Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Definitions

• MS Multiple sclerosis
• MS The diagnosis of MS is still defined primarily by clinical terms and relies on a combination of history, neurological examination and ancillary laboratory and neuro- imaging studies.
• an otherwise healthy person presents with the acute or sub acute onset of neurological symptomatology (attack) manifested by unilateral loss of vision, vertigo, ataxia, dyscoordination, gait difficulties, sensory impairment characterized by paresthesia, dysesthesia, sensory loss, urinary disturbances until incontinence, diplopia, dysarthria or various degrees of motor weakness until paralysis.
• the symptoms are usually painless, remain for several days to a few weeks, and then partially or completely resolve.
• a second attack will occur. During this period after the first attack, the patient is defined to suffer from probable MS.
• Probable MS patients may remain undiagnosed for years.
• CDMS clinically definite MS
• the diagnosis of clinically definite MS is made (Poser criteria 1983; C. M. Poser et al, Ann. Neural. 1983; 13, 227). These criteria have been revised in recent years to include radiological criteria for establishing the diagnosis (McDonald et al., Ann Neural 50:121-7; Polman et al., Ann Neural 2005; 58: 840-6) but even with these modifications, it may often take several years from the onset of clinical symptoms to establish the diagnosis.
• Laboratory tests for MS include: 1) cerebrospinal fluid (CSF) evaluation of IgG synthesis, oligoclonal bands; 2) MRI of the brain and spinal cord and; 3) exclusion of other autoimmune diseases by blood tests [e.g.; serum B 12 level; HTLV 1 or HIV 1 titers; sedimentation rate or C-reactive protein; RA latex (Rheumatoid arthritis); ANA, anti-DNA antibodies (systemic lupus erythematosus)].
• CSF cerebrospinal fluid
• MS-specific markers may further provide important insights into the pathogenesis of MS, including the precise mechanism of neuronal and myelin injury and the events leading to the onset of the disease.
• a method for determining whether a subject has or is likely to develop multiple sclerosis (MS) or a condition relating thereto is provided herein.
• This method may comprise determining in a biological sample of a subject, the ratio of the level of a cystatin C protein fragment lacking about 8 amino acids at its C-terminus to the level of cystatin C protein, wherein a ratio of at least about 2 indicates that the subject has or is likely to develop MS or a condition relating thereto.
• the cystatin C protein lacking about 8 amino acids at its C-terminus may consist essentially of the amino acid sequence SEQ ID NO: 2.
• the biological sample is cerebrospinal fluid (CSF), in which the ratio of the level of a cystatin C protein fragment lacking about 8 amino acids at its C-terminus to the level of cystatin C protein.
• CSF cerebrospinal fluid
• a method for determining whether a subject has or is likely to develop multiple sclerosis (MS) or a condition relating thereto comprises:
• the method comprises:
• a method for determining whether a subject has or is likely to develop MS or a condition relating thereto comprising determining in a biological sample of a subject, the level of a cystatin C protein fragment lacking about 8 amino acids at its C-terminus, wherein a level of at least about 2-fold indicates that the subject has or is likely to develop MS or a condition relating thereto.
• this method comprises: (a) contacting the biological sample or a portion thereof with an antibody that binds specifically to the cystatin C protein fragment to thereby obtain cystatin C protein fragment antibody complexes;
• composition enriched in cystatin C protein fragment to a size separation process, to thereby obtain a level of cystatin C protein fragment.
• a method for determining whether a subject has or is likely to develop MS or a condition relating thereto comprising determining in a biological sample of a subject the level of one or more biomarkers identified in Table 2 or Table 4, wherein a different level of one or more biomarkers relative to the level in a control, indicates that the subject has or is likely to develop MS or a condition relating thereto.
• an isolated protein comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO: 2, wherein the protein does not comprise the last 8 amino acids of full-length cystatin C.
• nucleic acid encoding a protein comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO: 2, wherein the protein does not comprise the last 8 amino acids of full-length cystatin C.
• hererin is an isolated nucleic acid comprising a nucleotide sequence that is at least about 95% identical to SEQ ID NO: 1, wherein the nucleic acid does not encode a protein comprising the last 8 amino acids of full-length cystatin C.
• the nucleic acid comprises a nucleotide sequence that is identical to SEQ ID NO: 1 or consists essentially of SEQ ID NO: 1.
• an isolated antibody that binds specifically to a cystatin C protein fragment lacking about 8 amino acids at its C-terminus and does not bind significantly to a full-length cystatin C protein.
• An isolated antibody that binds specifically to a cystatin C protein fragment consisting of the C-terminal 8 amino acids of cystatin C and does not bind significantly to a full length cystatin C protein is also provided.
• kits for use in an assay for determining whether a subject has or is likely to develop multiple sclerosis (MS) or a condition relating thereto may comprise an antibody that binds specifically to a cystatin C protein and a reagent for use in an assay provided herein.
• a kit comprises an antibody that binds specifically to a cystatin C protein and one or more comparative values to which the results of an assay using the antibody can be compared.
• a method may comprise administering to a subject in need thereof a therapeutically effective amount of an agent that decreases cathepsin activity.
• the cathepsin is cathepsin B.
• the agent may inhibit the activity of cystatin C.
• the agent inhibits the proteolytic cleavage of the last 8 amino acids of cystatin C.
• Figure 1 shows a representative CSF spectra generated by SELDI analysis.
• A Patient with multiple sclerosis showing a prominent peak at 12.5kD (arrow). The 13.4 kD peak is blunted.
• B The 12.5 kD peak is absent from the control patient. However the 13.4 kD peak is prominent (slanted arrow). Another small peak at 13.6 kD is also noted (vertical arrow) which is absent from the MS spectra.
• Figure 2 shows a comparison of the ratio of the 12.5 kD to 13.4 kD peak in CSF from different disease states.
• the 12.5/13.4 peak ratio was significantly elevated in the MS group compared to OND (P ⁇ 0.001), TM (P ⁇ 0.001) HIV-ND (P ⁇ 0.05) and HIV-D (P ⁇ 0.05).
• Figure 3 shows an effect of anatomical location of last clinical attack on 12.5 kD peak height in CSF of MS/CIS patients. The peak was significantly higher in patients with recent infratentorial disease activity compared to those with a supratentorial involvement (P ⁇ 0.05).
• Figure 4 shows a partial purification of the 12.5 kD protein from CSF.
• A CSF incubated with protein A beads to remove IgG and then analyzed by SELDI time of flight mass spectroscopy shows the presence of the 12.5 kD protein.
• B CSF was further exposed to strong anion exchange beads and reanalyzed by SELDI time of flight mass spectroscopy which shows the removal of the 11.6 kD and 13.8 kD complexes and relative enrichment of the 12.5 kD protein.
• Figure 5 shows immuno depletion of cystatin C from CSF.
• CSF was analyzed by SELDI time of flight mass spectroscopy following incubation with either (A) protein A beads alone, (B) protein A beads bound to rabbit anti-fusin antisera or (C) protein A beads bound to rabbit antisera to cystatin C. Both the 12.5 kD and the 13.4 kD proteins were selectively removed by the anti-cystatin antisera.
• Figure 6 shows a correlation of cystatin C levels and cathepsin B activity in the CSF of
• Figure 7 shows N and C terminal cleavage of cystatin C.
• A Site of cleavage of cystatin C at the N terminal fragment between the arginine and lysine residues and at the C terminal region between the lysine and serine residues are marked by arrows.
• Figure 8 shows inhibition of cystatin C cleavage by pepstatin A.
• a CSF sample that had only the 13.4 kD peak was incubated with a protease inhibitor cocktail (PIC), a MMP inhibitor (FN439) or a cathepsin D inhibitor (pepstatin A) for 48 hours at room temperature.
• PIC protease inhibitor cocktail
• MMP inhibitor FN439
• pepstatin A cathepsin D inhibitor
• Figure 9 shows modulation of cystatin C activity by cathepsin D and MMP-2.
• cystatin C shows significant inhibition of cathepsin B activity
• Figure 10 shows Table 1, which lists demographics of patients with MS/CIS.
• FIG. 11 shows Table 2, which lists peak intensities significantly altered in patients with
• Figure 12 shows Table 3, which lists peptides recovered from tryptic digestion of the 12.5 kDa protein band. Amino acid residues, observed molecular weight, and sequence are shown.
• Figure 13 shows cerebrospinal fluid spectra generated by surface-enhanced laser desorption/ionization analysis.
• MS multiple sclerosis
• CIS clinically isolated syndromes
• the 12.5kDa peak is absent from control patients with other neurological diseases (OND). However, the 13.4kDa peak is prominent.
• the scales in A and B are identical.
• Figure 14 shows a decision tree for identifying patients with MS.
• MS and CIS patients were analyzed as a single group.
• BPS analysis of 2 1 7 clusters shows that the 12.5kDa peak was the top splitter that correctly identified 19 of 29 MS/CIS patients. Of the remaining 29 samples, the 4.7 kDa peak correctly identified 16 OND patients. 3 of the OND patients but none of the MS/CIS patients were misclassified.
