HK1166368B - Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers - Google Patents

Description

Clinical diagnosis of liver fibrosis using a novel panel of low abundance human plasma protein biomarkers

Cross Reference to Related Applications

This application claims priority from U.S. provisional application No. 61/178,334 filed on 5,14, 2009.

Technical Field

The present application relates generally to methods of diagnosing liver fibrosis or cirrhosis using a panel of antibodies targeting a panel of novel hepatic scarring biomarkers that are very low in abundance. These novel proteins are also useful as biomarkers for hepatitis, hepatocellular carcinoma (HCC), and drug targets for hepatic scarring, hepatitis, and HCC.

Background

Hepatic fibrosis: liver fibrosis is a wound healing response characterized by excessive accumulation of scar tissue (i.e., extracellular matrix) in the liver. The normal structural elements of the tissue are replaced by an excess of non-functional scar tissue. Biopsy by puncturing the liver is the primary means of diagnosing and assessing fibrosis, but there is ample evidence that this technique has many limitations and disadvantages, including patient discomfort, pain, bleeding, and death in a very few cases. Furthermore, if fibrosis is not uniform throughout the liver, the biopsy may be unreliable. Liver fibrosis can be caused by a variety of factors including alcohol and viruses.

Liver cirrhosis: cirrhosis is the most severe form of scarring of the liver, and, unlike liver fibrosis, is generally considered to be irreversible and have nodules. In the uk, cirrhosis causes more than 6000 deaths per year, in the us approximately 27,000 deaths per year, and cirrhosis is the ninth leading cause of death (MacSween et al, (2002), pathoftheiver, fourth edition, churchlilllivingstone). Cirrhosis is a major risk factor for HCC, and the only treatment at this stage of liver cancer is liver transplantation. In the case of virus-induced liver cancer, liver scarring and HCC may recur after transplantation. It is necessary to diagnose fibrosis at an early stage of reversible hepatic scarring so that irreversible cirrhosis can be prevented.

Hepatitis c virus: about 1.7 million of the world's population (i.e., 3% of the world's population (see, e.g., WHO, j. viral. hepat.1999; 6: 35-47)) is infected with hepatitis c virus (HCV, HepC), and about four million of the united states are infected with hepatitis c virus. HCV is the major cause of liver fibrosis and cirrhosis. About 80% of individuals acutely infected with HCV become chronically infected. Thus, HCV is a major cause of chronic hepatitis. Once chronically infected with the virus, the virus is almost never cleared without treatment. In very few cases, HCV infection causes clinically acute disease and even liver failure. Chronic HCV infection can vary significantly among individuals, some of whom have clinically insignificant or the mildest degree of liver disease and never develop complications, while others have clinically significant chronic hepatitis and continue to progress to liver fibrosis and cirrhosis. About 20% of HCV-bearing individuals who develop cirrhosis will develop end-stage liver disease and an increased risk of developing primary liver cancer.

There is a need for improved methods for diagnosing liver fibrosis and cirrhosis in a patient.

Disclosure of Invention

The present invention provides methods for detecting fibrosis and cirrhosis.

In one embodiment, the present invention provides a method of detecting fibrosis and cirrhosis of the liver, the method comprising: (a) determining the level of an HF-ASSOCIATED (HF-ASSOCIATED) polypeptide in a biological sample taken from the patient; and (b) comparing said level (a) to a control level of said HF-related polypeptide to determine a positive or negative diagnosis of said fibrosis. These biomarkers may be applied to any disease exhibiting fibrosis, such as liver fibrosis, kidney fibrosis, heart fibrosis (cardiac fibrosis), skin fibrosis, pancreatic fibrosis, and the like, but in particular embodiments the fibrosis is liver fibrosis. The invention selects the polypeptides in the following groups: 14-3-3 protein ζ/, adiponectin, alpha-albumin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein C-III, apolipoprotein E, C4 b-binding protein β chain, intact/cleaved complement C3dg, corticosteroid-binding globulin, fibrinogen γ chain, β -binding globin at ph5.46 to ph5.49, binding globin-related protein, blood bindin, immunoglobulin J chain, leucine-rich alpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-binding protein 4, serum paraoxonase/aromatase 1, sex hormone-binding globulin, and zinc-alpha-2-glycoprotein. These biomarkers can be used in combination with the polypeptides of international publication WO/2008/031051, including inter-alpha-trypsin inhibitor heavy chain H4 fragment, alpha 1 antichymotrypsin, apolipoprotein L1, preprotein, albumin, CD5 antigen-like protein isoforms, beta 2 glycoprotein I, alpha 2 macroglobulin, and immunoglobulin components, globin-binding alpha 1, alpha 2, and beta chains, complement components (C3, C4, and factor H-related protein 1), adiponectin, ApoE, prothrombin, clusterin, and angiotensinogen. In other embodiments, fibrosis comprises differential regulation of HF-related polypeptides. In other embodiments, the sample is taken from serum or plasma.

In another embodiment, the invention provides a method of detecting an HF-related polypeptide, the method comprising: a) isolating a biological sample from a patient suffering from fibrosis or cirrhosis, b) isolating a biological sample from a patient not suffering from fibrosis, c) analyzing the samples from a) and b) with 2D-PAGE, and D) comparing the 2D-PAGE results to identify polypeptides differentially expressed between patients suffering from and not suffering from fibrosis or cirrhosis. Using 2D-PAGE, biomarkers can be detected using a wide range of pH commonly used, e.g., pH3 to pH10, pH3 to pH11, or pH4 to pH7. Very low abundance biomarkers can be detected with any narrow pH range from pH3 to pH11, such as but not limited to: pH3 to pH5.6, pH3 to pH6, pH3.9 to pH5.1, pH4.7 to pH5.9, pH5 to pH8, pH5.3 to pH6.5, pH5.5 to pH6.7, pH6 to pH11, pH6.2 to pH7.5, pH6.3 to pH8.3, pH7 to pH10, pH7 to pH11, pH3.5 to pH4.5, pH3 to pH7, and pH6 to pH 9. 2D-PAGE gels covering these narrow pH ranges can also be used to identify novel biomarkers and drug targets in other diseases.

In another embodiment, the present invention provides a method of grading the severity of fibrosis, the method comprising: a) determining the level of at least one HF-related polypeptide in a biological sample taken from the patient; and b) comparing the level of the HF-related polypeptide in the patient's biological sample with a predetermined level of the HF-related polypeptide in a patient population without fibrosis to cirrhosis.

