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WO2007065463A1 - UTILISATION DE C3a ET DE SES DÉRIVÉS COMME BIOMARQUEUR POUR DES ALTÉRATIONS BÉNIGNES DU TISSU PULMONAIRE ET DES TUMEURS PULMONAIRES MALIGNES, PROCÉDÉ DE DÉTECTION ET SYSTÈME DE TEST - Google Patents

UTILISATION DE C3a ET DE SES DÉRIVÉS COMME BIOMARQUEUR POUR DES ALTÉRATIONS BÉNIGNES DU TISSU PULMONAIRE ET DES TUMEURS PULMONAIRES MALIGNES, PROCÉDÉ DE DÉTECTION ET SYSTÈME DE TEST Download PDF

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Publication number
WO2007065463A1
WO2007065463A1 PCT/EP2005/013239 EP2005013239W WO2007065463A1 WO 2007065463 A1 WO2007065463 A1 WO 2007065463A1 EP 2005013239 W EP2005013239 W EP 2005013239W WO 2007065463 A1 WO2007065463 A1 WO 2007065463A1
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Prior art keywords
derivative
precursors
malign
benign
lung tissue
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PCT/EP2005/013239
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English (en)
Inventor
Hartmut Juhl
Kerstin David
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Indivumed Gmbh
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Priority to PCT/EP2005/013239 priority Critical patent/WO2007065463A1/fr
Publication of WO2007065463A1 publication Critical patent/WO2007065463A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of detection of benign lung tissue alterations and/or malign lung tumors as well as precursors thereof.
  • Lung cancer is the most common malignant disease worldwide, and is the major cause of death from cancer, particularly amongst men. It was a rare disease until the beginning of the 20th century. Since then, the worldwide occurence of lung cancer has increased rapidly and now accounts for an estimated 901 ,746 new cases each year among men and 337,115 among women.
  • lung cancer The association between lung cancer and smoking is probably the most intensively investigated relationship in epidemiology.
  • smoking lung cancer can be caused by chemicals like chromates, nickel, cadmium, beryllium, arsenic, polycyclic hydrocarbon, burning products of carbon like carbon black and tar, as well as asbest.
  • radionucleotides and ionizing radiation energy can cause lung cancer. Also genetic predispositions have been observed.
  • adenocarcinoma large cell carcinoma and small cell carcinoma.
  • Squamous cell carcinoma is very strongly associated with smoking and represents the most common type of lung cancer in many populations.
  • histogenesis and the putative precursor lesions of lung cancer are largely unknown for the different histological types, the presence of putative precursor lesions (dysplasia, metaplasia and carcinoma in situ) are commonly reported in resection specimens and/or sputum cytology for squamous cell carcinoma.
  • Diagnostic methods by apparatus like X-ray analysis, computed tomography, scintiscanning or invasive methods like bronchoscopy with biopsy and histology are available as options for lung carcinoma screening.
  • Such screening methods are time consuming, expensive, have side-effects and are less accepted by the patient.
  • CEA cancer epidermal growth factor
  • SCC squamous cell carcinoma antigen
  • NSE neuron specific enolase
  • CEA-(Carcinoembryonic antigen)-levels in blood samples have been used to detect lung carcinoma.
  • CEA levels are not specifically elevated in lung carcinoma and have been shown to be elevated also in patients with other malignant diseases, e.g., cancers of the stomach, pancreas, breast, and colon, and also with various non-malignant conditions, e.g., alcoholic liver disease, inflammatory bowel disease, heavy cigarette smoking, and pancreatitis.
  • malignant diseases e.g., cancers of the stomach, pancreas, breast, and colon
  • non-malignant conditions e.g., alcoholic liver disease, inflammatory bowel disease, heavy cigarette smoking, and pancreatitis.
  • An object of the invention is to provide means allowing a detection of benign lung tissue alterations and/or malign lung tumors as well as precursors thereof. It is a further object to provide means of allowing a selective and specific detection of benign lung tissue alterations and/or malign lung tumors by a non-invasive method.
  • Another object of the present invention is to provide a test system for detecting benign lung tissue alterations and/or malign lung tumors which is cost effective and can be widely used.
  • test system should be easy to handle and convenient for the individual to be examined for benign lung tissue alterations and/or malign lung tumors.
  • the objects underlying the present invention are solved by the use of C3a or a derivative thereof as a biomarker for the detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof in an individual.
