WO2009077157A1 - Procédé pour le diagnostic de tumeur colorectale - Google Patents

Procédé pour le diagnostic de tumeur colorectale Download PDF

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WO2009077157A1
WO2009077157A1 PCT/EP2008/010665 EP2008010665W WO2009077157A1 WO 2009077157 A1 WO2009077157 A1 WO 2009077157A1 EP 2008010665 W EP2008010665 W EP 2008010665W WO 2009077157 A1 WO2009077157 A1 WO 2009077157A1
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nfe2l3
colorectal tumor
tumor diagnosis
gene
prognosis
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PCT/EP2008/010665
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Tamara Maes
Elisabet Rosell I Vives
Olga DURANY TÜRK
Xavier Gregori
Cristina Alemany Boor
Simó SCHWARTZ NAVARRO
Gabriel CAPELLÀ MUNAR
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Oncnosis Pharma A.I.E.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/53—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • Colorectal cancer is one of the most common types of cancer in the European Union. In the year 2000, a total of 220,000 new cases were detected and 120,000 deaths were registered. Besides, incidence and mortality rates of this type of cancer have increased (by 2.4% and 6.1% respectively, from 1995 to 2000). Mortality is high and it represents the second most important tumor location in men and women, with a temporal upward trend (2.2% in men and 0.7% in women). Most nowadays, mortality is higher among men, although it was higher among women in the 1960s.
  • NFE2L3 gene also called Nrf3
  • Nrf3 is the third member of the family of NF-E2-related- factors, with a substantial homology to Nrf2 [Kobayashi et al. Molecular Cloning and Functional Characterization of a New Cap'n' Collar Family Transcription Factor Nrf3 J. Biol. Chem., 1999, 274: 6443-6452].
  • the antioxidant-response element (ARE) and the related NF-E2 nuclear factors are well-known for their participation in the basal expression and induction of defensive genes in response to antioxidants.
  • NF-E2- related-factors belong to the family of proteins-like basic leucine zipper. The basic region, in front of the leucine zipper region, is responsible for the union to the DNA.
  • NFE2L3 gene has been described as a negative regulator of ARE- mediated gene expression [Sankaranarayanan et al. Nrf3 negatively regulates antioxidant-response element-mediated expression and antioxidant induction of NAD (P)H:quinone oxidoreductasel gene. J Biol Chem., 2004, 279(49): 50810-]
  • NFE2L3 Changes in NFE2L3 expression have been related to transplant rejection diagnosis and follow-up. [Wohlgemuth, et al. Methods and compositions for diagnosing and monitoring transplant rejection, 2007. U.S.Pat. No. 7,235,358]. NFE2L3 has also been included in expression prints (broad gene selections used to control expression change profiles against reference samples) in relation to: a) ovarian serous papillary carcinoma diagnosis [Santin, Alessandro D. Gene expression profiling in primary ovarian serous papillary tumors and normal ovarian epithelium. 2005. U.S. Pat. No. Application: 2005004853] b) solid tumor formation in stem cells [Clarke, et al.
  • compositions and methods for treating and diagnosing cancer 2007 U.S. Pat. No Application: 20070099209] c) testicular seminoma diagnosis [Nakamura, et al. Method for diagnosing testicular seminomas. 2006. U.S. Pat. No Application: 20060194199] d) auto immune and chronic inflammatory diseases, SLE in particular [Wohlgemuth, et al. Methods and compositions for diagnosing or monitoring auto immune and chronic inflammatory diseases. 2005. U.S. Pat. No. 6,905,827] e) male infertility [Dix, et al. Genetic testing for male factor infertility. 2006. U.S. Pat. No Application: 20060199204].
  • the present invention offers a simple and reliable method for the diagnosis and/or prognosis of colorectal tumor, which is useful for early diagnosis and progression evaluation, and, therefore, it can be included in regular clinical practice.
