WO2005040813A1 - Utilisation de la proteine xag pour le diagnostic in vitro de l'adenocarcinome canalaire du pancreas - Google Patents

Utilisation de la proteine xag pour le diagnostic in vitro de l'adenocarcinome canalaire du pancreas Download PDF

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WO2005040813A1
WO2005040813A1 PCT/EP2004/012063 EP2004012063W WO2005040813A1 WO 2005040813 A1 WO2005040813 A1 WO 2005040813A1 EP 2004012063 W EP2004012063 W EP 2004012063W WO 2005040813 A1 WO2005040813 A1 WO 2005040813A1
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xag
protein
individual
gene
sample
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PCT/EP2004/012063
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English (en)
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Jokin Del Amo Iribarren
Corina Junquera Sanchez-Vallejo
Jorge Ochoa Garay
Pedro Escudero Garcia De Galdeano
Maria Eladia Argüelles Sanchez
Juan Ramon De Los Toyos Gonzalez
Simon Santa Cruz
Antonio Martinez Martinez
Luis Barneo Serra
Laureano Simon Buela
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Progenika Biopharma S.A.
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Publication of WO2005040813A1 publication Critical patent/WO2005040813A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention refers to an in vitro method to detect the presence of cancer of the pancreas in an individual, to determine the stage or severity of this cancer in an individual, or to monitor the effect of therapy administered to an individual with cancer of the pancreas; to seek, identify, develop and evaluate the efficacy of compounds for the treatment of cancer of the pancreas in an attempt to develop new medicinal products as well as agents that inhibit the expression and/or activity of the XAG protein, and/or the effects of its expression.
  • pancreatic cancer was the cause of more than 220,000 deaths world- ide and more than 3,600 in Spain during the year 2000 (GLOBOCAN) and 50,000 deaths per year in Europe - excluding the former USSR - (Medical library).
  • the clinical behaviour of pancreatic cancer is homogeneous and unfavourable, and there is no significant differences in survival rates by stage.
  • the number of patients with good-prognosis pancreatic cancer is negligible. A possible explanation for this is that even in patients with small tumours classified as stage I, the disease has spread beyond the pancreas. Diagnosis of pancreatic cancer at early stages, except for a few exceptions, is difficult: 75% of diagnosed patients are at a late stage (stage III or IV).
  • Pancreatic cancer is a highly aggressive neoplasia, resistant to cytostatic treatment. Only 1 to 4% of the patients remain alive five years after diagnosis, and these are patients with localized tumours which have been completely removed (Warshaw A.L., and Fernandes del Castillo C, N. Eng. J. Med., 1992, 326:455- 465; Ahlgren J.D., Semin. Oncol., 1996, 23:241-250). For these reasons, the development of very early diagnostic procedures and effective therapies are crucial to fight this disease (Byungwoo R., et al, Cancer Res., 2002, 62:819-826).
  • XAG plays a role in metastasis as a regulator of receptor adhesion and function
  • the authors of the present invention have discovered after thorough research and by using a number of techniques (DNA arrays and quantitative RT-PCR to quantitate mRNA levels), and Western blotting to quantitate protein levels), that the expression level of the xag gene is higher in pancreatic cancer than in non-tumour pancreatic tissue from the same patients or in pancreas tissue from pancreatitis patients.
  • the authors of the present invention have found that blocking the activity of XAG in pancreatic ductal adenocarcinoma cells by means of purified anti-XAG antibodies leads to a reduction in cell proliferation.
  • the in vitro detection of high levels of XAG or high levels of XAG protein, or a combination thereof in samples of pancreatic tissue or in other samples, preferably serum samples, of individuals will make possible an early detection of pancreatic cancer.
  • the development of new drugs targeted specifically against XAG gene or the XAG protein constitutes a new approach to treat pancreatic carcinoma.
  • the present invention therefore, provides a highly sensitive in vitro method to detect pancreatic ductal adenocarcinoma in an individual, to determine the stage or severity of said condition in an individual, or to monitor the effect of therapy administered to an individual with said condition, by detecting and/or quantifying the XAG protein, the XAG mRNA or the corresponding XAG cDNA, in a sample from said individual.
