WO2005015218A1 - Utilisation de la proteinase 3(prn3) et de l'inhibiteur de l'elastase leucocytaire (ileu) en tant que marqueurs pour le cancer colorectal - Google Patents

Utilisation de la proteinase 3(prn3) et de l'inhibiteur de l'elastase leucocytaire (ileu) en tant que marqueurs pour le cancer colorectal Download PDF

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WO2005015218A1
WO2005015218A1 PCT/EP2004/008082 EP2004008082W WO2005015218A1 WO 2005015218 A1 WO2005015218 A1 WO 2005015218A1 EP 2004008082 W EP2004008082 W EP 2004008082W WO 2005015218 A1 WO2005015218 A1 WO 2005015218A1
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prn3
ileu
colorectal cancer
diagnosis
binding agent
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PCT/EP2004/008082
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English (en)
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Michael Tacke
Peter Berndt
Marie-Luise Hagmann
Johann Karl
Hanno Langen
Stefan Palme
Markus Roessler
Wolfgang Rollinger
Werner Zolg
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
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Publication of WO2005015218A1 publication Critical patent/WO2005015218A1/fr

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    • 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
    • G01N33/57419—Specifically defined cancers of colon
    • 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
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8121—Serpins
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

Definitions

  • CRC colorectal cancer
  • the prognosis in advanced stages of tumor is poor. More than one third of the patients will die from progressive disease within five5 years after diagnosis, corresponding to a survival rate of about 40% for five years. Current treatment is only curing a fraction of the patients and clearly has the best effect on those patients diagnosed in an early stage of disease.
  • CRC colorectal cancer
  • a protein encoded by a rare mRNA may be found in very high amounts and a protein encoded by an abundant mRNA may nonetheless be hard to detect and find at all. This lack of correlation between mRNA-level and protein level is due to reasons like mRNA stability, efficiency of translation, stability of the protein, etc.
  • WO 02/078636 reports about nine colorectal cancer-associated spots as found by surface-enhanced laser desorption and ionization (SELDI). These spots are seen more frequently in sera obtained from patients with CRC as compared to sera obtained from healthy controls. However, the identity of the molecule(s) comprised in such spot, e.g., its (their sequence), is not known.
  • a new diagnostic marker as a single marker should be at least as good as the best single marker known in the art. Or, a new marker should lead to a progress in diagnostic sensitivity and/or specificity either if used alone or in combination with one or more other markers, respectively.
  • the diagnostic sensitivity and/or specificity of a test is best assessed by its receiver-operating characteristics, which will be described in detail below.
  • CEA carcinoembryonic antigen
  • a tumor-associated glycoprotein a tumor-associated glycoprotein
  • serum CEA determination possesses neither sensitivity nor the specificity to enable its use as a screening test for colorectal cancer in the asymptomatic population (Reynoso, G., et al., JAMA 220 (1972) 361-365; Sturgeon,
  • Samples taken from stool have the advantage that such sampling is easily possible by non-invasive means.
  • the guaiac test is currently most widely used as a screening assay for CRC from stool.
  • the guaiac test however, has both poor sensitivity as well as poor specificity.
  • the sensitivity of the guaiac-based fecal occult blood tests is ⁇ 26%, which means 74% of patients with malignant lesions will remain undetected (Ahlquist, D.A., Gastroenterol. Clin. North Am. 26 (1997) 41-55).
  • the hemoglobin assay has an unsatisfactory sensitivity for the detection of colorectal neoplasms. Whereas cancer in its progressed carcinoma stage is detected with a sensitivity of about 87% the earlier tumor stages are not detected with a sufficient sensitivity.
  • the hemoglobin-haptoglobin complex assay was more sensitive in the detection of earlier stages of CRC. This more sensitive detection was accompanied by a poor specificity. Since poor specificity, however, translates to a high number of unnecessary secondary investigations, like colonoscopy, an assay with a poor accuracy also does not meet the requirements of a generally accepted screening assay.
  • the present invention therefore relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual; b) contacting said sample with a first specific binding agent for proteinase 3 (PRN3) and a second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU) under conditions appropriate for formation of a complex between said first and second binding agent and PRN3 bound to ILEU (PRN3/ILEU); and c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • a preferred method uses a stool sample obtained from an individual.
  • the stool sample is processed to obtain a processed sample liquid which is more convenient to handle than a stool specimen.
  • Such processed sample is then incubated with the first specific binding agent for proteinase 3 (PRN3) and the second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU).