  • the present invention refers to a method for the diagnosis and/or prognosis of colorectal tumors comprising the analysis of a sample from a patient to determine NFE2L3 gene expression level and its comparison to a control value, where the change of gene expression level of NFE2L3 is indicative of colorectal tumor.
  • change of gene expression level of NFE2L3 refers to an increase of gene expression with respect to the control value in the control curve shown in Figure 1.
  • colorectal tumor refers to any premalignant lesion, such as adenoma, or malignant lesion like adenocarcinoma.
  • prognosis refers to the capacity to predict that the evolution of a premalignant lesion like colorectal adenoma into a malignant lesion like adenocarcinoma, based on the expression levels of NFE2L3 gene with respect to the control value obtained in the control curve shown in the examples, wherein the higher the level of expression of such gene, the greater the chances are that a premalignant lesson will become malignant.
  • NFE2L3 gene expression level is determined by measurement of mRNA codified by such gene, or by its fragments.
  • measurement of mRNA codified by NFE2L3 gene is performed by DNA microarrays.
  • measurement of mRNA codified by NFE2L3 gene is performed by PCR, and in a more particular embodiment it is performed by RT-PCR.
  • NFE2L3 gene expression level is determined by measurement of the amount of protein codified by said gene and/or by its fragments.
  • measurement of the amount of protein codified by NFE2L3 gene is performed by immunohistochemical, inmunoenzimatic or inmunocromatography techniques or by other conventional techniques known by experts on the subject.
  • immunohistochemical techniques are performed using anti-NFE2L3 antibodies or fragments of them.
  • antibodies are polyclonal antibodies.
  • antibodies are monoclonal antibodies.
  • measurement of the amount of protein codified by NFE2L3 is performed by diagnostic imaging techniques.
  • diagnostic imaging techniques are performed by nanoparticles detected by antibodies or any other means.
  • measurement of the amount of protein codified by NFE2L3 gene is performed by protein chips.
  • the sample is a tissue.
  • the sample is a fluid.
  • a second aspect of this invention refers to a colorectal tumor diagnosis and/or prognosis kit comprising the reagents needed to perform the method described in this invention.
  • Figure 1 NFE2L3 amplification curves.
  • the curve having the higher Ct corresponds to the normal sample, whereas the two curves with lower Ct correspond to the adenoma and adenocarcinoma samples.
  • the difference in Ct confirmed a high over expression.
  • the present invention offers a simple and reliable method for the diagnosis and/or prognosis of colorectal tumor, which is very important for early development of disease and patient evolution assessment.
  • An experiment on DNA microarrays was performed to identify genes with a differential expression level between paired samples of normal colorectal tissue and adenoma and carcinoma tissue from patients with colorectal cancer.
  • the study involved two sample cohorts: a- Diagnosis study samples: paired material from the same patient consisting of healthy tissue, one or several adenomas and carcinoma (See Table 1 )
  • b- Prognosis study samples tumoral samples (325 cases of carcinoma) non- paired with their normal tissue. Samples were classified according to their stage (Duke I, II, III and IV), location (right colon, left colon and rectum), recurrence (non recurrent, less than a year and more than a year) and hospital of origin. Healthy samples representative of the three locations were used (30 cases)
  • Table 1 Cases analyzed. Number of patients in diagnostic series with paired samples of healthy mucosa, adenoma and carcinoma, indicating stage and location.
  • RNA samples from the tissues were analyzed with genomic techniques using specifically designed DNA microarrays.
  • This microarray (Oncochip) had oligonucleotides representing a total of 4528 human genes, some of which were selected because they participate in neoplastic transformation processes: angiogenesis
  • Chips were scanned with an Axon Genepix 4000B and raw data was obtained with GenePix Pro software. Raw results were processed with a tool developed internally for compensation, normalization, statistical data analysis and interpretation of DNA microarrays data.