In another embodiment, the invention provides a kit useful in prognosis of fibrosis in an untreated individual and during treatment, the kit comprising an HF-related agent, wherein the agent specifically detects an HF-related polypeptide. In a particular embodiment, the agent is an antibody or functional equivalent thereof that binds to an HF-related polypeptide. These antibodies can be used to perform immunoassays, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, western blot, transmission turbidimetry, nephelometry, and the like. HF-related polypeptides can also be quantified using non-antibody approaches, such as, but not limited to, multiple reaction monitoring using mass spectrometry. The kit may further comprise at least one target that specifically detects another gene or gene product for use as a prognostic indicator.

In another embodiment, the present invention provides a method of determining the prognosis of fibrosis, the method comprising: (a) determining the level of an HF-related polypeptide in a biological sample taken from the patient; and (b) comparing said level of (a) to a control level of said HF-related polypeptide to determine a positive or negative diagnosis of said fibrosis.

Drawings

Several of the figures 1 to 4 show the changes observed in the expression of the major novel biomarkers. Each figure shows an enlarged region of a 2D-PAGE gel in which the corresponding positions of the identified proteins are circled. Representative plasma gel images of healthy individuals and patients with cirrhosis are shown.

Figure 1 shows that the lipid transfer inhibitor protein appears in normal plasma but is reduced in plasma of patients with cirrhosis.

FIG. 2 shows that zinc-alpha-2-glycoprotein appears in normal plasma but is reduced in plasma of patients with liver cirrhosis.

FIG. 3 shows a decrease in the characteristic points (features) of beta-haptoglobin at pH5.46 to pH5.49. Upper panel-evenly distributed array of beta-haptoglobin spots, which showed no significant difference in normal plasma and liver cirrhosis patient plasma. Lower panel-magnified image of beta-globin spots observed at about ph5.46 to ph5.49, showing that the beta-globin spots observed at about ph5.46 to ph5.49 appear in normal plasma but are reduced in plasma of cirrhosis patients.

Figure 4 shows a reduction in lysis of complement C3 in cirrhosis. Upper panel-shows complement C3dg not present in normal plasma but present in plasma of cirrhosis patients. Lower panel-shows that a fragment of complement C3 a chain preceding the thioester site of complement C3 a chain appears in normal plasma but not in the plasma of cirrhosis patients.

Detailed Description

The following description summarizes the invention summarized above. However, the invention is not limited to the particular methodology, protocols, cell lines, animal species, constructs, reagents described and as such may vary. Likewise, the terminology used herein describes particular embodiments only and is not intended to limit the scope of the present invention.

The present inventors found that various proteins are differentially expressed in human plasma samples of HCV-induced cirrhosis patients when compared to healthy individuals. This finding was achieved by comparing these serum samples using a technique of bi-directional separation of proteins in a gel matrix to give discrete protein spots. The method uses a narrow range of pH values from pH3 to pH5.6 for the immobilization of a pH gradient gel that is different from the immobilized pH gradient gel used in WO/2008/031051 from pH3 to pH 10.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.

For the purposes of description and disclosure, all publications and patents mentioned herein are incorporated by reference, e.g., the constructs and methodologies described in the publications that can be used in connection with the teachings of the present invention. The publications discussed above and their entire contents were provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

a. Definition of

For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below.

The singular forms ("a", "an" and "the") include the plural reference unless the context clearly dictates otherwise.

"2D-PAGE" refers to two-dimensional polyacrylamide gel electrophoresis.

"ELISA" refers to enzyme-linked immunosorbent assay.

"HCC" refers to hepatocellular carcinoma.

"HCV" refers to hepatitis C virus.

"HF" refers to liver fibrosis.

"kDa" means kilodaltons.

"PBS" refers to phosphate buffered saline.

"PBS-T" refers to PBS solution containing Tween.

"biological samples" include a variety of sample types taken from organisms that can be used in diagnostic or monitoring assays. The term encompasses blood and other liquid samples of biological origin, solid tissue samples (e.g., biopsy samples), or tissue cultures or cells derived therefrom and progeny thereof. In addition, the term may include circulating tumor cells or other cells. The term encompasses, inter alia, clinical samples and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluid, and tissue samples. The term also encompasses samples that are processed in any way after they have been obtained, e.g., by treatment with reagents, solubilization, enrichment of certain components.

"biomolecule sequence" or "sequence" refers to all or part of a polynucleotide or polypeptide sequence.

"BLAST" refers to the basic local alignment search tool (BasiclocalAlignmentSearchTool), a technique for detecting non-spaced subsequences that match a given query sequence. "BLASTP" is a BLAST program that compares amino acid query sequences to protein sequence databases. "BLASTX" is a BLAST program that compares the six-box conceptual translation products of a nucleic acid query sequence (double-stranded) to a protein sequence database.

"cancer," "neoplasm," and "tumor," as used interchangeably herein, refer to a cell or tissue exhibiting an abnormal growth phenotype characterized by a significant deregulation of cell proliferation. The methods and compositions of the invention find particular application in pre-cancerous (i.e., benign), malignant, pre-metastatic, and non-metastatic cells.

By "fibrosis is characterized by differential regulation of HF-related polypeptides" is meant a subject having tissue that exhibits scarring, and in which the HF-related proteins have differential expression.

"fibrotic phenotype" refers to a variety of biological phenomena characterized by fibrotic cells. This phenomenon may vary with the type of fibrosis, but the fibrotic phenotype is often identified by abnormalities in scar tissue formation.

By "cell type" is meant a cell from a given source (e.g., a tissue or organ) or in a given state of differentiation, or a cell associated with a given pathology or genetic makeup.

"complementary" refers to the topological compatibility or pairing of the probe molecule with its target interaction surface. The target and its probe may be described as complementary, and the features of the contact surface are complementary to each other.

The term "detectable" refers to the pattern of polypeptide expression observable using techniques described in this application and well known to those skilled in the art. For example, polypeptide expression can be "detected" by standard techniques, including immunoassays such as western blotting.

"diagnosis" and "diagnosing" generally includes determining a subject's susceptibility to a disease or disorder, determining whether a subject is currently suffering from a disease or disorder, prognosing a subject suffering from a disease or disorder (e.g., identification of pre-metastatic or metastatic cancer status or fibrosis), and diagnostic evaluation (therametrics) (e.g., monitoring a subject's symptoms to provide information about the effect or efficacy of a treatment).

"differential expression" refers to quantitative and qualitative differences in the temporal and tissue expression patterns of genes. For example, a differentially expressed gene may have its expression activated or not activated at all in the normal and disease states. Such qualitatively regulated genes may exhibit an expression pattern in a given tissue, cell type or serum/plasma of the subject that is detectable in a control state or a disease state, or detectable in both a control state and a disease state but with different expression. As used herein, "differentially expressed protein" refers to an amino acid sequence that uniquely identifies the differentially expressed protein, thereby correlating the detection of the differentially expressed protein in the sample with the presence of the differentially expressed protein in the sample.