  • PRN3 proteinase 3
  • ILEU serpin-type proteinase inhibitor leukocyte elastase inhibitor
  • the present invention therefore also relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual; b) processing said sample to obtain a processed liquid sample; c) contacting said processed liquid sample with a first specific binding agent for proteinase 3 (PRN3) and a second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU) under conditions appropriate for formation of a complex between said first and second binding agent and PRN3 bound to ILEU (PRN3/ILEU); and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • PRN3 proteinase 3
  • ILEU serpin-type proteinase inhibitor leukocyte elastase inhibitor
  • Another preferred embodiment of the invention is a method for the diagnosis of colorectal cancer comprising the steps of a) processing a stool sample obtained from an individual to obtain a processed liquid sample, b) contacting said processed liquid sample with a first specific binding agent for proteinase 3 (PRN3) and a second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU) under conditions appropriate for formation of a complex between said first and second binding agent and PRN3 bound to ILEU
  • PRN3 proteinase 3
  • ILEU serpin-type proteinase inhibitor leukocyte elastase inhibitor
  • the stool sample is processed to retrieve colonycytes which are then smeared on a microscopic slide. Such processed sample is then incubated with the first specific binding agent for proteinase 3 (PRN3) and a second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU) under conditions appropriate for formation of a complex between said first and second binding agent and PRN3 bound to ILEU (PRN3/ILEU).
  • PRN3 proteinase 3
  • ILEU serpin-type proteinase inhibitor leukocyte elastase inhibitor
  • the present invention therefore also relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual; b) processing said sample to retrieve colonycytes; c) contacting said processed sample with a first specific binding agent for proteinase 3
  • PRN3 and a second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU) under conditions appropriate for formation of a complex between said first and second binding agent and PRN3 bound to ILEU (PRN3/ILEU), and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • PRN3/ILEU serpin-type proteinase inhibitor leukocyte elastase inhibitor
  • the protein PRN3 proteinase 3, also known as myeloblastin precursor or Wegener's autoantigen; Swiss-PROT: P24158
  • proteinase 3 is a serine proteinase found in azurophilic granules of polymorphonuclear neutrophils (Baggiolini, M., et al.,Agents Actions 8 (1978) 3-10). Proteinase 3 has been characterized and named independently as p29b (Campanelli, D., et al, J. Clin. Invest.
  • PRN3 is a protein of about 29 kDa and exists as multiple Mr species, probably due to differences in glycosylation (Wilde, C.G., et al., J. Biol. Chem. 265 (1990) 2038-2041).
  • PRN3 has been associated with a number of human diseases. It is the target antigen of the cytoplasmic pattern of antineutrophil cytoplasmic autoantibodies (c-ANCA) detected in the circulation of Wegener's granulomatosis (WG) patients (Ludemann, J., et al., J. Exp. Med. 171 (1990) 357-362). PRN3 also is involved in growth and differentiation of leukemia cells (Bories, D., et al., Cell 59 (1989) 959-
  • PRN3 can be detected on the cell surface following activation by tumor necrosis factor alpha (TNF ⁇ ) in vitro (Charles, L.A., et al., J. Leuk. Biol. 50 (1991) 539-546), and PRN3 has been reported to release TNF ⁇ from cell surfaces in vitro (Robache- Gallea et al., 1995). PRN3 is also found on the surface of cells in patients with ANCA-associated vasculitides (Csernok, E., et al., Advan. Exp. Med. Biol. 336
  • ILEU Leukocyte elastase inhibitor, also known as monocyte/neutrophil elastase inhibitor; Swiss-PROT P30740
  • SEQ ID NO:2 The sequence of ILEU (Leukocyte elastase inhibitor, also known as monocyte/neutrophil elastase inhibitor; Swiss-PROT P30740) is given in SEQ ID NO:2. It is a 42 kDa protein of the serpin superfamily (Remold-O'Donnell, E., et al, PNAS 89 (1992) 5635-5639). Serpin-type inhibitors are described to bind covalently to their target proteinases and form stable serpin-proteinase complexes, which are not dissociated when treated with SDS (Olson, S., et al., JBC 270 (1995) 30007- 30017).
  • ILEU was shown to inhibit, amongst others, PRN3 (proteinase-3) fast and efficiently (Cooley, J., et al., Biochem. 40 (2001) 15762-15770). This inhibition is accomplished through a covalent binding in a 1:1 stoichiometry. SDS-PAGE revealed that the ILEU-PRN3-complex formed was resistant to SDS treatment (Cooley, J., et al., Biochem. 40 (2001) 15762-15770).
  • the present invention shall not be construed to be limited to the full-length protein PRN3 of SEQ ID NO :1 bound to the full-length protein ILEU of SEQ NO ID:2.
  • ILEU-PRN3-complexes also may comprise physiological or artificial fragments of PRN3 or ILEU, secondary modifications of PRN3 or ILEU, as well as allelic variants of PRN3 or ILEU. Therefore, PRN3 as well as fragments, modifications and variants of PRN3 being bound to ILEU or to fragments, modifications and variants therof are also encompassed by the present invention.