  • the present invention provides targets or tools for the screening, identification, development and evaluation of the efficacy of compounds for the treatment of pancreatic ductal adenocarcinoma.
  • this invention provides agents that inhibit the expression and/or the activity of the XAG protein in the treatment of pancreatic ductal adenocarcinoma
  • the object of the present invention is to develop an in vitro method to detect the presence of pancreatic ductal adenocarcinoma, to determine the stage or severity of said condition in an individual, or to monitor the effect of therapy administered to an individual with said condition.
  • a second object of the present invention is an in vitro method to screen for, identify, develop and evaluate the efficacy of compounds to treat pancreatic ductal adenocarcinoma.
  • An additional object of the invention is the use of sequences derived from the xag gene for the in vitro diagnosis and prognosis of pancreatic ductal adenocarcinoma, and for the screening, identification, development and evaluation of the efficacy of compounds for the treatment of said condition.
  • Another object of the present invention is to provide agents that inhibit expression and/or activity of the protein XAG to treat pancreatic ductal adenocarcinoma.
  • a further object of this invention is a pharmaceutical composition consisting of one or several therapeutic agents and an acceptable pharmaceutical excipient to treat pancreatic ductal adenocarcinoma.
  • a final object of the present invention consists in a kit for carrying out the present invention.
  • FIGURES DESCRIPTION OF THE FIGURES
  • Figure 2 Calculation of the amplification efficiency of PCR reactions of xag, in experiments to measure gene expression by real time quantitative RT-PCR in pancreas samples.
  • Figure 3 Calculation of the amplification efficiency of PCR reactions of ribllO, in experiments to measure gene expression by real time quantitative RT-PCR in pancreas samples.
  • Figure 4 Effect of anti-XAG purified serum on BxPC-3 viability/proliferation. BxPC-3 viability/proliferation, as quantified in an MTT assay, upon treatment with immnune and pre-immune sera.
  • the terms "subject” or “individual” refer to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans. The subject is preferably a male or female human of any age or race.
  • the term “cancer” or “carcinoma” refer to a disease characterised by abnormal or dysregulated proliferation of cells capable of invading adjacent tissues and spreading to distant organs.
  • the term “cancer of the pancreas” or “pancreatic ductal adenocarcinoma” refers to any malignant proliferative disorder of pancreatic ductal cells.
  • tissue refers to any abnormal mass of tissue generated by a neoplastic process, whether benign (non-cancerous) or malignant (cancerous).
  • gene refers to a region of a molecular chain of deoxyribonucleotides that encodes a protein and may represent a portion of a coding sequence or a complete coding sequence.
  • DNA refers to deoxyribonucleic acid.
  • a DNA sequence is a sequence of deoxyribonucleotides.
  • RNA refers to ribonucleic acid.
  • An RNA sequence is a sequence of ribonucleotides.
  • mRNA refers to messenger ribonucleic acid, which is the fraction of total RNA which is translated to protein.
  • cDNA refers to a nucleotide sequence complementary to a sequence of mRNA.
  • mRNA transcript of refers to the mRNA product transcribed from the corresponding gene (DNA), as a first step in the expression and translation to protein.
  • nucleotide sequence or “nucleotidic sequence” refers either to a sequence of ribonucleotides (RNA) or a sequence of deoxyribonucleotides (DNA).
  • protein refers to at least one molecular chain of amino acids linked through either covalent or non-covalent bonds.
  • the term includes all forms of post- translational protein modifications, for example glycosylation, phosphorylation or acetylation.
  • peptide and polypeptide refer to molecular chains of amino acids that represent a protein fragment.
  • protein and polypeptide are used interchangeably.
  • antibody refers to a glycoprotein that exhibits a specific binding activity for a target molecule called an "antigen”.
  • antibody includes monoclonal and polyclonal antibodies, either intact or fragments derived from them; it also includes human antibodies, humanised antibodies and antibodies of non-human origin.
