WO2006100446A2 - Method of diagnosing interstitial lung disease (ild) - Google Patents

Method of diagnosing interstitial lung disease (ild) Download PDF

Info

Publication number
WO2006100446A2
WO2006100446A2 PCT/GB2006/000981 GB2006000981W WO2006100446A2 WO 2006100446 A2 WO2006100446 A2 WO 2006100446A2 GB 2006000981 W GB2006000981 W GB 2006000981W WO 2006100446 A2 WO2006100446 A2 WO 2006100446A2
Authority
WO
WIPO (PCT)
Prior art keywords
ild
peptides
diagnostic test
peptide
protein
Prior art date
Application number
PCT/GB2006/000981
Other languages
French (fr)
Other versions
WO2006100446A3 (en
Inventor
Toshihide Nishimura
Atsushi Ogiwara
Takeshi Kawamura
Takao Kawakami
Yutaka Kyono
Mitsuhiro Kanazawa
Fredrik Nyberg
György MARKO-VARGA
Hisae Anyoji
Original Assignee
Astrazeneca Uk Limited
Medical Proteoscope Co. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0505960A external-priority patent/GB0505960D0/en
Priority claimed from GBGB0519759.5A external-priority patent/GB0519759D0/en
Application filed by Astrazeneca Uk Limited, Medical Proteoscope Co. Ltd. filed Critical Astrazeneca Uk Limited
Publication of WO2006100446A2 publication Critical patent/WO2006100446A2/en
Publication of WO2006100446A3 publication Critical patent/WO2006100446A3/en

Links

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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Definitions