  • Example 1 NFE2L3 expression data obtained with DNA microarrays analysis. Using trasncriptomic studies, a NFE2L3 gene differential expression was detected in adenomas and colorectal tumors when compared to normal tissue. Results were validated using biochemical methods as shown in the examples. The results of this study showed an increase in mRNA NFE2L3 expression, in adenomas as well as in colorectal adenocarcinomas when compared to normal tissue.
  • Nuclear factor erythroid-derived 2 like 3 member of Cap-n-collar family
  • FC (Fold Change) value results corresponding to the NFE2L3 gene obtained in the arrays.
  • T Tumor; AD: Adenoma: N: Normal R: Rectum; Cl: Left Colon; CD: Right Colon; El-Il: Dukes Stage I- II; EIII-IV: Dukes Stage IM-IV.
  • Example 2 mRNA isolation and results confirmation by cDNA synthesis and PCR TaqMan
  • Quantitative PCR with specific probes was used to validate changes in gene expression detected by oligonucleotides microarrays.
  • the NFE2L3 gene did not have a TaqMan probe on inventory, so it was necessary to ask for a specific probe and perform the trial in a conventional way, without using microfluidic plates. 384-well optic plates were used, with a 10 ⁇ l reaction. Relative quantification of the NFE2L3 gene was performed in the set of samples (diagnostic samples) of normal tissue, adenoma and adenocarcinoma from the same patient.
  • Applied Biosystems cDNA high capacity kit (Ref 4322171 ) was used for cDNA synthesis. In order to establish standard curves, 2.5 ⁇ g RNA was used and then changed to cDNA, and for the other samples, synthesis was performed from 1 ⁇ g total RNA. Per 100 ⁇ l reverse transcription reaction, human RNA was combined with master mix provided by the company which contains: random hexamers, MgCI 2 , 500 ⁇ M of each of the following dATP, dTTP, dCTP and dGTP, 0.4 U/ ⁇ l RNasa inhibitor and 1.25 U/ ⁇ l transcriptase in the right buffer.
  • Reactions were performed at 25° C during 10 minutes to maximize mold RNA and primer union, followed by 120 minutes at 37° C and 5-minute incubation at 95° C to cancel reverse transcriptase.
  • TaqMan PCR assays for NFE2L3 and internal control were performed twice on cDNA samples from tumoral and normal tissue. Parallel assays were performed with each sample normalizing the expression of the samples with cyclophilin, as it had been done with other genes. 200 ng mold cDNA were used for amplification. Reaction was performed according to the following parameters: 50° C during 2 minutes, 95° C during 10 minutes, and 40 cycles at 95° C during 15 seconds and 60° C during one minute.
  • Standard curves were prepared for NFE2L3 and internal control using dilutions in series of human RNA control samples.
  • TaqMan PCR data were obtained using Sequence Detector Software (SDS version 1.9; Applied Biosystems) Specifically, TaqMan Hs00852569_g1 probe was used to amplify NFE2L3 gene, based on the UniGene ID Hs.404741 and Hs.651505 gene sequence, which amplifies a 174 bp fragment between Chr. 7 - 26190678 - 26192432 chromosome locations.
  • Table 3 Relative quantification of NFE2L3 gene.
  • a set of samples was used which included normal tissue, adenoma and adenocarcinoma from the same patient. 200 ng of mold cDNA were used for amplification. Results were normalized with cyclophilin as endogenous control. Relative quantification was calculated with the Delta Delta Ct method, using the normal sample as reference. A 1 RQ value means no change was detected, below 1 means sub expression and over 1 means over expression.
  • Example 3 Obtaining a polyclonal antibody from NFE2L3 (30-694) recombinant protein and its use in colorectal cancer diagnosis and/or prognosis methods.
