with HepaRG cells suggested that this effect reflected an impaired capacity of FL-HepaRG cells to suppress IL-4+iNKT cell development and transwell experiments showed this suppression of IL-4+iNKT cells was contact dependent (Fig. 1c). Consistent with HepaRG cells specifically suppressing IL-4+iNKT cell development, the inventors observed no effect of FL- or UL-HepaRG cells on IL-4 production by conventional T cells (data not shown).

[0112] The results show that IL-4+iNKT cells are suppressed by HepaRG cells through a cell contact-dependent mechanism that is impaired by fat-loading. Therefore, the inventors inferred the existence of a cell surface ligand expressed by HepaRG cells that should bind to a receptor on iNKT cells to inhibit their expression of IL-4. Knowing that the putative ligand should be down-regulated in HepaRG cells by fat-loading narrowed the list of possible candidate receptors. The inventors first performed a bulk RNA-sequencing analysis searching for differentially expressed genes between FL- and UL-HepaRG cells. Surprisingly few genes were affected by fat-loading and, after filtering for transmembrane gene products, no credible candidates were found (data not shown). Therefore, the inventors next performed proteomic analyses of FL- or UL-HepaRG cells but again returned no candidate cell surface receptors (data not shown). These results suggested that fat-loading had little impact on the global proteomic profile of HepaRG cells.

[0113] To corroborate this conclusion, the inventors next performed a flow cytometry-based screen for expression of 361 markers in FL- and UL-HepaRG cells (Fig. 1d). Surprisingly, the inventors found a high proportion of significantly down-regulated cell-surface receptors, including CD46, that were not differentially expressed in the transcriptomic or proteomic studies (data not shown). CD46 is expressed by conventional T cells and influences their effector differentiation. Notably, the soluble form of CD46 (sCD46) was present in coculture supernatants and generally higher levels were detected in FL-HepaRG cocultures (Fig. le, Fig. 4). Furthermore, adding chimeric CD46-Ig protein to iNKT cell cultures suppressed IL-4+iNKT cells (Fig. 1f). To confirm that CD46 was functionally important for IL-4+iNKT cell development, the inventors showed that blocking CD46 in cocultures with neutralising antibodies (Fig. 1g) or knockdown of CD46 in HepaRG cells promoted IL-4+iNKT cell development (Fig. 1h). To establish whether loss of CD46 expression in FL-HepaRG cells reflected a shift in expression of CD46 isoforms, the inventors performed TaqMan PCR to measure alternative splice variants; however, no differences were detected (data not shown).

[0114] Consequently, loss of surface CD46 expression in FL-HepaRG appears to be a post- secretional effect that is consistent with shedding of immune receptors from the surface of HepaRG cells, possibly through enzymatic cleavage surface. Matrix metalloproteinases (MMPs)are zinc-dependent endopeptidases that are involved in remodeling of the extracellular matrix (Naim, A., Pan, Q. & Baig, M.S. J Clin Exp Hepatol 7, 367-372 (2017)). MMPs participate in the process of liver regeneration along with many liver diseases including hepatic inflammation. Therefore, the inventors measured the concentration of secreted MMPs in coculture supernatants. Expression of MMP-1, MMP-3, MMP-7 and MMP-10 was significantly higher in cocultures with FL-HepaRG cells compared to UL-HepaRG cells (data not shown). Treating HepaRG cells with a selection of MMP-inhibitors (TAPI-1, GI254023X and Batimastat) led to a dose-dependent increase in CD46 expression at the cell surface (Fig. 1j) and likewise to a suppression of IL-4+iNKT cell development (Fig. 1i). Taken together, these results show that MMP-mediated loss of cell surface CD46 expression after fat-loading of HepaRG cells leads to disinhibited IL-4+iNKT cell differentiation.

[0115] To establish the in vivo relevance of these discoveries, the inventors next measured sCD46 concentrations in patient plasma samples (Fig. 2a). It was found an association between steatosis grade and sCD46 concentrations, implying that MMP-mediated cleavage of immune receptors from hepatocytes in patients with fatty liver disease might explain the initial observation of IL-4+iNKT cell enrichment in steatotic livers. At present, steatosis grading in patients requires liver biopsy and histological evaluation, or imaging studies by ultrasound or magnetic resonance imaging (MRI).