  • Interstitial lung disease is a well-known co-morbidity in patients with non-small- cell lung cancer (NSCLC).
  • NSCLC non-small- cell lung cancer
  • ILD has been reported in patients with various chemotherapies and radiotherapy, as well as Gefitinib treatment. Frequencies of ILD in NSCLC from 3% to 30% following single or combination therapies for NSCLC have been reported. The mechanism causing and the risk factors contributing to ILD are unclear.
  • Gefitinib possesses anti-proliferative activity such as anti-cancer activity and, accordingly, is useful in methods of treatment of proliferative disease such as cancer in the human or animal body.
  • Gefitinib was approved in Japan on 5 July 2002 for treatment of inoperable or recurrent NSCLC. The early experience of gefitinib in Japan was encouraging, with responses seen in patients in phase I-II clinical trial and favorable safety profile compared with cytotoxic drugs.
  • proteins or fragments thereof can be used as makers for predicting the onset of ILD in a patient, particularly in a patient that is being treated with an EGFR tyrosine kinase inhibitor such as gefitinib.
  • upregulation or downregulation of the levels on these proteins, or their peptides, in biological fluids can be used as an indicator of susceptibility to ILD in a patient.
  • a diagnostic test to detect ILD or to identify the susceptibility of a patient to develop ILD which comprises measurement of one or more of the peptides shown below, or a protein comprising one or more of said peptides, in a biological sample obtained from the patient:
  • Peptides comprising Methionine include Oxidation of M (methionine) referring to the oxidation of sulphur to a sulphur oxide - see TABLE 1 hereinbelow.
  • Peptides comprising Oxidation of methionine include DLFGGRLTSDMIAER and
  • the diagnostic test is one wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of an elevated level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
  • a diagnostic test is one wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of a reduced level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
  • a diagnostic test detecting elevated peptide or protein levels as described above further comprises measurement of a reduced level of one or more of the peptides defined above or a protein comprising one or more of said peptides, compared to the level of said peptide or protein in patients without ILD.
  • a diagnostic test described hereinabove comprises measuring one or more proteins comprising one or more of the said peptides.
  • Preferred proteins are selected from
  • a diagnostic test for a protein is measured using an immunoassay.
  • suitable assay methodologies are described in the following references, which are incorporated herein by reference in their entirety:
  • the diagnostic test is to determine susceptibility to ILD following administration of a drug to a patient; preferably the drug is an erb receptor drug or a DMARD and especially the drug is gefitinib.
  • the drug is an erb receptor drug or a DMARD and especially the drug is gefitinib.
  • Alternative embodiments of the invention are contemplated as set out below:
  • a method of selecting a patient for therapy with a drug preferably gefitinib; or
  • a method of carrying out a clinical trial comprising patient selection and/or monitoring using a diagnostic method as described herein.
  • Intact proteins can be assayed in a number of ways utilizing both gel-based as well as liquid phase separation technologies. Two-dimensional gel electrophoresis is used with solubilised protein mixtures where the proteins are separated based upon charge and size [1-3]. The proteins are resolved such that both isomeric forms, as well as post-translational modifications, are resolved. Quantitation of the proteins is made by staining techniques, where both pre- and post staining techniques can be applied. Metabolic labelling also allows the linear range to be extended up to 5 orders of magnitude, offering sensitivities within the femtomolar range [4].
  • Protein identification is performed from excised gel spots.
  • the proteins are digested after chemical degradation and modification.
  • the resulting peptide mixtures are extracted from the isolated gel sample and subsequently identified by mass spectrometry [5-7].
  • Multidimensional HPLC High Performance Liquid Chromatography
  • HPLC High Performance Liquid Chromatography
  • the protein or peptide mixture is passed through a succession of chromato graphic stationary phases or dimensions which gives a higher resolving power.
  • HPLC is flexible for many experimental approaches and various stationary and mobile phases can be selected for their suitability in resolving specific protein or peptide classes of interest and for compatibility with each other and with downstream mass spectrometric methods of detection and identification.
  • High Performance Liquid Chromatography is currently the best methodology for solute separations which also allows for automated operation with a high degree of reproducibility [12-19]. On-line configurations of these types of multi-mechanism separation platforms are commonly applied within proteomics studies today [20-22].
  • MS Mass spectrometry
  • MS mass spectrometry
  • MALDI matrix-assisted laser desorption ionization
  • ESI electrospray ionization
  • TOF time-of-flight
  • peptides are co-crystallized with the matrix, and pulsed with lasers. This treatment vaporizes and ionizes the peptides. The molecular weights (masses) of the charged peptides are then determined in a TOF analyzer. In this device, an electric field accelerates the charged molecules toward a detector, and the differences in the length of time it takes ionized peptides to reach the detector (their time-of-flight) reveal the molecular weights of the peptides; smaller peptides reach the detector more quickly. This method generates mass profiles of the peptide mixtures - that is, profiles of the molecular weights and amounts of peptides in the mixture.
  • MS can therefore be used to measure a wide range of molecule types (proteins, peptide, or any other bio-molecules) and a wide range of sample types/biological materials.
  • Correct sample preparation is known to be crucial for the MS signal generation and spectra resolution and sensitivity. Sample preparation is therefore a crucial area for overall feasibility and sensitivity of analysis.
  • Proteins are bio-macro molecules that are difficult to separate by liquid phase chromatographic separation techniques, due to the unfavorable mass transfer within the particles of the chromatographic column material, the stationary phase [23].
  • proteins can be rendered into smaller unit form by breaking the peptide bond joining two adjacent amino acids. This can be accomplished by enzymatic cleavage by certain other proteins that are capable of interacting and dissolving peptide bonds on other proteins. Trypsin is the most commonly used protease, used in protein expression analysis studies. After the enzymatic degradation, a resulting complex mixture of peptides will be separated and fractionated by capillary chromatography. AU peptides that are the sum of the digested proteins in the sample will be unresolved at this stage.
  • the peptides that has generated from the corresponding protein will not be separated as one unit in the chromatographic fractionation step, but rather be separated together with the resulting peptides from all other proteins in the sample.
  • the high resolved and separated eluting peptides from the capillary will be fractioned most commonly based upon charge and hydrophobicity.
  • the separated peptides are introduced on-line from the chromatographic part of the platform into the mass spectrometer, thereby circumventing possible contaminations.
  • the peptides are then mass determined (m/z), in order to capture all the peptides present in that given time window.
  • a number of peptide masses are selected for sequencing (MS/MS), based upon their abundance in the given time window.
  • Electrospray is a gentle source that can ionize important analytes such as peptides, and proteins.
  • Time-delayed fragmentation product ion spectra typically display reduced sequential fragmentation products leading to spectra that are more easily interpreted.
  • Several important experimental parameters important to time-delayed fragmentation have been identified and are discussed. The technique has applications for both small precursor ions and multiply charged peptides [26-30].
  • Tandem mass spectrometry is at the heart of most of modern mass spectrometric investigations of complex mixtures.
  • the fragmentation involves activation of a precursor ion via collisions with a target gas and may produce charged and neutral fragments.
  • the nature of the fragment ions, as well as their intensities, is often indicative of the structure of the precursor ion and thus can yield useful information for the identification of unknown analytes, as well as providing a useful screening technique for different classes of analytes.
  • Activation via multiple collisions both prolongs the activation time and enables higher energies to be deposited into precursor ions.
  • Higher collision gas pressures also imply higher collision relaxation rates.
  • biomarkers Various biological markers, known as biomarkers, have been identified and studied through the application of biochemistry and molecular biology to medical and toxicological states. Biomarkers can be discovered in both tissues and biofluids, where blood is the most common biofluid used in biomarker studies [36-38].
  • a biomarker can be described as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention".
  • a biomarker is any identifiable and measurable indicator associated with a particular condition or disease where there is a correlation between the presence or level of the biomarker and some aspect of the condition or disease (including the presence of, the level or changing level of, the type of, the stage of, the susceptibility to the condition or disease, or the responsiveness to a drag used for treating the condition or disease). The correlation may be qualitative, quantitative, or both qualitative and quantitative.
  • a biomarker is a compound, compound fragment or group of compounds. Such compounds may be any compounds found in or produced by an organism, including proteins (and peptides), nucleic acids and other compounds.
  • Biomarkers may have a predictive power, and as such may be used to predict or detect the presence, level, type or stage of particular conditions or diseases (including the presence or level of particular microorganisms or toxins), the susceptibility (including genetic susceptibility) to particular conditions or diseases, or the response to particular treatments (including drug treatments). It is thought that biomarkers will play an increasingly important role in the future of drag discovery and development, by improving the efficiency of research and development programs. Biomarkers can be used as diagnostic agents, monitors of disease progression, monitors of treatment and predictors of clinical outcome. For example, various biomarker research projects are attempting to identify markers of specific cancers and of specific cardiovascular and immunological diseases.
  • references herein to ILD being associated with a particular treatment or drag therapy are not intended to in any way to suggest that the ILD is caused by a particular drag.
  • the exact cause of ILD is currently not understood and is the subject of ongoing research to determine causative factors.
  • ILD has been reported in cancer patients receiving a number of treatments for cancers such as non-small cell lung cancer. ILD has also been reported in patients receiving other therapies where ILD has been reported as a co-morbidity such as treatments for rheumatoid arthritis, particularly with patients treated with certain disease-modifying anti-rheumatic drugs (DMARDs). Accordingly the method of the invention is suitable for use in the treatment of medical conditions where ILD is a co-morbidity.
  • DMARDs disease-modifying anti-rheumatic drugs
  • agents which inhibit cancer cell invasion for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);
  • inhibitors of growth factor function for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [HerceptinTM] and the anti-erbbl antibody cetuximab [C225]), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amme (gefitinib, AZD1839), N-(3-ethynylphenylphenylphenyl
  • vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO00/40529, WO 00/41669, WO01/92224, WO02/04434 and WO02/08213;
  • antisense therapies for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense;
  • gene therapy approaches including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and
  • GDEPT gene-directed enzyme pro-drug therapy
  • immunotherapy approaches including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.
  • cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor
  • the invention is particularly useful in targeted therapies for the treatment of cancers, for example those that provide an anti-cancer effect by inhibiting growth factor function, such as erbB receptor drugs and anti-angiogenic agents, for example VEGF inhibitors, insulin growth factor (IGF) inhibitors and platelet derived growth factor inhibitors (PGDF).
  • growth factor function such as erbB receptor drugs and anti-angiogenic agents, for example VEGF inhibitors, insulin growth factor (IGF) inhibitors and platelet derived growth factor inhibitors (PGDF).
  • IGF insulin growth factor
  • PGDF platelet derived growth factor inhibitors
  • erbB receptor drug used herein includes drugs acting upon the erbB family of receptor tyrosine kinases, which include EGFR, erbB2 (HER), erbB3 and erbB4.
  • the erbB receptor drug is an erbB receptor tyrosine kinase inhibitor.
  • the erbB receptor drug is an EGFR tyrosine kinase inhibitor.