  • a rabbit polyclonal antibody was obtained from the NFE2L3 (30-694) recombinant protein following the immunization guidelines described below:
  • DAY 0 Intramuscular injection of 200 ⁇ g of the recombinant protein into the leg of the rabbit. An emulsion of 400 ⁇ g of recombinant protein in 0.5 ml PBS + 0.5 ml Freund's
  • the rabbit is immunized with the same dose and Freund's
  • the column is then washed with phosphate buffer and IgG is eluted in 0.1 M-HCI Glycine to pH 2.6. All the fractions are collected from the ice bath, mixed and neutralized with saturated Tris-HCI. Finally, dialysis is performed against PBS 1x and the IgG purity is tested using SDS-PAGE at 11%.
  • Purified anti NFE2L3 (349-2345) IgG are marked with peroxidase to be used in diagnostic trials.
  • peroxidase Activation of peroxidase (PO): Each peroxidase mg is dissolved in 100 ⁇ l NaHCO 3 0.1 M pH 9.2 and 100 ⁇ l of NaIO 4 10 mM are added per mg of peroxidase. This is incubated for two hours at room temperature in the dark. Conjugation: Every 3 mg IgG, 1 mg of activated peroxidase is mixed. The mixture was performed in a heat-sealed Pasteur pipette and with fiberglass at its base. Ephadex G- 25-80 is added to this mixture in a proportion of 1/6 of the combined volume of the solutions (1/6 g per each ml of solution), and this is kept during 3 hours at room temperature and in the dark.
  • the conjugate is eluted breaking the lower part of the pipette. 1/20 of the volume of the recently prepared conjugated NaBH 4 (5 mg NaBH 4 / ml de NaOH 0.1 mM) is added. This is kept during 30 min at room temperature in the dark. Later, 1/10 of the volume of the conjugated NaBH 4 is added and incubated for 1 hour at 4 0 C with stirring. Dialysis is performed against PBS during at least one night. Procedure: m/5 mg of dissolved BNHS is added to a V volume of IgG with an m (mg) mass when it is used in a volume/10 of DMSO. This is kept during 3-4 hours at room temperature.
  • Example 3 of this invention could be carried out in mice. Resides, mice spleens could be used for the development of monoclonal antibodies against NFE2L3 protein, which could be later used in diagnostic trials. Mice immunization was performed to obtain polyclonal antibodies according to the following procedure:
  • NFE2L3 (30-694) protein purified by CIGn-6M was quantified to perform the intraperitoneal immunization of five mice with 50 ⁇ g of it in presence of Freund's Complete Adjuvant in a first immunization or Freund's Incomplete Adjuvant in the following immunizations (Days 15, 30, 45).
  • DAY 37 One week after the third immunization, blood is obtained from the mice tails and the serum using indirect ELISA, against the NFE2L3 (30-694) protein and against another protein expressed using the same system as negative control, making different dilutions of it and developing it with TMB.
  • Table 6 shows the antibodies specific to NFE2L3 (30-694) protein present in the serum of immunized mice.
  • the antibodies of the present invention can be useful in diagnostic trials, for example, to detect NFE2L3 expression in specific cells, tissues or serum.
  • the antibodies of the present invention can be used in any known trial method, such as antigen-competition test, double antibody test (DAS-ELISA), immunoprecipitation tests, immuno- chromatography, immuno-PCR trials, etc.
  • Example 4 Development of DAS-ELISA trials based on rabbit polyclonal IgG for detecting NFE2 L3 protein.
  • a DAS-ELISA (double antibody sandwich) trial has been prepared to detect the NFE2L3 protein.
  • Antigen capture has been performed using rabbit purified IgGs anti- NFE2L3 (30-694) and the same polyclonal marked with peroxidase or Biotin for the detection of the protein and later development with TMB.
  • the following table shows the sensitivity of the trial to detect the NFE2L3 (30-694) recombinant protein.
  • NFE2L3 (30-694) recombinant protein.
  • an unrelated protein was used, expressed using the same E.coli system.
  • Example 5 Obtaining recombinant antibodies that recognize NFE2L3 and its use in colorectal cancer diagnosis and/or prognosis methods.