"expression" generally refers to the process by which a polynucleotide sequence undergoes successful transcription and translation, thereby expressing a detectable level of an amino acid sequence or protein. In some contexts, expression refers to the production of mRNA. In other contexts, expression refers to the production of a protein or fragment thereof. The fragments may be produced by enzymatic cleavage or by biological processes characteristic of the normal state or disease state.

An "expression product" or "gene product" is a biological molecule, such as a protein or mRNA, that is produced when a gene in the body is transcribed or translated or post-translationally modified.

"protein fragment" refers to a portion of a protein. For example, a protein fragment can comprise a polypeptide obtained by digestion of a full-length protein isolated from cultured cells. In one embodiment, a protein fragment comprises at least about 6 amino acids. In another embodiment, the fragment comprises at least about 10 amino acids. In yet another embodiment, a protein fragment comprises at least about 16 amino acids.

In the context of the present application, the term "functional equivalent" refers to a protein having functional or structural properties substantially similar to all or part of a native HF-related protein. The term "functional equivalent" is intended to include "fragments", "mutants", "derivatives", "alleles", "hybrids", "variants", "analogues" or "chemical derivatives" of the native HF-related protein.

In the context of immunoglobulins, the term "functional equivalent" refers to an immunoglobulin molecule that exhibits substantially similar immunological binding characteristics as the parent immunoglobulin. "immunological binding properties" refer to the type of noncovalent interaction that occurs between an immunoglobulin molecule and a specific antigen of the immunoglobulin. Indeed, for example, a functional equivalent of a monoclonal antibody immunoglobulin may inhibit the binding of a parent monoclonal antibody to its antigen. Functional equivalents may comprise F (ab') 2 fragments, F (ab) molecules, Fv fragments, single chain variable fragments (scFv) of phage display technology, single domain antibodies, chimeric antibodies, and the like, so long as the immunoglobulin exhibits the properties of a parent immunoglobulin.

The term "fusion protein" refers to a protein composed of two or more polypeptides that are joined by peptide bonds from their respective amino and carboxy termini to form a single contiguous polypeptide, although typically the polypeptides are not joined in the native state. It will be appreciated that two or more polypeptide components may be linked directly or indirectly via a peptide linker/spacer.

"Gene" refers to a polynucleotide sequence comprising the regulatory and coding sequences required for production of a polypeptide or precursor. A polypeptide can be encoded by a full-length coding sequence or by any portion of a coding sequence. A gene may constitute an uninterrupted coding sequence or a gene may comprise one or more introns bounded by suitable splice sites. In addition, a gene may comprise one or more modifications in the coding or untranslated region that may affect the biological activity or chemical structure of the expression product, the rate of expression, or the manner in which expression is regulated. Such modifications include, but are not limited to: mutations, insertions, deletions and substitutions of one or more nucleotides. In this regard, such modified genes may be referred to as "variants" of the "native" gene.

"Gene expression" refers to the process by which a polynucleotide sequence undergoes successful transcription and translation, thereby expressing a detectable level of the nucleotide sequence.

The term "homology" as used herein refers to the degree of complementarity. Partial homology or complete homology (i.e., identity) may exist. A partially complementary sequence is a sequence that at least partially inhibits hybridization of the same sequence to a target polynucleotide; partially complementary sequences are also referred to using the functional term "substantially homologous". Inhibition of hybridization of a perfectly complementary sequence to a target sequence under low stringency conditions can be detected using hybridization assays (southern blot) or northern blot (northern blot), solution phase hybridization, and the like). Under low stringency conditions, a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., hybridization) of a completely homologous sequence or probe to the target sequence. This is not to say that low stringency conditions are those which allow non-specific binding; low stringency conditions require that the binding of two sequences to each other be a specific (i.e., selective) interaction. The absence of non-specific binding can be tested by using a second target sequence that even lacks a partial degree of complementarity (e.g., less than 30% identity); without non-specific binding, the probe will not hybridize to a second non-complementary target sequence.

The terms "individual", "subject", "host" and "patient" are used interchangeably herein to refer to any mammalian subject in need of diagnosis, treatment or therapy. In a preferred embodiment, the individual, subject, host or patient is a human. Other subjects included woodchucks and ducks, which were known to develop HCC and hepatitis. Other subjects may include, but are not limited to: cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates, and mice.

"isolated" refers to a polynucleotide, polypeptide, immunoglobulin or host cell that is in an environment that is different from the environment in which the polynucleotide, polypeptide, immunoglobulin or host cell is naturally produced.

"Label" refers to an agent that is capable of providing a detectable signal directly or by interacting with one or more additional members of a signal producing system to provide a detectable signal. Labels that are directly detectable and that can be used in the present invention include fluorescent labels. Specific fluorophores include fluorescein, rhodamine, BODIPY, cyanine dyes, and the like. The present invention also contemplates the use of radioisotopes (e.g., as³⁵S、³²P、³H, etc.) as a marker. Chroma markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polyethylene, latex) beads may also be used. See, for example, U.S. Pat. nos. 4,366,241, 4,277,437, 4,275,149, 3,996,345, 3,939,350, 3,850,752, and 3,817,837.

The term "normal physiological conditions" refers to conditions typical of living organisms or cells. Although some organs or organisms provide extreme conditions, the intra-and intracellular environment typically varies around pH7 (i.e., from pH6.5 to pH7.5), contains water as the primary solvent, and is present at temperature conditions above 0 ℃ and below 50 ℃. The different salt concentrations depend on the organ, organism, cell or cell compartment used as reference.

"Polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length (either ribonucleotides or deoxyribonucleotides). Thus, these terms include, but are not limited to: single, double or multiple stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. These terms also include, but are not limited to: mRNA or cDNA comprising intron sequences. The backbone of the polynucleotide may comprise sugars and phosphate groups (as may typically be found in RNA or DNA) or modified or substituted sugar or phosphate groups. Alternatively, the polynucleotide scaffold may comprise a polymer of synthetic subunits such as phosphoramidites, and thus the polynucleotide scaffold may be a phosphoramidate or mixed phosphoramidate-phosphate diester oligomer of oligodeoxynucleotides. Polynucleotides may comprise modified nucleotides (e.g., methylated nucleotides and nucleotide analogs), uracils, other sugars and linking groups such as fluororiboses and thioesters, and nucleotide branches. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by binding to a labeling element. Other types of modifications included within this definition are capping, substitution of one or more of the naturally occurring nucleotides with an analog, and methods of introducing the polynucleotide to a protein, metal ion, labeling element, other polynucleotide, or solid support. The term "polynucleotide" also includes peptide nucleic acids. The polynucleotide may further comprise genomic DNA, cDNA or DNA-RNA hybrids.

"polypeptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include translated, untranslated, chemically modified, biochemically modified, and derivatized amino acids. The polypeptide or protein may be naturally occurring, recombinant or synthetic or any combination of these. In addition, the polypeptide or protein may comprise a fragment of a naturally occurring protein or peptide. The polypeptide or protein may be a single molecule or may be a multi-molecule complex. In addition, such polypeptides or proteins may have a modified peptide backbone. These terms include fusion proteins, which include fusion proteins comprising heterologous amino acid sequences, fusion proteins comprising heterologous and homologous leader sequences, with or without an N-terminal methionine residue, immunolabeling proteins, and the like.

"predisposition to a disease or disorder" refers to the susceptibility of an individual to the disease or disorder. For example, susceptible individuals are statistically more likely to develop cancer or fibrosis than normal/wild-type individuals.

The terms "prognosis (" prognosis "and" prognosie ")" refer to a behavior or technique that predicts the course of a disease. In addition, the term refers to the prospect of survival and recovery from disease as expected by the general course of disease or as shown by the specific characteristics of an individual case. In addition, the term refers to the act or technique of identifying the disease based on its signs and symptoms.

The term "prognostic indicator" or "indicator" refers to anything that can be, or is related to, the basis or foundation of prognosis. These terms further refer to any basis or basis for differential diagnosis, including the results of the tests and characterization of gene expression as described herein, as well as distinguishing a disease or disorder from other diseases or disorders exhibiting similar symptoms. In addition, the term "index" or "prognostic indicator" refers to any basis or basis, including the results of a test and the characteristics of gene expression as described herein, which can be used to distinguish between the likely course of a malignant disease.

"protein capture agent" refers to a molecule or multi-molecule complex that can bind a protein to itself. In one embodiment, the protein capture agents bind their binding partners in a substantially specific manner. In one embodiment, the protein capture agent may exhibit a dissociation constant (KD) of less than about 10 "6. The protein capture agent may comprise a biomolecule such as a protein or a polynucleotide. The biomolecule may further comprise a naturally occurring, recombinant or synthetic biomolecule. Examples of protein capture agents include immunoglobulins, antigens, receptors, or other proteins or portions or fragments thereof. Furthermore, it should be understood that a protein capture agent is not limited to agents that interact with their binding partners only through non-covalent interactions. Conversely, protein capture agents may also be covalently linked to the protein to which they are bound. For example, a protein capture agent can be photocrosslinked with its binding partner upon binding.

"sequence identity" refers to the degree of similarity or complementarity. There may be partial consistency or complete consistency. A partially complementary sequence is a sequence that at least partially inhibits hybridization of the same sequence to a target polynucleotide; it is also intended that the functional term "substantially consistent" be used. Inhibition of hybridization of a fully complementary sequence to a target sequence under low stringency conditions can be detected using hybridization assays (southern or northern blots, solution phase hybridization, etc.). Under low stringency conditions, a substantially identical sequence or probe will compete for and inhibit the binding (i.e., hybridization) of a completely identical sequence or probe to the target sequence. This is not to say that low stringency conditions are those which allow non-specific binding; low stringency conditions require that the binding of two sequences to each other be a specific (i.e., selective) interaction. The absence of non-specific binding can be tested using a second target sequence that even lacks a partial degree of complementarity (e.g., less than 30% identity), and without non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

Another method of observing sequence identity for two nucleic acid or polypeptide sequences herein involves reference to the same residues in the two sequences when aligned for maximum correspondence within a particular region. As used herein, percent sequence identity refers to a value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentages are calculated by: the number of identical nucleobase sites present in both sequences is determined to yield the number of matched sites, the number of matched sites is divided by the total number of sites in the comparison window, and the result is multiplied by 100 to yield the percentage of sequence identity.

"stringent conditions" refers to conditions under which a probe can hybridize to its target polynucleotide sequence but not to other sequences. Stringent conditions are sequence dependent (e.g., longer sequences hybridize specifically at higher temperatures). Typically, stringent conditions are selected to be about 5 ℃ below the thermal melting point (Tm) of the particular sequence under defined ionic strength and pH conditions. The Tm is the temperature (under defined conditions of ionic strength, pH, and polynucleotide concentration) at equilibrium at which 50% of a probe complementary to the target hybridizes to the target sequence. Typically, stringent conditions are conditions of about ph7.0 to about ph8.3 for short probes (e.g., 10 to 50 nucleotides), a salt concentration of at least about 0.01M to about 1.0M sodium ion concentration (or other salt), and a temperature of at least about 30 ℃. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

"substantially purified" refers to a compound that is isolated from the natural environment and is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 99.99% or more free of other components with which it is naturally associated.

"target protein" refers to a polypeptide that specifically hybridizes to or binds to a protein capture agent, which is typically derived from a biological sample. The presence or absence of the target protein may be detected or the target protein may be quantified. The target protein has a structure that is recognized by a corresponding protein capture agent directed to the target. The target protein or amino acid may also refer to a particular substructure of a larger protein to which the protein capture agent is directed or the entire structure (e.g., a gene or mRNA) whose expression level is desired to be detected.

The terms "treating", and the like "refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or a symptom thereof, and/or the effect may be therapeutic in terms of a partial or complete stabilization or partial or complete cure of the disease and/or adverse effects caused by the disease. "treatment" includes any treatment of a disease in a mammal (particularly a human) and includes: (a) preventing the development of a disease or condition in a subject who may be susceptible to infection with the disease or condition but has not yet been diagnosed as having the disease or condition; (b) inhibiting the disease symptoms, i.e., arresting their development; or (c) alleviating the symptoms of the disease, i.e., restoring the disease or symptoms.

HF-related polypeptides

In one aspect, the invention relates to "HF-related polypeptides" including polypeptides whose differential expression is associated with liver fibrosis. HF-related polypeptides also include variants of naturally occurring proteins, wherein such variants are the same as or substantially similar to the naturally occurring protein. In general, the sequence of a variant polypeptide has at least about 80% sequence identity, typically at least about 90% sequence identity, and more typically at least about 98% sequence identity with an HF-related polypeptide described herein, as determined by BLAST. Variant polypeptides may be naturally or non-naturally glycosylated.

In general, variants of an HF-related polypeptide described herein have a sequence identity of greater than at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or greater than at least about 99.99%, as determined using methods well known in the art (e.g., BLAST).