  • EGF receptor tyrosine kinase inhibitors include but are not limited to gefitinib, Erlotinib (OSI-774, CP-358774), PKI-166, EKB-569, HKI-272 (WAY-177820), lapatinib (GW2016, GW-572016), canertinib (CI-1033, PD183805), AEE788, XL647, BMS 5599626 or any of the compounds as disclosed in WO2004/006846, WO2003/082831, or WO2003/082290.
  • the erbB receptor drug is an anti-EGFR antibody such as for example one of cetuximab (C225), matuzumab (EMD-72000), panitumumab (ABX-EGF/ rHuMAb-EGFr), MRl-I, IMC-11F8 or EGFRLIl.
  • the erbB receptor drugs mentioned herein may be used as monotherapy or in combination with other drugs of the same or different classes.
  • the EGF receptor tyrosine kinase inhibitor is gefitinib.
  • DMARDs for use in the treatment of rheumatic conditions are well known to those of ordinary skill in the art.
  • a particular example of a DMARD which may be associated with ILD is Arava (leflunomide).
  • a pharmacoepidemiology nested case-control study was undertaken on NSCLC patients to measure the relative risk of ILD associated with gefitinib treatment, compared to conventional therapy. The study also set out to identify risk factor for ILD in order to offer safer use of gefitinib.
  • the CCS study is the first large-scale prospective pharmacoepidemiology study of patients in Japan.
  • the card was intended to alert them to symptoms that should prompt a clinic visit and work up for ILD.
  • the main purpose of the card was to increase awareness and attention to ILD symptoms in all NSCLC patients regardless of treatment, and to get patients with ILD to present early for diagnosis.
  • a Case Review Board consisting of Pulmonology, Oncology, and diagnostic Radiology specialists independently review all ILD patient clinical and radiological information.
  • a set of clinical and radiological criteria had been pre-defined and an overall assessment by scaling was made based on clinical and radiological findings ( Figure 2).
  • CRB review was carried out in a blinded manner for treatment. Provisional cases verified by the CRB were classified as index cases. So far approximately 72 provisional cases were reviewed by the CRB of which 54 were confirmed as index cases, giving an overall diagnosis rate of about 76% of reported provisional cases.
  • proteomics analysis was undertaken in gefitinib treated patients and its objective was to detect proteins/peptides (including known markers of lung damage) that were significantly of greater (or lesser) abundance in gefitinib-treated patients with ILD than in gefitinib-treated patients without ILD.
  • the outcome of the study is to identify proteins that show 'a significant difference' in the 'protein mass index' defined in the analytical methods between ILD and non-ILD patients treated with gifitinib.
  • Two blood samples were taken for proteomics.
  • One so-called 'pre' was obtained between 1 and 3 hours after the first dose of gefitinib at registration into the cohort.
  • another blood sample was collected promptly after a patient was registered as a provisional ILD case (post) and from patients selected as controls. The timing of the samples varied according to centre.
  • Proteins expressed in plasma have been quantified on a label-free basis. Proteins were extracted from plasma and were subjected to tryptic digestion after depleting abundant human serum albumin (HSA) and IgG. Both m/z-values (MS) of resulted tryptic peptides and their daughter ion spectra (MS/MS) reflecting their amino-acid sequences were obtained by a linear ion-trap mass spectrometric measurement with a ⁇ -electrospray-ionization interface following a capillary reverse-phase micro flow-liquid chromatography ( ⁇ -LC).
  • HSA human serum albumin
  • ⁇ -LC capillary reverse-phase micro flow-liquid chromatography
  • MTI maps corrected by the z-OPAL procedure can be compared to others and so one MTI map averaged within one group is then compared to other average map of a different group.
  • Peptide signals significantly associated to one group such as disease or normal can be identified with a certain statistical confidence by the method combined with the z-OPAL.
  • the corresponding proteins can be sequenced by the data-dependent consecutive tandem mass spectrometric (MS/MS) measurement for specific peptide ions. Meaningful proteins are thus identified from peptides ions with quantitative differences between the whole averaged MTI maps of two groups such as case vs control.
  • the capillary reversed-phase ⁇ LC -MS/MS system comprised a Paradigm MS4 dual solvent delivery system (Michrom BioResources, Inc., Auburn, CA, USA) for HPLC, an HTC PAL auto sampler (CTC Analytics, Zwingen, Switzerland), Finnigan LTQ linear ion-trap mass spectrometers (ITMS, Thermo Electron, San Jose, CA) equipped with nanoelectrospray ionization (NSI) sources (AMR Inc., Tokyo, Japan) 1 . Digested samples of 10 ⁇ L (equivalent to ca.
  • l ⁇ g proteins were automatically injected into a peptide CapTrap cartridge (2.0 x 0.5 mm i.d., Michrom) on an injector valve for concentration and desalting. After desalting with 0.1% TFA (aq.) containing 2% acetonitrile, the sample was loaded into a capillary reversed-phase column, MAGIC Cl 8 (3 ⁇ m, 200 A, 50 x 0.2 mm i.d., Michrom) for separation. The solutions of 2% and 90% acetnitrile (aq.) were used as mobile phase A and B, respectively, and both contained with 0.1% formic acid.
  • the gradient conditions in the chromatographic run were as follows: B from 5% (0 min) to 40% (70 min), and then up to 95% (80 min).
  • Effluent solvent at 1.0-1.2 ⁇ L/min from the HPLC was introduced into the mass spectrometer by the NSI interface via an injector valve with a CapTrap cartridge and the RP column.
  • the NSI needle (FortisTip, OmniSeparo-TJ, Hyogo, Japan), which was connected directly to the reverse-phase column outlet, was used as the NSI interface and the voltage was 2.0 IcV, while the capillary was heated to 200°C. No sheath or auxiliary gas was used.
  • the mass spectrometer was operated in a data-dependent acquisition mode in which MS acquisition with a mass range of m/z 450-2000 was automatically switched to MS/MS acquisition under the automated control of the Xcalibur software.
  • the three most intense ions of the full MS scan were selected as the parent ions and subjected to MS/MS scans with an isolation width of m/z 2.0; the activation amplitude parameter was set at 35%.
  • the trapping time was 100 ms under the auto gain control mode. Data was acquired using the dynamic mass-exclusion windows that had an exclusion of 2.0-min duration and exclusion mass widths of -0.5 and +1.5 Da.
  • Data analysis mainly consists of two individual approaches. One is quantitative analysis, and another is qualitative analysis. Quantitative analysis is used to discover the significant difference of peptide signals by comparing two different sample groups. This process uses the full MS data to quantify the amount of peptide molecular ions by their ion signal intensity. Qualitative analysis is used to identify peptide sequence of peptide molecular ions by using their MS/MS spectra. These two processes can proceed simultaneously and at the final stage, the results of these two analyses are combined.
  • LC-MS data are generated in RAW file format. They are firstly converted to the text format by XcaliburTM program (Thermo Electron), then converted into XML format. In the next step, the signals derived from the internal standards are detected and marked. Intensity is also normalized so that total signal intensity becomes the given constant value (IxIO 11 ). The marked internal standards play important role in the following data alignment process. Following step is to calculate distances between all the LC-MS data belong to the same group. This distance information is used in the next step to determine the order of clustering by the minimum spanning tree algorithm. This clustering process is to generate single aligned LC-MS profile from plural number of original LC-MS data within the same group.
  • LC-MS profiles from each group to be compared are further aligned to determine the correspondence of every data point between the groups.
  • the same alignment program named 'i-OPAL' is used.
  • i-OPAL is named after the "internal standard-guided optimum profile alignment" 2 . This is a program to align two time axes of different LC-MS measurement by non-linearly compensating the time axis. The correspondence of two time axes is calculated based on the dynamic programming algorithm, and information of the internal standards acts as landmarks to be aligned each other.
  • MGF file is employed as MASCOT input file
  • MASCOT search is executed by following parameters.
  • MASCOT result saves into a certain directory as
  • the MPSnr database is a non-redundant peptide sequence database that is generated by NCBI Nr, Refseq, Gene Ontology, HUGO and SwissProt database. This database comprises not only the sequence and unique id, but also HUGO Gene symbol, Sequence description, Gene Ontology localization and linkage to the original sequence database (such as NCBI Nr, Refseq and SwissProt). Those data are available in an Oracle database, and it is used for the annotation phase. After MASCOT search is completed, the Mascot result DAT file is parsed and re-stored into Oracle. The detail scoring algorithm of MASCOT is not opened, but its score is calculated for each identified peptide. Our approach checks the MASCOT peptide score distribution to generate an original score rank.
  • Identified peptide sequence tends to crowd together around Score 0 to 25; however, if those identified peptides are dispersed, we can assess the significance of score behind the normal MASCOT score by scaling distribution. The number of distribution above is rank for the score rank for peptide score.
  • Our approach creates a qualitative result list for each comparative group. This list consists of 9 categories: Order, Hit Title, Symbol, Plasma, Sample number, ID_SMP, SCOREJRANK, SCORE and DESCRIPTION. The qualitative analysis results and above-mentioned quantitative results are combined with each other by tracing back to the original data where the quantitative MS data and the qualitative MS/MS data are stored. Additional annotation on cellular localization is appended from the GO database. Thus all the data items required to generate the final result table are available. Data points and peptide entries were selected according to the following criteria:
  • MASCOT score of peptide identification is equal or greater than 40 Selection criterion 2:
  • MASCOT score of peptide identification is equal or greater than 20
  • Selection criterion 1 • Average normalized signal intensity of both group is equal or greater than lxlO 6 , and
  • MASCOT score of peptide identification is equal or greater than 40 Selection criterion 2:
  • signal points of that retention time is less than 5 min. or greater than 75 min., and those of which m/z is greater than 1500 were omitted from the selection.
  • Oxidation of M refers to the oxidation of sulphur - to a sulphur oxide which gives an addition of +16 in molecular mass of that peptide. 5
  • a peptide mass annotation assay that utilises isotope labelled peptides of one, several or all of the biomarkers that is added to the sample prior to analysis and used as an internal standard for both qualitative and quantitative analysis where the i-OPAL Software has been applied to select the biomarkers of significance.
  • a peptide sequence mass annotation assay that utilises isotope labelled peptides of one, several or all of the biomarkers that is added to the sample prior to analysis and used as an internal standard for both qualitative and quantitative analysis of peptide fragment sequences where the i-OPAL Software has been applied to select the biomarkers of significance.
  • a test kit can be used in all potential patients who would receive Drug for their NSCLC. Those with elevations in the range for diagnosis of susceptibility would not be offered therapy. Used in this way a test enables the clinician to exclude high-risk patient from Drug treatment.
  • An example of use of markers to characterize ILD is provided in Ishii et al, Thorax 2003: vol 58, 52- 57.
  • the test may be used for early diagnosis of ILD. As we all know, a definitive ILD diagnosis will often require a biopsy. If a blood test can replace a biopsy, it is highly advantageous.
  • ILD which comprises measurement of one or more of the peptides shown below, or a protein comprising one or more of said peptides, in a biological sample obtained from the patient:
  • diagnosis of ILD or determination of susceptibility to ILD comprises measurement of an elevated level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
  • diagnosis of ILD or determination of susceptibility to ILD comprises measurement of a reduced level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
  • a diagnostic test according to paragraph 2 further comprising measurement of a reduced level of one or more of the peptides defined in paragraph 3, or a protein comprising one or more of said peptides, compared to the level of said peptide or protein in patients without ILD.
  • a diagnostic test according to any one of the preceding paragraphs comprising measuring one or more proteins comprising one or more of the said peptides.
  • a diagnostic test according to paragraph 1 comprising measuring one or more proteins selected from AMBP, APOAl, APOA2, APOA4, APOB, BF, C3, FGA, FGG, FLJ38348, FNl, GNL2, HP, ICAM3, ITIHl, LOC388504, PZP, SERPINA3, SERPINGl, TF, TNFRSF8 as defined by reference to TABLE 1 hereinabove.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention provides a diagnostic test to detect ILD or to identify the susceptibility of a patient to develop ILD which comprises measurement of one or more of the peptides set forth in Table 1, or a protein comprising one or more of said peptides, in a biological sample obtained from the patient.