  • the detection can be carried out in vivo and in vitro. Pursuant to a preferred embodiment, the detection is carried out in vitro.
  • a method for detecting of benign lung tissue alterations, malign lung tumors and/or precursors thereof comprising the steps: a) providing an isolated sample material which has been taken from an
  • the objects are also solved by a method for monitoring the development and/or the course and/or the treatment of benign lung tissue alterations, malign lung tumors and/or precursors thereof comprising the steps: a) providing an isolated sample material which has been taken from an
  • the level of C3a or a derivative thereof is both an early and a general detection biomarker for benign or malign lung diseases.
  • the level of C3a or a derivative thereof is raised significantly compared to the level of C3a or a derivative thereof of non-diseased or healthy individuals.
  • the level can be increased by a factor of at least 2, preferably by a factor of at least 3, further preferred by a factor of at least 4.
  • the factor can also be in the range of 5 to 10, preferably of 6 to 8.
  • the level of C3a or a derivative thereof can be used as an early detection biomarker or generally as a biomarker in diagnostic methods or diagnostic test systems of benign or malign lung diseases.
  • the term "benign lung tissue alterations" as used in the present invention preferably refers to lung diseases which have to be treated by surgery.
  • the term "benign lung tissue alterations" refers to diseases such as chronic pneumonia, chronic bronchitis, focal chronic-organizing pneumonia, unspecific interstitial pneumonia, pleural effusion, abscess of lung, abscess of mediastinum, pseudo tumor caused by chronic pneumonia, aspergilloma, chronic pneumonia, inflammatory amyloid patients, chronic infection, tissue degeneration caused by tuberculosis, granuloma, like sarcoidosis, embryoplastic tumors, like harmartoma, haemangioma, fibroma, necrotic lung parenchyma and/or other benign tumors. Benign tumor means that the tumor grows non-invasive and does not metastasize.
  • malign lung tumors preferably refer to diseases such as squamous cell carcinoma, adenocarcinoma, large cell carcinoma and/or small cell carcinoma.
  • precursors thereof as used in the present invention refers to precursors of benign lung tissue alterations and/or precursors of malign lung tumors.
  • the precursor can be one or more altered cells and/or an altered tissue.
  • the alteration may be a genetic alteration, an altered gene expression and/or an altered histology.
  • the effectiveness of a surgical or therapeutical procedure is controlled in order to decide as to whether the benign lung tissue alterations and/or malign lung tumors are completely removed.
  • the therapy of benign lung tissue alterations and/or malign lung tumors with one or more chemical substances, antibodies, antisense-RNA, radiation, e.g. X-rays or combinations thereof is controlled in order to control the effectiveness of the treatment.
  • test system for detecting of benign lung tissue alterations and/or malign lung tumors in a sample of an individual comprising: a) an antibody or a receptor which binds to an epitope of C3a or a derivative thereof,
  • an array comprising detection molecules for detecting of benign lung tissue alterations and/or malign lung tumors in an individual comprising as detection molecule: a) a nucleic acid probe immobilized to a solid support for binding to and detecting mRNA encoding C3a or a derivative thereof and/or for binding to and detecting C3a proteins or derivatives thereof, or
  • a receptor immobilized to a solid support for binding to and detecting of an epitope of C3a or a derivative thereof, wherein preferably each different amounts of detection molecules are immobilized to the solid support to increase the accuracy of the quantification.
  • the nucleic acid probe is for example selected from the group consisting of single- stranded or double-stranded DNA or RNA, aptamers and combinations thereof.
  • Aptamers are single-stranded oligonucleotides that assume a specific, sequence- dependent shape and bind to protein targets with high specificity and affinity.
  • the objects are furthermore solved by a method for determining whether a compound is effective in the treatment of benign lung tissue alterations and/or malign lung tumors comprising the steps: a) treating of a patient suffering from benign lung tissue alterations and/or malign lung tumors with a compound,
  • sample material is also designated as “sample”.
  • biomarker is meant to designate a protein or protein fragment or a nucleic acid which is indicative for the incidence of benign lung tissue alterations, malign lung tumors and/or precursors thereof. That means the “biomarker” is used as a mean for detecting benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the term “individual” or “individuals” is meant to designate a mammal. Preferably, the mammal is a human being such as a patient.