[0116] sCD46 concentrations were determined in plasma from n=91 patients with varying grades of hepatic steatosis. Cases were randomly assigned to a training (n=45) or validation (n=46) set. First, training set data were used to set a cut-off for sCD46 of 26.19 ng / ml that discriminated between patients with no histologically evident steatosis and patients with any degree of steatosis (Fig. 2b). Applying this cut-off value to the validation set, sCD46 correctly classified 58.7 % patients in the validation set, as compared to 32.6 % under the no-information model (Fig. 2c). Because patients with <5 % steatosis are conventionally classified as non- steatotic, distinguishing between Grades ≤1 and ≥2 is clinically more relevant than distinguishing between Grades 0 and ≥1. Therefore, the inventors next asked whether plasma sCD46 concentrations discriminated between steatosis Grade ≤1 and ≥2. sCD46 was a very good discriminator in the training (AUC = 0.944) and validation (AUC = 0.886) sets (Fig. 2d-e). Taking a second sCD46 cut-off value of 45.55 ng / ml, the inventors constructed a decision tree to predict steatosis grade in patients (Fig. 2f) that achieved an overall correct prediction rate of 58.7 % in the validation set. Crucially, this model performs very well in classifying patients as non- steatotic (Grade ≤1) or steatotic (Grade ≥2) with a correct classification rate of 97.8 %, positivepredictive value (PPV) = 100 % and negative predictive value (NPV) = 97.6 % in the validation set (Fig. 2g-h).

[0117] Next, the inventors wanted to show that sCD46 is a potential clinically applicable, minimally invasive, diagnostic biomarker of steatosis by testing a second patient cohort, living liver donors, that are otherwise completely healthy. The inventors tested sCD46 concentrations in plasma from n=158 patients with varying levels of hepatic steatosis and confirmed the respective cut-off values (Fig 2i and o). Furthermore, plasma sCD46 concentrations discriminated between none vs. moderate steatosis (AUC = 0.795) as well as moderate vs. severe steatosis (AUC 0.934) and between none vs. any existing steatosis (AUC 0.831) (Fig. 2j-I). Finally, the model with sCD46 performs extremely well in classifying different steatosis levels (correct rate 69.6%) and especially in predicting existing steatosis (correct rate 79.1%) (Fig.2m&n).The invention is further chacterized by the following items:Items1. A method of diagnosing a subject with non-alcoholic fatty liver disease (NAFLD), the method comprising determining a soluble CD46 (sCD46) concentration in a peripheral blood (PB) sample obtained from a subject, wherein said sCD46 concentration is indicative for whether or not said subject suffers from NAFLD.2. The method of item 1 , wherein said PB sample is a plasma or a serum sample.3. The method of item 1 or 2, wherein said sCD46 concentration is detected using any one of an enzyme-linked immunosorbent assay (ELISA), a bead-based sandwich assay, a flow cytometry-based competitive binding assay or an immunoturbidimetric assay.4. The method of item 3, wherein a binding partner for sCD46 is used.5. The method of item 4, wherein said binding partner is any one of an immunoglobulin or a fragment thereof, a proteinaceous binding molecule with immunoglobulin-like functions, or a recombinant receptor.6. The method of any one of items 1-5, wherein said subject is a human.7. The method of any one of items 1-6, wherein said subject is an adult.8. The method of any one of items 1-7, wherein said subject is suspected to suffer from NAFLD.9. The method of any one of items 1-7, wherein said subject is suspected to be a liver transplant donor.10. The method of any one of items 1-9, the method further comprising comparing said sCD46 concentration determined in said PB sample with a cut-off value.11. The method of item 10, wherein if said sCD46 concentration is equal or above said cut-off value, it is indicative for whether or not said subject suffers from NAFLD.12. The method of any one of items 10-11, wherein said cut-off value is in the range between about 43 ng / ml and about 47 ng / ml.13. The method of item 12, wherein said cut-off value is about 45 ng / ml.14. A data processing system comprising a processor configured to perform a method comprising the steps of a) obtaining a determined soluble CD46 (sCD46) concentration from a peripheral blood (PB) sample obtained from a subject; b) comparing said sCD46 concentration obtained in step a) with a cut-off value; c) indicating whether or not said subject suffers from non-alcoholic fatty liver disease (NAFLD).15. The data processing system of item 14, the method further comprising indicating whether or not said subject suffers from NAFLD, if said sCD46 concentration is equal or above said cut-off value.16. A device capable of detecting a soluble CD46 (sCD46) concentration in a peripheral blood (PB) sample obtained from a subject, comprising the data processing system of any one of items 14-15.17. A computer program comprising instructions to cause the data processing system of any one of items 14-15 or the device of item 16 to execute the steps of a) obtaining a determined soluble CD46 (sCD46) concentration from a peripheral blood (PB) sample obtained from a subject; b) comparing said sCD46 concentration obtained in step a) with a cut-off value; c) indicating whether or not said subject suffers from non-alcoholic fatty liver disease (NAFLD).18. A computer-readable medium having stored thereon the computer program of item 17.