  • “Monoclonal antibodies” is a homogeneous, highly specific antibody population that can bind to a single antigenic site or “determinant” on the target molecule.
  • “Polyclonal antibodies” is a heterogeneous antibody population that can bind to multiple antigenic sites or “determinants” of the target molecule.
  • the term “epitope”, as it is used in the present invention, refers to an antigenic determinant of a protein recognised by a specific antibody.
  • An epitope may consist of a contiguous stretch of amino acids (linear epitope), of non-contiguous amino acids that are brought into proximity with one another by virtue of the three dimensional folding of the polypeptide chain (discontinuous epitopes), of post-translational modifications of a protein or of a combination thereof.
  • solid phase refers to a non- aqueous matrix to which the antibody can bind.
  • solid phase materials include but are not limited to glass, polysaccharides (for example agarose), polyacrylamide, polystyrene, polyvinylic alcohol and silicons. Examples of solid phase forms are the well of a plate and a purification column.
  • oligonucleotide primer and “primer” are used interchangeably in the present invention, and are used to refer to nucleotide sequences complementary to target nucleotide sequences of the xag or ribllO genes. Each primer hybridises to its target nucleotide sequence and acts as an initiation site for nucleotide polymerisation catalysed by DNA polymerase, RNA polymerase or reverse transcriptase.
  • probe refers to a nucleotide sequence complementary to a nucleotide sequence derived from the xag gene that can be used to detect the corresponding nucleotide sequence derived from the xag genes.
  • therapeutic target refers to nucleotide or peptide sequences against which a drug or therapeutic compound can be designed and applied.
  • antagonistic molecules refers to any molecule that inhibits the biological activity of the antagonised molecule. Examples of antagonistic molecules are the following: proteins, peptides, variations of natural peptide sequences and small organic molecules (usually molecules with a molecular weight under 500 Daltons).
  • normal reference values refers to the level of certain proteins, mRNA or other metabolites of the body which present a healthy individual.
  • the present invention is based on the finding that the expression of xag gene (anterior gradient 2 homolog -Xenopus laevis- aliases: agr2, ag2, gob-4, hag-2, xag-2. UniGene: Hs.226391, GeneBank: NM_006408) and the concentration of XAG protein are increased in pancreatic ductal adenocarcinoma but not in non-tumorous inflamed pancreatic tissue (pancreatitis) relative to non-tumorous non-inflamed pancreatic tissue.
  • the present invention provides firstly an in vitro method to detect pancreatic ductal adenocarcinoma in an individual, to determine the stage or severity of this condition in the individual, or to monitor the effect of the therapy administered to an individual with this condition, which involves: a) quantification of XAG protein, xag mRNA or the corresponding cDNA in a sample from an individual and; b) comparison of the amount of XAG protein, xag mRNA or the corresponding cDNA, in a sample from an individual with the amount of XAG protein, xag mRNA or the corresponding cDNA, or a combination thereof detected in samples from control individuals or in previous samples from the same individual or with normal reference values.
  • the method provided by the present invention is highly sensitive and specific and is based on the fact that subjects or individuals diagnosed with pancreatic ductal adenocarcinoma present high levels of mRNA transcribed from the xag gene (high levels of xag gene expression), or a high concentration of the protein encoded by the xag gene (XAG protein) relative to the levels in samples from subjects without a clinical history of this type of cancer.
  • the present method includes a step in which the sample is obtained from the individual.
  • a number of liquid samples can be used, such as urine, blood, plasma, serum, pleural fluid, ascitic fluid, synovial fluid, bile, semen, gastric exudate or cerebrospinal fluid.
  • the sample can also be pancreatic tissue, which can be obtained by conventional methods, preferably surgical resection. Samples can be obtained from subjects previously diagnosed or not with a specific type of cancer, or from subjects who are receiving or have previously received anti-cancer treatment, in particular pancreatic ductal adenocarcinoma.
  • the present method also includes a step in which either protein or total RNA is extracted from the sample. One of these two extracts provides the working material for the next phase.
  • the extraction protocols for protein or total RNA are well known by experts in the field (Chomczynski P. et al., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532).