  • the term "healthy individual(s)" is used only in respect of the pathological condition of benign lung tissue alterations, malign lung tumors and/or precursors thereof and does not exclude the individual to suffer from diseases other than benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the term "derivative thereof is meant to describe any modification on DNA, mRNA or protein level comprising e.g. the truncated gene, fragments of said gene, a mutated gene, or modified gene.
  • the term "gene” includes nucleic acid sequences, such as DNA, RNA, mRNA or protein sequences or oligopeptide sequences or peptide sequences.
  • the derivative can be a modification which is an result of a deletion, substitution or insertion of the gene.
  • the gene modification can be a result of the naturally occurring gene variability.
  • naturally occurring gene variability means modifications which are not a result of genetic engineering.
  • the gene modification can be a result of the processing of the gene or gene product within the body and/ or a degradation product.
  • the modification on protein level can be due to enzymatic or chemical modification within the body.
  • the modification can be a a glycosylation or phosphorylation or farnesylation.
  • the derivative codes for or comprises at least 5 amino acids, more preferably 10 amino acids, most preferably 20 amino acids of the unmodified protein.
  • the derivative codes for at least one epitope of the respective protein.
  • the term "C3a or a derivative thereof as used in the present invention also comprises truncated C3a, fragments of C3a, mutated C3a, modified C3a or the precursor C3 (Fig. 1, SEQ ID No.1) or fragments of C3.
  • the derivative has a protein sequence identity of 80%, preferably 90%, more preferably 98% with the sequence SEQ-ID-No. 2 (Fig 2A, SEQ ID No. 2).
  • the modification of "C3a” can be due to enzymatic or chemical modification.
  • the term C3a or a derivative thereof especially comprises a truncated C3a-protein preferably having a molecular weight in the range of 8,950 ⁇ 25 Da; more preferably in the range of 8,950 ⁇ 20 Da.
  • the truncated C3a-protein has a molecular weight of 8,939 Da.
  • the C3a-protein has no C-terminal Arginin and optionally a molecular weight in the range of 8,950 ⁇ 20 Da.
  • the C3a derivative is C3a-desArg (Fig 2B, SEQ ID No. 3).
  • the C3a derivative is obtained by cleavage of C3a by mastcell-chymase.
  • the C3a is obtained by cleavage of C3 by C3-convertase.
  • C3a belongs to the group of anaphylatoxins.
  • C3a, C4a and C5a are proteolytic products of serine proteases of the complement system.
  • C3a (SEQ-ID-No.2) is derived from the third component (C3) (SEQ-ID-NO.1) of the blood complement system during complement activation.
  • C3a is a hormone with local effectiveness. Approximately 40% of the amino acid residues in C3a are involved in a helical conformation. Serum anaphylatoxins are involved in a variety of cellular immune responses, as well as being potent proinflammartory agents. C3a produces powerful effects on blood vessel walls, contraction of smooth muscle and an increase in vascular permeability. The C-terminal arginine in C3a is of fundamental importance for its biological activity.
  • Anaphylatoxins are regulated by carboxypeptidase N (anaphylatoxin inactivator), which removes within seconds the carboxyterminal arginine. This mechanism converts the intact anaphylatoxin into a less active C3a- desArg form (SEQ ID No.3).
  • epipe is meant to designate any structural element of a protein or peptide or any proteinaceous structure allowing the specific binding of an antibody, an antibody fragment, a protein or peptide structure or a receptor.
  • sample material such as a body fluid or tissue sample which already has been isolated from the human body. Subsequently the sample material can be fractionated and/or purified. It is for example possible, to store the sample material to be tested in a freezer and to carry out the methods of the present invention at an appropriate point in time after thawing the respective sample material.
  • the protein C3a or a derivative, especially C3a-desArg, thereof can be used as a biomarker for the detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the inventors have now surprisingly found that the level protein C3a or a derivative thereof in a body fluid is elevated in individuals having benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • sample material can be tissue, cells or a body fluid.
  • the sample material is a body fluid such as blood, blood plasma, blood serum, bone marrow, stool, synovial fluid, lymphatic fluid, cerebrospinal fluid, sputum, urine, mother milk, sperm, exudate and mixtures thereof.
  • the body fluid has been isolated before carrying out the methods of the present invention.
  • the methods of the invention are preferably carried out in vitro by a technician in a laboratory.
  • C3a or a derivative thereof is measured in blood plasma or blood serum.