  • Artificial fragments of PRN3 preferably encompass a peptide produced synthetically or by recombinant techniques, which at least comprises one epitope of diagnostic interest consisting of at least 6 contiguous amino acids as derived from the sequence disclosed in SEQ ID NO: 1. Such fragment may advantageously be used for generation of antibodies or as a standard in an immunoassay. More preferred the artificial fragment comprises at least two epitopes of interest appropriate for setting up a sandwich immunoassay.
  • the present invention encompasses antibodies generated using a peptide as above for PRN3 but derived from the sequence disclosed in SEQ ID NO:2, whereby the peptide represents at least one ILEU epitope.
  • a preferred embodiment of the present invention is a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) contacting said sample with a first specific binding agent for proteinase 3 (PRN3) and a second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU) under conditions appropriate for formation of a complex between said first and second binding agent and PRN3 bound to ILEU (PRN3/ILEU), and c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • PRN3 proteinase 3
  • ILEU serpin-type proteinase inhibitor leukocyte elastase inhibitor
  • method is a sandwich immunoassay using either a PRN3-specific antibody for capturing and an ILEU-specific antibody for detecting PRN3/ILEU, or an ILEU- specific antibody for capturing and a PRN3-specific antibody for detecting
  • a further preferred embodiment of the present invention is a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) processing said sample to obtain a processed liquid sample, c) contacting said processed liquid sample with a first specific binding agent for proteinase 3 (PRN3) and a second specific binding agent for the serpin-type proteinase inhibitor leukocyte elastase inhibitor (ILEU) under conditions appropriate for formation of a complex between said first and second binding agent and PRN3 bound to ILEU (PRN3/ILEU), and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • PRN3 proteinase 3
  • ILEU serpin-type proteinase inhibitor leukocyte elastase inhibitor
  • PRN3/ILEU can serve as a marker for CRC but also free PRN3, alone or in combination with PRN3/ILEU.
  • the findings on which this invention is based in total provides for three different specific targets: (i) PRN3/ILEU alone, (ii) PRN3/ILEU and free PRN3, and (iii) unbound PRN3 alone.
  • the three targets can be used individually as markers or a ratio of the amounts of at least two of the targets (i), (ii) and (iii) can be determined. The ratio can serve as a parameter characterizing CRC.
  • Another preferred embodiment of the invention is a method for the diagnosis of colorectal cancer comprising the steps of (a) providing a stool sample obtained from an individual, b) contacting said sample with a binding agent against an epitope shared by PRN3 and PRN3/ILEU under conditions appropriate for formation of a complex between said binding agent and PRN3 as well as PRN3/ILEU, and (c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • a) providing a stool sample obtained from an individual b) contacting said sample with a binding agent against an epitope shared by PRN3 and PRN3/ILEU under conditions appropriate for formation of a complex between said binding agent and PRN3 as well as PRN3/ILEU, and (c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • a further preferred embodiment of the invention is a method for the diagnosis of colorectal cancer comprising the steps of (a) providing a stool sample obtained from an individual, b) processing said sample to obtain a processed liquid sample, c) contacting said processed liquid sample with a binding agent against an epitope shared by PRN3 and PRN3/ILEU under conditions appropriate for formation of a complex between said binding agent and PRN3 as well as PRN3/ILEU, and (d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • total PRN3 all PRN3 present in the liquid sample
  • Another preferred embodiment of the invention is a method for the diagnosis of colorectal cancer comprising the steps of (a) providing a stool sample obtained from an individual, b) contacting said sample with a binding agent against an epitope present on unbound PRN3 but not on PRN3/ILEU under conditions appropriate for formation of a complex between said binding agent and PRN3, and (c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • at least one binding reagent is required which recognizes an epitope on PRN3 absent, e.g., masked, when PRN3 is bound to ILEU.
  • at least one such reagent is either used as capture or as detection reagent.
  • a further preferred embodiment of the invention is a method for the diagnosis of colorectal cancer comprising the steps of (a) providing a stool sample obtained from an individual, b) processing said sample to obtain a processed liquid sample, c) contacting said processed liquid sample with a binding agent against an epitope present on unbound PRN3 but not on PRN3/ILEU under conditions appropriate for formation of a complex between said binding agent and PRN3, and (d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • at least one binding reagent is required which recognizes an epitope on PRN3 absent, e.g., masked if PRN3 is bound to ILEU.
  • any of the novel markers PRN3/ILEU, total PRN3 and/or unbound PRN3 alone or in combination may be used for monitoring as well as for screening purposes.
  • the novel marker PRN3 bound to ILEU may be used for monitoring as well as for screening purposes. Its use for screening purposes is most preferred.