  • any conventional assay can be used in the context of the invention to detect cancer, provided that it measures in vitro the levels of mRNA transcribed from the xag gene or its complementary cDNA, the concentration of the XAG protein in samples collected from the individuals under study and from control individuals. Therefore, this invention provides a method to detect pancreatic ductal adenocarcinoma in an individual, to determine the stage or severity of said condition in an individual, or to monitor the effect of the therapy administered to individuals with these conditions, as based on the measurement of the expression of the xag gene, or the concentration of the XAG protein or a combination thereof.
  • the method of the invention includes a first step in which the protein extracted from the sample is placed in contact with a composition of one or more antibodies specific for one or more epitopes of XAG protein, and a second step in which the complexes formed by antibodies and the XAG protein are quantified.
  • immunological assays available to detect and to quantify the formation of specific antigen-antibody complexes; a number of protein- binding assays, competitive and non-competitive, have been previously described, and several of these are commercially available.
  • the XAG protein can be quantified by means of specific antibodies in the form of monoclonal antibodies, polyclonal antibodies, intact or recombinant fragments of antibodies, combibodies and Fab or scFv of antibody fragments.
  • These antibodies can be human, humanised or non-human in origin.
  • the antibodies used in these assays can be labelled or unlabelled; the unlabelled antibodies can be used in agglutination assays; the labelled antibodies can be used in a wide range of assays.
  • Antibody labels include radionucleotides, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, colorants and derivatives.
  • assays well known to those skilled in the art that can be applied to the present invention, which use unlabelled antibodies as primary reagents and labelled antibodies as secondary reagents.
  • these techniques include but are not limited to Western-blot or Western transfer, ELISA (Enzyme-linked immunosorbent assay), RIA (Radioimmunoassay), Competitive EIA (Competitive enzyme immunoassay), DAS-ELISA (Double antibody sandwich-ELISA), immunocytochemical and immunohistochemical techniques, techniques based on biochips or protein microarrays that use specific antibodies, and colloidal precipitation-based assays in formats such as dipsticks.
  • ELISA Enzyme-linked immunosorbent assay
  • RIA Radioimmunoassay
  • Competitive EIA Competitive enzyme immunoassay
  • DAS-ELISA Double antibody sandwich-ELISA
  • immunocytochemical and immunohistochemical techniques techniques based on biochips or protein microarrays that use specific antibodies, and colloidal precipitation-based assay
  • the preferred immunoassay for the invention is a double antibody sandwich ELISA (DAS-ELISA).
  • DAS-ELISA double antibody sandwich ELISA
  • any antibody or combination of antibodies can be used, specific for one or more epitopes on the XAG protein.
  • a monoclonal or polyclonal antibody, or a fragment thereof, or a combination thereof is attached to the surface of a solid phase; the test sample containing antigen is then placed in contact with it and incubated under appropriate conditions to allow for the formation of antigen-antibody complexes.
  • an indicator reagent is added to and incubated with the antigen-antibody complexes.
  • This reagent is usually a monoclonal or polyclonal antibody, or a fragment thereof, or a combination thereof bound to a signal- generating compound and specific for one or more epitopes on the antigen.
  • the signal is quantified. The signal generated is proportional to the amount of antigen (XAG) in the test sample. If the aim is to quantify mRNA or the corresponding cDNA of the xag gene and not the protein encoded by said gene, for the detection of carcinoma in vitro, the method of the invention consists of multiple steps.
  • the method of the invention comprises a first step of amplification of the total RNA extract or the corresponding cDNA synthesised by reverse transcription from the mRNA, and a second step of quantification of the amplification product of mRNA or the corresponding cDNA of the xag gene.
  • mRNA amplification consists of reverse transcription (RT) of mRNA into cDNA, followed by Polymerase Chain Reaction (PCR) that uses oligonucleotide primers with the following sequences: SEQ ID NO: 1 and SEQ ID NO: 2 for xag.
  • PCR is a technique for the amplification of a nucleotide sequence (target) contained in a mixture of nucleotide sequences.
  • target a nucleotide sequence contained in a mixture of nucleotide sequences.