  • FAB-310 expresses the antibodies in Fab fragments on the surfaces of M13 filamentous phages, fused with g3p protein. These Fab fragments have a human part, from healthy donors DNA, and a synthetic part, which enables a diversity of 3.5x10 10 (1.5 x10 10 of kappa and 2 x 10 10 of lambda).
  • NFE2L3(30-694) The selection of antibodies against NFE2L3(30-694) was carried out following the methodology recommended in FAB-310. Phages showing affinity with the NFE2L3(30- 694) were captured through consecutive rounds of bonding, washing, elution, and amplification, lmmunotubes (Nunc, Denmark) were coated with NFE2L3(30-694) (200 ⁇ g/ immunotube and selection round) during the night at a temperature of 4 0 C and blocked during 1 hour with 3% of BSA.
  • Phages from an aliquot of the 200 ⁇ l (10 12 phages) library were precipitated with PEG/ NaCI, and then, they were suspended in 2 ml phosphate buffered saline (PBS) which had BSA at 3%. The solution was incubated at room temperature during 30 minutes before being added to the coated immunotubes. After one hour of incubation at room temperature, the immunotubes were washed 5 times with PBS Tween 20 at 0.1% (PBST) and 5 times with PBS. Bonded phages were eluted with 1.0 ml 0.1 M trietilamine during 10 minutes and the phage solution was immediately neutralized with 0.5 ml Tris-HCI 1.0 M pH 8.0.
  • PBS phosphate buffered saline
  • E. coli TG-1 The eluted phages were amplified in E. coli TG-1 and used for additional selection rounds. 3 selection rounds were performed against NFE2L3(30-694). To obtain phage-producing individual colonies, E. coli TG-1 was infected with the eluted phages from the second and third selection rounds, then, they were plated in serial dilutions on 2 X TY plates complemented with ampicillin (100 ⁇ g/ml) and glucose (1-2%) (2XTYAG). 94 individual randomly chosen colonies (47 from each selection round) were grown in a 96-well plate during the night at 37° with 2xTYAG.
  • the plate was used to inoculate new cultures in 96-well plates until they reached a cellular density of 0.4 (abs 600nm) on a stirrer at 37 0 C. Then, enough auxiliary M13K07 phages (Marks et al. J. MoI. Biol. 222, 581 (1991 )) were added to provide a MOI of 20 and they were grown for 30 minutes at 37 0 C. 50 ⁇ l of 2XTY complemented with ampicillin (100 ⁇ g/ml) and Kanamycin (100 ⁇ g/ml)(2XTYAK) were added to each well and this was grown on a stirrer at 3O 0 C during the night.
  • Phages were separated from the bacterial sediment through centrifugation and supematants were used directly in phage ELISAs to detect clones that bonded with plates covered with NFE2L3(30-694), but not with plates covered with a protein used as negative control. CTHRC1 protein was used as negative control. Phages fixed to NFE2L3(30- 694) were detected with anti-M13-HRP. Specific fragments (antibodies) were defined as the phage clones showing a sign of ELISA at least 3 times higher in the plate covered with NFE2L3(30-694) than in the plate covered with the negative control. Clones that produce Fab fragments with soluble expression (sFab) were also isolated.
  • the gene that codifies g3p protein of isolated pMID21 plasmid from bacteria infected with phages from the second and third selection round was eliminated.
  • the plasmid was digested with Mlu ⁇ enzyme, bonded and transformed into E. coli TG-1.
  • 94 randomly chosen individual colonies (47 from each selection round) were grown in a 96-well plate during the night at 37 0 C with 2xTYAG. The following day, the plate was used to inoculate new cultures in a 96-well plate with 2xTYA complemented with 0.1% glucose, which were grown until they reached a cellular density of 0.9 (abs 600nm) on a stirrer at 37 0 C.