In one embodiment, the variant HF-related polypeptide may be a mutant polypeptide. Mutations in HF-related polypeptides can result from, but are not limited to, amino acid substitutions, additions or deletions. Amino acid substitutions may be conservative amino acid substitutions or substitutions that remove non-essential amino acids. In general, conservative amino acid substitutions are those that preserve the conventional charge, hydrophobicity, hydrophilicity, and/or steric effects of the substituted amino acid.

In some mutated HF-related polypeptides, amino acids may be substituted to alter phosphorylation sites or acetylation sites.

Importantly, variant polypeptides can be designed to retain or have increased biological activity of a particular region of a protein (e.g., a functional domain and/or a region associated with a consensus sequence in which the polypeptide is a member of a protein family). The choice of amino acid changes used to generate the variant can be based on the accessibility of the amino acids (internal versus external), the thermostability of the variant polypeptide, the intended glycosylation site, the intended disulfide bridge, the intended metal binding site, and the intended substitution within the proline loop. Cysteine-depleting muteins can be produced according to the disclosure of U.S. Pat. No. 4,959,314.

Variants also include fragments of the HF-related polypeptides disclosed herein, particularly biologically active fragments and fragments corresponding to functional domains. Typically, the target fragment will be at least about 10aa to at least about 15aa long, usually at least about 50aa long, and may be as long as 300aa or longer. Protein variants described herein are encoded by polynucleotides within the scope of the present invention.

The HF-related polypeptides of the invention are provided in a non-naturally occurring environment, e.g., isolated from the environment in which they are naturally occurring. In some embodiments, the HF-related protein is present in a substantially purified form.

Hf-related agents: modulators and binding partners

In another aspect, the invention provides "HF-related agents," which refer to a class of molecules comprised of "HF-related polypeptide binding partners.

An HF-related polypeptide binding partner is a molecule that binds to an HF-related polypeptide. Exemplary polypeptide binding partners are immunoglobulins. The binding partner may, but need not, modulate the biological activity of the HF-related polypeptide.

d. Immunoglobulins

HF-related agents for identifying HF-related proteins include immunoglobulins and functional equivalents of immunoglobulins that specifically bind to HF-related polypeptides. The terms "immunoglobulin" and "antibody" are used interchangeably and are used herein in their broadest sense. Thus, "immunoglobulin" and "antibody" include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. In one embodiment, the subject immunoglobulin comprises at least one human constant domain. In another embodiment, the HF-related agent immunoglobulin comprises a constant domain that exhibits at least about 90% to 95% sequence identity to a human constant domain, but retains human effector function. The immunoglobulin HF-related agent or functional equivalent thereof can be human, chimeric, humanized, murine, CDR-grafted, phage-displayed, bacterial-displayed, yeast-displayed, transgenic mouse-generated, mutagenized, and randomized.

i. General antibodies

The terms "antibody" and "immunoglobulin" include fully assembled antibodies and antibody fragments (e.g., Fab ', F' (ab)₂Fv, single chain antibody, diabody), including recombinant antibodies and antibody fragments. Preferably, the immunoglobulin or antibody is chimeric, human or humanized.

The variable domains of the heavy and light chains recognize or bind to specific epitopes of the cognate antigen. The term "epitope" refers to a specific binding site or antigenic determinant on an antigen to which the variable ends of an immunoglobulin bind. Epitopes may be linear, i.e., consist of a sequence of amino acid residues found in the original HF-related sequence. Epitopes can also be conformational, allowing the immunoglobulin to recognize 3-D structures found in folded HF-related molecules. Epitopes can also be a combination of linear and conformational elements. Furthermore, the carbohydrate moiety of the molecule as expressed by the tumor cells at the target site may also be an epitope.

If: 1) an immunoglobulin exhibits a threshold level of binding activity, and/or 2) the immunoglobulin does not significantly cross-react with a known related polypeptide molecule, then the immunoglobulin is considered "specifically binding". The binding affinity of an immunoglobulin can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NYAcad. Sci.51: 660-. At one endIn some embodiments, the immunoglobulin of the invention is at least 10 times higher than other proteins³Times, more preferably at least 104 times, more preferably at least 10 times⁵Times, even more preferably at least 10⁶Binding to HF-related proteins at a doubling of the level.

Polyclonal and monoclonal antibodies

The immunoglobulins of the present invention may be polyclonal or monoclonal and may be produced by any method well known in the art.

Preferably, polyclonal antibodies are produced in animals by multiple subcutaneous (sc), intraperitoneal (ip), or intramuscular (im) injections of the relevant antigen and adjuvant. It may be useful to bind the relevant antigen to a protein that is immunogenic in the species to be immunized. In addition, aggregating agents such as alum or other agents are suitable for enhancing the immune response.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, unlike the production of polyclonal antibodies, which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant of the antigen.

In addition to their specificity, monoclonal antibodies also have the advantage that they can be synthesized uncontaminated by other immunoglobulins. For example, monoclonal antibodies can be produced by hybridoma methods or by recombinant DNA methods. Monoclonal antibody HF-related agents can also be isolated from phage antibody libraries.

Chimeric and humanized antibodies

An HF-related polypeptide binding immunoglobulin or antibody may be "chimeric" according to the definition that the variable region may be from one species (e.g., rodent) and the constant region may be from a second species (e.g., human).

A "humanized" form of a non-human HF-related protein binding antibody is a chimeric antibody that contains minimal sequences from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some cases, Framework Region (FR) residues of the human immunoglobulin are substituted with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or donor antibody.

In general, a humanized antibody will comprise substantially all of at least one variable domain, and typically a humanized antibody will comprise substantially all of two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In one embodiment, a humanized antibody comprises humanized FRs that exhibit at least 65% sequence identity to recipient (non-human) FRs (e.g., murine FRs). The humanized antibody may further comprise at least a portion of an immunoglobulin constant region (Fc), particularly a human immunoglobulin.

Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody contains one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. Humanization can be substantially achieved by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Thus, such humanized antibodies are chimeric in that regions substantially smaller than the entire human variable domain are substituted with the corresponding sequences in the non-human species. In fact, humanized antibodies are typically human antibodies in which some hypervariable region residues, and possibly some FR residues, are substituted by residues in analogous sites in rodent antibodies. The choice of human variable domains (light and heavy chains) to be used to make humanized antibodies is very important to reduce antigenicity.

Other methods generally involve conferring donor CDR binding affinity to the antibody acceptor variable region backbone. One method involves simultaneous grafting and optimization of the binding affinity of the variable region binding fragments. Another approach involves optimizing the binding affinity of the antibody variable regions.

Antibody fragments

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')²Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment. The Fab fragment also contains the constant region of the light chain and the first constant region of the heavy Chain (CHI).

