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WO2025008404A1 - Utilisation d'une concentration de cd46 soluble dans du sang périphérique en tant que paramètre pour le diagnostic d'une stéato-hépatopathie non alcoolique - Google Patents

Utilisation d'une concentration de cd46 soluble dans du sang périphérique en tant que paramètre pour le diagnostic d'une stéato-hépatopathie non alcoolique Download PDF

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WO2025008404A1
WO2025008404A1 PCT/EP2024/068737 EP2024068737W WO2025008404A1 WO 2025008404 A1 WO2025008404 A1 WO 2025008404A1 EP 2024068737 W EP2024068737 W EP 2024068737W WO 2025008404 A1 WO2025008404 A1 WO 2025008404A1
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scd46
subject
concentration
nafld
cut
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Jens M. WERNER
Florian BITTERER
James Alexander Hutchinson
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Universitaet Regensburg
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Universitaet Regensburg
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/20—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for electronic clinical trials or questionnaires
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/70—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for mining of medical data, e.g. analysing previous cases of other patients
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • 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.
  • 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.
  • Nonalcoholic fatty liver disease 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.
  • Hepatic steatosis 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 to hepatocytes, which ultimately leads to chronic inflammation of the liver (Ertunc, M.E. & Hotamisligil, G.S. J Lipid Res 57, 2099-2114 (2016)).
  • 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.
  • steatohepatitis is determined as non-alcoholic steatohepatitis (short: NASH) within NAFLD.
  • NASH non-alcoholic steatohepatitis
  • this NASH may even drive hepatic fibrosis, which can eventually progress to cirrhosis and hepatocellular carcinoma.
  • NAFLD non-invasive liver biopsy
  • biopsies are performed these days if the patient simultaneously suffers from a liver tumor which will then also be removed surgically.
  • NAFLD is only diagnosed in an advanced stage.
  • 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.
  • sCD46 soluble CD46
  • PB peripheral blood
  • sCD46 concentration in the PB 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.
  • 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.
  • 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.
  • 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.
  • ROC Receiving Operator Characteristic
  • AUC area under curve
  • Figure 3 Recruitment of patients undergoing liver surgery for a prospective, non- randomised observational clinical study.
  • Figure 4 Competitive cell-based flow cytometry assay to measure the concentration of soluble sCD46.
  • 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.
  • NAFLD Nonalcoholic fatty liver disease
  • HS hepatic steatosis
  • monogenic hereditary disorders 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 (2016)).
  • hepatocyte injury e.g., ballooning
  • 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.
  • diagnosis / diagnosing 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.
  • 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.
  • 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.
  • the subject being used in the present invention is a human. More preferably, said subject is an adult.
  • 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”.
  • 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).
  • the present invention 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.
  • 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.
  • the present invention comprises the method of the invention, wherein said subject is suspected to be a liver transplant donor.
  • sonographical determination may be applied for classifying a non-steatotic or steatotic liver.
  • 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.
  • 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.
  • 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
  • CD46 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 (2016)).
  • 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. MMPs participate in the process of liver regeneration along with many liver diseases including NAFLD.
  • MMPs Matrix metalloproteinases
  • 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).
  • TIMPs tissue inhibitor of metalloproteinases
  • 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.
  • 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)).
  • NK natural killer
  • NKT natural killer T
  • MAIT mucosal-associated invariant T
  • NKT cells are a minor subpopulation of TCR ⁇ -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 are characterised by co-expression of classic NK cell markers and the invariant TCR-V ⁇ 24-J ⁇ 18 chain, often in conjunction with TCR-V ⁇ 11, which allows them to recognise ⁇ -galactosylceramide ( ⁇ -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.
  • iNKT cells 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.
  • IL-4 interleukin-4
  • 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.
  • shedding of immune regulatory molecules 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.
  • 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.
  • concentration of sCD46 can be measured using any detection method known to a person skilled in the art.
  • 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.