• Figure 15 shows the results of an analysis of CSF from patients with Multiple Sclerosis.
• Figure 16 and Figure 17 show Table 4, which lists peaks differentially expressed between controls and MS samples using Biomarker Wizard, including the two cystatin C peaks.
• Table 4A lists peaks A-I, which are elevated in MS.
• Table 4B lists peaks J-S, which are diminished in MS. Each of these peaks was identified using the same the weak cation CMlO chip as was used for the cystatin C peaks, hence these peaks have binding properties that are distinct in their identification.
• Figure 18 shows a spectrum that shows one of the peaks listed in Table 4.
• MS multiple sclerosis
• the present invention is based at least in part on the identification of biological markers for MS.
• Samples of CSF from patients with CIS or MS were analyzed using the protein array technology surface-enhanced laser desorption/ionization (SELDI) time-of- flight mass spectroscopy, and the resulting mass spectra profiles were compared to those obtained from patients who did not have the disease. This resulted in the identification of several biomarkers of the disease.
• SELDI protein array technology surface-enhanced laser desorption/ionization
• One of the biomarkers is a novel protein of 12.5 kD that was 100% specific for MS/CIS, as compared to non-MS patients. Tandem mass spectroscopy of a tryptic digest of this 12.5 kD protein identified it as a cleavage product of full-length cystatin C (13.4 kD), an important inhibitor of cysteine proteases including the cathepsins. While total cystatin C levels in the MS patients was not different compared to controls, the patients with the highest 12.5/13.4 peak ratios also had the greatest cathepsin B inhibitory activity. This suggests that cleavage of cystatin C may be an adaptive host response that identifies a subgroup of patients with MS.
• MS is intended to refer to all types and stages of MS.
• Types of MS include but are not limited to the following: benign MS, relapsing remitting MS, secondary chronic progressive MS, and primary progressive MS, Progressive Relapsing Multiple Sclerosis, Chronic Progressive MS, Transitional/Progressive MS, malignant MS, also known as Marburg's Variant, and acute multiple sclerosis.
• Early stages of MS include an acute or sub acute onset of neurological symptomatology (attack); first attack, probable stage, second attack, early relapsing-remitting stages, and clinically isolated syndromes (CIS).
• Constants relating to MS include, e.g., Devic's Disease, also known as Neuromyelitis Optica; and Balo's concentric sclerosis.
• the methods described herein may also predict the presence or likelihood of development of the early stages of MS and conditions relating to MS. For example, the methods described herein may determine: the likelihood of a symptomless subject to develop MS or an early stage thereof or a condition relating thereto; the likelihood of a subject having symptoms, e.g., symptoms which resemble those present in early stage MS, to have or to develop MS or an early stage thereof or a condition relating thereto; the likelihood of a subject having early stage MS symptoms to develop MS; or the likelihood of a subject having early stage MS symptoms to develop a particular type of MS.
• the methods described herein may also be used to determine the prognosis of a subject having MS, an early stage thereof or a condition relating thereto.
• the methods may allow the prognosis of a subject that is being treated, e.g., with interferon-beta.
• the methods may also be used for determining the severity of the disease.
• the method may be used to determine whether a subject is more likely than not to have MS, an early stage thereof, or a condition relating thereto, or is more likely to have MS, an early stage thereof, or a condition relating thereto than to have another disease, based on the difference between the measured and standard level or reference range of the biomarker.
• a patient with a putative diagnosis of MS or a condition relating thereto may be diagnosed as being "more likely” or "less likely” to have MS in light of the information provided by a method of the present invention.
• a plurality of biomarkers are measured, at least one and up to all of the measured biomarkers must differ, in the appropriate direction, for the subject to be diagnosed as having (or being more likely to have) MS or a condition relating thereto. Preferably, such difference is statistically significant.
• Biomarker measurements are taken of a biological sample from a patient, e.g., suspected of having the disease, and compared with a standard level or reference range.
• the standard biomarker level or reference range is obtained by measuring the same marker or markers in a set of controls, such as from subject(s) that do not have MS or a condition relating thereto.
• the standard level can be obtained from one or more subjects, e.g., 1-5; 5-10; 10-50; or more subjects.
• Measurement of the standard biomarker level or reference range need not be made contemporaneously; it may be a historical measurement or a data set to which to compare the marker measurements.
• the normal control is matched to the patient with respect to some attribute(s) (e.g., age or sex).
• the patient can be diagnosed, e.g., as having MS or a condition related thereto; as being likely to develop MS or a condition related thereto; or as not having MS or a condition related thereto.
• a significant difference may be a statistically significant difference.
• a statistically significant difference between the measured and standard (control) level may be determined by first adding or substracting at least one, at least two, at least three, or at least four standard deviations to a standard or reference level to obtain a reference range. A measured level may then be compared to this reference range. For markers (e.g., Table 4A) whose levels are elevated in MS or a condition related thereto, the measured level is greater than the control level plus at least one, at least two, at least 3, or at least 4 standard deviations above the reference level. For markers (e.g., Table 4B) whose levels are diminished in MS or a condition related thereto, the measured level is less than the control level minus at least one, at least two, at least 3, or at least 4 standard deviations below the reference level.
• a significant difference may mean a difference of at least 2- fold, 3-, A-, 5-, 10- or more fold, with respect to the reference value
• a method for determining whether a subject has or is likely to develop MS, a precursory condition thereof or a condition relating to MS comprises determining in a biological sample of the subject the level or amount of a marker set forth in Table 4A. If the level is more than the control mean intensity plus the standard deviation for that marker in the Table, then the result is considered positive, i.e., the subject has or is likely to develop MS, a precursory condition thereof or a condition relating to MS. A level that is at least about the control mean intensity plus 2, 3, 4 or 5 standard deviations for that marker in the Table is also considered a positive result.
• a different set of control values and optionally standard deviations can be obtained according to methods known in the art.
• a result may be considered positive if the value measured is higher than the mean control value plus 1, 2, 3, 4 or 5 standard deviations calculated for that mean contol value.
• a method may comprise first obtaining a mean control value and a standard deviation, to which a value measured in a subject can be compared.
• Mean control values and standard deviations can be determined according to methods known in the art and may be based on, e.g., 2, 3, 5, 10, 20, 50 or more individuals who are not known to have MS, a precursory condition thereof or a condition relating thereto.
• a method may comprise one or more of the following: obtaining a biological sample from the subject; determining the level of a marker in the sample, wherein the marker is selected from the group consisting of the markers set forth in Table 2 and Table 4; comparing the level of the marker in the sample to a reference value; and determining whether the level of the marker is increased (Table 4A) or decreased (Table 4B) by at least about 1, 2, 3, or 4 standard deviations, or by at least about 50%, 2-fold, 3-, A-, 5-, 10- or more fold, with respect to the reference value.
• the reference value may be the level of the marker in at least one sample from a non-multiple sclerosis subject.
• the level of the marker may be determined by detecting the presence of the polypeptide in the sample. Also in all embodiments, the presence of the marker may also be determined by assessing the gene expression or activity of the marker present in the sample, as compared to a reference value.
• biomarkers may be more predictive of the disease activity or aid in diagnosis compared to a single biomarker.
• the methods of the present invention may be used to make the diagnosis of MS or a condition relating thereto, independently from other information such as the patient's symptoms or the results of other clinical or paraclinical tests. However, the methods of the present invention are preferably used in conjunction with such other data points.
• Biomarkers described herein may be measured in combination with other signs, symptoms and clinical tests of MS, such as MRI scans or MS biomarkers reported in the literature. Likewise, more than one of the biomarkers of the present invention may be measured in combination. Measurement of the biomarkers of the invention along with any other markers known in the art, including those not specifically listed herein, falls within the scope of the present invention.
• the description of the methods herein makes reference to measuring "a marker.”
• the methods of the invention may involve measuring two markers, three markers, or four or more markers, or ratios of the same.
• the two markers, three markers, or four or more markers may comprise any combination of markers seleceted from Table 2 or Table 4.
• a method for diagnosing multiple sclerosis in a subject may comprise obtaining one or more biological samples from the subject; determining the level of a plurality of markers in the one or more biological samples, wherein at least one of the plurality of markers is selected from the group consisting of the markers listed in Table 2 and Table 4; and comparing the level of at least one of the plurality of markers to a reference value.
• the biological sample to be tested for biomarkers may be of any tissue or fluid.
• the sample is a CSF or serum sample, but other biological fluids or tissue may be used.
• Possible biological fluids to be tested for biomarkers include, but are not limited to, plasma, saliva, urine, and neural tissue.
• CSF represents a preferred biological sample to analyze for MS markers as it bathes the brain and removes metabolites and molecular debris from its liquid environment.
• biomolecules associated with the presence and/or progression of MS are expected to be present at highest concentrations in this body fluid.
• molecules initially identified in CSF may also be present, presumably at lower concentrations, in more easily obtainable fluids such as serum, urine, and saliva.