  • the diagnostic method according to the present invention may help to assess tumor load, efficacy of treatment and tumor recurrence in the follow-up of patients.
  • Increased levels of PRN3/ILEU are directly correlated to tumor burden. After chemotherapy a short term (few hours to 14 days) increase in PRN3/ILEU may serve as an indicator of tumor cell death. In the follow-up of patients (from 3 months to 10 years) an increase of PRN3/ILEU can be used as an indicator for tumor recurrence in the colorectum.
  • the diagnostic method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of CRC by measuring the level of PRN3/ILEU in a stool sample and correlating the level measured to the presence or absence of CRC.
  • the staging of cancer is the classification of the disease in terms of extent, progression, and severity. It groups cancer patients so that generalizations can be made about prognosis and the choice of therapy.
  • TNM system Today, the TNM system is the most widely used classification of the anatomical extent of cancer. It represents an internationally accepted, uniform staging system.
  • T the extent of the primary tumor
  • N the status of regional lymph nodes
  • M the presence or absence of distant metastases
  • TNM criteria are published by the UICC (International Union against Cancer), Sobin, L.H., Wittekind, Ch. (eds): TNM Classification of Malignant Tumours, fifth edition, 1997. What is especially important is, that early diagnosis of CRC translates to a much better prognosis. Malignant tumors of the colorectum arise from benign tumors, i.e. from adenoma. Therefore, best prognosis have those patients diagnosed at the adenoma stage.
  • early diagnosis of CRC refers to a diagnosis at a pre-malignant state (adenoma) or at a tumor stage where no metastases at all (neither proximal nor distal), i.e., adenoma, Ti S , NO, M0 or Tl-4; NO; M0 are present.
  • T ⁇ s denotes carcinoma in situ.
  • the detection of PRN3/ILEU is used to diagnose CRC as early as in the adenoma stage.
  • CRC is diagnosed when it has not yet fully grown through the bowel wall and thus neither the visceral peritoneum is perforated nor other organs or structures are invaded, i.e., that diagnosis is made at stage T fS , NO, M0 or Tl-3; NO; M0 NO; M0).
  • the diagnostic method according to the present invention is based on a stool sample which is derived from an individual.
  • the stool sample is extracted and PRN3/ILEU is specifically measured from this processed stool sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for PRN3, ILEU or PRN3/ILEU, a lectin binding to PRN3, ILEU or PRN3/ILEU or an antibody to PRN3, ILEU or PRN3/ILEU.
  • a specific binding agent has at least an affinity of 10 7 1/mol for its corresponding target molecule.
  • the specific binding agent preferably has an affinity of 10 1/mol or even more preferred of 10 1/mol for its target molecule, i.e. PRN3, ILEU or PRN3/ILEU.
  • PRN3, ILEU or PRN3/ILEU i.e. PRN3, ILEU or PRN3/ILEU.
  • the level of binding to a biomolecule other than the target molecule results in a binding affinity which is only 10%, more preferably only 5% of the affinity of the target molecule or less.
  • a most preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity.
  • a pair of specific binding agents preferably comprises a first antibody reactive with PRN3 and a second antibody reactive with ILEU such that the pair of antibodies is capable of forming a complex with PRN3/ILEU.
  • a specific binding agent preferably is an antibody specifically reactive with PRN3/ILEU but not PRN3 or ILEU alone.
  • a specific binding agent directed against unbound PRN3 whereby the specific binding agent preferably is an antibody reactive with an epitope of PRN3 which is masked when PRN3 is bound to ILEU.
  • antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as genetic constructs comprising the binding domain of an antibody. Any antibody fragment retaining the above criteria of a specific binding agent can be used.
  • Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam). In addition, the skilled artisan is well aware of methods based on immunosorbents that can be used for the specific isolation of antibodies. By these means the quality of polyclonal antibodies and hence their performance in immunoassays can be enhanced. (Tijssen, P., supra, pages 108-115).
  • polyclonal antibodies raised in rabbits have been used.
  • polyclonal antibodies from different species e.g. rats or guinea pigs
  • monoclonal antibodies can also be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.
  • the generation and use of monoclonal antibodies to PRN3, ILEU and PRN3/ILEU in a method according to the present invention is yet another preferred embodiment.
  • PRN3/ILEU has been identified as a marker which is useful in the diagnosis of CRC
  • alternative ways may be used to reach a result comparable to the achievements of the present invention.
  • alternative strategies to generate antibodies may be used.
  • Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of PRN3, ILEU or PRN3/ILEU for immunization.
  • DNA Immunization also known as DNA vaccination may be used.
  • the stool sample is obtained from an individual.
  • An aliquot of the stool sample may be used directly.