  • detection method e.g. flow cytometry
  • Such detection method 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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), a bead-based sandwich assay, a flow cytometry-based competitive binding assay or an immunoturbidimetric assay.
  • 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.
  • 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.
  • Fluorescent signals used in flow cytometry 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).
  • any desired detectable marker or combination of detectable markers can be detected by the optics and/or electronics of a flow cytometer.
  • 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.
  • 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.
  • 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.
  • 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.
  • an immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions may be chosen.
  • 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.
  • beads such as LUMINEX® microspheres
  • 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.
  • 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.
  • 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.
  • glubodies see e.g.
  • 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.
  • An immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions may be PEGylated or hyperglycosylated if desired.
  • 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.
  • 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.
  • 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.
  • An immunoglobulin may be monoclonal or polyclonal.
  • 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.
  • 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.
  • 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 or immunoglobulin fragments and proteinaceous binding molecules with immunoglobulin-like functions, in both prokaryotic and eukaryotic organisms.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • KrO Krome Orange
  • fluorescent proteins examples include, but are not limited to, EGFP, emerald, EYFP, a phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC), Monomeric Red Fluorescent Protein (mRFP), mOrange, mPlum and mCherry.
  • PE phycoerythrin
  • APC allophycocyanin
  • mRFP Monomeric Red Fluorescent Protein
  • mOrange Monomeric Red Fluorescent Protein
  • mPlum mCherry
  • a reversibly photoswitchable fluorescent protein such as Dronpa, bsDronpa and Padron may be employed (Andresen, M., et al., Nature Biotechnology (2008) 26, 9, 1035).
  • suitable enzymes alkaline phosphatase, soybean peroxidase, or horseradish peroxidase may serve as a few illustrative examples.
  • tandem conjugates such as PE-Cy5.5 or PE-Cy7 may also be used.
  • 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.
  • 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.
  • 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.
  • anticoagulants i.e., EDTA, citrate, heparin
  • the respective method according to the present invention may also involve analysis of one or more PB samples from the subject in vitro.
  • 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.
  • 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.
  • 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 on the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ROC Receiver Operating Characteristic
  • 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 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).
  • TPR true positive rate
  • FPR false positive rate
  • 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.
  • 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 “first” subpopulation e.g., which is diagnosed with NAFLD, grade ⁇ 2
  • 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.
  • 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.
  • the comparison to a cut-off value may be carried out manually, semi-automatically or in a fully automated manner.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 (2016), 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.
  • 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.
  • 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).
  • 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.
  • concentration of sCD46 determined in the PB sample of the subject as defined herein is expressed preferably in ng/ml.
  • 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)).
  • 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 and particularly, 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 (2016), 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.
  • the cut-off value is for example 4.5
  • the sCD46 concentration is 4.49 (e.g. in ng/ml) or below.
  • 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.
  • 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 (2016), 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”.
  • 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).
  • the same most preferred another cut-off value has been established (about 26 ng/ml, in particular 26.19 ng/ml).
  • 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, even more preferably about 45 ng/ml, most preferably about 45.55 ng/ml.
  • 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.
  • 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.
  • 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.
  • 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.
  • the step of obtaining said determined concentration as defined above may be an optional step.
  • obtaining / obtain the determined CD46 concentration 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.