• biomarkers may be valuable for monitoring all stages of the disease and response to therapy.
• Serum also represents a preferred biological sample as it is expected to be reflective of the systemic manifestations of the disease.
• the level of a marker may be compared to the level of another marker or some other component in a different tissue, fluid or biological "compartment.” Thus, a differential comparison may be made of a marker in CSF and serum. It is also within the scope of the invention to compare the level of a marker with the level of another marker or some other component within the same compartment.
• some of the marker measurement values are higher in samples from MS patients, while others are lower.
• a significant difference in the appropriate direction in the measured value of one or more of the markers indicates that the patient has (or is more likely to have) MS or a condition relating thereto. If only one biomarker is measured, then that value must increase or decrease to indicate MS or a condition relating thereto.
• multiple markers are measured, and a diagnosis of MS, or early stage thereof, or a condition relating thereto is indicated by changes in multiple markers.
• Measurements can be of (i) a biomarker of the present invention, (ii) a biomarker of the present invention and another factor known to be associated with MS or a condition relating thereto (e.g., MRI scan); (iii) a plurality of biomarkers comprising at least one biomarker of the present invention and at least one biomarker reported in the literature, or (iv) any combination of the foregoing.
• the amount of change in a biomarker level may be an indication of the relatively likelihood of the presence of the disease.
• a method of diagnosing or pro gno sing or determining whether a subject has or is likely to develop multiple sclerosis may comprise obtaining a sample from an individual and determining the level, amount, or activity of a C-terminal cystatin C polypeptide cleavage product, and e.g., compare it to the amount of full-length cystatin C polypeptide in the sample.
• the C-terminal cystatin C polypeptide cleavage product may function as an internal standard for diagnosing MS, an early stage thereof, or a condition relating thereto.
• An increase in the C-terminal cystatin C polypeptide cleavage product relative to the amount of full-length cystatin C polypeptide in the sample is predictive of individuals that are afflicted with or at risk of developing MS or an early stage thereof or a condition relating thereto.
• those skilled in the art may measure a ratio of the level, amount or activity of the C-terminal cystatin C polypeptide cleavage product relative to the amount of full-length cystatin C polypeptide in the sample. This may be useful to diagnose individuals that are afflicted with or at risk of developing MS, an early stage thereof, or a condition relating thereto when the results indicate an increased ratio of a C-terminal cystatin C polypeptide cleavage product relative to the amount of full-length cystatin C polypeptide in the sample, relative to a ratio calculated from a reference sample.
• the comparison of said ratios reflects a significant increase in the level, amount, or activity of the C-terminal cystatin C polypeptide cleavage product or products thereof relative to the cystatin C polypeptide, said individual is identified as being afflicted with or at risk of developing multiple sclerosis, an early stage thereof or a condition related thereto.
• the comparison of said ratios reflects an increase in the level, amount, or activity of the C-terminal cystatin C polypeptide cleavage product thereof relative to the cystatin C polypeptide by at least about 1.5, 1.8, 2.0, or 2.5, -fold, said individual is identified as being afflicted with or at risk of developing multiple sclerosis, an early stage thereof or a condition related thereto.
• the comparison of said ratios reflects an identical or decrease in the level, amount, or activity of the C-terminal cystatin C polypeptide cleavage product or products thereof relative to the cystatin C polypeptide, said individual is identified as not being afflicted with or at risk of developing multiple sclerosis, an early stage thereof or a condition related thereto.
• a method may comprise:
• a method comprises one or more of the following steps, not necessarily in the order provided: (a) contacting the biological sample or a portion thereof with an antibody that binds specifically to a cystatin C protein and to the cystatin C protein fragment to thereby obtain cystatin C and cystatin C protein fragment antibody complexes; (b) isolating cystatin C and cystatin C protein fragment antibody complexes from the biological sample, to thereby obtain a composition enriched in cystatin C and cystatin C protein fragment; (c) subjecting the composition enriched in cystatin C and cystatin C protein fragment to a size separation process, to thereby obtain a level of cystatin C protein and a level of cystatin C protein fragment; and (d) determining the ratio of the level of cystatin C protein fragment to the level of cystatin C protein.
• One skilled in the art may measure a ratio of the level, amount or activity of a cystatin C protein fragment lacking about 8 amino acids at its C-terminus (SEQ ID NO: 2) relative to a cystatin C protein that does not lack about 8 amino acids at its C-terminus (e.g., a full-length cystatin C polypeptide).
• SEQ ID NO: 2 a cystatin C protein fragment lacking about 8 amino acids at its C-terminus
• a method may comprise: (a) contacting a biological sample or a portion thereof with an antibody that binds specifically to a cystatin C protein and to the cystatin C protein fragment to thereby obtain cystatin C and cystatin C protein fragment antibody complexes; (b) isolating cystatin C and cystatin C protein fragment antibody complexes from the biological sample, to thereby obtain a composition enriched in cystatin C and cystatin C protein fragment; (c) subjecting the composition enriched in cystatin C and cystatin C protein fragment to a size separation process, to thereby obtain a level of cystatin C protein and a level of cystatin C protein fragment; and (d) determining the ratio of the level of cystatin C protein fragment to the level of cystatin C protein.
• a size separation process can be chromatography, e.g, gel chromatography, and others further described herein.
• a method of diagnosis of having of likelihood of developing a disease or condition as described herein may also comprise a combination of methods, e.g., determining the level of one or more biomarkers, e.g., described herein, and the ratio between the cystatin C cleavage product and cystatin C. Measurement and Detection of Biomarkers
• biomarker levels are measured using conventional techniques.
• a wide variety of techniques are available, including mass spectroscopy, immunoprecipitation, chromatographic separations, 2-D gel separations, binding assays (e.g., immunoassays), competitive inhibition assays, and so on. It is within the ability of one of ordinary skill in the art to determine which method would be most appropriate for measuring a specific marker.
• a robust ELISA assay may be best suited for use in a physician's office while a measurement requiring more sophisticated instrumentation may be best suited for use in a clinical laboratory. Regardless of the method selected, it is important that the measurements be reproducible.
• the markers of the invention can be measured by mass spectroscopy, which allows direct measurements of analytes with high sensitivity and reproducibility.
• mass spectrometric methods are available and could be used to accomplish the measurement.
• Electrospray ionization (ESI) allows quantification of differences in relative concentration of various species in one sample against another; absolute quantification is possible by normalization techniques (e.g., using an internal standard).
• Matrix-assisted laser desorption ionization (MALDI) or the related SELDI technology (Ciphergen, Inc.) also could be used to make a determination of whether a marker is present, and the relative or absolute level of the marker.
• mass spectrometers that allow time-of- flight (TOF) measurements have high accuracy and resolution and are able to measure low abundant species, even in complex matrices like serum or CSF.
• the level of the markers may be determined using a standard immunoassay, such as sandwiched ELISA using matched antibody pairs and chemiluminescent detection.
• a standard immunoassay such as sandwiched ELISA using matched antibody pairs and chemiluminescent detection.
• Commercially available or custom monoclonal or polyclonal antibodies are typically used.
• the assay can be adapted for use with other reagents that specifically bind to the marker. Standard protocols and data analysis are used to determine the marker concentrations from the assay data.
• quantification can be based on derivatization in combination with isotopic labeling, referred to as isotope coded affinity tags ("ICAT").
• ICAT isotope coded affinity tags
• one- and two-dimensional gels have been used to separate proteins and quantify gels spots by silver staining, fluorescence or radioactive labeling. These differently stained spots have been detected using mass spectroscopy, and identified by tandem mass spectroscopy techniques.
• the markers are measured using mass spectroscopy in connection with a separation technology, such as liquid chromatography-mass spectroscopy or gas chromatography-mass spectroscopy.
• Reverse-phase liquid chromatography may be coupled to high resolution, high mass accuracy ESI time-of- flight (TOF) mass spectroscopy. This allows spectral intensity measurement of a large number of biomolecules from a relatively small amount of any complex biological material without sacrificing sensitivity or throughput. Analyzing a sample will allow the marker (specified by a specific retention time and m/z) to be determined and quantified.
• separations may be performed using custom chromatographic surfaces (e.g., a bead on which a marker specific reagent has been immobilized). Molecules retained on the media subsequently may be eluted for analysis by mass spectroscopy.
• an antibody may be used to isolate a protein marker provided herein in a biological sample (e.g., by immunoprecipitation).
• a sample is contacted with an antibody affixed to a solid support (such as a bead or solid surface) to a biomarker of the invention, and the marker becomes tethered to the support by virtue of being bound to the antibody affixed to the solid support.
• the solid support containing the antibody-biomarker complex is washed under conditions which allow the antibody to remain bound to the biomarker. Non-specific components of the sample are thus separated and removed from the presence of the biomarker, with the biomarker remaining tethered to the support.
• the resulting composition thus becomes enriched with biomarker as a result of the concentration of the marker in the sample and the removal of non-marker components of the sample.
• the level of the marker may then be determined by any of a number of methods.