Pepsin treatment produced F (ab') containing two antigen binding sites²Fragments and the fragments are still capable of cross-linking with antigen. Fab 'fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CHI domain of the Fab' fragment, including one or more cysteines of the antibody hinge region. Fab '-SH is the name herein for Fab' in which the cysteine residues of the constant domains have at least one free thiol group. F (ab')²Antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are well known in the art.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. The region is composed of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define the antigen-binding site of the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) is capable of recognizing and binding antigen, although with lower affinity than the entire binding site.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains that enables the scFV to form the desired structure for antigen binding. See Plucklthun, 113 Theepharmalcoogyocolonnabilides 269-315(Rosenburg and Moore eds 1994). See also international publication WO93/16185, U.S. patent nos. 5,587,458 and 5,571,894.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of the intact antibody. However, these fragments can now be produced directly by recombinant host cells.

v. binding and labelling

anti-HF-related protein antibodies may be administered in their "naked" form or unconjugated form, or may have other agents bound to them.

For example, the antibody may be in detectably labeled form. The antibody can be detectably labeled using radioisotopes, affinity labels (e.g., biotin, avidin, etc.), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (e.g., FIFC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for achieving such labeling are well known in the art.

Bispecific antibodies

The bispecific antibodies of the invention are small antibody fragments containing two antigen binding sites. Each fragment comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short in length to pair the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites.

Methods for making bispecific antibodies are well known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities.

In another approach, antibody variable domains (antibody-antigen binding sites) with the desired binding specificities can be fused to immunoglobulin constant domain sequences. In particular, the variable domain is fused to an immunoglobulin heavy chain constant domain comprising at least part of a hinge region, a CH2 region, and a CH3 region. In one embodiment, the fusion protein comprises a first heavy chain constant region (CHI) as it contains the sites necessary for light chain binding. Polynucleotides encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain can be inserted into separate expression vectors and co-transfected into a suitable host organism. The above method provides a high degree of flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment, when unequal proportions of the three polypeptide chains used in the construct provide for optimal yield. However, it is possible to insert the coding sequences of two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains at equal rates leads to high performance, or when said rates are of no particular significance.

Bispecific antibodies were also produced using leucine zippers and single chain fv (sfv) dimers.

e. Diagnosis, prognosis and assessment of fibrosis treatment

Thus, in another aspect, the invention provides methods for the diagnosis and prognosis of fibrosis using the HF-related polypeptides described herein. In specific non-limiting embodiments, the methods are useful in detecting HF-related polypeptides in cells or serum/plasma, facilitating diagnosis of and severity of fibrosis in a subject, facilitating determination of the prognosis of a subject, determining susceptibility of a subject to fibrosis, and assessing responsiveness of a subject to treatment (e.g., by providing a measure of the effect of treatment (e.g., assessing tumor burden during or after a chemotherapy regimen)). These methods may include detecting the level of an HF-related polypeptide in a biological sample (e.g., serum/plasma or fibrotic tissue or cells suspected of or about to be produced) of a patient. The detection methods of the invention can be performed in vitro or in vivo on isolated cells or in whole tissues or body fluids (e.g., blood, plasma, serum, urine, etc.). In one embodiment, HF-related polypeptides can be used to detect and assess fibrosis. These biomarkers can be applied to any disease exhibiting fibrosis, such as liver fibrosis, kidney fibrosis, heart fibrosis, skin fibrosis, pancreas fibrosis, etc., but more preferably, the fibrosis is liver fibrosis.

f. Detection of HF-related Polypeptides

The present invention provides methods for detecting HF-related polypeptides in serum or plasma. Any of a variety of known methods can be used for detection, including but not limited to: immunoassays using antibodies specific for the encoded polypeptide, e.g., by enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), western blot, transmission turbidimetry, scattering turbidimetry, and the like, and functional detection of the encoded polypeptide; such functional detection of the encoded polypeptide, e.g., biological activity. HF-related polypeptides can also be quantified using non-antibody approaches such as, but not limited to, multiple reaction monitoring using mass spectrometry.

It will be apparent to those of ordinary skill in the art, after reading the present specification, that the detection methods and other methods described herein can be readily modified. Such variations are within the intended scope of the invention. For example, in the above detection schemes, the probes used in the detection may be immobilized on a solid support, and the test sample is contacted with the immobilized probes. Binding of the test sample to the probes can then be detected in a variety of ways, for example, by detecting a detectable label bound to the test sample, thereby facilitating detection of the test sample-immobilized probe complexes.

The invention further provides methods for detecting the presence of and/or measuring the level of an HF-related polypeptide in a biological sample using antibodies specific for the HF-related polypeptide. In particular, the method for detecting the presence of an HF-related polypeptide in a biological sample may comprise the steps of: contacting the sample with the monoclonal antibody and detecting binding of the antibody to the HF-related polypeptide in the sample. More specifically, antibodies can be labeled with compounds that generate detectable signals, including but not limited to: radiolabels, enzymes, chromophores, and fluorophores.

The detection of specific binding of an antibody or functional equivalent thereof specific for the HF-related polypeptide when compared to a suitable control is an indication that the HF-related polypeptide is present in the sample. Suitable controls include samples known to be free of HF-related polypeptides and samples contacted with an antibody that is not specific for the encoded polypeptide (e.g., an anti-idiotypic antibody). Various methods of detecting specific antibody-antigen interactions are known in the art and may be used in methods including, but not limited to, standard immunohistological methods, immunoprecipitation, enzyme immunoassay, and radioimmunoassay. Generally, a specific antibody will be detectably labeled, either directly or indirectly. Direct labeling includes radioisotopes; product-detectable enzymes (e.g., luciferase, 3-galactosidase, etc.); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, etc.); a fluorescent emitting metal (e.g., 112Eu or other member of the lanthanide series linked to an antibody via a metal chelating group such as EDTA); chemiluminescent compounds (e.g., luminol, isoluminol, acridinium salts, and the like); bioluminescent compounds (e.g., fluorescein, aequorin (green fluorescent protein), etc.). The antibody may be attached (conjugated) to an insoluble support, such as a polystyrene plate or bead. Indirect labels include a specific secondary antibody to the antibody specific for the encoded polypeptide ("specific primary antibody"), wherein the secondary antibody is labeled as described above, and a member of a specific binding partner, e.g., biotin-avidin, and the like. The biological sample may be contacted and immobilized on a solid support or carrier, such as nitrocellulose, which is capable of immobilizing cells, cell particles, or soluble proteins. The support may then be washed with a suitable buffer and then contacted with a detectably labeled specific primary antibody. Detection methods are known in the art and will be selected based on the signal emitted by the detectable label. Detection is generally achieved by comparison with suitable controls and suitable standards.

g. Reagent kit

The detection method may be provided as part of a kit. Thus, the present invention also provides kits for detecting the presence and/or detecting the level of an HF-related polypeptide in a biological sample. The steps of using these kits can be performed by a clinical laboratory, a laboratory, a physician, or an individual. The kit of the invention is used for detecting HF-related polypeptides differentially expressed during fibrosis. The kit may provide additional components useful in the steps including, but not limited to, buffers, developers, labels, reaction surfaces, detection methods, control samples, standards, instructions, and explanatory information.