  • the obtained sCD46 concentration is compared with 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.
  • a more meaningful characterization of modem digital data processing systems is the functional classification as either computational or input/output (“I/O") oriented.
  • 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.
  • 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.
  • the present invention further comprises the interaction between the data processing steps and other technical means such as a flow cytometry device.
  • 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.
  • 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.
  • said flow cytometry device as defined above may be a FACS.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a computer-readable medium having stored thereon the computer program as defined above is also comprised 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.
  • 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.
  • PBMC peripheral blood mononuclear cells
  • IHL Intrahepatic lymphocytes
  • hepatocytes were removed by filtration through a 40 ⁇ m 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 lOO ⁇ g/ml DNase I (Applichem Cat. A3778,0100) and slowly diluted.
  • V ⁇ 24-J ⁇ 18 + 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 GlutaMAXTM, 10% HyClone FetalClone II serum, 1% sodium pyruvate, 1% MEM non-essential amino acids, 7.5% NaHCO 3 , 1% penicillin/streptomycin and 50 mM 2-mercaptoethanol.
  • Enriched iNKT cells were seeded at 5 x 10 4 cells/cm 2 in 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.
  • 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.
  • HepaRG cell line was obtained from Biopredic International, France. For two weeks, HepaRG cells were cultured in 12-well plates (3.9 cm 2 - TPP) or in T75 flasks (TPP) at 2.5 x 10 4 cells/cm 2 in 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 ⁇ g/ml hydrocortisone and 80 ⁇ g/ml gentamycin).
  • William E medium supplemented with 10% HyClone FetalClone II serum, 1% penicillin/streptomycin, 1% L-glutamine, 0.023 lE/ml insulin, 4.7 ⁇ g/ml hydrocortisone and 80 ⁇ g/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).
  • BSA bovine serum albumin
  • FL-HepaRG fat loading
  • the inventors used unloaded (UL)-HepaRG as control cells that were generated using only isopropanol, which was used as a solvent for fatty acids.
  • cytokines For intracellular detection of cytokines, cultured iNKT cells were harvested, washed and then stimulated with 50 ng/ml PMA [Cat. Pl 585] and 1 ⁇ g/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 by calculating background (isotype)-subtracted geometric mean fluorescence intensities.
  • Stable CD46 knockdown of HepaRG cells was performed using Lipofectamine 3000 (ThermoFisher - Cat. L300000) according to the manufacturer's instructions.
  • the shRNA plasmids used (CD46 knockdown and random with a scrambled sequence) were obtained from SantaCruz (Cat. sc-35004-SH). After 1.5 ⁇ g/ml puromycin selection, low CD46-expressing transfectants were sorted using a BD FACSAriaTM IL
  • Example 1 Steatosis is associated with intrahepatic enrichment of IL-4 + iNKT cells.
  • PBMC peripheral blood
  • IHL intrahepatic lymphocytes
  • the inventors next performed a flow cytometry-based screen for expression of 361 markers in FL- and UL-HepaRG cells (Fig. 1d).
  • 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.
  • 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).
  • MMPs matrix metalloproteinases
  • MMP-1, MMP-3, MMP-7 and MMP-10 were 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 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).
  • 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.
  • steatosis grading in patients requires liver biopsy and histological evaluation, or imaging studies by ultrasound or magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • 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).
  • 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 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.
  • the invention is further chacterized by the following items:
  • a method of diagnosing a subject with non-alcoholic fatty liver disease 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.
  • sCD46 soluble CD46
  • PB sample is a plasma or a serum sample.
  • binding partner is any one of an immunoglobulin or a fragment thereof, a proteinaceous binding molecule with immunoglobulin-like functions, or a recombinant receptor.
  • 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).
  • sCD46 soluble CD46
  • 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.
  • 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).
  • sCD46 soluble CD46

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Abstract

La présente invention concerne une méthode de diagnostic d'un sujet atteint d'une stéato-hépatopathie non alcoolique (NAFLD), la méthode consistant à déterminer une concentration de CD46 soluble (sCD46) dans un échantillon de sang périphérique (PB) obtenu d'un sujet, ladite concentration de sCD46 indiquant si ledit sujet souffre ou non de NAFLD. De plus, la présente invention concerne également un système de traitement de données comprenant un processeur configuré pour exécuter un procédé comprenant les étapes consistant à obtenir une concentration de sCD46 déterminée d'un échantillon de PB obtenu d'un sujet, à comparer ladite concentration de sCD46 à une valeur de coupure, puis à indiquer si ledit sujet souffre ou non de NAFLD.
PCT/EP2024/068737 2023-07-04 2024-07-03 Utilisation d'une concentration de cd46 soluble dans du sang périphérique en tant que paramètre pour le diagnostic d'une stéato-hépatopathie non alcoolique Pending WO2025008404A1 (fr)

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