• the antibody-marker complex may be detected, or the marker may be eluted from the antibody and detected.
• the antibody-marker complex or eluted marker may be subjected to any number of methods for determining size, such as spectroscopy, chromatographic separations, or 2-D gel separations.
• Analysis by liquid chromatography-mass spectroscopy produces a mass intensity spectrum, the peaks of which represent various components of the sample, each component having a characteristic mass-to-charge ratio (m/z) and retention time (r.t.).
• the presence of a peak with the m/z and retention time of a biomarker indicates that the marker is present.
• the peak representing a marker may be compared to a corresponding peak from another spectrum (e.g., from a control sample) to obtain a relative measurement.
• Any normalization technique in the art e.g., an internal standard
• deconvoluting software is available to separate overlapping peaks.
• the retention time depends to some degree on the conditions employed in performing the liquid chromatography separation.
• the mass spectrometer selected for this purpose preferably provides high mass accuracy and high mass resolution.
• the mass accuracy of a well-calibrated Micromass TOF instrument, for example, is reported to be approximately 2 mDa, with resolution m/Am exceeding 5000.
• a number of the assays discussed above employ a reagent that specifically binds to the marker ("marker specific reagent"). Any molecule that is capable of specifically binding to a marker is included within the invention.
• the marker specific reagents are antibodies or antibody fragments. In other embodiments, the marker specific reagents are non-antibody species.
• a marker specific reagent may be an enzyme for which the marker is a substrate. The marker specific reagents may recognize any epitope of the targeted markers.
• a marker specific reagent may be identified and produced by any method accepted in the art. Methods for identifying and producing antibodies and antibody fragments specific for an analyte are well known. Examples of other methods used to identify marker specific reagents include binding assays with random peptide libraries (e.g., phage display) and design methods based on an analysis of the structure of the marker.
• the chromatographic separation techniques described above also may be coupled to an analytical technique other than mass spectroscopy such as fluorescence detection of tagged molecules, NMR, capillary UV, evaporative light scattering or electrochemical detection.
• analytical technique other than mass spectroscopy such as fluorescence detection of tagged molecules, NMR, capillary UV, evaporative light scattering or electrochemical detection.
• a method for monitoring an MS patient over time to determine whether the disease is progressing.
• the specific techniques used in implementing this embodiment are similar to those used in the embodiments described above.
• the method is performed by obtaining a biological sample, such as serum or CSF, from the subject at a certain time (ti); measuring the level of at least one of the biomarkers in the biological sample; and comparing the measured level with the level measured with respect to a biological sample obtained from the subject at an earlier time (to). Depending upon the difference between the measured levels, it can be seen whether the marker level has increased, decreased, or remained constant over the interval (ti-to).
• a further deviation of a marker in the direction indicating MS or a condition relating thereto, or the measurement of additional increased or decreased MS markers, would suggest a progression of the disease during the interval.
• Subsequent sample acquisitions and measurements can be performed as many times as desired over a range of times t 2 to t n .
• tracking a marker level in a patient can be used to predict exacerbations or indicate the clinical course of the disease.
• biomarkers of the present invention could be further investigated to distinguish between any or all of the known forms of MS (CIS, benign MS, relapsing remitting MS, secondary chronic progressive MS, and primary progressive MS) or any described types or subtypes of the disease.
• the sensitivity and specificity of any method of the present invention could be further investigated with respect to distinguishing MS or a condition relating thereto from other diseases of autoimmunity, or other nervous system disorders, or to predict relapse and remission.
• the markers of the present invention can be used to assess the efficacy of a therapeutic intervention in a subject.
• the same approach described above would be used, except a suitable treatment would be started, or an ongoing treatment would be changed, before the second measurement (i.e., after to and before ti).
• the treatment can be any therapeutic intervention, such as drug administration, dietary restriction or surgery, and can follow any suitable schedule over any time period.
• the measurements before and after could then be compared to determine whether or not the treatment was effective.
• the determination may be confounded by other superimposed processes (e.g., an exacerbation of the disease during the same period).
• a marker may also be used to screen a candidate drug in a clinical trial to determine whether a candidate drug is effective in treating MS or a condition relating thereto.
• a biological sample is obtained from each subject in population of subjects diagnosed with MS or a condition relating thereto.
• assays are performed on each subject's sample to measure levels of a biological marker. In some embodiments, only a single marker is monitored, while in other embodiments, a combination of markers is monitored.
• a predetermined dose of a candidate drug is administered to a portion or sub- population of the same subject population. Drug administration can follow any suitable schedule over any time period. In some cases, varying doses are administered to different subjects within the sub-population, or the drug is administered by different routes.
• a biological sample is acquired from the sub-population and the same assays are performed on the biological samples as were previously performed to obtain measurement values.
• subsequent sample acquisitions and measurements can be performed as many times as desired over a range of times t 2 to t n .
• a different sub-population of the subject population may serve as a control group, to which a placebo is administered.
• the same procedure may be followed for the control group: obtaining the biological sample, processing the sample, and measuring the biological markers to obtain a measurement chart.
• Specific doses and delivery routes can also be examined.
• the method is performed by administering the candidate drug at specified dose or delivery routes to subjects with MS or a condition relating thereto; obtaining biological samples, such as serum or CSF, from the subjects; measuring the level of at least one of the biomarkers in each of the biological samples; and, comparing the measured level for each sample with other samples and/or a standard level.
• the standard level is obtained by measuring the same marker or markers in the subject before drug administration.
• the drug can be considered to have an effect on MS or a condition relating thereto. If multiple biomarkers are measured, at least one and up to all of the biomarkers must change, in the expected direction, for the drug to be considered effective. Preferably, multiple markers must change for the drug to be considered effective, and preferably, such change is statistically significant.
• the above description is not limited to a candidate drug, but is applicable to determining whether any therapeutic intervention is effective in treating MS or a condition relating thereto.
• Measurements can be of both biomarkers of the present invention and other measurements and factors associated with MS or a condition relating thereto (e.g., measurement of biomarkers reported in the literature and/or MRI imaging). Furthermore, the amount of change in a biomarker level may be an indication of the relatively efficacy of the drug.
• cystatin C is a cleavage product of cystatin C.
• a composition comprising a C- terminal cleavage product of cystatin C.
• the C-terminal cleavage product comprises a deletion of amino acids from the C-terminus.
• the cystatin C cleavage product is a cystatin C polypeptide that lacks exactly 8, about 8, or at least 8 amino acids at its C-terminus.
• Human cystatin C is a 146 amino acid polypeptide with Accession number NP 000090 on the NCBI website. The full-length human cystatin C polypeptide sequence is shown below:
• SEQ ID NO: 2 which consists of SEQ ID NO: 4 lacking exactly 8 contiguous amino acid residues from the C-terminus is shown below:
• polypeptides comprising, consisting of, or consisting essentially of an amino acid sequence that is at least about 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 2.
• the polypeptides preferably do not comprise the last 8 amino acids that are normally present in full-length cystatin C, i.e., the polypeptide does not comprise all or a portion of the amino acid sequence SKSTCQDA at its C-terminus.
• Certain polypeptides comprise an amino acid sequence that differs from SEQ ID NO: 2 in one or more, e.g., 1, 2, 3, 4, 5 or 10, amino acid substitutions, additions or deletions. The amino acid changes may be conservative amino acid changes.
• Polypeptides comprising at least 4, 10, 20, 30, 50, 100 or 130 contiguous amino acids of SEQ ID NO: 2 are also encompassed. Polypeptides may have a molecular weight of approximately between 7 kDa and 12.5 kDa.
• the polypeptides are preferably biologically active, i.e., they retain at least one biological activity of wild-type cystatin C, e.g., inhibition of cathepsin B.
• the polypeptides described in the previous paragraph may exhibit a biological function of a protein comprising an amino acid sequence consisting of SEQ ID NO: 2.
• Certain polypeptides have a stronger biological activity than wild-type cystatin C, e.g., at least about 50%, 2 fold, 3 fold, 5 fold or more stronger.
• Other polypeptides may have an activity that is similar or identical to that of wild-type cystatin C.
• Biological activity of cystatin C may be determined as described in the examples.
• cystatin C fragments lacking the N-terminal 8 amino acids
• cystatin C fragments lacking both the N-terminal 8 amino acids and the C-terminal 8 amino acids, e.g.,
• Proteins lacking the 8 N-terminal amino acids generally have a lower biological activity relative to the wild-type cystatin C.
• Homo logs of such protein e.g., comprising, consisting of, or consisting essentially of an amino acid sequence that is at least about 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 5 or 7 are also encompassed.
• a cystatin C polypeptide cleavage product may be linked, directly or indirectly to one or more amino acids or to one or more heterologous peptides, e.g., to form a fusion protein.
• Heterologous peptides may be peptides that can be used for detecting; purifying; stabilizing; or solubilizing the endostatin peptide.
• a heterologous peptide may be a TAG peptide or a His6 tag.
• a peptide or protein may by linked to an immunoglobulin (Ig) constant heavy or light chain domain or portion thereof.