  • an aliquot of the stool sample is processed to yield a liquid sample.
  • the stool sample is preferably used or processed directly after sampling or stored cooled or more conveniently stored frozen.
  • Frozen stool samples can be processed by thawing, followed by dilution in an appropriate buffer, mixing and centrifugation. Supernatants are used as liquid sample for subsequent measurement of marker PRN3/ILEU.
  • An aliquot of the processed stool sample is incubated with the specific binding agent or pair of binding agents for PRN3, ILEU or PRN3/ILEU under conditions appropriate for formation of a complex of the binding agent or pair of binding agents and PRN3, ILEU or PRN3/ILEU.
  • Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.
  • the amount of complex is measured and correlated to the diagnosis of CRC.
  • the skilled artisan will appreciate there are numerous methods to measure the amount of formed complex consisting of the specific binding agent or pair of specific binding agents and PRN3, ILEU or PRN3/ILEU, all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis, et al., eds. (1996) Immunoassay, Academic Press, Boston).
  • PRN3/ILEU is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture PRN3/ILEU on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable is used on the other side.
  • PRN3/ILEU can be measured from a stool sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker PRN3/ILEU in the diagnosis of CRC.
  • Antibodies to PRN3/ILEU with great advantage can also be used in established procedures, e.g., to detect colorectal cancer cells in situ, in biopsies, or in immunohistological procedures.
  • an antibody to PRN3/ILEU is used in a qualitative (PRN3/ILEU present or absent) or quantitative (PRN3/ILEU amount is determined) immunoassay. Based on quantitative determination of PRN3/ILEU and total PRN3 or unbound PRN3 the ratio between these markers can be correlated to the diagnosis of colorectal cancer.
  • the present invention relates to use of PRN3/ILEU as a marker molecule in the diagnosis of colorectal cancer from a stool sample obtained from an individual.
  • marker molecule is used to indicate that an increased level of the analyte PRN3/ILEU or total PRN3 as measured from a bodily fluid or especially a processed stool sample obtained from an individual marks the presence of CRC.
  • novel marker PRN3/ILEU in the early diagnosis of colorectal cancer.
  • PRN3/ILEU protein PRN3/ILEU itself
  • PRN3/ILEU as a marker molecule for colorectal cancer in combination with one or more other marker molecules for colorectal cancer in the diagnosis of colorectal cancer from a stool sample obtained from an individual.
  • PRN3/ILEU may be combined are hemoglobin and/or the hemoglobin-haptoglobin complex.
  • Diagnostic reagents in the field of specific binding assays like immunoassays, usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for PRN3/ILEU and auxiliary reagents for measurement of PRN3/ILEU.
  • the ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision thresh-hold over the entire range of data observed.
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true-positive test results) (number of true-positive + number of false- negative test results)].
  • This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • the false-positive fraction or 1 - specificity [defined as (number of false- positive results) / (number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination (no overlap in the two distributions of results) has an
  • One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • the diagnostic method based on measurement of PRN3/ILEU alone in comparison to the established marker hemoglobin alone has been found to have an at least as good a diagnostic accuracy (sensitivity/specificity profile) as demonstrated by the area under the curve.
  • Figure 1 Identification of intact PRN3/ILEU with an apparent MW of 54 kDa in colon tumor tissue.
  • Figure 1 shows a typical example of a 2D-gel, loaded with a tumor sample (left side), and a gel, loaded with a matched control sample (right side) obtained from adjacent healthy mucosa. The circle in the enlarged section of these gels indicates the position for PRN3/ILEU.
  • the PRN3/ILEU complex was not detectable by the same method in healthy mucosa.
  • Figure 2 shows a typical example of a Western-Blot.
  • the gel was loaded with tissue lysates from colorectal tumor tissue and adjacent healthy control tissue from 5 patients (subject 22: colon ca, Dukes B; subject 23: colon ca, Dukes B; subject 27: rectum ca, Dukes B; subject 29: rectum ca, Dukes A, and subject 34: colon ca, Dukes B).
  • Presence of the PRN3-ILEU complex (upper clipping) or of uncomplexed PRN3 (lower clipping) in the samples was tested using a polyclonal rabbit anti-ILEU (upper clipping) or polyclonal anti-PRN3 (lower clipping) serum.
  • Lanes containing tumor lysates are indicated with "T", lanes containing normal control tissue with "N".
  • the arrows indicate the position in the gels of the PRN3-ILEU (apparent MW of -70 kDa) and the uncomplexed PRN3 (apparent MW of ⁇ 28 kDa) band. All tumor samples gave a strong signal at the positions of PRN3-ILEU and PRN3, whereas only a weak or no signal could be detected in the lysates from adjacent normal control tissue.