  • an excess of a pair of oligonucleotide primers is used that hybridise to complementary strands of the target nucleotide sequence.
  • an enzyme with polymerase activity (DNA Taq Polymerase) extends each primer, using the target nucleotide sequence as a template. Therefore, upon dissociation from the original strand, the extension product becomes a target sequence.
  • New primer molecules hybridise to original or new target nucleotide sequences, and the polymerase extends them again. This cycle is repeated multiple times to exponentially increase the number of target nucleotide sequences.
  • the amplified product is detected by agarose gel electrophoresis as follows: five micro liters of amplification product are separated by electrophoresis on a 2% agarose gel in 0.5 x Tris-Borate-EDTA (TBE) buffer at 100 volts DC for one hour. After electrophoresis the gel is submerged in a solution of ethidium bromide, which binds to nucleic acids and allows visualization of the PCR product upon illumination with ultraviolet (uv) light.
  • TBE Tris-Borate-EDTA
  • the PCR product can be transferred to a nylon membrane by the Southern blotting or Southern transfer technique and detected with a labelled cDNA probe specific for the xag.
  • mRNA can be electrophoresed, transferred to a nylon membrane by means of the Northern blotting or Northern transfer technique, and detected with a probe specific for the mRNA or the corresponding cDNA of the xag gene.
  • amplification and quantification of the mRNA corresponding to the xag gene can be carried out by quantitative real time RT-PCR (Q-PCR).
  • the final step of the method of the invention to detect in vitro pancreatic ductal adenocarcinoma in a sample from an individual involves comparing the amount of XAG protein, the amount of mRNA or the corresponding cDNA of the xag gene detected in a sample from an individual, with the amount of XAG protein, the amount of mRNA or the corresponding cDNA of the xag gene detected in samples from control subjects or in non- tumorous samples from the same individual or with normal reference values.
  • the invention also provides an in vitro method to identify and evaluate the efficacy of therapeutic agents against pancreatic ductal adenocarcinoma, which comprises: a) placing in contact a culture of immortalized pancreas cells with the candidate compound under the appropriate conditions and for the required period of time for them to interact, b) detecting and quantifying the expression levels of the xag gene or the XAG protein or a combination thereof, and c) comparing the above expression levels with those of a control culture of immortalized pancreas cells not treated with the candidate compound.
  • the quantification of the expression levels of the xag gene or the XAG protein is performed in a similar manner to that described in the method of the invention to detect in vitro pancreatic ductal adenocarcinoma in an individual.
  • Another object of this invention refers to the use of nucleotide or peptide sequences derived from the xag gene to detect pancreatic ductal adenocarcinoma, to determine the stage or severity of said cancer in an individual or to monitor the effect of the therapy administered to an individual with said cancer.
  • a further object of this invention involves providing agents characterised by their ability to inhibit expression and/or activity of the XAG protein.
  • agents which can be identified and evaluated according to the present invention, can be selected from the following: a) small interfering RNAs or any other interfering RNA that silences the expression of the xag gene, and b) other cytotoxic agents, such as toxins, molecules with radioactive atoms or chemotherapeutic agents, including but not limited to small organic and inorganic molecules, peptides, phosphopeptides, antisense molecules, ribozymes, triple-helix molecules, double stranded RNA etc., which inhibit the expression and/or the activity of the XAG protein, and c) an antibody, or combination of antibodies, specific for one or more epitopes on the XAG protein, preferably a human or humanised monoclonal antibody; or a fragment of an antibody, a single-chain antibody or an anti-idiotype antibody, and d) antagonistic compounds of the XAG protein which inhibit one or more of the functions of the XAG protein.
  • cytotoxic agents such as toxins, molecules with radio
  • Another object of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising an agent of this invention, or a pharmaceutically acceptable salt, derivative or prodrug thereof together with a pharmaceutically acceptable carrier, adjuvant, or vehicle, for administration to a patient.
  • pharmaceutical compositions include any solid (tablets, pills, capsules, granules etc.) or liquid (solutions, suspensions or emulsions) composition for oral, topical or parenteral administration, etc.