  • IPTG Isopropyl-beta-d- thiogalactopyranoside
  • each sFab or Fab fragment carries its genotype in the interior of the phage particle, once the positive clones to NFE2L3(30-694) were isolated, the sequences that codify them were established.
  • the reverse sequencing primer M13/pUC oligonucleotide New England Biolabs M13/pUC Reverse Sequencing Primer (-48) (24-mer), ref # S 1233S was used to arrange the variable domain (CDRs).
  • the aminoacid sequences of the variable domains of the VH and VL chains of the clones obtained were compared to determine the number of different selected clones. A total of 7 different clones of sFab were obtained.
  • a Fab clone was then sequenced to be used as capture antibody in an ELISA which is later described. The 8 clones obtained are shown in Table 8.
  • sFab fragments from the present invention were purified to be used in different diagnosis methods.
  • sFab producing clones were grown until they reached a cellular density of 0.9 (abs ⁇ OOnm) on a stirrer at 37 0 C.
  • sFab production was induced with a final IPTG concentration of 1 mM and the cultures were grown on a stirrer at 3O 0 C during the night.
  • sFabs were separated from bacterial sediment through centrifugation and purified by affinity with protein-A sepharose. Between 0.5 and 2.0 mg sFabs were obtained per liter of culture. Purified sFab fragments were marked with biotin to be used in different adhesion trials.
  • each sFab was dialyzed during the night with a sodium bicarbonate 0.1 M pH 8.5.
  • the volume of each sFab was adjusted to 1 ml with sodium bicarbonate and 5 mg of dissolved 'biotinyl-N-hidroxysuccinimide ester (BNHS)(Pierce) were added at the moment of usage in 100 ⁇ l of DMSO (Sigma).
  • BNHS 'biotinyl-N-hidroxysuccinimide ester
  • the reaction was kept rotating for 4 hours at room temperature. Subsequently, the reaction was stopped by adding 20 ⁇ l of ammonium chloride.
  • sFabs were dialyzed 48 hours against PBS with several changes.
  • titers obtained varied a lot, for example, the highest titer was that of E5sFab ⁇ NFE2L3 clone with a 50000 titer and the lowest one was that of E10sFab ⁇ NFE2L3 clone with a titer of 50.
  • sFabs bond with other non-related proteins, such as lysozyme, fibrinogen, albumin, ovalbumin, human IgG or other biomarker, such as CTHRC1 , was also determined. Results demonstrated that the sFabs obtained only recognized the NFE2L3(30-694), and did not recognize unrelated proteins; consequently, obtained sFabs are specific to NFE2L3.
  • Biotinylated sFabs Clones (1) Titer w
  • Biotinylated sFabs had an original concentration of 500 ⁇ g/ml.
  • Titers (2) correspond to dilutions indicating an absorbance (450nm) of 1.0-
  • Antigen-competition tests were carried out to test whether different ⁇ -NFE2L3 sFab clones recognized different epitopes in the antigen.
  • NFE2L3(30-694) was coated in Maxisorp NUNC 96-well immuno plates in a 1-5 ⁇ g/ml concentration. The biotinylated sFab bond in presence of growing concentrations of non-botinylated sFabs was measured with strep-HRP. According to the results obtained, three antigenic groups were defined based on the inhibition percentage.
  • Antibodies of this invention can be useful in diagnosis trials, for example to detect NFE2L3 expression in specific cells, tissues or serum.
  • Antibodies of this invention can be used in any known trial method, such as competitive bonding tests, double antibody test (DAS-ELISA), immunoprecipitation tests, immuno-PCR trials, etc. Based on the antigenic groups obtained, a sandwich ELISA or capture ELISA (DAS- ELISA) was adjusted to detect TNFRSF12A using the sFab combinations from different antigenic groups.
  • DAS-ELISA double antibody test
  • DAS- ELISA capture ELISA
  • the trial comprised the following steps:
  • NFE2L3(30-694) to be analyzed (10-100 ng / well) was diluted in PBST BSA at 3% (PBSTB) and 100 ⁇ l of this solution were added to the wells, and then, incubated during 60 minutes at 3O 0 C. The plate was washed again with PBST.