Description

METHOD
The present invention relates to a method for diagnosing interstitial lung disease (ILD) and to a method for predicting the susceptibility of a patient to developing ILD during a therapeutic treatment, for example chemotherapy for a cancer. More particularly the method of the invention relates to the detection of the presence or of levels of certain proteins or peptides in patients susceptible to developing ILD. The methods are suitable for detecting ILD in patients that may be treated with an erb inhibitor, especially patients with advanced Non-small Cell Lung
Cancer (NSCLC), for example adenocarcinoma, which are being treated with an epidermal growth factor tyrosine kinase inhibitor.
Interstitial lung disease (ILD) is a well-known co-morbidity in patients with non-small- cell lung cancer (NSCLC). ILD has been reported in patients with various chemotherapies and radiotherapy, as well as Gefitinib treatment. Frequencies of ILD in NSCLC from 3% to 30% following single or combination therapies for NSCLC have been reported. The mechanism causing and the risk factors contributing to ILD are unclear.
® Gefitinib (also known as Iressa , by way of the code number ZD1839 and Chemical
Abstracts Registry Number 184475-35-2) is disclosed in International Patent Application WO 96/33980 (Example 1) and is a potent inhibitor of the epidermal growth factor receptor (EGFR) family of tyrosine kinase enzymes such as erbBl. Gefitinib possesses anti-proliferative activity such as anti-cancer activity and, accordingly, is useful in methods of treatment of proliferative disease such as cancer in the human or animal body. Gefitinib was approved in Japan on 5 July 2002 for treatment of inoperable or recurrent NSCLC. The early experience of gefitinib in Japan was encouraging, with responses seen in patients in phase I-II clinical trial and favorable safety profile compared with cytotoxic drugs. This led to early approval of gefitinib in Japan. However an increasing number of cases of ILD have been reported post launch of gefitinib along with debate regarding the appropriateness of gefitinib therapy for patients having ILD or a significant risk thereto. Diagnosis of ILD is presently complex (see Figures 1 and 2). Accordingly there is a need for more convenient ways to diagnose ILD or risk thereof in the presence or absence of ongoing drug therapy.
Summary of the Invention
We have surprisingly found that certain proteins or fragments thereof can be used as makers for predicting the onset of ILD in a patient, particularly in a patient that is being treated with an EGFR tyrosine kinase inhibitor such as gefitinib.
In particular, upregulation or downregulation of the levels on these proteins, or their peptides, in biological fluids can be used as an indicator of susceptibility to ILD in a patient.
According to a first aspect of the present invention there is provided a diagnostic test to detect ILD or to identify the susceptibility of a patient to develop ILD which comprises measurement of one or more of the peptides shown below, or a protein comprising one or more of said peptides, in a biological sample obtained from the patient:
Peptide
VKSPELQAEAK
SARLGATRGPR
ATEHLSTLSEK
VRPQQLVK
ATEHLSTLSEK
QLEQVIAK
ESSSHHPGIAEFPSR
QKVEPLRAELQEGAR
QFTSSTSYNRGDSTFESK
LRTEGDGVYTLNDK
FISLGEACKK
IDVHLVPDR
SSGRR
VGYVSGWGR
FISLGEACK
CVLFPYGGCQGNGNK
VDLSFSPSQSLPASHAHLR
SETKDLLFRDDTVCLAK
CSTSSLLEACTFR
LYGSEAFATDFQDSAAAKK
DSGRDYVSQFEGSALGK
LPGQDNLVK
ASTPNGYDNGIIWATWK
MLEEIMKYEASILTHDSSIR
KNPREDYLDVYVFGVGPLVNQVNINALASK DLPEPR
SELTQQLNALFQDKLGEVNTYAGDLQK FSVPAGIVPSFQALTAR
FRVVSMDENFHPLNELIPLVYIQDPK
ADQEGAREIVCNVTLGGERR
YTHDQLFFIAFAQNWCΓKRR
DLATVYVDVLKDSGRDYVSQFEGSALGK VTWAPPPSDDLTNFLVR
SAGWNIPIGLLYCDLPEPR
NIQVPR
LLDSLPSDTRLVLLNAIYLSAK
DLFGGRLTSDMIAER
SWVPHTFESELSDPVELLVAES
EPCVESLVSQYFQTVTDYGKDLMEK
ILGDMQPGDYFDLVLFGTR
Peptides comprising Methionine include Oxidation of M (methionine) referring to the oxidation of sulphur to a sulphur oxide - see TABLE 1 hereinbelow. Peptides comprising Oxidation of methionine) include DLFGGRLTSDMIAER and
EPCVESLVSQYFQTVTDYGKDLMEK.
Preferably the diagnostic test is one wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of an elevated level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
Peptide
VKSPELQ AgAK
SARLGATRGPR
ATEHLSTLSEK
VRPQQLVK
ATEHLSTLSEK
ESSSHHPGIAEFPSR
QKVEPLRAELQEGAR
QFTSSTSYNRGDSTFESK
LRTEGDGVYTLNDK
FISLGEACKK
ID VHLVPDR
SSGRR
FISLGEACK
SETKDLLFRDDTVCLAK
CSTSSLLEACTFR
LYGSEAFATDFQDSAAAKK
DSGRDYVSQFEGSALGK
LPGQDNLVK
ASTPNGYDNGIIWATWK
MLEEΓMKYEASILTHDSSIR
DLPEPR SAGWNIPIGLLYCDLPEPR
NIQVPR
EPCVESLVSQYFQTVTDYGKDLMEK
Preferably a diagnostic test is one wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of a reduced level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
Peptide
QLEQVIAK
VGYVSGWGR
CVLFPYGGCQGNGNK
VDLSFSPSQSLPASHAHLR
KNPRED YLD VYVFGVGPLVNQVNrNALASK
SELTQQLNALFQDKLGEVNTYAGDLQK FSVPAGIVIPSFQALTAR
FRVVSMDENFHPLNELIPLVYIQDPK
ADQEGAREΓVCNVTLGGERR
YTHDQLFFIAFAQNWCIKRR
DLATVYVDVLKDSGRDYVSQFEGSALGK VTWAPPPSIDLTNFLVR
LLDSLPSDTRLVLLNAIYLSAK
DLFGGRLTSDMIAER
SWVPHTFESELSDPVELLVAES
ILGDMQPGDYFDLVLFGTR
Preferably a diagnostic test detecting elevated peptide or protein levels as described above further comprises measurement of a reduced level of one or more of the peptides defined above or a protein comprising one or more of said peptides, compared to the level of said peptide or protein in patients without ILD.
Preferably, a diagnostic test described hereinabove comprises measuring one or more proteins comprising one or more of the said peptides. Preferred proteins are selected from
AHSG, AMBP, APOAl, APOA2, APOA4, APOB, BF, C3, CFH, FGA, FGG, FLJ38348, FNl, GNL2, HP, ICAM3, ITIHl, ITIH4, LOC388504, MRPL27, PZP, SERPINA3, SERPINGl, TF and TNFRSF8 as defined by reference to TABLE 1 herein.
In one embodiment of the invention a diagnostic test for a protein is measured using an immunoassay. Examples of suitable assay methodologies are described in the following references, which are incorporated herein by reference in their entirety:
• Goddard J. R,Reymond J. L.,
Enzyme assays for high-throughput screening. Curr Opin Biotechnol. 2004 Aug;15(4):314-22.
• J. Werner ZoIg and Hanno Langen; How Industry Is Approaching the Search for New Diagnostic Markers and Biomarkers, MoI. Cell. Proteomics, Apr 2004; 3: 345 - 354.
• Alberti K. G, The future of the measurement of intermediary metabolites in clinical Medicine. Ann Clin Biochem. 1975 Sep; 12(5): 192-5. • Nielsen, U. B., Geierstanger B. H., Multiplexed sandwich assays in microarray format. J
Immunol Methods. 2004 Jul;290(l-2):107-20.
In another embodiment of the invention a diagnostic test for a peptide is measured using mass spectrometry. The mass or sequence of the peptide may be measured. A preferred biological sample is a biological fluid or bio-fluid. Suitable bio-fluids include any one of blood, serum, plasma, urine and saliva.
Preferably the diagnostic test is to determine susceptibility to ILD following administration of a drug to a patient; preferably the drug is an erb receptor drug or a DMARD and especially the drug is gefitinib. Alternative embodiments of the invention are contemplated as set out below:
A method of selecting a patient for therapy with a drug, preferably gefitinib; or
A method of monitoring a patient receiving therapy with a drug, preferably gefitinib, for risk of developing ILD; or
A diagnostic kit comprising a means for measuring one or more of the peptides or proteins of the invention as described herein; or
A method of carrying out a clinical trial comprising patient selection and/or monitoring using a diagnostic method as described herein.
Detailed Description of the Invention Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods. See, generally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel et al, Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), McPherson et al., PCR Volume 1, Oxford University Press, (1991), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N. Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.). These documents are incorporated herein by reference.
PROTEIN ANALYSIS
Intact proteins can be assayed in a number of ways utilizing both gel-based as well as liquid phase separation technologies. Two-dimensional gel electrophoresis is used with solubilised protein mixtures where the proteins are separated based upon charge and size [1-3]. The proteins are resolved such that both isomeric forms, as well as post-translational modifications, are resolved. Quantitation of the proteins is made by staining techniques, where both pre- and post staining techniques can be applied. Metabolic labelling also allows the linear range to be extended up to 5 orders of magnitude, offering sensitivities within the femtomolar range [4].
Protein identification is performed from excised gel spots. The proteins are digested after chemical degradation and modification. The resulting peptide mixtures are extracted from the isolated gel sample and subsequently identified by mass spectrometry [5-7].
Multidimensional HPLC (High Performance Liquid Chromatography) can be used as a good alternative for separating proteins or peptides [8-11]. The protein or peptide mixture is passed through a succession of chromato graphic stationary phases or dimensions which gives a higher resolving power. HPLC is flexible for many experimental approaches and various stationary and mobile phases can be selected for their suitability in resolving specific protein or peptide classes of interest and for compatibility with each other and with downstream mass spectrometric methods of detection and identification. High Performance Liquid Chromatography is currently the best methodology for solute separations which also allows for automated operation with a high degree of reproducibility [12-19]. On-line configurations of these types of multi-mechanism separation platforms are commonly applied within proteomics studies today [20-22].
Mass spectrometry (MS) is also an essential element of the proteomics field. In fact MS is the major tool used to study and characterize purified proteins in this field. The interface link in proteomics and MS, displaying hundreds or thousands of proteins, is made by gel technology where high resolution can be reached on a single gel. Researchers are successfully harnessing the power of MS to supersede the two-dimensional gels that originally gave proteomics its impetus.
The application and development of mass spectrometry (MS) to identify proteins or peptides separated via liquid phase separation techniques and/or gel-based separation techniques have led to significant technological advance in protein and peptide expression analysis. There are two main methods for the mass spectrometric characterization of proteins and peptides: matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). Using various approaches, MALDI and ESI ion sources can be combined with time-of-flight (TOF) or other types of mass spectrometric analyzers to determine the mass or the sequence of peptides.
In MALDI, peptides are co-crystallized with the matrix, and pulsed with lasers. This treatment vaporizes and ionizes the peptides. The molecular weights (masses) of the charged peptides are then determined in a TOF analyzer. In this device, an electric field accelerates the charged molecules toward a detector, and the differences in the length of time it takes ionized peptides to reach the detector (their time-of-flight) reveal the molecular weights of the peptides; smaller peptides reach the detector more quickly. This method generates mass profiles of the peptide mixtures - that is, profiles of the molecular weights and amounts of peptides in the mixture. These profiles can then be used to identify known proteins from protein sequence databases. In ESI and a technique called liquid chromatography (LC)/MS/MS, a voltage is applied to a very fine needle that contains a peptide mixture, generating peptide sequences, eluting from the LC-column. The needle then sprays droplets into a mass spectrometric analyzer where the droplets evaporate and peptide ions are released. In LC/MS/MS, researchers use microcapilliary LC devices to initially separate peptides. Mass spectrometry (MS) is a valuable analytical technique because it measures an intrinsic property of a bio-molecule, its mass, with very high sensitivity. MS can therefore be used to measure a wide range of molecule types (proteins, peptide, or any other bio-molecules) and a wide range of sample types/biological materials. Correct sample preparation is known to be crucial for the MS signal generation and spectra resolution and sensitivity. Sample preparation is therefore a crucial area for overall feasibility and sensitivity of analysis.
TOP-DOWN ANALYSIS OF PROTEINS