• Ig immunoglobulin
• a peptide may be linked to a CHl, CH2 and/or CH3 domain of a heavy chain.
• the constant region is from a light chain, it may be from a kappa or lambda light chain. If the constant region is from a heavy chain, it may be from an antibody of any one of the following classes of antibodies: IgG, IgA, IgE, IgD, and IgM.
• IgG may be IgGl, IgG2, IgG3 or IgG4.
• the constant domain may be an Fc fragment.
• the constant domain may be from a mammalian antibody, e.g., a human antibody. Soluble receptor-IgG fusion proteins are common immunological reagents and methods for their construction are known in the art (see e.g., U.S. Pat. Nos.
• immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where dimerization between two heavy chains takes place at the hinge region. It is recognized that inclusion of the CH2 and CH3 domains of the Fc region as part of the fusion polypeptide increases the in vivo circulation half-life of the polypeptide comprising the Fc region, and that of the oligomer or dimer comprising the polypeptide.
• Constant Ig domains may also contain one or more mutations that reduce or eliminate one or more effector function, e.g., binding to Fc receptors and complement activation (see, e.g., S. Morrison, Annu. Rev. Immunol, 10, pp. 239-65 (1992); Duncan and Winter (1988) Nature 332: 738-740; and Xu et al. (1994) J Biol. Chem. 269: 3469- 3474).
• effector function e.g., binding to Fc receptors and complement activation
• the constant Ig domain may be linked to the N-terminus or C-terminus of a peptide.
• a peptide and heterologous peptides or moeity may also be linked through a linker sequence, which may be degradable, e.g., hydrolyzable.
• a linker may comprise a thrombin cleavage site.
• An exemplary nucleotide sequence encoding such a site has the following nucleotide sequence: 5' TCT AGA GGT GGT CTA GTG CCG CGC GGC AGC GGT TCC CCC GGG TTG CAG 3', which encodes a peptide having the amino acid sequence: Ser Arg GIy GIy Leu VaI Pro Arg GIy Ser GIy Ser Pro GIy Leu GIn.
• a peptide may also be fused to a signal sequence.
• a nucleic acid encoding the peptide may be linked at its 5' end to a signal sequence, such that the peptide is secreted from the cell.
• a peptide or protein may also be linked to a moiety, such as a polymer.
• the polymer need not have any particular molecular weight, but it is preferred that the molecular weight be between about 300 and 100,000, more preferably between 10,000 and 40,000. In particular, sizes of 20,000 or more are best at preventing protein loss due to filtration in the kidneys.
• Exemplary polymers include water-soluble degradable or non- degradable polymer.
• the polymer may be a copolymer comprising an acrylic polymer, alkene polymer, urethane polymer, amide polymer, polyimine, polysaccharide, or ester polymer.
• the polymer is polyglutamate, a polysaccharide such as dextran or dextrin-2-sulphate, polyvinylpyrolidone, a copolymer of divinylether and maleic anhydride (DIVEMA), or a copolymer of polethylene glycol and aspartic acid.
• the polymer is a linear or branched polyethylene glycol.
• a polymer may be a homopolymer of polyethylene glycol (PEG) or is a polyoxyethylated polyol, wherein, preferably, the polymer is soluble in water at room temperature.
• PEG polyethylene glycol
• Non-limiting examples of such polymers include polyalkylene oxide homopolymers such as PEG or polypropylene glycols, polyoxyethylenated glycols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymer is maintained.
• polyoxyethylated polyols include, for example, polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, or the like.
• a peptide may be bonded directly to a polymer or bonded to a polymer via a linking group.
• the polymer may be bonded to an amino acid at the N-terminus or the C-terminus of the peptide. In certain instances, the polymer is bonded to the nitrogen atom of the N- terminus amino acid of the peptide. Alternatively, the polymer may be bonded to the sulfur atom of a cysteine residue or to a lysine or arginine residue. Other sites are also possible.
• a protein, peptide or analog thereof may be labeled, such as with a marker that may be directly or indirectly detectable.
• An indirect marker is a marker that cannot be detected by itself but needs a further directly detectable marker specific for the indirect marker.
• Exemplary detectable labels include enzymes, dyes, radioisotopes, digoxygenin, biotin, and radioisotopes.
• a protein described herein may also be fused to a peptide that may, e.g., facilitate labeling of the protein or linking it to another moiety.
• an 11 -residue peptide with the sequence DSLEFIASKLA (“YBBR tag”) may be fused to the N- or C- terminus of the protein, or inserted, e.g., in a flexible loop, in the middle of the protein (Yin et al. (2005) PNAS 102:15815).
• Functionally homologous peptides which preferable form an alp ha- helix, may also be used.
• This peptide can then be labeled site specifically by Sfb- catalyzed small-molecule CoA modification.
• the following labels may be attached: biotin, glutathione, fluorescent probes such as fluorescein, Alexa Fluor dyes, and redox probes such as porphyrin. Labeling can be performed as described in Yin et al., supra.
• polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
• Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.
• Peptides and proteins may also comprise one or more non-naturally occurring amino acids.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into peptides.
• Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma- Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalan
• Peptidomimetics are compounds based on, or derived from, peptides and proteins. Peptidomimetics can be obtained by structural modification of known peptide sequences using unnatural amino acids, conformational restraints, isosteric replacement, and the like.
• the subject peptidomimetics constitute the continum of structural space between peptides and non-peptide synthetic structures; peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent peptides.
• Peptidomimetics based on more substantial modifications of the backbone of a peptide can be used.
• Peptidomimetics which fall in this category include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called peptoids).
• Peptides may comprise at least one amino acid or every amino acid that is a D stereoisomer.
• Other peptides may comprise at least one amino acid that is reversed.
• the amino acid that is reversed may be a D stereoisomer. Every amino acid of a peptide may be reversed and/or every amino acid may be a D stereoisomer.
• a peptidomimetic in another illustrative embodiment, can be derived as a retro- enantio analog of a peptide.
• Retro-enantio analogs such as this can be synthesized with commercially available D-amino acids (or analogs thereof) and standard solid- or solution- phase peptide-synthesis techniques, as described, e.g., in WO 00/01720. The final product may be purified by HPLC to yield the pure retro-enantio analog.
• peptide derivatives which are differentially modified during or after synthesis, e.g., by benzylation, glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
• the peptides are acetylated at the N-terminus and/or amidated at the C-terminus.
• Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or pho spho threonine .
• a peptide may also be fused to a signal sequence.
• a nucleic acid encoding the peptide may be linked at its 5' end to a signal sequence, such that the peptide is secreted from the cell.
• Peptides may be used as a substantially pure preparation, e.g., wherein at least about 90% of the peptides in the preparation are the desired peptide. Compositions comprising at least about 50%, 60%, 70%, or 80% of the desired peptide may also be used. The polypeptides may also be encompassed in pharmaceutical compositions, e.g., comprising a pharmaceutically acceptable vehicle.
• Polypeptides described herein may be used as immunogens for the production of antibodies.
• the polyptides may be in a composition with an adjuvant.
• Proteins corresponding to the other peaks described in Table 2 and Table 4 are also encompassed.
• Exemplary polypeptides are those associated with the 12.5 kDa peak (Table 2 and Table 4 (peak A)), the 3.9 kDa peak (Table 2 and Table 4 (peak F)), the 13.4 kDa peak (Table 2 and Table 4 (peak J)), the 13.6 kDa peak (Table 2 and Table 4 (peak N)), and the 4.7 kDa peak (Table 4 (peak Q)). Fragments and variants of such polypeptides are also included within the scope of the invention. Nucleic acids
• nucleic acids encoding a C-terminal cleavage product of cystatin C.
• the C-terminal cleavage product comprises a deletion of amino acids from the C-terminus.
• the cystatin C cleavage product is a cystatin C polypeptide that lacks exactly 8, about 8, or at least 8 amino acids at its C-terminus.
• one embodiment of the present invention is directed to an isolated polynucleotide which encodes a cleavage product of a cystatin C polypeptide, or fragment thereof, e.g., those described herein.
• Isolated polynucleotides that encode polypeptides with higher sequence homologies of, for example, 70%, 80%, 90%, 95% or 98%, which have the ability to inhibit cathepsin B activity moreso than full-length cystatin C are also contemplated by this invention.
• an isolated polynucleotide encodes a cystatin C cleavage fragment comprising an amino acid sequence consisting essentially of SEQ ID NO: 2.
• the present invention is directed to an isolated polynucleotide which encodes a polypeptide comprising at least 4, 10, 20, 30, 50, 100 or 130 contiguous amino acids of SEQ ID NO: 2, wherein said polypeptide does not comprise the last 8 amino acids of full-length cystatin C and has the ability to inhibit cathepsin B activity moreso than full-length cystatin C.
• the isolate polynucleotide encodes a polypeptide representing a cystatin C cleavage product with activity similar to or identical to SEQ ID NO: 2.