  • Blood serum can be easily obtained by taking blood from an individual to be medically examined and separating the supernatant from the clotted blood.
  • the present invention provides both an early stage and a general biomarker which allows to detect the neoplastic disease at an early and still benign stage, neoplastic disease at an early stage or benign stage and/or early tumor stages.
  • the early detection enables the physician to timely remove the early lung tissue alterations and to dramatically increase the chance of the individual to survive.
  • the biomarker of the present invention can of course be used in diagnostic methods or diagnostic test systems for generally detecting or monitoring of benign or malign lung diseases or in the follow-up of patients diseased or cured of benign or malign lung diseases.
  • the present invention allows to monitor the level of C3a or a derivative thereof in a body fluid such as blood serum over an extended period of time, such as years.
  • the long term monitoring allows to differentiate between the group of healthy individuals and the group of benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • a routine check e.g. twice or three times a year, is particularly recommended for high risk groups such as smokers.
  • the level of C3a or a derivative thereof can be routinely checked, for example, once, twice or thrice a year. If an increase of the level of C3a or a derivative thereof is detected this can be indicative for benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the course of the disease and/or the treatment can be monitored. If the level of C3a or a derivative thereof further increases, for example after removal of the benign lung tissue alterations, malign lung tumors and/or precursors thereof, this can be indicative for exacerbation of the pathological condition. That means, the level of C3a or a derivative thereof is a valuable clinical parameter for detecting and/or monitoring of benign lung tissue alterations, malign lung tumors and/or precursors thereof. The level of C3a or a derivative thereof in body fluids is higher after incidence of benign lung tissue alterations, malign lung tumors and/or precursors thereof. Therefore, the level of C3a or a derivative thereof is an important clinical parameter to allow an early diagnosis and, consequently, an early treatment of the disease.
  • the level of C3a or a derivative thereof can surprisingly be 2 to 5 times higher in benign lung tissue alterations and/or malign lung tumors than in healthy individuals or other tumors such as prostate. Therefore, the quantification of C3a or a derivative thereof provides a mean to detect benign lung tissue alterations, malign lung tumors and/or precursors thereof from other tumors.
  • the method of the invention for detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof comprises the step of providing an isolated sample material which has been taken from an individual, then determining the level of C3a or a derivative thereof in the isolated sample material, and finally comparing the determined level of C3a or a derivative thereof with one or more reference values.
  • one or more further biomarker(s) is/are additionally detected in an isolated sample material which has been taken from an individual, the level of the biomarker(s) is/are determined and compared with one or more respective reference values.
  • the reference value can be calculated as the average level of C3a or a derivative thereof determined in a plurality of isolated samples of healthy individuals or individuals suffering from benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • This reference value can be established as a range to be considered as normal meaning that the person is healthy or suffers from benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • a specific value within a range can then be indicative for healthy condition or the pathological condition of benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • This range of reference value(s) can be established by taking a statistically relevant number of body fluid samples, such as serum samples, of healthy individuals as it is done for any other medical parameter range such as, e.g., blood sugar.
  • two reference values are calculated which are designated as negative control and positive control.
  • the reference value of the negative control is calculated from healthy individuals and the positive control is calculated from individuals suffering from benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the reference values can be individual reference values calculated as the average level of C3a or a derivative thereof determined in a plurality of isolated samples taken from the individual over a period of time.
  • the C3a or a derivative thereof level can be measured, for example, from the same blood serum sample when measuring blood sugar and can be used to establish an individual calibration curve allowing to specifically detect any individual increase of the level of C3a or a derivative thereof.
  • the reference value for further biomarkers can also be calculated in the same way as described for C3a or a derivative thereof.
  • the average levels of C3a or a derivative thereof or further biomarkers may be the mean or median level.
  • the present invention further provides a test system for detecting benign lung tissue alterations, malign lung tumors and/or precursors thereof in an isolated sample material of an individual.
  • the test system is based either on the specificity of an antibody or a receptor to specifically bind to an epitope or a suitable structural element of C3a or a derivative thereof or a fragment of thereof.
  • a receptor can be any structure able to bind specifically to C3a or a derivative thereof.
  • the receptor can be, for example, an antibody fragment such as an Fab or an F(ab') 2 fragment or any other protein or peptide structure being able to specifically bind to C3a or a derivative thereof.