  • _ Detection Biotinylated sFabs were added to the wells (optimum dilution according to table 2) and incubated during 60 minutes at 3O 0 C. The plate was washed three times with PBST and once with PBS. A solution prepared through dilution of strep-HRP at 1 :8000 in PBSTB was added to each well and incubated at room temperature during 60 minutes. The plate was washed three times with PBST.
  • sFab fragments cannot efficiently capture NFE2L3(30-694) in this type of plates because they are small and when they are immobilized on the plate surfaces, their domains may be affected.
  • This problem could be solved using a different type of plates that allow Fab fragments to be oriented, for example estreptavidine plates, plates covered with A protein, plates that allow covalent bond through the amino region of the sFab fragments to the plates surfaces. Due to the high titer (50000 titer, see table 9) of E5sFab ⁇ NFE2L3 clone, it was used in a NFE2L3 detection trial.
  • the biotinylated E5sFab ⁇ NFE2L3 sFab was immobilized in an estreptavidine plate to capture NFE2L3(30-694), and other anti-NFE2L3 phage clones were used to detect it. The phages were later detected with ⁇ M13 HRP. Capture NFE2L3(30-694) sign that were at least 2 times the sign without NFE2L3(30- 694) were considered positive. As shown in table 10, one of the four phage clones (clone F11 ) recognized NFE2L3(30-694) captured by E5sFab ⁇ NFE2L3. In this trial, up to ng NFE2L3(30-694) per well were detected.
  • the sensitivity of this trial can be improved by converting the trial in an immuno-PCR, since each sFab or Fab fragment carries its genotype in the interior of the phage particle. It has been described that phages in immuno-PCR can increase trial sensitivity up to 10000 times compared to the same trial using the ELISA format (Nucleic Acids Research, 2006, Vol.34, No .8). In another attempt to improve this trial, comparisons were made using F11 clone phages, the periplasmatic extract of this clone and the purified Fab protein (F11 Fab ⁇ NFE2L3). F11 Fab ⁇ NFE2L3 protein is a Fab fragment bonded with the g3p protein, which was purified from bacteria periplasma.
  • the grey cells indicate that c/f is higher than 2.
  • NFE2L3(30-694) with botinylated E5sFab ⁇ NFE2L3 represented by absorbance values at 450 nm.
  • sFab E5sFab ⁇ NFE2L3 is a good antibody to capture NFE2L3 in plates where antibodies are oriented (for example, estreptavidine plate), and also to detect captured NFE2L3 using other forms of capture.
  • F11 phage clone could be useful to detect captured NFE2L3 and it could be applied in immuno-PCR when high sensitivity is required.
  • a NFE2L3 detection (DAS-ELISA) trial was optimized using purified polyclonal antibodies to capture NFE2L3, combined with botinylated E5sFab ⁇ NFE2L3 detection sFab, detecting up to 4 ng NFE2L3(30-694)/ well.
  • An immuno-chromatographic trial was developed comprising only one step based on the lateral flow technique.
  • the device where the antigen-antibody reaction takes place has a conjugate area, where the sample is dispensed for its analysis and a test area, where the results are interpreted.
  • the diagnosis trial was based on the immuno capture of colored latex microparticles coated with a specific antibody for NFE2 L3 (30-694) protein while it goes through a membrane on which said antibody or another antibody specific to IgG anti-NFE2 L3
  • the sample to be analyzed contains the desired protein, it will be captured by the antibodies in a covalent bond with the colored latex microparticles (conjugate area).
  • test line the complex formed by latex-antibody-protein particles will migrate by chromatography to the test area.
