Patent Application
20100104581
The invention relates the identification of TRIB1 as (i) a marker for the diagnosis, in particular in blood, and prognosis of inflammatory conditions, such as chronic graft rejection, and (ii) as a therapeutic target for these conditions.
More than 250 000 Europeans live with an organ transplant and 80 000 remain on transplant waiting lists. Increasing the life of a transplanted organ remains a major challenge in transplantation. Indeed, despite an increase in graft survival and a decrease in the risks of rejection, the leading cause of graft loss, chronic rejection (CR), is only poorly influenced by currently used immunosuppressors.
There remains a need for a reliable diagnostic or prognostic test applicable to a blood sample or a graft biopsy, to monitor and adapt the therapeutic treatment available to each patient. The identification of a new marker is also a major objective in the search for novel immunosuppressors.
In this context, the literature reports a few studies of transcriptome profiling in renal transplant biopsies (Flechner et al, 2004a; Scherer et al, 2003), and one study in the peripheral blood lymphocytes of renal transplant patients (Flechner et al, 2004b).
International patent application WO2005/070086 also describes expression profiles of genes that are determinative of graft tolerant phenotypes (see also Brouard et al, 2005; Louis et al 2006; Baeten et al 2006).
The inventors have now identified that TRIB1 (“Tribbles homolog 1”) is a marker of chronic rejection in clinical renal transplantation, and more generally of any inflammatory condition, such as joint inflammation, multiple sclerosis, and inflammatory bowel disease. More particularly the inventors showed that TRIB1 is a peripheral blood marker of such pathologies.
This new marker can serve to distinguish CR from other types of chronic dysfunction, thereby enabling therapeutic adaptation and more individualized immunosuppression. It further enables earlier detection of CR and thus improves graft outcome by allowing for early therapeutic intervention before the onset of irreversible graft damage.
On this basis, the invention provides an in vitro method of diagnosis and/or prognosis of an inflammatory condition, which method comprises determining the expression level of TRIB1 in a biological sample of a patient, wherein a higher expression of TRIB1 in the patient, compared to a reference value, is indicative of an inflammatory condition.
TRIB1 marker is all the more useful that its expression level can be easily assessed from any blood or plasma sample, as well as in tissue biopsies, using standard kits.
The expression level may be determined by any method known in the art, e.g. by measuring the level of TRIB1 mRNA in the biological sample, or by measuring the level of TRIB1 protein, e.g. by an immunoassay of the TRIB1 protein.
The invention further provides a method for screening compounds useful for treating an inflammatory condition, which method comprises determining the ability of a test compound to modulate the expression or activity of TRIB1. More particularly, the test compound that inhibits the expression or activity of TRIB1 is a candidate for treating an inflammatory condition.
Another subject of the invention is a method of treating an inflammatory condition, which method comprises modulating TRIB1 expression or activity.
In a preferred embodiment, this method comprises administering to a patient in need thereof a modulator of TRIB1 expression or activity selected from the group consisting of an antagonist of TRIB1, an antisense or a RNAi of TRIB1, and an antibody or a fragment or a derivative thereof specific to a TRIB1 polypeptide.
TRIB1 can act as prognostic marker, thereby enabling the early detection of chronic rejection before the apparition of lesions within the organ transplant or chronic inflammatory conditions.
The invention provides methods for diagnosis or prognosis, as well as treatments, of an inflammatory condition.
“Inflammatory condition” refers to any inflammatory disease or disorder known in the art whether of a chronic or acute nature, including, but not limited to, rheumatoid arthritis, inflammatory skin diseases, such as, psoriasis and eczema, restenosis, multiple sclerosis, surgical adhesion, tuberculosis, inflammatory lung diseases such as asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps and pulmonary fibrosis, inflammatory bowel disease, such as, Crohn's disease and ulcerative colitis, graft rejections, inflammatory diseases that affect or cause obstruction of a body passageway, such as, vasculitis, Wegener's granulomatosis and Kawasaki disease, systemic lupus erythematosus, inflammation of the eye, nose or throat, such as, neovascular diseases of the eye including neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroblasia, mascular degeneration, reduction of intraocular pressure, corneal neovascularization, such as, corneal infections, immunological processes, such as, graft rejection and Steven-Johnson's syndrome, alkali burns, trauma and inflammation (of any cause).
The inflammatory condition is more particularly a graft rejection, more particularly an allograft rejection. It can be a graft of any organ, in particular solid organs such as kidney, lung, liver, heart, etc. In a specific embodiment, the graft rejection is chronic rejection in clinical renal transplantation.