Examples

Differentially expressed proteins established when comparing plasma from healthy individuals to plasma from cirrhosis patients

The present inventors found that various proteins are differentially expressed in human plasma samples from HCV-induced cirrhosis patients when compared to healthy individuals. This finding was achieved by comparing these plasma samples using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), a technique that separates proteins in two dimensions on a gel matrix resulting in discrete protein spots. 2D-PAGE using a broad range of pH3 to pH10 has previously been used in International publication WO/2008/031051 to identify biomarkers in the serum of liver fibrosis. The identification of the fibrotic biomarker in the present invention is different from international publication WO/2008/031051 in that a narrow range of pH3 to pH5.6 is used. This pH range was chosen because it is outside the range of the main isoforms of the three most abundant plasma/serum proteins (albumin, IgG, transferrin). This is the first use of this pH3 to pH5.6 range for biomarker discovery.

The discovery of novel biomarkers in disease is limited by the range of dynamic protein concentrations in serum and plasma that span ten orders of magnitude. Thus, high abundance proteins (especially albumin, immunoglobulins and transferrin) limit the detection of low abundance proteins. To overcome this problem in the search for biomarkers, many have attempted antibody-based and dye affinity-based primary separation strategies to deplete high-abundance proteins from samples prior to electrophoresis to improve the expression of low-abundance proteins. However, immunoprecipitation is expensive because large amounts of antibody are required to deplete these highly abundant proteins, whereas dye-affinity methods are less efficient and less specific, with unnecessary removal of large amounts of non-albumin, and thus potentially removing potential biomarkers in proteomic analysis. Unlike the large amount of protein used in the present invention (2mg), multiple samples were loaded with similar high levels of protein after depletion, which is challenging due to recovery problems during removal of the high abundance protein and losses during concentration.

Although there is a problem with high abundance proteins in plasma, several novel candidate biomarkers of liver fibrosis have been successfully identified in international publication WO/2008/031051 using 2D-PAGE over a wide range of pH3 to pH 10. In the present application, this panel of biomarkers of liver fibrosis was increased by using 2D-PAGE with a narrow range of pH3 to pH5.6, outside the main isotype range of albumin, immunoglobulin and transferrin, thus allowing four times more protein to be loaded than International publication WO/2008/031051. This allows for improved expression of low abundance feature points and improved separation. The use of this pH range achieves a significant improvement in gel-based separation of serum and plasma acidic proteomes and allows the identification of low abundance liver fibrosis biomarkers that are not visible in international publication WO/2008/031051 using a wide range of pH3 to pH 10.

Example 1

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)

To identify biomarkers for HCV-induced hepatic scarring, plasma samples of healthy individuals and plasma samples of HCV-induced cirrhosis patients (6 individuals per group) were analyzed in 2D-PAGE based proteomic studies. Whole plasma samples were collected in P100 tubes (BD, Oxford, UK). These P100 plasma tubes were chosen because, unlike other blood collection tubes, they contain proprietary protein stabilizers that dissolve immediately when the blood is collected, which enhance recovery and preservation of the protein, making them suitable for proteomic analysis and biomarker discovery. Unlike international publication WO/2008/031051, which analyzes sera from patients with different degrees of liver fibrosis, the present invention focuses on discussing proteins differentially expressed between samples from healthy individuals and samples from patients with cirrhosis. This approach was taken because all differentially expressed proteins previously identified in mild and moderate fibrosis were also found in cirrhosis, indicating that analysis of these intermediate stages in this study will not yield any other candidate fibrosis biomarkers. 2mg of plasma protein was separated by charge in the first dimension gel using a nonlinear gradient from pH3 to pH5.6, and then by molecular weight (size) in the second dimension using a 9% to 16% (w/v) SDS-PAGE gradient. Electrophoresis, fluorescent staining and gel scanning were performed as described in gandaharan et al, (2007) in clin.

Example 2

Differential image analysis and protein identification

The resulting two-dimensional array of spots was compared between normal plasma samples and cirrhosis plasma samples by computer-assisted image analysis. Scanned images of all 2D-PAGE gels were analyzed by computer-assisted image analysis as described by gangadaran et al, (2007), clin. Differential expression changes greater than or equal to 2-fold difference are considered significant. A total of 57 differentially expressed signatures were cleaved, trypsinized and mass-analyzed as described by Gangadharan et al, (2007), clin.

Example 3

Human plasma biomarkers for identifying liver fibrosis caused by cirrhosis

Differential image analysis showed preliminary evidence of potential plasma biomarkers. See fig. 1-4. This analysis shows that the expression of lipid transfer inhibitory protein, zinc-alpha-2-glycoprotein, beta-haptoglobin at ph5.46 to ph5.49, haptoglobin-related protein, apolipoprotein C-III, apolipoprotein E, C4 b-binding protein beta chain, paraoxonase/aromatase 1, retinol binding protein 4, alpha-albumin, alpha-2-HS-glycoprotein, corticosteroid binding globulin, leucine rich alpha-2-glycoprotein and fibrinogen gamma chain is reduced in cirrhosis serum, while the expression of the complete complement C3dg, immunoglobulin J chain, sex hormone binding globulin, 14-3-3 protein zeta/, adiponectin and alpha-1-antitrypsin is increased. Post-translational modification of glycoprotein hemopexins is also observed.

The fibrotic biomarker that has been described in International publication WO/2008/031051 has also been identified: CD5 antigen-like protein and increased Ig α/κ chains; alpha-1-antichymotrypsin, clusterin, complement C4, inter-alpha-trypsin inhibitor heavy chain H4 and transthyretin decrease. In the case of the m-alpha-trypsin inhibitor heavy chain H4, analysis of the peptide sequence by mass spectrometry confirmed that the uncleaved form of the protein was reduced, whereas only 35kDa and 70kDa cleaved fragments were previously identified as reduced in International publication WO/2008/031051.

All of the above proteins were identified by mass spectrometry as the highest scoring protein. Other lower scoring proteins were identified in the gel feature points, which, although not excluded, were less likely to be responsible for the differential expression changes. The lower scoring proteins identified were increased Ig λ chain and apolipoprotein AI, and decreased albumin, AMBP, prothrombin, pigment epithelium derived factor and serum amyloid P-component.