  • the pharmaceutical compositions may be in oral form, either solid or liquid.
  • Suitable dose forms for oral administration may be tablets, capsules, syrops or solutions and may contain conventional excipients known in the art such as binding agents, for example syrup, acacia, gelatin, sorbitol, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate.
  • the solid oral compositions may be prepared by conventional methods of blending, filling or tabletting.
  • the tablets may for example be prepared by wet or dry granulation and optionally coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
  • the pharmaceutical compositions may also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the apropriate unit dosage form. Adequate excipients can be used, such as bulking agents, buffering agents or surfactants.
  • the mentioned formulations will be prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.
  • Administration of the compounds or compositions of the present invention may be by any suitable method, such as intravenous infusion, oral preparations, and intraperitoneal and intravenous administration. Oral administration is preferred because of the convenience for the patient and the chronic character of the diseases to be treated. Generally an effective administered amount of a compound of the invention will depend on the relative efficacy of the compound chosen, the severity of the disorder being treated and the weight of the sufferer. However, active compounds will typically be administered once or more times a day for example 1 , 2, 3 or 4 times daily, with typical total daily doses in the range of from 0.1 to 1000 ⁇ g/mg/kg/day. The compounds and compositions of this invention may be used with other drugs to provide a combination therapy.
  • kits for carrying out the present invention provides a kit that comprises an antibody that specifically recognizes either the XAG protein and a carrier in suitable packing.
  • the kit of the invention comprises a primer pair designed to specifically amplify a nucleic acid having a sequence that is specific to the xag gene.
  • the sequence of the primer pair can be determined from the sequence of the corresponding xag gene by employing bioinformatic tools.
  • the sequence of said primer pair is preferably selected from SEQ ID NO:l and SEQ ID NO:2.
  • kits can be employed to detect the presence of pancreatic ductal adenocarcinoma in an individual, to determine the stage or severity of said conditions in an individual or to monitor the effect of the therapy administered to the individual with said conditions.
  • the following examples serve to illustrate the invention.
  • Example 1 Differential expression analysis of the xag gene in pancreatic tissue samples by means of Human Genome U133 DNA microarrays 1.1. Material and methods Microarrays. GeneChip Test 3 (Affymetrix, Santa Clara) microarrays were used, that permit the quality of RNA to be tested before expression analysis with the GeneChip Human Genome U133A microarray (Affymetrix, Santa Clara), which includes 13,220 complete sequences of annotated genes; the xag gene is represented in the microarray by the Affymetrix 209173_at probe set, which is a set of sense oligonucleotides 25 nucleotides long designed after the Unigene Hs.91011 or the GeneBank AF088867 sequences (Table 1). Table 1. Description of the probes corresponding to the 209173_at probe set.
  • Affymetrix 209173_at probe set is a set of sense oligonucleotides 25 nucleotides long designed after the Unigene Hs
  • Stage III tumour disseminated to regional lymph nodes.
  • Stage IVB metastases in distant organs or tissues. All samples were clinically and histologically classified (grade and stage) at the Hospital Central de Asturias, the same hospital where they had been collected by following the precepts of the Helsinki declaration. Samples were frozen in liquid nitrogen immediately after extraction and stored at -80°C until analysis. For each tumour stage the following samples were analysed: -Stage I tumour: 7 samples -Stage III tumour: 1 sample -Stage IV tumour: 2 samples
  • RNA from biopsies was obtained by homogenising the tissue in TRIzol® Reagent (Life Technologies) and by following the manufacturer's instructions. Total RNA was cleaned with the RNeasy kit (QIAGEN) (Chomczynski P. et al., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). From each total RNA preparation 10 Dg were used as starting material for the synthesis of the first cDNA strand with the reverse transcriptase SuperscriptTM II RNase (Life Technologies), and by using as a primer an oligonucleotide oligo-dT containing the sequence of phage T7 RNA polymerase promoter.