Most preferably, the condition is a chronic humoral rejection. Chronic humoral rejection (CHR) is characterized by transplant glomerulopathy accompanied by deposition of the complement split product C4d in graft biopsies and the presence of circulating anti-donor HLA antibody (Mauiyyedi S et al, 2001).
Other preferred inflammatory conditions include joint inflammation, multiple sclerosis, spondyloarthropathy or inflammatory bowel disease.
Tribbles homolog 1 (TRIB1) is a known gene which is a member of the Tribbles family that controls MAPK cascades (Kiss-Toth et al, 2004). SEQ ID NO:1 shows the cDNA sequence, and SEQ ID NO:2 shows the protein sequence, in humans. TRIB1 is referred to as a “marker” or “biomarker” in that it enables to diagnose or predict the development of an inflammatory condition.
The expression level of said marker is determined in a biological sample of a patient and compared to the expression level of said marker in a control subject.
The term “biological sample” means any biological sample derived from a patient, preferably a sample which contains nucleic acids. Examples of such samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma, saliva, urine, seminal fluid, tissue such as graft or diseased tissue biopsies etc. Peripheral blood or tissue biopsies are preferred, and mononuclear cells (PBMCs) are the preferred cells. Total RNAs can be easily extracted therefrom. The biological sample may be treated prior to its use, e.g. in order to render nucleic acids available. Techniques of cell or protein lysis, concentration or dilution of nucleic acids, are known by the skilled person.
The term “patient” refers to any subject to be tested. It is preferably a human, but includes any mammal, e.g. horses, cats, dogs, cattle, sheep, etc. The patient may be asymptomatic or may show early or advanced symptoms of an inflammatory condition.
The “diagnosis” means the identification of the condition or the assessment of the severity of the condition.
The term “prognosis” means the assessment of the outcome of the condition, i.e. to determine the evolution of the condition, and the risk of worsening.
The term “reference” generally refers to a control subject who is either healthy or is a subject who is not affected with an inflammatory condition but who may be optionally affected with a different condition. It may also be a statistic control value. In order to determine the evolution of the inflammatory condition, it may further be very useful to test a patient for the expression the marker, and to monitor the effect of a drug or the evolution of the condition, by testing him/her a second time, e.g. a few days later. In that case the results of the second test are compared with the results of the first test, and in general also with the results obtained with a “healthy” subject, i.e. a subject who is not affected with an inflammation or a subject who has not undergone grafting. The “reference” then refers either to the same test patient or to a “healthy subject”.
“A higher expression of TRIB1”, in the patient, compared to a reference value, means that the expression level of TRIB1 in the patient is significantly increased versus the reference value. The increase can be e.g. at least 5%, or at least 10%, or at least 20%, more preferably at least 30%.
Determination of the expression level of a gene can be performed by a variety of techniques.
More preferably, the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.
In a preferred embodiment, the expression level may be determined by determining the quantity of mRNA. Generally the expression level is a relative expression level.
Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Preferably quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e.g. avidin/biotin).
Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50% formamide, 5× or 6×SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
In a particular embodiment of the invention, the method for diagnosis or prognosis of an inflammatory condition comprises the steps of providing total RNAs extracted from PBMCs obtained from a blood sample or tissue biopsy of the patient, and subjecting the RNAs to amplification and hybridization to specific probes.
Preferably the expression level is determined by real-time quantitative or semi-quantitative RT-PCR.
In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
The expression level of the TRIB1 marker may also be determined by assaying the marker protein. Various methods for detecting and/or quantifying the expression of the marker proteins are described in greater detail below.
Such methods comprise contacting a biological sample with a binding partner capable of selectively interacting with a marker protein present in the sample. The binding partner is generally an antibody, that may be polyclonal or monoclonal, preferably monoclonal.
Procedures for raising polyclonal antibodies are well known. Typically, such antibodies can be raised by administering the marker protein subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. Each injected material contains adjuvants with or without pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988), that further discloses procedures for preparing monoclonal antibodies.
Laboratory methods for preparing monoclonal antibodies are well known. Monoclonal antibodies (Mabs) may be prepared by immunizing the purified marker protein into a mammal, e.g. a mouse, rat, rabbit, goat, human. The antibody-producing cells in the immunized mammal are isolated and fused with myeloma or heteromyeloma cells to produce hybrid cells (hybridoma). The hybridoma cells producing the monoclonal antibodies are utilized as a source of the desired monoclonal antibody. Also contemplated is the use of Mabs produced by an expressing nucleic acid cloned from a hybridoma.