Proteins are bio-macro molecules that are difficult to separate by liquid phase chromatographic separation techniques, due to the unfavorable mass transfer within the particles of the chromatographic column material, the stationary phase [23]. However, proteins can be rendered into smaller unit form by breaking the peptide bond joining two adjacent amino acids. This can be accomplished by enzymatic cleavage by certain other proteins that are capable of interacting and dissolving peptide bonds on other proteins. Trypsin is the most commonly used protease, used in protein expression analysis studies. After the enzymatic degradation, a resulting complex mixture of peptides will be separated and fractionated by capillary chromatography. AU peptides that are the sum of the digested proteins in the sample will be unresolved at this stage. The peptides that has generated from the corresponding protein will not be separated as one unit in the chromatographic fractionation step, but rather be separated together with the resulting peptides from all other proteins in the sample. The high resolved and separated eluting peptides from the capillary, will be fractioned most commonly based upon charge and hydrophobicity. The separated peptides are introduced on-line from the chromatographic part of the platform into the mass spectrometer, thereby circumventing possible contaminations. The peptides are then mass determined (m/z), in order to capture all the peptides present in that given time window. Next, a number of peptide masses are selected for sequencing (MS/MS), based upon their abundance in the given time window. This is performed by a new ion sampling interface by an electrospray ionization ion trap mass spectrometer system [24-34]. The interface uses linear quadrupoles as ion guides and ion traps to enhance the performance of the trap. Trapping ions in the linear quadrupoles is demonstrated to improve the duty cycle of the system. Dipolar excitation of ions trapped in a linear quadrupole is used to eject unwanted ions. After the first appearance of successful instrumentation in 1990, [35] ion trap mass spectrometry with electro- spray ionization (ESI) has become a widely used tool for trace analysis. Electrospray is a gentle source that can ionize important analytes such as peptides, and proteins. Highly charged ions produced in ESI can extend the range of mass analyzers. Trap mass spectrometers have favorable capabilities such as flexible tandem MS capability (MS n ). hi this ionization process, the precursor ion is activated by acceleration into a mass-selective linear ion trap under conditions whereby some of the fragment ions formed are unstable within the trap. After a time delay the stability parameters of the ion trap are changed to allow capture of fragments that that were previously unstable. The result is a product ion spectrum that originates from precursor ions with a modified internal energy distribution. It is possible to follow the evolution of the precursor internal energy distribution for many milliseconds after admittance of the precursor ions into the linear ion trap. Time-delayed fragmentation product ion spectra typically display reduced sequential fragmentation products leading to spectra that are more easily interpreted. Several important experimental parameters important to time-delayed fragmentation have been identified and are discussed. The technique has applications for both small precursor ions and multiply charged peptides [26-30].
Tandem mass spectrometry (MS/MS) is at the heart of most of modern mass spectrometric investigations of complex mixtures. The fragmentation involves activation of a precursor ion via collisions with a target gas and may produce charged and neutral fragments. The nature of the fragment ions, as well as their intensities, is often indicative of the structure of the precursor ion and thus can yield useful information for the identification of unknown analytes, as well as providing a useful screening technique for different classes of analytes.
Activation via multiple collisions both prolongs the activation time and enables higher energies to be deposited into precursor ions. Higher collision gas pressures also imply higher collision relaxation rates.
BIOMARKERS
Various biological markers, known as biomarkers, have been identified and studied through the application of biochemistry and molecular biology to medical and toxicological states. Biomarkers can be discovered in both tissues and biofluids, where blood is the most common biofluid used in biomarker studies [36-38].
A biomarker can be described as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention". A biomarker is any identifiable and measurable indicator associated with a particular condition or disease where there is a correlation between the presence or level of the biomarker and some aspect of the condition or disease (including the presence of, the level or changing level of, the type of, the stage of, the susceptibility to the condition or disease, or the responsiveness to a drag used for treating the condition or disease). The correlation may be qualitative, quantitative, or both qualitative and quantitative. Typically a biomarker is a compound, compound fragment or group of compounds. Such compounds may be any compounds found in or produced by an organism, including proteins (and peptides), nucleic acids and other compounds.
Biomarkers may have a predictive power, and as such may be used to predict or detect the presence, level, type or stage of particular conditions or diseases (including the presence or level of particular microorganisms or toxins), the susceptibility (including genetic susceptibility) to particular conditions or diseases, or the response to particular treatments (including drug treatments). It is thought that biomarkers will play an increasingly important role in the future of drag discovery and development, by improving the efficiency of research and development programs. Biomarkers can be used as diagnostic agents, monitors of disease progression, monitors of treatment and predictors of clinical outcome. For example, various biomarker research projects are attempting to identify markers of specific cancers and of specific cardiovascular and immunological diseases.
DRUGS
ILD has been observed during the treatment of a number of medical conditions, for example as discussed in Linda C. Koo et al, (published online 14 Jan 2005) Pharmacoepidemiology and Drag Safety. The method of the present invention may be used to test for the susceptibility of a patient to develop ILD prior to or after administration of a drag. Accordingly the test may be used to determine if a patient will develop ILD during therapy or is likely to develop ILD following the administration of a drag. The method may be used to detect ILD early in patients and provide appropriate medical intervention to control or mitigate the effects of the ILD. Alternatively, the method may be used to identify patients that may be prone to developing ILD such that an appropriate treatment can be administered to those patients at risk of developing ILD.
For the avoidance of any doubt, references herein to ILD being associated with a particular treatment or drag therapy are not intended to in any way to suggest that the ILD is caused by a particular drag. As discussed herein, the exact cause of ILD is currently not understood and is the subject of ongoing research to determine causative factors.
ILD has been reported in cancer patients receiving a number of treatments for cancers such as non-small cell lung cancer. ILD has also been reported in patients receiving other therapies where ILD has been reported as a co-morbidity such as treatments for rheumatoid arthritis, particularly with patients treated with certain disease-modifying anti-rheumatic drugs (DMARDs). Accordingly the method of the invention is suitable for use in the treatment of medical conditions where ILD is a co-morbidity.
The method of the invention may be used as part of a wide range of cancer therapies including conventional chemotherapy and more novel cancer therapies such as targeted therapies. Examples of cancer therapies for which the present method may be useful include but are not limited to: (i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5-fiuorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea; antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); (ii) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α-reductase such as finasteride;
(iii) agents which inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function); (iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [Herceptin™] and the anti-erbbl antibody cetuximab [C225]), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amme (gefitinib, AZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amme (erlotinib, OSI- 774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-moφholinopropoxy)quinazolm-4- amine (CI 1033)), for example inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family;
(v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™], compounds such as those disclosed in International Patent Applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin αvβ3 function and angiostatin);
(vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO00/40529, WO 00/41669, WO01/92224, WO02/04434 and WO02/08213; (vii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense;
(viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and
(ix) immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.
The invention is particularly useful in targeted therapies for the treatment of cancers, for example those that provide an anti-cancer effect by inhibiting growth factor function, such as erbB receptor drugs and anti-angiogenic agents, for example VEGF inhibitors, insulin growth factor (IGF) inhibitors and platelet derived growth factor inhibitors (PGDF).
The term "erbB receptor drug" used herein includes drugs acting upon the erbB family of receptor tyrosine kinases, which include EGFR, erbB2 (HER), erbB3 and erbB4. In an embodiment the erbB receptor drug is an erbB receptor tyrosine kinase inhibitor. In a further embodiment the erbB receptor drug is an EGFR tyrosine kinase inhibitor. Examples of EGF receptor tyrosine kinase inhibitors include but are not limited to gefitinib, Erlotinib (OSI-774, CP-358774), PKI-166, EKB-569, HKI-272 (WAY-177820), lapatinib (GW2016, GW-572016), canertinib (CI-1033, PD183805), AEE788, XL647, BMS 5599626 or any of the compounds as disclosed in WO2004/006846, WO2003/082831, or WO2003/082290.
In another embodiment the erbB receptor drug is an anti-EGFR antibody such as for example one of cetuximab (C225), matuzumab (EMD-72000), panitumumab (ABX-EGF/ rHuMAb-EGFr), MRl-I, IMC-11F8 or EGFRLIl. The erbB receptor drugs mentioned herein may be used as monotherapy or in combination with other drugs of the same or different classes. hi a particular embodiment the EGF receptor tyrosine kinase inhibitor is gefitinib.
DMARDs for use in the treatment of rheumatic conditions are well known to those of ordinary skill in the art. A particular example of a DMARD which may be associated with ILD is Arava (leflunomide).
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.
EXAMPLES
Example 1
Nested Case-control (CCS) pharmaco-vigilance study
A pharmacoepidemiology nested case-control study (CCS) was undertaken on NSCLC patients to measure the relative risk of ILD associated with gefitinib treatment, compared to conventional therapy. The study also set out to identify risk factor for ILD in order to offer safer use of gefitinib. The CCS study is the first large-scale prospective pharmacoepidemiology study of patients in Japan.
Approximately 5000-6000 advanced/recurrence NSCLC patients who have received at least one chemotherapy regimen are registered to the study cohort. Patients receive the most appropriate regimen for treatment of advanced/recurrence NSCLC as judged by the Principle
Investigator. Patients in the cohort are followed for 12 weeks from the start of the treatment.
Those who develop ILD according to clinical judgement aided by a diagnostic algorithm are registered as 'provisional cases' to the case-control study nested within the cohort. For each case, four patients are randomly selected from among the patients who have not yet developed ILD to serve as appropriate controls. The study commenced in September 2003 and proceeded smoothly. By the end of February 2005 approximately 2757 NSCLC patients had been registered and 95 provisional ILD cases had been diagnosed with 358 controls selected.
Principal Investigators in this study are instructed to assess all patients for possible ILD using a diagnostic algorithm (Figure 1). It is important that patients receive the same intensity of work up for ILD irrespective of their treatment with gefitinib or not. The clinical diagnostic algorithm for the diagnosis of ILD has been designed to enable the investigators to apply a validated and consistent approach to the diagnosis of ILD and exclusion of alternative diagnosis in advanced/recurrent NSCLC patients.
Patients participated to the study cohort were provided an information card. The card was intended to alert them to symptoms that should prompt a clinic visit and work up for ILD. The main purpose of the card was to increase awareness and attention to ILD symptoms in all NSCLC patients regardless of treatment, and to get patients with ILD to present early for diagnosis.
A Case Review Board (CRB) consisting of Pulmonology, Oncology, and diagnostic Radiology specialists independently review all ILD patient clinical and radiological information. A set of clinical and radiological criteria had been pre-defined and an overall assessment by scaling was made based on clinical and radiological findings (Figure 2). CRB review was carried out in a blinded manner for treatment. Provisional cases verified by the CRB were classified as index cases. So far approximately 72 provisional cases were reviewed by the CRB of which 54 were confirmed as index cases, giving an overall diagnosis rate of about 76% of reported provisional cases. The proteomics analysis was undertaken in gefitinib treated patients and its objective was to detect proteins/peptides (including known markers of lung damage) that were significantly of greater (or lesser) abundance in gefitinib-treated patients with ILD than in gefitinib-treated patients without ILD. hi other words, the outcome of the study is to identify proteins that show 'a significant difference' in the 'protein mass index' defined in the analytical methods between ILD and non-ILD patients treated with gifitinib.
Two blood samples were taken for proteomics. One so-called 'pre' was obtained between 1 and 3 hours after the first dose of gefitinib at registration into the cohort. In addition, another blood sample was collected promptly after a patient was registered as a provisional ILD case (post) and from patients selected as controls. The timing of the samples varied according to centre.
Example 2
Measurement of protein markers as ILD diagnostic tests