• the isolated polynucleotide encodes a cleavage product, or fragment thereof, of cystatin C having a molecular weight of approximately between 7 kDa and 12.5 kDa and exhibiting a similar or identical biological function of SEQ ID NO: 2.
• the polynucleotide encodes a polypeptide as set forth above having the ability to modulate, e.g. capable of inhibiting cathepsin B activity moreso than full-length cystatin C.
• the invention is also directed to polynucleotides encoding derivatives or analogs of the cystatin C cleavage products which are functionally active, i.e., capable of inhibiting cathepsin B activity moreso than full-length cystatin C.
• the polynucleotide encodes a polypeptide cleavage product in the form of a fusion protein.
• a further aspect of the invention is the use of the polynucleotide as set forth above for the production of a polypeptide to be used as an immunogen for the production of antibodies. Methods of production of the cleavage product, e.g. by recombinant means, are provided.
• the invention relates to a recombinant cell harboring a polynucleotide described herein, capable of producing a polypeptide described above, (e.g. SEQ ID NO: 2).
• the present invention relates to use of such a recombinant cell for the production of recombinant cystatin C cleavage product.
• a cystatin C cleavage product may be expressed from a polynucleotide encoding a cleavage product, or from a nucleic acid expressing a cystatin C polypeptide and thereafter cleaving the cystatin C by proteolytic digest to produce the cleavage product.
• the human cystatin C cDNA is an 818 nucleotide sequence with Accession number NM 000099 on the NCBI website.
• the open reading frame encoding full-length cystatin C consists of nucleotides 76 to 490 and is shown below:
• the invention relates to an isolated polynucleotide, or fragment thereof, encoding any of the cystatin C polypeptides described above.
• the invention relates to an isolated polynucleotide having a polynucleotide sequence with at least 60% identity to the polynucleotide sequence of SEQ ID NO: 1, wherein the polynucleotide sequence does not encode a polypeptide comprising the last 8 amino acids of full-length cystatin C.
• the polypeptide has the ability to inhibit cathepsin B activity moreso than full-length cystatin C. Isolated nucleic acids with higher sequence homologies of, for example, 70%, 80%, 90%, 95% or 98% with similar function are also contemplated by this invention.
• the invention in another embodiment, relates to an isolated polynucleotide encoding a fragment of a cystatin C polypeptide, which fragment comprises a contiguous stretch of at least 4, 10, 20, 50, 100 or 130 amino acids of SEQ ID NO: 2, wherein said polypeptide does not comprise the last 8 amino acids of full-length cystatin C.
• the recited fragment has the ability to inhibit cathepsin B activity more than full-length cystatin C.
• the polynucleotide may encode a fragment that has a molecular weight between about 7 kDa and 12.5 kDa.
• the polynucleotide as set forth above relates to the nucleic acid sequence for a cystatin C cleavage product as described above, including the genomic sequence, mRNA or cDNA, polymorphic, allelic, isoforms and mutant forms thereof, and nucleic acid constructs of the gene, including vectors, plasmids and recombinant cells and transgenic organisms containing or corresponding to cystatin C cleavage product.
• Nucleic acids include vectors, such as expression vectors for producing a peptide, e.g., viral vectors. Also encompassed herein are cells comprising a nucleic acid encoding a peptide described herein and methods for producing peptides comprising culturing these cells to produce a peptide. These methods can be used of producing recombinant peptides or for expression of a petpide in a cell, e.g., in a cell of a subject.
• Appropriate vectors may be introduced into host cells using well known techniques such as infection, transduction, transfection, transvection, electroporation and transformation.
• the vector may be, for example, a phage, plasmid, viral or retroviral vector.
• Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
• the vector may contain a selectable marker for propagation in a host.
• a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• Preferred vectors comprise cis-acting control regions to the polynucleotide of interest.
• Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
• the vectors provide for specific expression, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
• Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
• vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses and vectors derived from combinations thereof, such as cosmids and phagemids.
• the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• Other suitable promoters will be known to the skilled artisan.
• the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating site at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
• the expression vectors will preferably include at least one selectable marker.
• markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin, or ampicillin resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• vectors preferred for use in bacteria include pQE70, pQE60 and pQE9, pQElO available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A available from Stratagene; pET series of vectors available fromNovagen; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
• eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
• Other suitable vectors will be readily apparent to the skilled artisan.
• bacterial promoters suitable for use in the present invention include the E. coli lad and lacZ promoters, the T3, T5 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, the trp promoter and the xyl/tet chimeric promoter.
• Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
• Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals (for example, Davis, et al., Basic Methods In Molecular Biology (1986)).
• Enhancers are cis-acting elements of DNA, usually about from 10 to 300 nucleotides that act to increase transcriptional activity of a promoter in a given host cell-type.
• enhancers include the SV40 enhancer, which is located on the late side of the replication origin at nucleotides 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
• a recombinant soluble form of a polypeptide of the present invention may also be produced, e.g., by deleting at least a portion of the transmembrane domain, such that the protein is not capable to localize itself to a cell membrane.
• nucleic acids encoding splice variants or nucleic acids representing transcripts synthesized from an alternative transcriptional initiation site such as those whose transcription was initiated from a site in an intron.
• Such homologues can be cloned by hybridization or PCR using standard methods known in the art.
• the polynucleotide sequence may also encode for a leader sequence, e.g., the natural leader sequence or a heterologous leader sequence.
• the nucleic acid can be engineered such that the natural leader sequence is deleted and a heterologous leader sequence inserted in its place.
• leader sequence is used interchangeably herein with the term "signal peptide”.
• the desired DNA sequence may be fused in the same reading frame to a DNA sequence which aids in expression and secretion of the polypeptide from the host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of the polypeptide from the cell.
• the protein having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the protein.
• secretion signals may be incorporated into the expressed polypeptide, for example, the amino acid sequence KDEL.
• the signals may be endogenous to the polypeptide or they may be heterologous signals.
• the invention relates to a recombinant cell producing a polypeptide described above, (e.g. SEQ ID NO: 2), preferably a recombinant form of the polypeptide.
• the present invention relates to use of such a recombinant cell for the production of recombinant cystatin C cleavage product.
• a cystatin C cleavage product may be expressed from a nucleic acid expressing the same, or from a nucleic acid expressing a cystatin C polypeptide and thereafter cleaving the cystatin C by proteolytic digest to produce the cleavage product.
• Antibodies binding specifically to the biomarkers described herein, e.g., in Table 2 and Table 4, are also encompassed herein.
• the present invention provides antibodies that bind with high specificity to the cystatin C cleavage product polypeptides provided herein.
• antibodies that bind to a polypeptide consisting of SEQ ID NOs: 2 or 4 are provided.
• antibodies generated against the full length polypeptide or cleavage product antibodies may also be generated in response to smaller constructs comprising epitopic core regions.
• Antibodies that bind to any of the polypeptides described above are also provided.
• Antibodies may bind essentially only to a full-length cystatin C, e.g., an antibody may bind specifically to an epitope that is absent in the cleavage product (e.g. SEQ ID NO: 2), e.g., an antibody to an cystatin C epitope within the C-terminal most 8 amino acids of the full-length polypeptide, without significant cross-hybridization to the cleavage product. Other antibodies may detect both types of proteins. An antibody that binds to both a cystatin C full-length and C-terminal cleavage product may bind to an epitope within the region of amino acids 1-138 of the full-length, such as an N-terminal region.
• an antibody may bind specifically to an epitope that is absent in the cleavage product (e.g. SEQ ID NO: 2), e.g., an antibody to an cystatin C epitope within the C-terminal most 8 amino acids of the full-length polypeptide, without significant cross-
• An antibody may specifically recognize the C-terminal cystatin C cleavage product.
• the antibody may only bind to the C-terminal cystatin C cleavage product (e.g., SEQ ID NO: 2), without significant cross-hybridization to the full-length cystatin C polypeptide (e.g, SEQ ID NO: 4).
• the antibody may be used to determine the level of the C-terminal cleavage product.
• the term "antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
• the term “antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), hybrid antibodies, chimeric antibodies, humanized antibodies and the like.
• the techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
• Humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
• Monoclonal antibodies are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred.
• the invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.
• a wide range of animal species can be used for the production of antisera.
• the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
• the present invention further provides antibodies against cystatin C cleavage products, generally of the monoclonal type, that are linked to one or more other agents to form an antibody conjugate. Any antibody of sufficient selectivity, specificity and affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art.
• antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
• Detectable labels are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired.
• Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, as may be termed "immunotoxins" (described in U.S. Pat. Nos. 5,686,072, 5,578,706, 4,792,447, 5,045,451, 4,664,911 and 5,767,072, each incorporated herein by reference).
• Antibody conjugates are thus preferred for use as diagnostic agents in the methods described herein.
• Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging.” Again, antibody-directed imaging is less preferred for use with this invention.
• Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated herein by reference). Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the antibody (U.S. Pat. No.
• Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
• a coupling agent such as glutaraldehyde or periodate.
• Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
• Antibody conjugates may be used in vivo or in vitro.