  • the apparent molecular weight of the PRN3-ILEU complex in this experiment was about 70 kDa and therefore different to the apparent molecular weight of the PRN3-ILEU complex as determined on the 2D -gels where an apparent molecular weight of about 54 kDa was determined (see Figure 1).
  • the determination of the apparent molecular weight in the Western- blot experiment is regarded as more precise since in this experiment a molecular weight marker is included.
  • the 2D-gels in contrast, do not contain a molecular weight standard and therefore estimations of the molecular weight of a given spot are clearly less precise.
  • the molecular weight of the PRN3-ILEU complex as determined in the Western-Blot experiment is well in accordance with the theoretical molecular weight.
  • tissue specimen from 10 patients suffering from colorectal cancer are analyzed. From each patient three different tissue types are collected from therapeutic resections: tumor tissue (> 80% tumor) (T), adjacent healthy tissue (N) and stripped mucosa from adjacent healthy mucosa (M). The latter two tissue types serve as matched healthy control samples. Tissues are immediately snap frozen after resection and stored at - 80°C before processing. Tumors are diagnosed by histopathological criteria.
  • 0.8-1.2 g of frozen tissue are put into a mortar and completely frozen by liquid nitrogen.
  • the tissue is pulverized in the mortar, dissolved in the 10-fold volume (w/v) of lysis buffer (40 mM Na-citrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete ® EDTA-free [Roche Diagnostics GmbH, Mannheim,
  • Each patient is analyzed separately by image analysis with the ProteomeWeaver® software (Definiens AG, Germany, M ⁇ nchen).
  • ProteomeWeaver® software Detiens AG, Germany, M ⁇ nchen.
  • all spots of the gel are excised by a picking robot and the proteins present in the spots are identified by
  • MALDI-TOF mass spectrometry (Ultraflex TM Tof/Tof, Bruker Daltonik GmbH, Bremen, Germany).
  • 4 gels from the tumor sample are compared with 4 gels each from adjacent normal and stripped mucosa tissue and analyzed for distinctive spots corresponding to differentially expressed proteins.
  • protein PRN3/ILEU is found to be specifically expressed or strongly overexpressed in tumor tissue and not detectable or less strongly expressed in healthy control tissue. It therefore - amongst many other proteins - qualifies as a candidate marker for use in the diagnosis of colorectal cancer.
  • Polyclonal antibody to the colorectal cancer marker protein PRN3/ILEU is generated for further use of the antibody in the measurement of serum, and plasma, and blood and stool levels of PRN3/ILEU, total PRN3 and/or unbound PRN3 by immunodetection assays, e.g. Western Blotting and ELISA.
  • PRN3/ILEU recombinant expression of each protein is performed for obtaining immunogens. Expression is done applying a combination of the RTS 100 expression system and E.coli.
  • the DNA sequences encoding PRN3 and ILEU are analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY" system. This is a commercial web based service (www.proteoexpert.com).
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No.
  • the construct is transformed into E. coli BL 21 (DE 3) (Studier, F.W., et al., Methods Enzymol. 185 (1990) 60-89) and the transformed bacteria are cultivated in a 1 1 batch for protein expression.
  • PRN3 and ILEU are expressed separately. Purification of His-PRN3 and His-ILEU fusion proteins is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His- His-PRN3 or the His-ILEU fusion protein is pelleted by centrifugation.
  • the cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidium chloride, imidazole and thioglycerole, followed by homogenization using an Ultra-Turrax ® .
  • Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and Urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acid conditions.
  • Two sets of 12 week old A/J mice are initially immunized intraperitoneally, one set with with 100 ⁇ g PRN3, the other with 100 ⁇ g ILEU. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g PRN3 or ILEU adsorbed to aluminum hydroxide and 10 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g PRN3 or ILEU, respectively, in PBS buffer for each.
  • Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C, Methods in Enzymology 73 (1981) 3-46. In this process ca. 1*10 8 spleen cells of the immunized mouse are mixed with 2xl0 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at
  • the cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 400 g in a 50 ml conical tube. The supernatant is discarded, the cell sediment is gently loosened by tapping, 1 ml PEG (molecular weight 4000, Merck, Darmstadt) is added and mixed by pipetting. After 1 min in a water-bath at 37°C, 5 ml RPMI 1640 without FCS is added drop-wise at room temperature within a period of 4-5 min. Afterwards 5 ml RPMI 1640 containing 10% FCS is added drop-wise within ca.
  • FCS fetal calf serum
  • 100 U/ml is added to the medium as a growth factor.
  • PRN3- and ILEU-positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter.
  • interleukin 6 at 100 U/ml is added to the medium as a growth additive.