Proteins expressed in plasma have been quantified on a label-free basis. Proteins were extracted from plasma and were subjected to tryptic digestion after depleting abundant human serum albumin (HSA) and IgG. Both m/z-values (MS) of resulted tryptic peptides and their daughter ion spectra (MS/MS) reflecting their amino-acid sequences were obtained by a linear ion-trap mass spectrometric measurement with a μ-electrospray-ionization interface following a capillary reverse-phase micro flow-liquid chromatography (μ-LC).1 Resultant m/z-retention time- signal intensity (MTI) data were aligned so that individual MTI data can be compared each other using the informatics software referred as the internal-standard guided optimal-profile alignment (abbreviated as /-OPAL) strategy.2 Whereas raw μLC-MS data themselves can not be directly compared because of fluctuations in both ion intensities and nonlinear behavior under μLC gradient separation, MTI maps corrected by the z-OPAL procedure can be compared to others and so one MTI map averaged within one group is then compared to other average map of a different group. Peptide signals significantly associated to one group such as disease or normal can be identified with a certain statistical confidence by the method combined with the z-OPAL. The corresponding proteins can be sequenced by the data-dependent consecutive tandem mass spectrometric (MS/MS) measurement for specific peptide ions. Meaningful proteins are thus identified from peptides ions with quantitative differences between the whole averaged MTI maps of two groups such as case vs control.
Sample preparation. From each patient ca. 6-ml blood was taken into a 10-ml sampling tube containing Heparin-Na and was 2-3 times upside-down mixed. Then, it was subjected to centrifugation at 3,000 rpm for 10 minutes at 4g. A 3-ml plasma was obtained from the supernatant. Its individual 1.5-ml plasmas were separated into two 2-ml sterile polypropyrene tubes for their freeze storage at -800C.
Depletion of both HSA and IgG and in-solution tryptic digestion. Both abundant HSA and IgG were depleted from plasma using HSA and IgG antibody resins. The solution containing 200-μg proteins was subjected to the cycle of reduction and alkylation of cysteine residues by iodoacetamide at pH 9.0 in order to modify the cycteine thiol group into carboxyamidomethyl group. Proteins extracted from plasma and a certain amount of lysozyme, the internal standard, was digested together with trypsin (Sequence-grade modified, Promega, Madison, WI, USA), except for omission of the reduction and alkylation cycle. Resultant peptide mixtures were dried up under vacuum and stored at -2O0C until use.
Mass spectro metric measurements for pep tide-signal quantification and protein identification
The capillary reversed-phase μLC -MS/MS system comprised a Paradigm MS4 dual solvent delivery system (Michrom BioResources, Inc., Auburn, CA, USA) for HPLC, an HTC PAL auto sampler (CTC Analytics, Zwingen, Switzerland), Finnigan LTQ linear ion-trap mass spectrometers (ITMS, Thermo Electron, San Jose, CA) equipped with nanoelectrospray ionization (NSI) sources (AMR Inc., Tokyo, Japan)1. Digested samples of 10 μL (equivalent to ca. lμg proteins) were automatically injected into a peptide CapTrap cartridge (2.0 x 0.5 mm i.d., Michrom) on an injector valve for concentration and desalting. After desalting with 0.1% TFA (aq.) containing 2% acetonitrile, the sample was loaded into a capillary reversed-phase column, MAGIC Cl 8 (3 μm, 200 A, 50 x 0.2 mm i.d., Michrom) for separation. The solutions of 2% and 90% acetnitrile (aq.) were used as mobile phase A and B, respectively, and both contained with 0.1% formic acid. The gradient conditions in the chromatographic run were as follows: B from 5% (0 min) to 40% (70 min), and then up to 95% (80 min). Effluent solvent at 1.0-1.2 μL/min from the HPLC was introduced into the mass spectrometer by the NSI interface via an injector valve with a CapTrap cartridge and the RP column. The NSI needle (FortisTip, OmniSeparo-TJ, Hyogo, Japan), which was connected directly to the reverse-phase column outlet, was used as the NSI interface and the voltage was 2.0 IcV, while the capillary was heated to 200°C. No sheath or auxiliary gas was used. Further, the mass spectrometer was operated in a data-dependent acquisition mode in which MS acquisition with a mass range of m/z 450-2000 was automatically switched to MS/MS acquisition under the automated control of the Xcalibur software. The three most intense ions of the full MS scan were selected as the parent ions and subjected to MS/MS scans with an isolation width of m/z 2.0; the activation amplitude parameter was set at 35%. The trapping time was 100 ms under the auto gain control mode. Data was acquired using the dynamic mass-exclusion windows that had an exclusion of 2.0-min duration and exclusion mass widths of -0.5 and +1.5 Da.
Data analysis
Data analysis mainly consists of two individual approaches. One is quantitative analysis, and another is qualitative analysis. Quantitative analysis is used to discover the significant difference of peptide signals by comparing two different sample groups. This process uses the full MS data to quantify the amount of peptide molecular ions by their ion signal intensity. Qualitative analysis is used to identify peptide sequence of peptide molecular ions by using their MS/MS spectra. These two processes can proceed simultaneously and at the final stage, the results of these two analyses are combined.
LC-MS data are generated in RAW file format. They are firstly converted to the text format by Xcalibur™ program (Thermo Electron), then converted into XML format. In the next step, the signals derived from the internal standards are detected and marked. Intensity is also normalized so that total signal intensity becomes the given constant value (IxIO11). The marked internal standards play important role in the following data alignment process. Following step is to calculate distances between all the LC-MS data belong to the same group. This distance information is used in the next step to determine the order of clustering by the minimum spanning tree algorithm. This clustering process is to generate single aligned LC-MS profile from plural number of original LC-MS data within the same group. Finally the aligned LC-MS profiles from each group to be compared are further aligned to determine the correspondence of every data point between the groups. From the pairwise distance calculation, intra-group clustering to the inter-group comparison, the same alignment program named 'i-OPAL' is used. i-OPAL is named after the "internal standard-guided optimum profile alignment"2. This is a program to align two time axes of different LC-MS measurement by non-linearly compensating the time axis. The correspondence of two time axes is calculated based on the dynamic programming algorithm, and information of the internal standards acts as landmarks to be aligned each other. Signal peak picking is applied after the final alignment, and statistical test is applied to all the picked signals to determine which signal points are significantly changed between the groups. In this study, we have four sample groups: case-pre, case-post, control-pre, and control-post. Inter-group comparisons were applied to the following 4 ways: case-pre vs. control-pre, case-post vs. control-post, case-pre vs. case-post, and control-pre vs. control-post. For each comparison, we adopted Student's t-test of both sided with the confidence level 0.1% to screen initial candidate signals. In order to assess the possibility to discriminate the groups using these signals, another index called 'overlap' is also calculated. It is defined as the ratio of the overlapped (or separated) range of the distribution of signal intensity of the two groups against the whole range of the signal distribution of both groups. If this value is negative, it means the signal intensity of the two groups does not overlap. Fold change is also calculated based on the average intensity of both groups. Note that the value of fold is represented as the log-to-base 2, and in the comparison of case-control, positive value means the signal is up-regulated in the case group, and in the comparison of pre-post, positive means that post is up-regulated. These data are finally assessed after combined with the result of qualitative analysis.
Meanwhile, the MS/MS spectra derived from the same RAW data are processed by the qualitative analysis. The file format of measured sample data by Finnigan LTQ is RAW file that can be managed by MASCOT, but its data size is too big to apply MASCOT. Therefore our approach employs lcq_dta program (extract_ms.exe) to create MASCOT input file as DTA format. However, lcq_dta program (extract_ms.exe) also generates MS spectrum, our approach gets rid of MS spectrums from the converted DTA files by each RAW file. After creation of
MS/MS spectrum DTA data, we bundle those DTA files up by 3,000 DTA files each as
MASCOT Generic File (MGF File). MGF file is employed as MASCOT input file, MASCOT search is executed by following parameters. MASCOT result saves into a certain directory as
DAT file.
MASCOT version: 1.9.02
Taxonomy: All entries
Report top: Auto Database: MPSnr (2004.11.09)
Enzyme: Trypsin
Max. Missed cleavages: 2
Fixed modifications: Carbamidomethyl (C)
Variable modifications: Oxidation (M) Search Mass type: Mono-isotopic
Peptide tol.: ± 2.0 Da
Peptide charge: 1+, 2+ and 3+ Search Type: MS/MS Ions search Data format: Mascot generic (MGF) Report Type: Peptide summary MS/MS: toL: ± 0.8 Da Instrument: ESI-TRAP
The MPSnr database is a non-redundant peptide sequence database that is generated by NCBI Nr, Refseq, Gene Ontology, HUGO and SwissProt database. This database comprises not only the sequence and unique id, but also HUGO Gene symbol, Sequence description, Gene Ontology localization and linkage to the original sequence database (such as NCBI Nr, Refseq and SwissProt). Those data are available in an Oracle database, and it is used for the annotation phase. After MASCOT search is completed, the Mascot result DAT file is parsed and re-stored into Oracle. The detail scoring algorithm of MASCOT is not opened, but its score is calculated for each identified peptide. Our approach checks the MASCOT peptide score distribution to generate an original score rank. Identified peptide sequence tends to crowd together around Score 0 to 25; however, if those identified peptides are dispersed, we can assess the significance of score behind the normal MASCOT score by scaling distribution. The number of distribution above is rank for the score rank for peptide score. Our approach creates a qualitative result list for each comparative group. This list consists of 9 categories: Order, Hit Title, Symbol, Plasma, Sample number, ID_SMP, SCOREJRANK, SCORE and DESCRIPTION. The qualitative analysis results and above-mentioned quantitative results are combined with each other by tracing back to the original data where the quantitative MS data and the qualitative MS/MS data are stored. Additional annotation on cellular localization is appended from the GO database. Thus all the data items required to generate the final result table are available. Data points and peptide entries were selected according to the following criteria:
From the comparison of case-pre vs. control-pre: Selection criterion 1 :
• Average normalized signal intensity of both group is equal or greater than
IxIO6, and • The overlap of the distribution of signal intensity of the two groups is less than
0 (i.e. two groups are completely separated), and
• MASCOT score of peptide identification is equal or greater than 40 Selection criterion 2:
• Average normalized signal intensity of both group is equal or greater than
IxIO7, and
• The overlap of the distribution of signal intensity of the two groups is less than 0 (i.e. two groups are completely separated), and
• MASCOT score of peptide identification is equal or greater than 20
From other comparisons:
Selection criterion 1 : • Average normalized signal intensity of both group is equal or greater than lxlO6, and
• The difference of the average normalized signal intensity between the two groups is equal or greater than IxIO6, and
• The overlap of the distribution of signal intensity of the two groups is less than 10%, and
• MASCOT score of peptide identification is equal or greater than 40 Selection criterion 2:
• Average normalized signal intensity of both group is equal or greater than
1x107, and • The difference of the average normalized signal intensity between the two groups is equal or greater than Ix 107, and
• The overlap of the distribution of signal intensity of the two groups is less than
10%, and
• MASCOT score of peptide identification is equal or greater than 20
Besides the above conditions, signal points of that retention time is less than 5 min. or greater than 75 min., and those of which m/z is greater than 1500 were omitted from the selection.
Finally the signals that clear both criteria were included in the table.
1. Fujii K, Nakano T, Kanazawa M, Akimoto S, Hirano T, Kato H, Nishimura T. Clinical-scale high-throughput human plasma proteome analysis: Lung adenocarcinoma. Proteomics 2005; 5:1150-59.
2. International application No. PCT/JP2004/004621
TABLE 1
Figure imgf000022_0001
Figure imgf000023_0001
* Note - Oxidation of M (methionine) refers to the oxidation of sulphur - to a sulphur oxide which gives an addition of +16 in molecular mass of that peptide. 5
Results
The chromatographs described for each sample in this study clearly show that there were significant differences in the LC-MS profiles between the Case and Control groups.
Furthermore, comparison between the Case and Control groups immediately after dosing show 0 that this change was observed not only at the time of ILD onset but at the time of administration of Iressa. Accordingly the results suggest it is possible to identify those patients with a risk of ILD prior to administration of Iressa by quantifying the proteins to become markers or monitoring the LC-MS profiles. Changed protein levels can be used to predict a greater chance of ILD whereby to predict patients who will develop ILD after treatment.
Example 3
In an alternative embodiment, the assay is performed using standards, which are identical to the candidate peptides with one difference, namely the inclusion of isotope in the sequence.
This allows the development of specific assays for any one or more of the candidate peptides identified. It also allows parallel multiplex assays utilising mass spectrometry technology which gives high throughput and good sensitivity. Such assays do not require the use of antibodies, as per ELISA assays which can also be performed.
Accordingly, there is provided a peptide mass annotation assay that utilises isotope labelled peptides of one, several or all of the biomarkers that is added to the sample prior to analysis and used as an internal standard for both qualitative and quantitative analysis.