The presence of the marker protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
The aforementioned assays generally involve separation of unbound marker protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
Typically, a solid support is first reacted with a solid phase component (e.g., one or more anti-TRIB1 antibody) under suitable binding conditions such that the component is sufficiently immobilized to the support according to methods well known to those skilled in the art. After reacting the solid support with the solid phase component, any non-immobilized solid-phase components are removed from the support by washing, and the support-bound component is then contacted with a biological sample suspected of containing ligand moieties (e.g., TRIB1 molecules toward the immobilized antibodies) under suitable binding conditions. After washing to remove any non-bound ligand, a secondary binder moiety is added under suitable binding conditions, wherein the secondary binder is capable of associating selectively with the bound ligand. The presence of the secondary binder can then be detected using techniques well known in the art.
The present invention also provides novel target and methods for the screening of drug candidates or leads. The methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems, in animals, etc.
The invention thus provides a method for screening compounds useful for treating an inflammatory condition, which method comprises determining the ability of a test compound to modulate the expression or activity of TRIB1. Such method is typically performed in vitro.
In a particular embodiment of the methods of screening, the modulation is an inhibition.
Such tests of activity of the protein may include testing the ability of TRIB1 to phosphorylate and/or activate MAPK (see Kiss-Toth et al, 2004).
The screening methods of the invention may comprise preliminary steps of contacting in vitro a test compound with a TRIB1 gene or polypeptide and determining the ability of said test compound to bind said TRIB1 gene or polypeptide. Binding to said gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to an inflammatory condition in a subject.
In a further particular embodiment, the method comprises contacting a recombinant host cell expressing a TRIB1 polypeptide according to the present invention with a test compound, and determining the ability of said test compound to bind said TRIB1 and to modulate the activity of TRIB1 polypeptide. The determination of binding may be performed by various techniques, such as by labeling of the test compound, by competition with a labeled reference ligand, etc.
The screening method of the invention may also comprise contacting in vitro a test compound with a TRIB1 polypeptide and determining the ability of said test compound to modulate the activity of said TRIB1 polypeptide.
A further object of this invention resides in a method of screening, said method comprising contacting in vitro a test compound with a TRIB1 gene and determining the ability of said test compound to modulate the expression of said TRIB1 gene.
In an other embodiment, this invention relates to a method of screening, said method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a TRIB1 gene promoter, and selecting the test compounds that modulate expression of the reporter gene.
The above screening assays may be performed in any suitable device, such as plates, tubes, dishes, flasks, etc. Typically, the assay is performed in multi-wells plates. Several test compounds can be assayed in parallel. Furthermore, the test compound may be of various origin, nature and composition. It may be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc., in isolated or in mixture with other substances. The compounds may be all or part of a combinatorial library of products, for instance.
It is further described a method of treating an inflammatory condition in a patient in need thereof, which method comprises modulating TRIB1 expression or activity in said patient.
The invention also relates to the use of a modulator of TRIB1 expression or activity for the manufacture of a medicament intended for the treatment of an inflammatory condition in a patient in need thereof.
In the context of the invention, the term “treating” or “treatment”, as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the condition to which such term applies, or one or more symptoms of condition.
According to the invention, the term “patient” or “patient in need thereof”, is intended for a human or non-human mammal affected or likely to be affected with the inflammatory condition.
The term “modulator of TRIB1 expression or activity” means any compound or substance that is capable of regulating, more particularly inhibiting, i.e. diminishing, the expression of TRIB1 or the activity of the protein. Such modulators include the compounds identified by means of the screening methods described above. It can be an antagonist of TRIB1, an antisense or a RNAi of TRIB1, an antibody or a fragment or a derivative thereof specific to a TRIB1 polypeptide.
Such modulators may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.
The below example illustrates the invention without limiting its scope.
All patients are informed of the study protocol and give informed consent. All animals are treated in accordance with standard European and Institutional Guidelines.
The inventors performed a meta-analysis by comparing a candidate gene list with those published in the literature by other groups having performed microarray studies in the context of renal transplantation (Flechner et al 2004a; Scherer et al 2003; Akalin et al 2001; Flechner et al 2004b; Donauer et al 2003). The published studies concern only rejection or stable function (not tolerance) and primarily involve graft biopsies and not blood. The aim of the present approach was to identify gene candidates present in the blood in the present study and also present in the biopsies in the studies in the literature. To make such a meta-analysis possible, more than 3000 genes have been first converted into a single identifier, the so-called <<GENE ID>>, using the gene accession conversion tool available at the NIH website: http://david.abcc.ncifcrf.gov/.