• the antibody may be linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
• suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
• Preferred secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
• a method may comprise contacting cystatin C or a biologically active fragment thereof with an agent and determining whether (a) the agent binds to cystatin C or the biologically active fragment thereof and/or (b) the agent inhibits the activity of cystatin C.
• a biologically active fragment may be a cystatin fragment lacking one or more amino acids at its C-terminus, e.g., as further described herein.
• a method may further comprise determining whether it can prevent or treat MS, an early stage thereof, or a condition relating thereto, e.g., in an animal model.
• An agent may be, e.g., an antibody or a molecule, e.g., a small molecule.
• kits for treating or preventing multiple sclerosis comprising administering to an individual afflicted therewith or in need thereof a therapeutically effective amount of an agent that modulates the expression, levels, or activity of a biomarker provided herein.
• methods of treating multiple sclerosis comprising administering to an individual afflicted therewith or in need thereof a therapeutically effective amount of an agent which decreases cathepsin activity.
• the cathepsin activity decreased is cathepsin B activity.
• the agent inhibits the activity of cystatin C.
• the agent inhibits the activity of cystatin C by inhibiting the proteolytic cleavage of cystatin C.
• the agent may be a protease inhibitor, which prevents cleavage of the 8 C-terminal most amino acids of cystatin C, thereby inhibiting the activity of cystatin C.
• the agent may inhibit the production of SEQ ID NO: 2.
• the agent may be an antibody, peptide, small molecule, or mimetic which binds to the cleavage site, thereby blocking cleavage of the C terminus of cystatin C, which in turn inhibits the production of SEQ ID NO: 2. Kits
• kits for diagnosing MS, an early stage thereof, or a condition relating thereto, monitoring progression of the disease or assessing response to therapy may comprise an agent for detecting or measuring one marker or a combination of two or more markers.
• a kit may comprise a reagent that specifically binds to a molecule selected from the group consisting of the molecules set forth in Table 2 and Table 4.
• Kit development may require specific antibody development, evaluation of the influence (if any) of matrix constituent ("matrix effects"), and assay performance specifications.
• a kit may comprise a container for sample collected from a patient and a marker specific reagent.
• MS multiple sclerosis
• Tandem mass spectroscopy of a tryptic digest of this 12.5 kD protein identified it as a cleavage product of full-length cystatin C (13.4 kD), an important inhibitor of cysteine proteases including the cathepsins. While total cystatin C levels in the MS patients was not different compared to controls, the patients with the highest 12.5/13.4 peak ratios also had the greatest cathepsin B inhibitory activity. This suggests that cleavage of cystatin C may be an adaptive host response and may identify a subgroup of patients with MS.
• MS multiple sclerosis
• MRI magnetic resonance imaging
• CSF cerebrospinal
• CSF from five patients was analyzed by two dimensional gel electrophoresis. Despite the small sample size, 15 proteins were found to be differentially expressed in the CSF of MS patients compared to controls 2 .
• CSF samples by SELDI time of flight mass spectroscopy from a larger sample size of well-characterized patients and controls. Analysis of CSF has several advantages over serum for biomarker discovery in neurological disease. CSF better represents local events in the brain as compared to serum. Further, high abundance proteins in serum may mask the low abundant, low molecular weight proteins that are the likely candidates for biomarkers.
• CSF was considered inflammatory in the control samples if one or more of the following abnormalities were present: white cell count >5 cells/mm 3 , detectable oligoclonal bands or IgG index >0.8.
• CSF samples from another 27 patients with acute transverse myelitis (TM) and 50 patients with HIV infection (22 without dementia and 28 with dementia) were used as other controls. All patients except three with TM had an inflammatory CSF but none had oligoclonal bands or an elevated IgG index.
• Samples from HIV infected patients were taken from the prospectively followed North Eastern AIDS dementia cohort 5 . None of the patients had opportunistic infections.
• T2 hyperintense lesions, Tl hypointense lesions, and gadolinium-enhancing Tl lesions meeting a > 3 mm size cutoff criteria was determined from each scan by a single blinded examiner. Each scan was also judged as to whether it met the formal requirements for an abnormality consistent with MS according to published criteria 3 ' 4 .
• CSF samples were handled equally and placed immediately on ice and centrifuged at 3000 rpm for 10 min. The cell free samples were then stored at -8O 0 C in 0.5 ml aliquots.
• a single aliquot of CSF was thawed and immediately realiquoted into 50 ⁇ l volumes and refrozen at -8O 0 C. Each sample was thawed once more before analysis.
• CSF samples were initially analyzed using the weak cation exchange (CMlO) and the hydrophobic chip (H50) protein chips (Ciphergen Biosystems, Freemont, CA).
• CMlO chips bound proteins with specific physio-chemical properties, which were then resolved by SELDI time of flight mass spectroscopy (Ciphergen Biosystems, Freemont CA). Spectra derived from CMlO chips showed a greater number of peaks and a better resolution of low molecular mass species and were used in all subsequent assays.
• Each spot on the array was then incubated with 15 ⁇ l ofCSF diluted in binding buffer to a final volume of 150 ⁇ l with gentle agitation for one hour at room temperature.
• the spots were washed in the same buffer three times, after which l ⁇ l of 50% saturated sinapinic acid (SPA) dissolved in 50% acetonitrile, 0.5% trifluro acetic acid solution was added.
• SPA sinapinic acid
• the chips were air-dried and SPA reapplied.
• the protein chips were analyzed in the ProteinChip® biology systems reader (model PBSIIc, Ciphergen Biosystems) using a laser intensity of 2.6 microJoules and a sensitivity setting of 5.
• Resulting spectra were noise filtered, baseline substracted, and calibrated with Ciphergen's "All-in-One Protein standard" consisting of cytochrome C (12,360.2 Daltons), myoglobin (16,951.5 Daltons), and GAPDH (35,688 Daltons).
• Biochemical properties of the unique peaks identified in CSF samples were further characterized by changing the pH of the binding buffer (range 4.0-9.0). The stability of these peaks was also determined by monitoring the effects of freeze/ thaw cycles on the CSF, heating of samples to 5O 0 C for 30 min or leaving them at room temperature for 16 hrs. Each sample was analyzed in duplicate.
• a single CSF sample (MS267) that had a prominent 12.5 kD peak was selected for further study.
• Tricine sample buffer Biorad, Hercules, CA.
• Tricine sample buffer Biorad, Hercules, CA.
• the anode buffer consisted of 0.2 M Tris-HCl, pH 8.9
• the cathode buffer consisted of 0.1 M Tris-HCl, 0.1 M Tricine, 0.1% SDS, pH 8.3.
• Samples were diluted in 10 mL of 50 mM Tris-HCl, 4% w/v SDS, 12% w/v sucrose, 5% v/v ⁇ -mercaptoethanol, and a trace of bromophenol blue, pH 6.8. After denaturation at 97 0 C for 5 min, samples were loaded onto the gel with 30 ⁇ l/lane. Gels were run at 200 mamps for 3 hr. After electrophoresis, gels were fixed, stained with a Silver Stain Plus Kit (Biorad, Hercules, CA), and dried between 2 pieces of cellophane.
• a Silver Stain Plus Kit Biorad, Hercules, CA
• the 12.5 kD band was excised following silver staining of the gel. Tryptic digestion and peptide extraction were performed on the excised band 6 .
• the gel band was destained in 15 mM potassium ferricyanide/ 50 mM sodium thio sulfate followed by washing with water and dehydration with acetonitrile.
• the isolated gel band was then incubated for 45 min at 55°C with 10 mM dithithreitol followed by incubation with 55 mM iodoacetamide for 30 min at room temperature. The sample was then washed and dehydrated with alternating washes of 5mM ammonium bicarbonate followed by acentonitrile.
• tryptic digestion was performed with 12.5 ⁇ g/ml trypsin in 5 mM ammonium bicarbonate overnight at 37°C.
• Peptides were extracted with successive incubations of 25 mM ammonium bicarbonate, followed by 5% formic acid and then acetonitrile. Samples were dried, cleaned and concentrated using an OMIX C 18 pipette tip according to manufacturer's instructions (Varian, Palo Alto, CA).
• An Axima CFR MALDI-TOF mass spectrometer (Kratos, Manchester, UK) was used for protein identification and accurate mass measurements. 2 ⁇ l of the cleaned peptides along with 125 fmol of a three-point calibrant mixture were spotted via the dried droplet method with 0.3 ⁇ l saturated ⁇ -Cyano-4-hydroxycinnamic acid (CHCA) (Sigma, St. Louis, MO) in 50% ethanol/50% ddF ⁇ O. Internal calibration was applied and the monoisotopic masses of the tryptic digest peaks were acquired. Tandem mass spectrometry (MS/MS) was performed on selected peaks.
• CHCA saturated ⁇ -Cyano-4-hydroxycinnamic acid
• a sandwich ELISA was used to measure cystatin C levels in the CSF samples according to the manufacturers instructions (Alexis Biochemicals, San Diego, CA). Each CSF sample and standard was analyzed in duplicate. Concentration of cystatin C in each CSF sample was determined using a standard curve and expressed as relative fluorescence units.