A similar approach can be applied to a peptide sequence mass annotation assay that utilises isotope labelled peptides for both qualitative and quantitative analysis of peptide fragment sequences.
Accordingly, there is also provided a peptide sequence mass annotation assay that utilises isotope labelled peptides of one, several or all of the biomarkers that is added to the sample prior to analysis and used as an internal standard for both qualitative and quantitative analysis of peptide fragment sequences
As above, the i-OPAL Software can been applied to select the biomarkers of significance.
Accordingly, there is also provided a peptide mass annotation assay that utilises isotope labelled peptides of one, several or all of the biomarkers that is added to the sample prior to analysis and used as an internal standard for both qualitative and quantitative analysis where the i-OPAL Software has been applied to select the biomarkers of significance.
Accordingly, there is also provided a peptide sequence mass annotation assay that utilises isotope labelled peptides of one, several or all of the biomarkers that is added to the sample prior to analysis and used as an internal standard for both qualitative and quantitative analysis of peptide fragment sequences where the i-OPAL Software has been applied to select the biomarkers of significance.
Example 4 Clinical application of novel ILD tests
Although analysis techniques using mass spectrometers are extremely useful for the accurate diagnosis of ILD appearance in a patient, it is difficult to apply such a technique to a clinical site for reasons of cost and the requirement for immediate results. It is extremely important clinically that the measurement of the proteins related to ILD can be established in a manner which allows the results to be obtained in a short time even at the clinic level in case of a university hospital and not only in general hospitals. Therefore, where as technique based on mass spectrometry cannot be used, preferred clinical assays include ELISA methods, in which antibodies directed to each of the peptide fragments identified herein are use to assay protein levels, and the use of polypeptide arrays.
Clinical use of the test is as follows:
a) A diagnostic test for susceptibility to develop ILD
A test kit can be used in all potential patients who would receive Drug for their NSCLC. Those with elevations in the range for diagnosis of susceptibility would not be offered therapy. Used in this way a test enables the clinician to exclude high-risk patient from Drug treatment. An example of use of markers to characterize ILD is provided in Ishii et al, Thorax 2003: vol 58, 52- 57.
b) A marker for accurate diagnosis of ILD
If the protein profile changed after the onset of ILD, the test may be used for early diagnosis of ILD. As we all know, a definitive ILD diagnosis will often require a biopsy. If a blood test can replace a biopsy, it is highly advantageous.
c) A marker for follow up during treatment
Patients with high level of protein expression will be at high risk of developing ILD, but there may be little or no treatment choice for them. They could still receive treatment with careful follow up on ILD markers during treatment. This would enable an early diagnosis of ILD, potentially removing much of the initial excess risk.
d) A marker for understanding of ILD from any known cause or for idiopathic disease The mechanism of drug induced ILD is not clear. Discovery of proteins that associated with the disease that might lead to a new explanation of the disease.
FURTHER ASPECTS
Further aspects of the present invention are presented in the following numbered paragraphs.
1. A diagnostic test to detect ILD or to identify the susceptibility of a patient to develop
ILD which comprises measurement of one or more of the peptides shown below, or a protein comprising one or more of said peptides, in a biological sample obtained from the patient:
Peptide
VKSPELQAEAK
SARLGATRGPR
ATEHLSTLSEK
QLEQVIAK
QKVEPLRAELQEGAR
LRTEGDGVYTLNDK
FISLGEACKK
VGYVSGWGR
CVLFPYGGCQGNGNK
VDLSFSPSQSLPASHAHLR
SETKDLLFRDDTVCLAK
CSTSSLLEACTFR
LYGSEAFATDFQDSAAAKK
DSGRDYVSQFEGSALGK
MLEEIMKYEASILTHDSSIR
KNPREDYLDVYVFGVGPLVNQVNINALASK
DLPEPR SELTQQLNALFQDKLGEVNTYAGDLQK
FSVPAGIVIPSFQALTAR
FRVVSMDENFHPLNELIPLVYIQDPK
ADQEGAREIVCNVTLGGERR
YTHDQLFFIAFAQNWCIKRR
DLATVYVDVLKDSGRDYVSQFEGSALGK
VTWAPPPSIDLTNFLVR
SAGWNIPIGLLYCDLPEPR
NIQVPR
LLDSLPSDTRLVLLNAIYLSAK
DLFGGRLTSDMIAER
SWVPHTFESELSDPVELLVAES
EPCVESLVSQYFQTVTDYGKDLMEK
ILGDMQPGDYFDLVLFGTR
2. A diagnostic test according to paragraph 1 wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of an elevated level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
Peptide
VKSPELQAEAK
SARLGATRGPR
ATEHLSTLSEK
QKVEPLRAELQEGAR
LRTEGDGVYTLNDK
FISLGEACKK
SETKDLLFRDDTVCLAK
CSTSSLLEACTFR
LYGSEAFATDFQDSAAAKK
DSGRDYVSQFEGSALGK
MLEEIMKYEASILTHDSSIR
DLPEPR
SAGWNIPIGLLYCDLPEPR
NIQVPR
EPCVESLVSQYFQTVTDYGKDLMEK
3. A diagnostic test according to paragraph 1 wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of a reduced level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
Peptide
QLEQVIAK
VGYVSGWGR
CVLFPYGGCQGNGNK VDLSFSPSQSLPASHAHLR
KNPREDYLDVYVFGVGPLVNQVNINALASK
SELTQQLNALFQDKLGEVNTYAGDLQK
FSVPAG I Vl PS FQALTAR
FRVVSMDENFHPLNELIPLVYIQDPK
ADQEGAREIVCNVTLGGERR
YTHDQLFFIAFAQNWCIKRR
DLATVYVDVLKDSGRDYVSQFEGSALGK
VTWAPPPSIDLTNFLVR
LLDSLPSDTRLVLLNAIYLSAK
DLFGGRLTSDMIAER
SWVPHTFESELSDPVELLVAES
ILGDMQPGDYFDLVLFGTR
4. A diagnostic test according to paragraph 2 further comprising measurement of a reduced level of one or more of the peptides defined in paragraph 3, or a protein comprising one or more of said peptides, compared to the level of said peptide or protein in patients without ILD.
5. A diagnostic test according to any one of the preceding paragraphs comprising measuring one or more proteins comprising one or more of the said peptides.
6. A diagnostic test according to any preceding paragraph, wherein one or more of said peptides is isotope labelled, added to the sample prior to analysis and used as an internal standard for qualitative and/or quantitative analysis.
7. A diagnostic test according to paragraph 1 comprising measuring one or more proteins selected from AMBP, APOAl, APOA2, APOA4, APOB, BF, C3, FGA, FGG, FLJ38348, FNl, GNL2, HP, ICAM3, ITIHl, LOC388504, PZP, SERPINA3, SERPINGl, TF, TNFRSF8 as defined by reference to TABLE 1 hereinabove.
8. A diagnostic test according to paragraph 5 or paragraph 7 wherein a protein is measured using an immunoassay.
9. A diagnostic test according to any preceding paragraph wherein a peptide is measured using mass spectrometry.
10. A diagnostic test according to any one of the present paragraphs wherein the biological sample is a bio-fluid.
11. A diagnostic test according to any one of the preceding paragraphs wherein said diagnostic test is to determine susceptibility to ILD following administration of a drug to a patient.
12. A diagnostic test according to paragraph 11 wherein the drug is an erb receptor drug or a DMARD.
13. A diagnostic test according to paragraph 11 wherein the drug is gefitinib.
REFERENCES
I. Bjellkvist B., Ek K., RMgetti P. G, Gianaza E., Gδrg A.,Westermeier R., Postel W., J Biochem Biophys Methods. 1982, 63, 17-39 2. Gδrg A., Methods MoI Biol. 1999, 112, 197-209
3. Gόrg A, Weiss, W., Dunn M. J., Proteomics. 2005, 5, 826-7.
4. Malmstrδm J., Bratt C, Lindberg H, Lindberg C, Wieslander E., Delander L., Sarnstrand B., Bums J. S., Mose-Larsen P., Fey S., and Marko-Varga G, Mol.Cell.
Proteomics, 2004, 3, 466-477 5. Mann, M., Hendrickson,R.C, & Pandey,A. Annu. Rev. Biochem, 2001, 70, 437-473
6. R. Aebersold and M. Mann, Nature, 2003, 422, 198-211.
7. AndersenJ.S., Lyon,C.E., Fox,A.H., Leung,A.K., Lam,Y.W., Steen,H., Mann,M., Lamond,A.L, Curr. Biol. 2002, 12, 1-11
8. R. I. Yates, J. Mass Spectrom., 1998, 33 1. 9. Gygi S. R, Rist B., Griffm T. 1, Eng J., Aebersold R., J. Proteome Res., 2002, 1, 47-54
10. Wolters,D.A., Washburn,M.R, & YatesJ.R., III., Anal. Chem. 2001, 73, 5683-5690
II. Washburn,M.R, WoltersJD., & YatesJ.R., Ill, Nat. Biotechnol. 2001, 19, 242-247 12. 1. Molnar and C. Horvath, J. Chromatogr., 1977, 142 , 623.
13. K. Kalghatgi and C. Horvath, J. Chromatogr., 1988 , 443, 343. 14. H. Chen and C. Horvath, J. Chromatogr. A, 1995, 705 3.
15. J. Frenz, Ph. Van der Schrieck and Cs. Horvath, J. Chromatogr. 1985, 1.
16. K. Kalghatgi, I. Fellegvari and C. Horvath, J. Chromatogr., 1992, 604, 47.
17. H. Chen and C. Horvath, Anal. Meth. Instr., 1993, 1 213.
18. D. Corradini, K. Kalghatgi and C. Horvath, J Chromatogr A, 1996, 728, 225. 19. Y. Shi, R. Xiang, J. K. Crawford, C. M. Colangelo, C. Horvath and J. A. Wilkins, J.
Proteome Res., 2004, 3 104. 20. Aebersold, R., Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422, 198- 207
21. Steen, H. and M. Mann., Nat Rev MoI Cell Biol 2004, 5, 699-711.
22. Olsen, J. V. and M. Mann.,. Proc Natl Acad Sci U S A2004 101, 13417-22 23. Marko-Varga G, and Fehniger T. E., J. Proteome Res., 2004, 3, 167-178
24. Blake TA, Ouyang Z, Wiseman JM, Takats Z, Guymon AJ, Kothari S, et al.,
Analytical Chemistry 2004;76(21):6293-305.
25. Campbell JM, Collings BA, Douglas D J., Rapid Communications in Mass
Spectrometry 1998;12(20):1463-74. 26. Cha BC, Blades M, Douglas DJ., Analytical Chemistry 2000;72(22):5647-54.
27. Hager JW., Rapid Communications in Mass Spectrometry 2003;17(13):1389- 98.
28. Kocher F, Favre A, Gonnet F, Tabet JC, Journal of Mass Spectrometry
1998;33(10):921-35.
29. Mao DM, Ding CF, Douglas DJ., Rapid Communications in Mass Spectrometry 2002;16(20): 1941-45.
30. Olsen JV, Mann M., Proceedings of the National Academy of Sciences of the United States of America 2004;101(37):13417-22.
31. Rocher F, Favre A, Gonnet F, Tabet JC, Journal of Mass Spectrometry
1998;33(10):921-35. 32. Settineri TA, Williamson BL, Hunter CL., Molecular & Cellular Proteomics 2003;2(7
Supplement):S75.
33. Wu J, Hager JW, Xia Y, Londry FA, McLuckey SA.,. Analytical Chemistry
2004;76(17):5006-15.
34. Zhang S, Chelius D., Journal of Biomolecular Techniques: JBT 2004;15(2):120-33. 35. Berkel, G. J. V.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-
1295 36. Anderson L, Anderson J., Proteomics 2000, 1, 1-13 37. Anderson, NX. ,Anderson, NG, . MoI Cell Proteomics. 2003 Jan;2(l):50
38. Anderson L, J. Physiol, 563, (2005) 23-60
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A diagnostic test to detect ILD or to identify the susceptibility of a patient to develop
ILD which comprises measurement of one or more of the peptides shown below, or a protein comprising one or more of said peptides, in a biological sample obtained from the patient:
Peptide
VKSPELQAEAK
SARLGATRGPR
ATEHLSTLSEK
VRPQQLVK
ATEHLSTLSEK
QLEQVIAK
ESSSHHPGIAEFPSR
QKVEPLRAELQEGAR
QFTSSTSYNRGDSTFESK
LRTEGDGVYTLNDK
FISLGEACKK
IDVHLVPDR
SSGRR
VGYVSGWGR
FISLGEACK
CVLFPYGGCQGNGNK
VDLSFSPSQSLPASHAHLR
SETKDLLFRDDTVCLAK
CSTSSLLEACTFR
LYGSEAFATDFQDSAAAKK
DSGRDYVSQFEGSALGK
LPGQDNLVK
ASTPNGYDNGIIWATWK
MLEEIMKYEASILTHDSSIR
KNPREDYLDVYVFGVGPLVNQVNINALASK DLPEPR
SELTQQLNALFQDKLGEVNTYAGDLQK FSVPAGIVIPSFQALTAR
FRVVSMDENFHPLNELIPLVYIQDPK
ADQEGAREIVCNVTLGGERR
YTHDQLFFIAFAQNWCIKRR
DLATVYVDVLKDSGRDYVSQFEGSALGK
VTWAPPPSIDLTNFLVR
SAGWNIPIGLLYCDLPEPR
NIQVPR
LLDSLPSDTRLVLLNAIYLSAK DLFGGRLTSDMIAER
SWVPHTFESELSDPVELLVAES
EPCVESLVSQYFQTVTDYGKDLMEK
ILGDMQPGDYFDLVLFGTR
2. A diagnostic test according to claim 1 wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of an elevated level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD:
Peptide
VKSPELQAEAK
SARLGATRGPR
ATEHLSTLSEK
VRPQQLVK
ATEHLSTLSEK
ESSSHHPGIAEFPSR
QKVEPLRAELQEGAR
QFTSSTSYNRGDSTFESK
LRTEGDGVYTLNDK
FISLGEACKK
IDVHLVPDR
SSGRR
FISLGEACK
SETKDLLFRDDTVCLAK
CSTSSLLEACTFR
LYGSEAFATDFQDSAAAKK
DSGRDYVSQFEGSALGK
LPGQDNLVK
ASTPNGYDNGIIWATWK
MLEEIMKYEASILTHDSSIR
DLPEPR
SAGWNIPIGLLYCDLPEPR
NIQVPR
EPCVESLVSQYFQTVTDYGKDLMEK
3. A diagnostic test according to claim 1 wherein diagnosis of ILD or determination of susceptibility to ILD comprises measurement of a reduced level of one or more of the peptides shown below, or a protein comprising one or more of said peptides, compared with the level of said peptide or protein in patients without ILD: Peptide QLEQVIAK
VGYVSGWGR
CVLFPYGGCQGNGNK
VDLSFSPSQSLPASHAHLR
KNPREDYLDVYVFGVGPLVNQVNINALASK SELTQQLNALFQDKLGEVNTYAGDLQK
FSVPAGΓVIPSFQALTAR
FRVVSMDENFHPLNELIPLVYIQDPK ADQEGAREIVCNVTLGGERR
YTHDQLFFIAFAQNWCIKRR
DLATVYVDVLKDSGRDYVSQFEGSALGK VTWAPPPSIDLTNFLVR
LLDSLPSDTRLVLLNAIYLSAK
DLFGGRLTSDMIAER
SWVPHTFESELSDPVELLVAES
ILGDMQPGDYFDLVLFGTR
4. A diagnostic test according to claim 2 further comprising measurement of a reduced level of one or more of the peptides defined in claim 3, or a protein comprising one or more of said peptides, compared to the level of said peptide or protein in patients without ILD.
5. A diagnostic test according to any one of the preceding claims comprising measuring one or more proteins comprising one or more of the said peptides.
6. A diagnostic test according to any preceding claim, wherein one or more of said peptides is isotope labelled, added to the sample prior to analysis and used as an internal standard for qualitative and/or quantitative analysis.
7. A diagnostic test according to claim 1 comprising measuring one or more proteins selected from AHSG, AMBP, APOAl, APOA2, APOA4, APOB, BF, C3, CFH, FGA, FGG, FLJ38348, FNl, GNL2, HP, ICAM3, ITIHl, ITIH4, LOC388504, MRPL27, PZP, SERPINA3, SERPINGl, TF and TNFRSF8 as defined by reference to TABLE 1 hereinabove.
8. A diagnostic test according to claim 5 or claim 7 wherein a protein is measured using an immunoassay.
9. A diagnostic test according to any preceding claim wherein a peptide is measured using mass spectrometry.
10. A diagnostic test according to any one of the present claims wherein the biological sample is a bio-fluid.
11. A diagnostic test according to any one of the preceding claims wherein said diagnostic test is to determine susceptibility to ILD following administration of a drug to a patient.
12. A diagnostic test according to claim 11 wherein the drug is an erb receptor drug or a DMARD.
13. A diagnostic test according to claim 11 wherein the drug is gefitinib.
PCT/GB2006/000981 2005-03-23 2006-03-20 Method of diagnosing interstitial lung disease (ild) WO2006100446A2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0505960A GB0505960D0 (en) 2005-03-23 2005-03-23 Method
GB0505960.5 2005-03-23
GB0519759.5 2005-09-28
GBGB0519759.5A GB0519759D0 (en) 2005-09-28 2005-09-28 Method