  • the antibody, antibody fragment or receptor is bound to a solid support such as, e.g., a plastic surface or beads, for example latex beads, to allow binding and detection of C3a or a derivative thereof.
  • a solid support such as, e.g., a plastic surface or beads, for example latex beads
  • a conventional microtiter plate can be used as a plastic surface.
  • the detection of the binding of C3a or a derivative thereof can be performed, for example, by using a secondary antibody labelled with a detectable group.
  • the detectable group can be, for example, a radioactive isotope or an enzyme like horseradish peroxidase or alkaline phosphatase detectable by adding a suitable substrate to produce, for example, a color or a fluorescence signal.
  • the test system can be an immunoassay such as an enzyme-linked
  • ELISA immunosorbentassay
  • RIA radio immunoassay
  • LIA luminescence immunossay
  • any other immunological test system using the specificity of antibodies or fragments of antibodies can be used such as Western blotting or immuno precipitation.
  • the test system can also be an array comprising detection molecules for detecting benign lung tissue alterations, malign lung tumors and/or precursors thereof in an individual, wherein the detection molecule can be a nucleic acid probe immobilized on a solid support for binding to and detecting of mRNA encoding C3a, fragments, mutations, variants or derivatives thereof, or an antibody immobilized on a solid support for binding to and detecting of an epitope of C3a or a derivative thereof, or a receptor immobilized on a solid support for binding to and detecting of an epitope of C3a or a derivative thereof.
  • the detection molecule can be a nucleic acid probe immobilized on a solid support for binding to and detecting of mRNA encoding C3a, fragments, mutations, variants or derivatives thereof, or an antibody immobilized on a solid support for binding to and detecting of an epitope of C3a or a derivative thereof, or a receptor immobilized on a solid support for binding to
  • the array comprises further detection molecules which are biomarkers for detecting benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the level of C3a or a derivative thereof is determined by mass spectroscopy.
  • Mass spectroscopy allows to specifically detect C3a or a derivative thereof via its molecular weight and to quantify the amount of C3a or a derivative thereof very easily.
  • mass spectroscopy allows to simultaneously detect other proteins which can have a relevance with respect to the detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the sensitivity and/or specificity of the detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof is enhanced additionally by detection of one or more further biomarker(s).
  • the sensitivity and/or specificity of the detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof is.
  • the sensitivity and/or specificity of the detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof is enhanced by additionally detection of CEA (carcinoembryonic antigen), M 2 PK (pyruvate kinase type tumor M2), pro GRP (pro gastrin releasing peptide), PTHrP (parathormone related peptide), SCC (squamous cell carcinoma antigen), p53, Cyfra 21-1 (cytokeratin 19 fragment), NSE (neuron specific enolase), CgA (chromogranin A), Ca 125, CA 72-4, CA 19-9, TPA (tissue polypeptide antigen), ADAM8 (ADAM metallopeptidase domain 8) or derivatives thereof or combinations thereof in combination with C3a or a derivative thereof.
  • the use of the level of ADAM8 in a biological sample e.g., M2 PK (pyruvate kinase type tumor M2), pro GRP (pro gastrin
  • the present invention allows the detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof with an increased sensitivity and/or specificity. Furthermore, the detection method is well accepted by the patients, since the detection method is non-invasive and can be performed using a sample material already taken from the individual.
  • the sensitivity and specificity are defined as follow:
  • the sensitivity is the number of true positive patients (%) with regard to the number of all patients (100 %).
  • the patients are individuals having benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the specificity is the number of true negative individuals (%) with regard to the number of all healthy individuals (100%).
  • TN True negative (test negative, diagnosis correct);
  • the sensitivity is calculated by the following formula: TP/(TP + FN) and the specificity is calculated by the following formula: TN/ (TN + FP)
  • each analysis group which is selected from TP, FP, TN, FN 1 is calculated for a plurality of isolated samples selected from the group consisting of healthy individuals or patients suffering from benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • TP, FP, TN, FN relates to number of individuals that are correlated with the status true positive, false positive, true negative, false negative, respectively.
  • the methods of the present invention can be carried out in combination with other diagnostic methods for detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof to increase the overall sensitivity and/or specificity.
  • the detection of C3a allows an very early detection of benign lung tissue alterations, malign lung tumors and/or precursors thereof and can therefore be used as an very early marker.