Example 4

Measurement of these novel proteins in serum/plasma would facilitate reliable diagnosis of fibrosis and cirrhosis, reducing the need for liver biopsy. These measurements can be achieved using immunoassays for antibodies that target these proteins.

A fragment of complement C3 was found to increase in cirrhosis and was observed at 39kDa, about the isoelectric point of pI4.9 on a 2D-PAGE gel. The amino acids of this fragment identified by mass spectrometry ranged from 955 to 1201. Complement C3dg has amino acids ranging from 955 to 1303 and its theoretical molecular weight and isoelectric point (39kDa and pI5) coincides with the observed gel characteristic point, demonstrating that complement C3 in this characteristic point is complement C3 dg. Complement C3dg is known to have a thioester site at amino acids 1010 to 1013 that is cleavable by plasmin (a fibrolytic-related enzyme). Plasmin is known to decrease in fibrosis, consistent with the increased levels of uncleaved complement C3dg observed in cirrhosis. A fragment of complement C3 was found to be reduced in fibrosis and cirrhosis in international publication WO/2008/031051, the fragment was observed at 49kdapi6.9, and mass-identified amino acids indicated that it was the alpha chain of complement C3 before the thioester site, demonstrating a reduction in plasmin-mediated cleavage of complement C3 in fibrosis (Gangadharan et al, (2007), clin. chem., 53, 1792). Thus, in the present invention, an antibody that targets the plasmin cleavage site of complement C3 (i.e., overlaps the thioester site at amino acids 1010 to 1013) would help determine the extent of cleavage. There does not appear to be a commercially available antibody directed against the thioester site region of complement C3. Antibodies directed against the lytic region can more reliably help determine increased levels of complement C3 that are not lysed in hepatic scarring.

Total haptoglobin is known to be reduced in fibrosis and is currently used with other proteins to diagnose liver fibrosis (see International publication WO 0216949). In the present invention, it was found that 2D-PAGE characteristic points containing beta-haptoglobin at pH5.46 to pH5.49 were reduced in cirrhosis and appeared more reliable than total haptoglobin. Haptoglobin is known to contain four potential glycosylation sites, all on its beta chain. Beta-haptoglobin is usually glycosylated in plasma/serum. When plasma/serum was separated by 2D-PAGE, beta-haptoglobin was found to be an array of equally spaced characteristic points between ph4.7 and ph5.8, which could be shown to be reduced in liver scarring. In the present invention, the globin-bound 2D-PAGE gel characteristic points at pH5.46 to pH5.49 are reduced in cirrhosis and appear more reliable than the other characteristic points for beta globin-bound between pH4.7 and pH 5.8.

Example 5

The fibrosis grade of each of the novel biomarkers can be formulated. The mean concentration of these biomarkers in serum from different stages of liver fibrosis is determined. Liver fibrosis is currently assessed clinically using a scale of 0 to 6, where 0 represents no fibrosis, 1-5 represents an intermediate stage of fibrosis with increasing severity from mild to moderate/severe, and 6 is cirrhosis (Ishak, (1995), jhepal, 22, 696). By determining the concentration range of the novel biomarker in these seven stages, a similar scoring system from 0 to 6 can be assigned. The result of adding the scores of all novel biomarkers will give a more reliable indicator of the degree of fibrosis, rather than detecting a single biomarker.

Example 6

Immunoassay method

The present invention provides kits for assessing fibrosis, which detect HF-related polypeptides. This is achieved by using antibodies that bind to HF-related polypeptides. These antibodies can be used to perform immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, western blots, transmission turbidimetry, nephelometry, and the like. HF-related polypeptides can also be quantified using non-antibody approaches such as, but not limited to, multiple reaction monitoring using mass spectrometry.

For ELISA, the detection can be performed in 96-well plates. One option is to use a non-competitive single-site binding ELISA. In this assay, known concentrations of antigen (in this example, novel biomarkers and serum samples) are prepared in bicarbonate buffer, added to 96-well plates and incubated overnight at 4 ℃. The plates were then washed three times with Phosphate Buffered Saline (PBS) and Tween solutions (PBS-T) and blocked with bovine serum albumin in PBS. After 1 hour incubation at 37 ℃, primary antibody to the antigen (in this example, the target biomarker) was added, and the plates were incubated for 1 hour at 37 ℃ and washed three times with PBS-T. Horseradish peroxidase-conjugated secondary antibodies (animal origin for primary antibody) were then added and the plates were incubated at 37 ℃ for 1 hour and then washed three times with PBS-T. Finally, a peroxidase substrate such as 2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was added to each well, and the absorbance at 405nm was read on a microplate reader after 30 minutes.

Alternatively, a sandwich ELISA may be used. In this type of assay, an antibody is bound to the bottom of a well. Antigen (in this case, biomarker protein) is added and unbound product is removed by washing. Then another antibody, which is labeled, is added that binds to the antigen. The amount of bound further antibody is quantified (usually by colorimetry). In addition to novel biomarkers, the inventors of the present invention propose that haptoglobin can be measured by ELISA. Haptoglobin levels in serum (in conjunction with a clinician-determined liver function test) will establish a more reliable score for liver fibrosis.

While the foregoing is directed to certain preferred embodiments, it will be understood that the invention is not so limited. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art and such modifications are intended to be within the scope of the present invention.

All publications, patent applications, and patents cited in this specification are herein incorporated by reference in their entirety.

Claims (3)

1. Use of a lipid transfer inhibitor protein in the preparation of a kit for detecting and assessing liver fibrosis in a serum or plasma sample of a human patient, wherein the level of lipid transfer inhibitor protein in a serum or plasma sample of the human patient is compared to a control level of the lipid transfer inhibitor protein for detecting and assessing liver fibrosis in a serum or plasma sample of the human patient.

2. Use of a lipid transfer inhibitor protein in the preparation of a kit for the severity stratification of liver fibrosis in a serum or plasma sample of a human patient, wherein the level of lipid transfer inhibitor protein in a serum or plasma sample of said human patient is compared to a control level of said lipid transfer inhibitor protein for the severity stratification of liver fibrosis in a serum or plasma sample of said human patient.

3. Use of a lipid transfer inhibitor protein in the manufacture of a kit for determining the prognosis of liver fibrosis in a serum or plasma sample of a human patient, wherein the level of the lipid transfer inhibitor protein in the serum or plasma sample of the human patient is compared to a control level of the lipid transfer inhibitor protein for determining the prognosis of liver fibrosis in the serum or plasma sample of the human patient.