Establishing and Screening of a “Bank” of cDNA Samples from Several Cohorts of Renal Transplant Patients and Healthy Individuals
In order to further ascertain the relevance of the chosen gene candidates to chronic rejection, a bank of cDNA samples from blood was established to compare the expression of the genes in chronic rejection, not only to tolerance, but also to other outcomes (stable function, renal failure, chronic rejection without immunosuppression) and also to age-matched healthy individuals. For this purpose approximately 50 patients with the relevant kidney function and healthy individuals have been recruited, blood samples collected on EDTA tubes and RNA and cDNA prepared from PBMC (Ficoll PBMC preparation and TRIZOL RNA extraction). In parallel, cDNA was prepared from RNA extracted from graft biopsies taken from renal transplant patients with normal graft histology or different diagnoses of late graft failure such as chronic allograft nephropathy, calcineurin inhibitor toxicity or chronic humoral rejection. Expression of the chosen molecules have been measured by quantitative RT-PCR using TaqMan primer and probe sets. Cell sorting was performed by multicolor flow cytometry on the PBMC of 16 transplant patients and healthy individuals to obtain RNA and cDNA from monocytes, CD4 T cells, CD8 T cells, NK cells and B cells (using antibodies labeled with different fluorochromes enabling separation from a single blood sample using FACSAria).
The meta-analysis led to the identification of 14 genes common between at least two of the published studies. An additional gene, Tribbles homolog 1 (TRIB1), was significantly upregulated in CR biopsies in a published microarray study (Flechner et al, 2004a) as well as in the blood of CR patients in the present study.
Expression of TRIB1 was measured by qPCR in the PBMC and peripheral blood cell subtypes of several cohorts of renal transplant patients and its expression correlated to renal histology (Banff and C4d staining) and function. The relevance of this gene was additionally studied in several rat models of allotransplantation and its function analyzed in the relevant cell subtype from healthy volunteers.
TRIB1 was significantly up-regulated in the PBMC of CR patients compared to those with stable renal function (
Interestingly, TRIB1 was also significantly upregulated in heart allografts presenting CR vs. tolerance in the rat (
The present study is thus the first one to show that TRIB1 is significantly upregulated in the peripheral blood of patients with CR. The inventors herein show that TRIB1 is a peripheral blood marker of CR.
Given the high expression of TRIB1 by monocytes and B cells, the blood formulae of the patients were analyzed in more detail. No significant correlation was found between total monocyte numbers and TRIB1 (p=0.23), indicating that the differences in TRIB1 observed in total PBMC do not simply reflect monocyte numbers.
Finally, because CHR is likely to be associated with chronic activation of endothelial cells (EC), the inventors studied TRIB1 mRNA accumulation in primary human renal artery-derived EC. The inventors showed that TRIB1 is also a marker of activated EC, being upregulated upon activation with the pro-inflammatory cytokine TNFa over 6, 12 and 24 hrs.
Thus, TRIB1 is a marker of antigen presenting cells where it is regulated differentially according to both cell and activation type.
Monocytes/macrophages and dendritic cells play a pathogenic role in a number of autoimmune inflammatory diseases including those of the joints and of the brain. Since they found TRIB1 to be expressed primarily by monocytes/dendritic cells, the inventors additionally analyzed TRIB1 expression in the PBMC of patients suffering from two such diseases: Spondyloarthropathies (SpA) and Multiple Sclerosis (MS).
TRIB1 was shown to be a marker of chronic inflammation since it is significantly upregulated in the blood of patients suffering from Spondyarthropathy and in a subgroup of patients suffering from relapsing Multiple Sclerosis (See Examples 2, 3 below).
SpA is a group of chronic inflammatory arthritides classified according to common features of peripheral and spinal arthritis.
To measure TRIB1 in the peripheral blood of patients with SpA compared to healthy individuals, PBMC were prepared from the peripheral blood of 10 patients with clinically defined SpA, and 10 healthy age-matched controls. The PBMC were subsequently subjected to RNA extraction and TRIB1 expression was measured by quantitative RT-PCR using commercialized TaqMan primers and probes. TRIB1 was shown to be significantly upregulated in the blood of patients suffering from Spondylarthropathy (
Multiple sclerosis (MS) is an inflammatory demyelinating disease characterized by immune abnormalities in the central nervous system (CNS) as well as in the periphery.
To measure TRIB1 in the peripheral blood of patients with MS compared to healthy individuals, PBMC were prepared from the peripheral blood of 6 patients with clinically defined MS during relapse, and 5 healthy age-matched controls. RNA extraction and TRIB1 expression was measured using commercialized TaqMan primers and probes.
TRIB1 was shown to be significantly upregulated in the blood of a sub-group of patients suffering from relapsing Multiple Sclerosis (
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP07/55100 | 5/25/2007 | WO | 00 | 12/3/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60808122 | May 2006 | US |