  • rabbit anti-NFE2L3 purified IgG have been immobilized, which could capture the latex-antibody-protein particles complex, which will produce a red/pink transverse band (test line). Whether the sample analyzed is positive or negative, the trial will be validated by a second blue transverse band (control line), which indicates that the chromatography has taken place correctly.
  • a solution of the same polyclonal antibody was prepared with a 1 mg/ml concentration.
  • the test line was obtained placing this solution on a nitrocellulose membrane at a 1 ⁇ l/cm dose.
  • Immunoreactive latex was prepared with a covalent bond of the rabbit polyclonal IgG anti-NFE2L3 antibody and 300 nm red latex microparticles. The concentration of the union of the antibody to the latex surface was 1 mg/m 2 .
  • the test containing 0.2% of conjugated latex with rabbit anti-NFE2L3 IgG was the one that showed greater sensitivity, detecting up to 0.15-0.078 ⁇ g/ml.
  • This example shows the capacity of immuno chromatographic trials to detect the NFE2L3(30-694) recombinant protein.

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Abstract

L'invention concerne un procédé pour le diagnostic et/ou le pronostic de tumeur colorectale, ce procédé comprenant l'analyse d'un échantillon prélevé sur un patient afin de déterminer le niveau d'expression du gène NFE2L3.
PCT/EP2008/010665 2007-12-14 2008-12-15 Procédé pour le diagnostic de tumeur colorectale WO2009077157A1 (fr)

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Cited By (1)

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WO2017159739A1 (fr) * 2016-03-16 2017-09-21 学校法人同志社 Agent anticancéreux

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WO2004018647A2 (fr) * 2002-08-26 2004-03-04 Case Western Reserve University Methodes et compositions pour categoriser des patients
US20040110712A1 (en) * 2002-08-26 2004-06-10 Markowitz Sanford D. Methods for treating patients and identifying therapeutics
US20070099209A1 (en) * 2005-06-13 2007-05-03 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing cancer

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WO2004018647A2 (fr) * 2002-08-26 2004-03-04 Case Western Reserve University Methodes et compositions pour categoriser des patients
US20040110712A1 (en) * 2002-08-26 2004-06-10 Markowitz Sanford D. Methods for treating patients and identifying therapeutics
US20070099209A1 (en) * 2005-06-13 2007-05-03 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing cancer

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CHIU SOU-TYAU ET AL: "Clinicopathologic correlation of up-regulated genes identified using cDNA microarray and real-time reverse transcription-PCR in human colorectal cancer.", CANCER EPIDEMIOLOGY, BIOMARKERS & PREVENTION : A PUBLICATION OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, COSPONSORED BY THE AMERICAN SOCIETY OF PREVENTIVE ONCOLOGY FEB 2005, vol. 14, no. 2, February 2005 (2005-02-01), pages 437 - 443, XP002521419, ISSN: 1055-9965 *
CRONER ROLAND S ET AL: "Common denominator genes that distinguish colorectal carcinoma from normal mucosa.", INTERNATIONAL JOURNAL OF COLORECTAL DISEASE JUL 2005, vol. 20, no. 4, July 2005 (2005-07-01), pages 353 - 362, XP002521418, ISSN: 0179-1958 *
DATABASE EMBASE [online] ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL; 16 September 2007 (2007-09-16), WANG Q ET AL: "Constructing a fusion protein eukaryotic expression vector of colorectal cancer related gene Nrf3 and its initial functional study on transfection in vitro", XP002521420, Database accession no. EMB-2007551230 *
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SABATES-BELLVER JACOB ET AL: "Transcriptome profile of human colorectal adenomas.", MOLECULAR CANCER RESEARCH : MCR DEC 2007, vol. 5, no. 12, 1 December 2007 (2007-12-01), pages 1263 - 1275, XP002521417, ISSN: 1541-7786 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017159739A1 (fr) * 2016-03-16 2017-09-21 学校法人同志社 Agent anticancéreux

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AR069697A1 (es) 2010-02-10

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