  • the second cDNA strand was synthesised using the enzymes DNA polymerase I of E. coli (Invitrogen Life Technologies), DNA ligase of E. coli (Invitrogen Life Technologies), RNase H of E. coli (Invitrogen Life Technologies), and phage T4 DNA polymerase (Invitrogen Life Technologies).
  • the biotin-labelled cRNA was synthesised using the ENZO BioArrayTM HighYieldTM Transcript Labelling Kit (Enzo Diagnostics Inc). After in vitro transcription, the nucleotides not incorporated were removed by running the samples on RNeasy columns (QIAGEN).
  • Array Hybridisation and scanning 15 ⁇ g of each biotinylated cRNA were fragmented at 94°C for 35 minutes in a buffer solution containing 40 mM Tris-Acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate. Fragmented cRNA was mixed with hybridisation buffer (100 mM MES, IM NaCl, 20 mM EDTA, 0.01% Tween 20), heated to 99° for 5 minutes, heated to 45° for 5 minutes, and loaded into the Affymetrix array. The first array type in which the hybridisation was carried out was Test 3 of Affymetrix.
  • This array allows testing of the RNA quality before expression analysis on the Affymetrix® GeneChip® Human Genome 133 A (HG-U133 A) microarray.
  • arrays were incubated in a rotary oven at 45° for 16 hours at a constant speed of 60 rpm. Array washing and staining was performed in the Affymetrix® Fluidics Station.
  • the washing/staining programme used includes: - 10 x 2 wash cycles in SSPE-T 6x (0.9 m NaCl, 60 mM NaH 2 PO4, 6 mM EDTA, 0.01% Tween 20) at 25°C, - 4 x 15 cycles in 0.1 mM MES, 0.1M NaCl, 0.01% Tween 20 at 50°C, - Staining of biotinylated cRNA with a phycoerythrin-streptavidin conjugate (10 ⁇ g/ml, Molecular Probes) - 10 x 4 wash cycles with SSPE-T at 25C° - Incubation with an anti-streptavidin antibody for 10 minutes -Staining with a phycoerythrin-streptavidin conjugate (1 mg ml, Molecular Probes) for 10 minutes - 15 x 4 wash cycles with SSPE-T at 30C° Arrays were scanned at 560 nm
  • Detection This indicates whether the transcript is Present (P), Absent (A) or Marginal (M), ii) Change: This indicates whether expression of a specific transcript Increases (I), Decreases (D), Does not change (NC), Increases marginally (IM), or decreases marginally (DM), iii) Signal Log Ratio (SLR): This indicates the intensity of the change in expression between the baseline (control) and the test sample. SLR is expressed as the log of the ratio ox fold change (number of times that gene expression is increased or decreased in the test - tumour- sample versus the control -non-tumorous- sample).
  • Example 2 Analysis of differential expression of the xag gene in samples of pancreas tissue using real-time quantitative RT-PCR 2.1.
  • Materials and methods The method involves reverse transcription of mRNA to cDNA followed by amplification in a LightCycler (Roche) thermal cycler, using SYBR Green to detect the amplified product. Quantification was performed in real time which permits calculation of the relative expression of the sequence in the samples during the linear phase of the reaction.
  • RNA from biopsies was obtained by homogenising the tissue in TRIzol® Reagent (Life Technologies) and by following the manufacturer's instructions. Total RNA was cleaned with the RNeasy kit (QIAGEN) (Chomczynski P. et al, Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). RNA was quantified by spectrophotometry and 5 ⁇ g of total RNA were digested with DNase I.
  • RNA 1 ⁇ g of DNase I- treated RNA was used as starting material for the synthesis of the first strand of cDNA with SuperscriptTM II reverse transcriptase (Life Technologies), using as a primer an oligo- dT oligonucleotide that contained the sequence of the phage T7 RNA polymerase promoter.
  • cDNA was diluted to the working concentration and aliquoted.
  • Amplification Synthesised cDNA was amplified with specific primers for the human xag gene SEQ ID NO: 1 and SEQ ID NO: 2, and the gene that codes for the human ribosomal protein L10, SEQ ID NO: 14 and SEQ ID NO: 15.