Claims

CLAIMS1. A method of diagnosing a subject with non-alcoholic fatty liver disease (NAFLD), the method comprising determining a soluble CD46 (sCD46) concentration in a peripheral blood (PB) sample obtained from a subject, wherein said sCD46 concentration is indicative for whether or not said subject suffers from NAFLD.

2. The method of claim 1, wherein said PB sample is a plasma or a serum sample.

3. The method of claim 1 or 2, wherein said sCD46 concentration is detected using any one of an enzyme-linked immunosorbent assay (ELISA), a bead-based sandwich assay, a flow cytometry-based competitive binding assay or an immunoturbidimetric assay.

4. The method of claim 3, wherein a binding partner for sCD46 is used.

5. The method of claim 4, wherein said binding partner is any one of an immunoglobulin or a fragment thereof, a proteinaceous binding molecule with immunoglobulin-like functions, or a recombinant receptor.

6. The method of any one of claims 1-5, wherein said subject is a human.

7. The method of any one of claims 1-6, wherein said subject is an adult.

8. The method of any one of claims 1-7, wherein said subject is suspected to suffer from NAFLD.

9. The method of any one of claims 1-7, wherein said subject is suspected to be a liver transplant donor.

10. The method of any one of claims 1-9, the method further comprising comparing said sCD46 concentration determined in said PB sample with a cut-off value.

11. The method of claim 10, wherein if said sCD46 concentration is equal or above said cut-off value, it is indicative for whether said subject suffers from NAFLD.

12. The method of any one of claims 10-11, wherein said cut-off value is in the range between 43 ng / ml and 47 ng / ml.

13. The method of claim 12, wherein said cut-off value is about 45 ng / ml.

14. A data processing system comprising a processor configured to perform a method comprising the steps of a) obtaining a determined soluble CD46 (sCD46) concentration from a peripheral blood (PB) sample obtained from a subject; b) comparing said sCD46 concentration obtained in step a) with a cut-off value; c) indicating whether or not said subject suffers from non-alcoholic fatty liver disease (NAFLD).

15. The data processing system of claim 14, the method further comprising indicating whether said subject suffers from NAFLD, if said sCD46 concentration is equal or above said cut- off value.

16. A device capable of detecting a soluble CD46 (sCD46) concentration in a peripheral blood (PB) sample obtained from a subject, comprising the data processing system of any one of claims 14-15.

17. A computer program comprising instructions to cause the data processing system of any one of claims 14-15 or the device of claim 16 to execute the steps of a) obtaining a determined soluble CD46 (sCD46) concentration from a peripheral blood (PB) sample obtained from a subject; b) comparing said sCD46 concentration obtained in step a) with a cut-off value; c) indicating whether or not said subject suffers from non-alcoholic fatty liver disease (NAFLD).

18. A computer-readable medium having stored thereon the computer program of claim 17.

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