  • the hybridoma cells obtained are sown at a density of lxlO 5 cells per ml in RPMI 1640 medium containing 10% FCS and proliferated for 7 days in a fermenter
  • Two sets of rabbits are immunized, one with PRN3 and one with ILEU.
  • a fresh emulsion of the protein solution 100 ⁇ g/ml PRN3 or ILEU, respectively
  • complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90.
  • Blood is drawn and resulting anti-PRN3 and anti-ILEU serum used for further experiments as described in Examples 3 to 7.
  • IgG immunoglobulin G
  • rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ⁇ l/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 x g, 30 min, 4°C), the pellet discarded and the supernatant collected. The pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered (0.2 ⁇ m).
  • the immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.
  • the precipitated immunoglobulins are collected by centrifugation (8,000 x g, 15 min, 4°C).
  • the supernatant is discarded.
  • the pellet is dissolved in 10 mM NaH 2 PO /NaOH, pH 7.5, 30 mM NaCl and exhaustively dialyzed.
  • the dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl. Per ml IgG solution 50 ⁇ l Biotin -N-hydroxysuccinimide (3.6 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim, Germany, Cat. No. 1 333 054) (3.8 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex® 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl). The fractions containing digoxigenylated IgG are collected. Monoclonal antibodies are labeled with digoxigenin according to the same procedure.
  • Tissue lysates from tumor samples and healthy control samples were prepared as described in Example 1, "Tissue preparation”.
  • the primary antibody polyclonal rabbit anti-IELU or polyclonal rabbit anti-PRN3 serum (generation described in Example 2), was diluted 1:10,000 in Roti®-Block blocking buffer and incubated with the membrane for 1 h.
  • the membranes were washed 6 times in PBS/0.05% Tween-20.
  • the specifically bound primary rabbit antibody was labeled with a POD-conjugated polyclonal sheep anti-rabbit IgG antibody, diluted to 10 mU/ml in 0.5 x Roti ® - Block blocking buffer. After incubation for 1 hour, the membranes were washed 6 times in PBS/0.05% Tween-20.
  • the membrane was incubated with the Lumi-Light PLUS Western Blotting Substrate (Order-No. 2015196, Roche Diagnostics GmbH, Mannheim,
  • the stool samples are thawed, tenfold diluted with a phosphate buffer, pH 7.4 and thoroughly mixed in order to yield a suspension of the stool sample in the extraction buffer. After centrifugation for 15 min at 12,000 g, the upper portion of the supernatant is saved for further analysis. An aliquot of this processed stool sample is used for quantification of PRN3 by ELISA.
  • a sandwich ELISA For detection of total PRN3 in a human processed stool sample, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-PRN3 polyclonal antibody (see example 2) are conjugated with biotin and digoxygenin, respectively.
  • Streptavidin-coated 96-well microtiter plates are incubated with 100 ⁇ l biotinylated anti-PRN3 polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween 20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted stool samples from patients. After binding of PRN3, plates are washed three times with 0.9% NaCl, 0.1% Tween 20.
  • wells are incubated with 100 ⁇ l of digoxygenylated anti-PRN3 polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. Thereafter, plates are washed three times to remove unbound antibody.
  • wells are incubated with 20 mU/ml anti-digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60 min in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. Plates are subsequently washed three times with the same buffer.
  • ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 11685767) and OD is measured after 30-60 min at 405 nm with an ELISA reader.
  • Accuracy is assessed by analyzing individual stool samples obtained from well- characterized patient cohorts, i.e., 30 patients having undergone colonoscopy and found to be free of adenoma or CRC, 30 patients diagnosed and staged as Tl-3, NO, M0 of CRC, and 30 patients diagnosed with progressed CRC, having at least tumor infiltration in at least one proximal lymph node or more severe forms of metastasis, respectively.
  • Total PRN3 is measured as described above in a stool sample obtained from each of these individuals.
  • ROC-analysis is performed according to Zweig, M. H., and Campbell, supra.
  • Discriminatory power for differentiating patients in the group T fs -3, NO, M0 from healthy individuals as measured by the area under the curve is found to be at least as good for total PRN3 as compared to the established marker hemoglobin (Hamoglobin ELISA; Catalogue No.: K7816; Immundiagnostik AG, Wiesenstr. 4, D 64625 Bensheim, Germany).
  • a sandwich ELISA For detection of PRN3/ILEU in a human processed stool sample, a sandwich ELISA is developed. Anti-PRN3 polyclonal antibody and anti-ILEU polyclonal antibody are used for capture and detection, respectively, or vice versa. For capture of the antigen, aliquots of the anti-PRN3 polyclonal antibody or the anti-ILEU polyclonal antibody (see Example 2) are conjugated with biotin. For detection of the antigen, aliquots of the anti-PRN3 polyclonal antibody or the anti-ILEU polyclonal antibody (see Example 2) are conjugated with digoxygenin.