  • the methods of the present invention are carried out as an early detection and/or monitoring method. If the results of the methods of the present invention should indicate the incidence of benign lung tissue alterations, malign lung tumors and/or precursors thereof further examinations such as bronchoscopy could be carried out.
  • Anti-C3a-desArg can be used when practicing the invention: • Anti-C3a-desArg: available with the Quidel immunoassay (Quidel Corporation, 10165 McKellar Court, San Diego, CA 92121, USA). Of course, other anti-C3a-desArg antibodies can be used as well.
  • the present invention further provides a method for determining whether a
  • the method for determining whether a compound is effective in the treatment of benign lung tissue alterations, malign lung tumors and/or precursors thereof comprises the steps of: a) treating of a patient suffering from benign lung tissue alterations, malign lung tumors and/or precursors thereof with a compound
  • the term "patient” as used in the present application covers humans as well as non- human beings such as animals.
  • the animals are preferably selected from the group consisting of rodents, e.g. mouse, rat, hamster, and other animals, e.g. guinea-pig, rabbit, hare, dog and pig.
  • These animals can be used to specifically induce certain disease states, like early benign lung tissue alterations, malign lung tumors and/or precursors thereof, for research purposes.
  • the induction of said disease states can, for example, be effected by treatment of the animals, for example, with radioactive or chemical substances known to induce benign lung tissue alterations, malign lung tumors and/or precursors thereof.
  • the disease states can also be induced using viral transfection systems. It is also possible to use genetically modified animals, in which one or more specific gene function(s) has/have been altered, or knock-out animals such as knock-out mice in which a specific gene function has been deleted.
  • the "compound” can be one or more chemical substances, an antibody, protein, peptide, antisense mRNA, small molecular drug, or combinations thereof.
  • the compound can also be replaced by irradiation, e.g. X-ray, or combinations of compounds and radiation can be used.
  • the level of C3a or a derivative thereof in a sample material of said patient can be determined by the above described detection techniques.
  • Fig. 1 shows the C3 protein sequence.
  • Fig. shows (A) the C3a protein sequence and (B) the C3a-desArg protein sequence.
  • Fig. 3 shows the quantification of C3a-desArg using an ELISA.
  • B benign lung diseases group
  • N healthy control group
  • T lung cancer group
  • the value of the y-axis refers to the rank sum order obtained by performance of the Mann-Whitney U Test.
  • the value of the y-axis refers to the rank sum order obtained by performance of the Mann-Whitney U Test.
  • Group 1 Serum from five groups of human patients were collected and investigated.
  • Group 1 consisted of 30 patients which were surgical patients treated for noncancerous diseases and non-lung diseases such as inguinal hernia, gall bladder stones or diverticulitis. These individuals of group 1 were taken as the group of healthy individuals, i. e. those, who did not suffer from benign lung tissue alterations, malign lung tumors and/or precursors thereof, wherein age and gender were matched to the other groups.
  • Group 2 consisted of 30 patients, who were treated for cancer unrelated lung diseases, wherein age and gender were matched to the other groups.
  • Group 3 consisted of 30 patients, who were patients having lung carcinoma. These patients are lung cancer patients with stadium HIA, IHB, IV, wherein age and gender were matched to the other groups.
  • Group 4 consisted of 30 patients which were surgical patients treated for non- prostate diseases wherein age and gender were matched to the other groups.
  • Group 5 consisted of 30 patients, who were patients having prostate carcinoma. These patients are prostate cancer patients with tumor Gleason scores of 3 + 4 or 4 + 3, wherein age and gender were matched to the other groups.
  • the microtiter strips included in the kit are coated with a monoclonal antibody (included in the immunoassay from Quidel) specific for human C3a-desArg.
  • a monoclonal antibody included in the immunoassay from Quidel
  • Samples were diluted and incubated for one hour at 18-25°C. During this incubation, C3a- desArg in the specimen was bound to the monoclonal antibody. After rinsing off the unbound native C3, peroxidase-conjugated rabbit anti-C3a antibody was used for the detection of bound C3a-desArg. Excess conjugate was removed through a washing step, and the amount of C3a-desArg in the serum sample was quantified using the peroxidase reaction and a standard curve.
  • Cut-off values are calculated by the CSRT(CART) algorithmus on the basis of decision-tree analysis (Breiman, L.,
  • the statistical data are evaluated on the basis on the median value.