Publications (2)

Publication Number Publication Date
WO2006100446A2 true WO2006100446A2 (en) 2006-09-28
WO2006100446A3 WO2006100446A3 (en) 2006-12-07

Family

ID=36649820

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2006/000981 WO2006100446A2 (en) 2005-03-23 2006-03-20 Method of diagnosing interstitial lung disease (ild)

Country Status (2)

Country Link
TW (1) TW200716978A (en)
WO (1) WO2006100446A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008128029A2 (en) * 2007-04-12 2008-10-23 Battelle Memorial Institute Solution fragmentation systems and processes for proteomics analysis
WO2011151461A3 (en) * 2010-06-04 2012-07-05 B.S.R.C. "Alexander Fleming" Autotaxin pathway modulation and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000060117A2 (en) * 1999-04-02 2000-10-12 Interleukin Genetics, Inc. Prediction of risk of interstitial lung disease
US20040039553A1 (en) * 2000-08-21 2004-02-26 Thierry Poynard Diagnosis method of inflammatory, fibrotic or cancerous disease using biochemical markers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000060117A2 (en) * 1999-04-02 2000-10-12 Interleukin Genetics, Inc. Prediction of risk of interstitial lung disease
US20040039553A1 (en) * 2000-08-21 2004-02-26 Thierry Poynard Diagnosis method of inflammatory, fibrotic or cancerous disease using biochemical markers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [Online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; January 2005 (2005-01), KONISHI JUN ET AL: "Analysis of the response and toxicity to gefitinib of non-small cell lung cancer." XP002391375 Database accession no. NLM15816608 & ANTICANCER RESEARCH. 2005 JAN-FEB, vol. 25, no. 1B, January 2005 (2005-01), pages 435-441, ISSN: 0250-7005 *
OHNISHI H ET AL: "Circulating KL-6 levels in patients with drug induced pneumonitis." THORAX. OCT 2003, vol. 58, no. 10, October 2003 (2003-10), pages 872-875, XP002391371 ISSN: 0040-6376 *
SUGA M ET AL: "Clinical significance of MCP-1 levels in BALF and serum in patients with interstitial lung diseases." THE EUROPEAN RESPIRATORY JOURNAL : OFFICIAL JOURNAL OF THE EUROPEAN SOCIETY FOR CLINICAL RESPIRATORY PHYSIOLOGY. AUG 1999, vol. 14, no. 2, August 1999 (1999-08), pages 376-382, XP002391373 ISSN: 0903-1936 *
TAKAHASHI H ET AL: "Diagnostic significance of surfactant proteins A and D in sera from patients with radiation pneumonitis." THE EUROPEAN RESPIRATORY JOURNAL : OFFICIAL JOURNAL OF THE EUROPEAN SOCIETY FOR CLINICAL RESPIRATORY PHYSIOLOGY. MAR 2001, vol. 17, no. 3, March 2001 (2001-03), pages 481-487, XP002391372 ISSN: 0903-1936 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008128029A2 (en) * 2007-04-12 2008-10-23 Battelle Memorial Institute Solution fragmentation systems and processes for proteomics analysis
WO2008128029A3 (en) * 2007-04-12 2008-12-11 Battelle Memorial Institute Solution fragmentation systems and processes for proteomics analysis
WO2011151461A3 (en) * 2010-06-04 2012-07-05 B.S.R.C. "Alexander Fleming" Autotaxin pathway modulation and uses thereof

Also Published As

Publication number Publication date
TW200716978A (en) 2007-05-01
WO2006100446A3 (en) 2006-12-07

Similar Documents

Publication Publication Date Title
Schey et al. Proteomics characterization of exosome cargo
AU2003262824B2 (en) Method for characterizing biomolecules utilizing a result driven strategy
EP3511973B1 (en) Quantitation of insulin by mass spectrometry
Helfer et al. Direct analysis of the mushroom poisons α-and β-amanitin in human urine using a novel on-line turbulent flow chromatography mode coupled to liquid chromatography–high resolution-mass spectrometry/mass spectrometry
US11428696B2 (en) Mass spectrometry analysis of mutant polypeptides in biological samples
AU2007258970A1 (en) Mass spectrometry biomarker assay
EP2851688B1 (en) Use of glycoprotein C4BPA as marker for detecting pancreatic cancer
Sun et al. Efficient enrichment and identification of phosphopeptides by cerium oxide using on‐plate matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis
EP3333571B1 (en) Method and kit for parallel quantification of splice variants of vascular endothelial growth factor (vegf-a)
JP7076075B2 (en) Diagnosis method of Alzheimer's disease using signal peptide as an index
WO2006100446A2 (en) Method of diagnosing interstitial lung disease (ild)
WO2015026909A2 (en) Msia-srm assay for biomarker analysis
US20140051105A1 (en) Mutant Proteins as Cancer-Specific Biomarkers
Sogawa et al. The measurement of a fibrinogen α C-chain 5.9 ákDa fragment (FIC 5.9) using MALDI-TOF MS and a stable isotope-labeled peptide standard dilution
Kodera et al. Establishment of a strategy for the discovery and verification of low-abundance biomarker peptides in plasma using two types of stable-isotope tags
JP2022517414A (en) Automated sample workflow for LC-MS-based HbA1c measurements at the intact protein level
Kienzl-Wagner et al. Proteomics in transplantation
WO2013166343A2 (en) Mrm-ms signature assay
EP3164703B1 (en) Systems, assays, and methods for determining risk factors for alzheimer's disease
WO2008074769A1 (en) Direct mass spectrometric analysis of aggregates of therapeutic proteins
da Silva Menegasso et al. In situ proteomic analysis of glioblastoma multiforme: a translational approach to improve prognostic/diagnostic routines
CN112881578A (en) Kit for liver cancer prognosis risk assessment and application
Nicolardi Development of ultrahigh resolution FTICR mass spectrometry methods for clinical proteomics

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

NENP Non-entry into the national phase in:

Ref country code: RU

WWW Wipo information: withdrawn in national office

Country of ref document: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06726423

Country of ref document: EP

Kind code of ref document: A2

WWW Wipo information: withdrawn in national office

Ref document number: 6726423

Country of ref document: EP