  • Streptavidin-coated 96-well microtiter plates are incubated with 100 ⁇ l biotinylated anti-PRN3 or anti-ILEU polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0,9% NaCl and 0.1% Tween 20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween 20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted stool samples from patients.
  • Accuracy is assessed by analyzing individual stool samples obtained from well- characterized patient cohorts, i.e., 30 patients having undergone colonoscopy and found to be free of adenoma or CRC, 30 patients diagnosed and staged as Tl-3, NO, M0 of CRC, and 30 patients diagnosed with progressed CRC, having at least tumor infiltration in at least one proximal lymph node or more severe forms of metastasis, respectively.
  • PRN3/ILEU is measured as described above in a stool sample obtained from each of these individuals.
  • ROC-analysis is performed according to Zweig, M.

Abstract

L'invention concerne le diagnostic du cancer colorectal et, plus spécifiquement, l'utilisation de la protéine PRN3/ILEU et/ou PRN3 non liée dans le diagnostic du cancer colorectal. L'invention concerne également une méthode de diagnostic du cancer colorectal à partir d'un échantillon de selles prélevé chez un sujet. Cette méthode consiste à mesurer le taux de PRN3/ILEU et/ou de PRN3 non liée dans ledit échantillon. La mesure de PRN3/ILEU et/ou de PRN3 non liée peut, par exemple, être utile dans la détection ou le diagnostic précoces du cancer colorectal.
PCT/EP2004/008082 2003-07-21 2004-07-20 Utilisation de la proteinase 3(prn3) et de l'inhibiteur de l'elastase leucocytaire (ileu) en tant que marqueurs pour le cancer colorectal WO2005015218A1 (fr)

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US8445211B2 (en) 2007-07-19 2013-05-21 Biomerieux I-Plastin assay method for the in vitro diagnosis of colorectal cancer
WO2009024691A3 (fr) * 2007-07-19 2009-05-22 Biomerieux Sa Procede de dosage du leucocyte elastase inhibitor pour le diagnostic in vitro du cancer colorectal
AU2008290466B2 (en) * 2007-07-19 2014-04-03 Biomerieux Method of assaying leukocyte elastase inhibitor for the in vitro diagnosis of colorectal cancer
US8735078B2 (en) 2007-07-19 2014-05-27 Biomerieux Apolipoprotein AII assay method for the in vitro diagnosis of colorectal cancer
CN101855555A (zh) * 2007-07-19 2010-10-06 生物梅里埃公司 体外诊断结肠直肠癌的白细胞弹性蛋白酶抑制剂测定方法
US8361731B2 (en) 2007-07-19 2013-01-29 Biomerieux Ezrin assay method for the in vitro diagnosis of colorectal cancer
FR2919063A1 (fr) * 2007-07-19 2009-01-23 Biomerieux Sa Procede de dosage du leucocyte elastase inhibitor pour le diagnostic in vitro du cancer colorectal.
US8367362B2 (en) 2007-07-19 2013-02-05 Biomerieux Aminoacylase 1 assay method for the in vitro diagnosis of colorectal cancer
US10591482B2 (en) 2007-07-19 2020-03-17 Biomerieux Method of assaying Apolipoprotein AI for the in vitro diagnosis of colorectal cancer
WO2009024691A2 (fr) * 2007-07-19 2009-02-26 bioMérieux Procede de dosage du leucocyte elastase inhibitor pour le diagnostic in vitro du cancer colorectal
US20100129828A1 (en) * 2007-07-19 2010-05-27 Biomerieux Method of assaying leukocyte elastase inhibitor for the in vitro diagnosis of colorectal cancer
US9890196B2 (en) 2007-07-19 2018-02-13 Biomerieux Ezrin assay method for the in vitro diagnosis of colorectal cancer
US9726670B2 (en) 2007-07-19 2017-08-08 Biomerieux Method for the assay of liver fatty acid binding protein, ACE and CA 19-9 for the in vitro diagnosis of colorectal cancer
US9891223B2 (en) 2007-07-19 2018-02-13 Biomerieux Method of assaying leukocyte elastase inhibitor for the in vitro diagnosis of colorectal cancer
US9388404B2 (en) 2008-07-10 2016-07-12 Biomerieux Protein disulfide isomerase assay method for the in vitro diagnosis of colorectal cancer
US8367806B2 (en) 2008-07-10 2013-02-05 Biomerieux Protein disulfide isomerase assay method for the in vitro diagnosis of colorectal cancer
CN117169519B (zh) * 2023-10-26 2024-01-30 艾康生物技术(杭州)有限公司 用于检测样本中tt3和/或tt4的解离剂和试剂盒
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