  • the median is the limit between two equivalent halfs, i.e. 50% of the values are lower and 50 % of the values are higher than the median value.
  • the Figures show a 25% to 75% interval, i.e. 25 % of the values are lower than the 25% value and 25 % of the values are higher than the 75% value.
  • the statistical evaluation is performed by the Mann Whitney U test (Rosner B. (2000): Fundamentals of biostatistics (5 th edition) Pacific Grove: Duxbury). The tests were considered as significant at p values p ⁇ 0.05.
  • the whiskers of the box plots show the minimum and maximum values.
  • the intensity of the concentration of C3a-desArg [ng/ml] measured by ELISA differs significantly between healthy individuals and patients suffering from benign lung alterations and/or malign lung tumors (Example 1). 30 serum samples in each group were measured.
  • N healthy patients
  • B benign lung disease
  • T malign lung disease (tumor) Serum concentrations of C3a-desArg measured by ELISA were significantly higher in patients suffering from benign lung alterations and malign lung tumors than in the healthy controls. According to these data, a discrimination of healthy individuals from patients suffering from benign lung alterations or malign lung tumors was possible. The results are illustrated in Fig. 3.
  • the level of C3a-desArg was determined in serum samples obtained from patients (Group 5) with prostate tumor.
  • N 30
  • ELISA measurement values Mean ⁇ Standard-Deviation for N: 2223 ⁇ 4495 and T: 2150 ⁇ 972
  • the ranges of N (healthy) and T (tumor) of C3a-desArg are strongly overlapping and do not allow a reliable discrimination between healthy and diseased patients.
  • the median value of C3a-desArg determined by ELISA in patients having lung tumor is 4579 ng/ml whereas the respective median value of C3a-desArg in patients having prostate tumor is 1964 ng/ml.
  • Table 2 shows the comparison of sensitivity and specificity of C3a-desArg measured by ELISA (as described above) for the discrimination between healthy controls and benign lung alterations and malign lung tumors. Cut-off values are shown in brackets.
  • C3a is an excellent biomarker for the detection of benign lung alterations or malign lung tumors.
  • C3a-desArg In contrast to the already known biomarkers such as CEA the sensitivity and specificity of the C3a-desArg test is high and allows an early specific detection of benign lung alterations or malign lung tumors without a bronchoscopy. In contrast thereto C3a-desArg is not a specific marker for prostate related diseases.

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Abstract

La présente invention concerne un procédé de détection d'altérations bénignes du tissu pulmonaire, de tumeurs pulmonaires malignes et/ou de leurs précurseurs, comprenant les étapes consistant à : a) obtenir un matériau échantillon isolé qui a été prélevé chez un individu, b) déterminer le taux de C3a ou d'un de ses dérivés dans ledit matériau échantillon isolé, c) comparer le taux déterminé de C3a ou d'un de ses dérivés à une ou plusieurs valeurs de référence. L'invention concerne en outre un procédé de suivi de l'évolution d'altérations bénignes du tissu pulmonaire, de tumeurs pulmonaires malignes et/ou de leurs précurseurs et/ou le traitement d'altérations bénignes du tissu pulmonaire, de tumeurs pulmonaires malignes et/ou de leurs précurseurs. L'invention concerne également un système de test et un réseau destinés à être utilisés dans ces procédés. En outre, l'invention concerne l'utilisation de C3a en tant que biomarqueur pour la détection d'altérations bénignes du tissu pulmonaire, de tumeurs pulmonaires malignes et/ou de leurs précurseurs chez un individu. Enfin, l'invention concerne un procédé permettant de déterminer si un composé est efficace dans le traitement d'altérations bénignes du tissu pulmonaire, de tumeurs pulmonaires malignes et/ou de leurs précurseurs.
PCT/EP2005/013239 2005-12-09 2005-12-09 UTILISATION DE C3a ET DE SES DÉRIVÉS COMME BIOMARQUEUR POUR DES ALTÉRATIONS BÉNIGNES DU TISSU PULMONAIRE ET DES TUMEURS PULMONAIRES MALIGNES, PROCÉDÉ DE DÉTECTION ET SYSTÈME DE TEST WO2007065463A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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WO2015115922A1 (fr) * 2014-01-29 2015-08-06 Gdański Uniwersytet Medyczny Profil de marqueurs protéiques du sang utilisé comme test pour la détection du cancer du poumon

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