• Cathepsin B activity Cathepsin B activity, a known substrate of cystatin C, was measured using an activity assay kit (Bio vision Research Products, Mountain View, CA). This fluorescence-based assay utilizes the preferred cathepsin-B substrate sequence Arg-Arg labeled with amino-4-trifluoromethyl coumarin (AFP). Cathepsin-B cleaves the synthetic substrate RR-AFC to release free AFC.
• THP-I cells (a monocytic cell line) were used as a source of cathepsin B.
• Cell lysates were prepared using a lysis buffer provided with the assay kit. Cell lysates from 1x10 6 cells were added to 50 ⁇ l of CSF in a microtiter plate (q.s. 100 ⁇ l). Two ⁇ l of substrate Ac- Arg-Arg- AFC was added to each well and incubated for 1 hr at 37 0 C. Absorbance was measured using a fluorescent plate reader with a 400 nm excitation filter and 505 nm emission filter. Controls included reaction buffer alone and a cathepsin B inhibitor provided in the kit. All samples were analyzed in duplicate.
• a peak at 3.9 kD (Table 2 provided on line) was also significantly elevated in the patients with MS/CIS, however the peak height was small and had only a two-fold increase in the MS/CIS patients compared to controls. Hence we have not pursued the identity of these proteins at this point.
• the 12.5 kD peak was present in 19/29 MS/CIS patients and in none of the patients with OND or TM. Its presence alone provided 100% specificity but only 66% sensitivity for diagnosis of MS when compared to these diseases.
• the 12.5 kD peak was found in some patients with HIV infection, the levels were small and significantly lower when compared to the MS/CIS patients.
• cystatin C is a protease inhibitor that specifically blocks cathepsin B activity
• cathepsin B activity was found suggesting that the cystatin C in the CSF of MS/CIS patients is bioactive (Figure 6A).
• CSF samples are a reliable biological specimen for SELDI analysis in search for biomarkers of MS.
• a distinct technical advantage of using CSF over serum is that it does not require pre-clearing of large and abundant proteins in serum that may mask the proteins of interest, which are usually present at much lower concentrations.
• the samples are also more likely to represent local events within the CNS compared to serum.
• the 12.5 kD peak is a breakdown product of the 13.4 kD peak.
• the intensity of the 12.5 kD peak and that of the 13.4 kD peak seem to be reciprocally related to each other and the sequence analysis of the 12.5 kD peak revealed that it corresponds to cystatin C, which is known to have a molecular mass of 13.4 kD 7 .
• Heating the CSF had no effect on the levels of the 12.5 kD and 13.4 kD peaks, while repeated freeze thaw cycles and overnight storage of CSF at room temperature resulted in a slight increase in the 12.5 peak intensity which suggests that heat treatment may denature the protease that cleaves the 13.4 kD protein into the 12.5 kD form (the 12.5 kDa peak resulting from the freeze/thaw is a different species than the peak in CSF of MS patients).
• Cystatin C is an inhibitor of cysteine proteases including cathepsins B, H, K, L and S. 8 It is present in high concentrations in CSF compared to serum and other body fluids 9 .
• the protein is a non-glycosylated molecule of 120 amino acids formed after removal of a 26 amino acid signal peptide 10 .
• cystatin C would also result in dysregulation of cathepsin function which have been implicated in a variety of effects including degranulation of cytotoxic lymphocytes ⁇ and in processing of MHC class II antigen in monocytes 12 .
• a previous study that measured cystatin C levels in CSF of MS patients by ELISA also found diminished levels in patients compared to healthy controls.
• cystatin C levels are increased in the CSF of patients with Alzheimer's disease 7 and Creutzfeldt- Jacob disease 15 .
• CSF was analyzed by SELDI and the 13.4 kD protein was further sequenced to identify it as cystatin C.
• a mutated form of cystatin C (Leu68Gln substitution) has been found.
• This protein accumulates in the amyloid deposits and is truncated by 10 amino acids at the amino terminal 16 . This region is critical for the functional activity of cystatin C 10 .
• Leukocyte elastase has been shown to cleave cystatin C at Valio-Gly ⁇ resulting in loss of its ability to bind to cathepsins 17 .
• one of the peptides from the tryptic digest of the 12.5 kD protein that matched to cystatin C contained an intact Leucr VaI 10 - Glyn and an intact amino terminal region suggesting the presence of a novel cleavage site at the carboxy terminal in the MS patients.
• the mass differences between the 12.5 kD and 13.4 kD proteins suggest that the cleavage site is at eight amino acids from the carboxy terminal end of the protein.
• cystatin C The role of cystatin C in the pathogenesis of MS is not understood. Elevated serum cystatin C levels have recently been shown to be a strong predictor of death in patients with cardiovascular disease 18 . We did not find any significant difference in the total cystatin levels in the MS/CIS patients compared to controls. Our data suggest that the total levels of cystatin C are inversely proportional to cathepsin B activity. Furthermore it appears that cleavage of cystatin C did not lead to any augmentation of cathepsin B activity. In fact, the patients with the highest 12.5/13.4 ratios seemed to have the highest cathepsin B inhibition activity as well. This raises the possibility that cleavage of cystatin C at the carboxy terminus may lead to enhanced activity of this protein.
• cystatin C and its breakdown product in the CSF of MS patients may identify a subtype of MS.
• larger sample sizes from MS patients at different stages of disease are needed to further validate our observations.
• the absence of the cystatin C cleavage product in the CSF of patients with TM and other neuroinflammatory diseases suggests that inflammation alone is not sufficient for cleavage of this protein. Therefore, this cleavage product may not only identify a subgroup of patients with MS/CIS but it may also be able to separate these patients from other inflammatory diseases.
• Example 2 Novel Cystatin C cleavage site in patients with Multiple Sclerosis Abstract: The effect of storage and freeze thaw cycles on cystatin C was examined in CSF. This resulted in cleavage of the cystatin C at eight amino acids from the N terminal and a resulting loss in its effect on cathepsin B activity. In contrast, a distinct smaller peak was noted in the CSF of patients with remitting relapsing Multiple Sclerosis resulting from the cleavage of eight amino acids from the C terminal region of the protein. When recombinant cystatin C was cleaved the C terminal region, an enhancement of the cystatin C activity was noted. Thus the cystatin C fragment in patients with Multiple Sclerosis is molecularly and functionally distinct.
• MMPs 5 ' 6 and cathepsins 7 have been shown to be altered in patients with remitting relapsing MS.
• All CSF samples used in our work were centrifuged immediately upon collection and then frozen at -8O 0 C within 2 hours until used for this analysis. Each sample was collected from the same clinic at our institution and handled and stored by the personnel in our laboratories using the same protocol.
• we examined the endogenous degradation of cystatin C No changes in cystatin C were noted for up to 4 hours at room temperature. While cleavage of cystatin C was noted by leaving the sample overnight, no changes were noted in the protein with heating at 6O 0 C for one hour.
• the protein inhibitor cocktail (Sigma) had no significant effect on the cleavage of cystatin C, while both FN-439 (500ug/ml), a MMP specific inhibitor, and pepstatin A (500ug/ml), a cathepsin D specific inhibitor, showed inhibition of the breakdown of cystatin C.
• Recombinant cystatin C was produced and the ability of MMP-2 and cathepsin D to cleave the recombinant protein was confirmed.
• cathepsin D cleaved cystatin C at four different sites, and yielded a 12.5 kD fragment following cleavage from the C terminal region 9 .
• MMP-2 cleaved cystatin C at three unique sites (GK, FC, and GT which correspond to amino acids 4,5; 96,97 and 108,109).
• Recombinant cystatin C was treated with either MMP-2 or cathepsin D and its activity monitored by a cathepsin B functional assay using a kit from Biovision Research Products, Mountain View, CA.
• cystatin C showed significant inhibition of cathepsin B.
• Treatment of cystatin C with cathepsin D showed further decrease in cathepsin B activity; in keeping with our previous observation that CSF of MS patients with the 12.5 kD fragment also showed a similar enhancement of cystatin C activity.
• treatment of cystatin C with MMP-2 lead to a decrease in its ability to inhibit cathepsin B activity.
• Cathepsin D and MMP-2 alone had no effect on cathepsin B activity (Figure 9).
• This cleavage product also has an apparent mass of 12.5 kD which is similar to the mass of the fragment generated by N terminal cleavage and the resolution of the mass spectrometer by Ciphergen is not sufficient to clearly distinguish between the two peaks. This may explain the rather broad base of the peaks seen by both Nakashima et al, and Hansson et al, which could represent the combination of the N and C terminal products 13 . In contrast, the peaks that we found with the CMlO chip were sharper, much larger and more distinct 4 . We further used an Axima CFR MALDl-TOF mass spectrometer to distinguish between the two peaks, since this instrument provides a much greater mass accuracy.

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