  • Real time PCR reactions were prepared using the LightCycler-FastStart DNA master SYBR Green I kit (Roche) and by following the manufacturer's instructions.
  • the amplification programme consisted of 1 10-min cycle at 95°C ("hot start") followed by 45 cycles at 95°C (denaturation) for 10 s, 60°C (annealing) for 5 s, 72°C (amplification and acquisition of fluorescence) for 10 s.
  • the programme for the analysis of denaturation curves is one cycle consisting of a 95°C-pulse, 65°C for 15 s, and a 95°C pulse during the amplification and acquisition step.
  • the specificity of the PCR products are determined by analysing the denaturation curves.
  • the ratio of xag transcripts to ribllO transcripts was calculated. This ratio was normalised for each and every tumour sample relative to the values of the control sample.
  • a reference curve was generated for each gene sequence by using serial dilutions of cDNA. The concentrations of template cDNA for the reference curve reactions were given the arbitrary values 10, 5, 2.5, 1.25 and 0.625. The efficiency was calculated by applying the following equation:
  • E 10 _1 p
  • E the efficiency of the amplification
  • p the slope of the reference curve.
  • the ratio of the values of gene expression was determined by applying the following equation, which relates the experimental data of the amplification and corrects for efficiency differences in the PCR reactions: E -(Cp target control -Cp target sample ) target Ratio -(Cp reference control - Cp reference sample) • refe ⁇ where E is the amplification efficiency, Cp is the Crossing point, target is xag, reference is ribllO, control is the control sample (non-tumorous or pancreatitis) and sample is the tumour sample. 2.2.
  • control wells had their culture medium replaced with fresh culture medium.
  • control wells contained the same dilutions of HiTrap purified pre- immune rabbit serum. Plates were incubated under the same conditions as above for a further 3 days. On day 5, 10 ml of MTT (thiazolyl blue tetrazolium bromide) (Sigma Chemical Co., St Louis, USA) at 5 mg/ml in PBS were added to each well and cells were incubated for an additional 2 h. Next, medium was removed by inverting and vigorously shaking the plates, 200 ml of DMSO were added to each well and the plates were kept at room temperature for 20 min. Optical density was determined on a plate reader (Labsystems, Ashford, UK) fitted with 570 nm (signal) and 650 nm (background) filters.
  • MTT thiazolyl blue tetrazolium bromide

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Abstract

L'invention porte sur une méthode de détection in vitro de l'adénocarcinome canalaire du pancréas pour: en déterminer l'évolution ou la sévérité; suivre les effets d'une thérapie dans l'un et/ou l'autre cas; cribler, identifier, développer et évaluer l'efficacité, de composés de traitement en vue du développement de nouveaux médicaments et d'agents inhibant l'expression et/ou l'activité de la protéine XAG (gradient antérieur 2 homologue-Xenopus laevis-aliases: AGR2, AG2, GOB-4, HAG-2, XAG-2. UniGene: Hs.226391, GeneBank: NM_006408), et/ou les effets de ladite expression.
PCT/EP2004/012063 2003-10-22 2004-10-22 Utilisation de la proteine xag pour le diagnostic in vitro de l'adenocarcinome canalaire du pancreas WO2005040813A1 (fr)

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CA2960499A1 (fr) * 2014-09-09 2016-03-17 Board Of Regents, The University Of Texas System Anticorps monoclonaux bloquants diriges contre agr2 et son recepteur c4.4a

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008025964A2 (fr) * 2006-08-26 2008-03-06 The University Of Liverpool Anticorps, essais et hybridomes
WO2008025964A3 (fr) * 2006-08-26 2008-06-12 Univ Liverpool Anticorps, essais et hybridomes
US8329875B2 (en) 2006-08-26 2012-12-11 The University Of Liverpool Antibodies to an epitope of AGR2, assays and hybridomas
WO2008088322A2 (fr) * 2007-01-12 2008-07-24 Biomoles, Inc. Système de détection du cancer à microréseau intelligent
WO2008088322A3 (fr) * 2007-01-12 2008-12-31 Biomoles Inc Système de détection du cancer à microréseau intelligent

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