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WO2024251754A1 - Kits et procédés pour immuno-pcr multiplex - Google Patents

Kits et procédés pour immuno-pcr multiplex Download PDF

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Publication number
WO2024251754A1
WO2024251754A1 PCT/EP2024/065375 EP2024065375W WO2024251754A1 WO 2024251754 A1 WO2024251754 A1 WO 2024251754A1 EP 2024065375 W EP2024065375 W EP 2024065375W WO 2024251754 A1 WO2024251754 A1 WO 2024251754A1
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WIPO (PCT)
Prior art keywords
mir
nucleic acid
pcr
hsa
ykl
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Inventor
Zouher Majd
Sylvie DELEDICQUE
Virginie GALOSEAU
Jérémy MAGNANENSI
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Genfit SA
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Genfit SA
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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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • NASH non-alcoholic steatohepatitis
  • NASH non-alcoholic steatohepatitis
  • NASH non-alcoholic steatohepatitis
  • Non-alcoholic steatohepatitis is also a progressive disease of the liver characterized histologically by fatty acid accumulation, hepatocyte damage and inflammation resembling alcoholic hepatitis.
  • NASH is a critical stage in the process that can lead to cirrhosis, liver failure and/or HCC (Hepatocellular Carcinoma).
  • HCC Hepatocellular Carcinoma
  • NASH is one of the most common causes of elevated aminotransferases in patients referred for evaluation to hepatologists. Because this disease can be potentially reversed or at least its consequences can be limited if the patient is diagnosed early enough, it is crucial to be able to provide the medical field with adapted tools allowing such an early, rapid and precise diagnostic.
  • liver biopsy is a very invasive procedure that may be cumbersome, worrisome and painful for the patient, and which raises concerns about morbidity and mortality.
  • liver biopsy cannot reasonably be proposed as a routine procedure for determining whether a person has a significant liver fibrosis.
  • NITS non invasive tests
  • WO2017046181 and WO2017167934 provide non-invasive diagnosis methods based on the measure of the levels of circulating biomarkers. These methods measure several independent circulating biomarkers which are either proteins or miRNAs.
  • WO2017167934 describes a non-invasive NASH diagnostic method which measures the levels of YKL-40 (or chitinase 3-like protein 1, CHI3L1) and hsa-miR-34.
  • the biomarkers especially a protein biomarker and a miRNA biomarker, need to be measured separately by different assays, e.g. polymerase chain reaction (PCR) assays for detecting a miRNA biomarker and an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA), for detecting a protein marker.
  • PCR polymerase chain reaction
  • ELISA enzyme-linked immunosorbent assay
  • immuno-PCR Compared to ELISA, immuno-PCR (IPCR) assay is a more rapid and sensitive method of detecting a protein.
  • Immuno-PCR relies on the use of an antibody, which has been conjugated to an oligonucleotide. This oligonucleotide can be then amplified by PCR and this conjugate acts as a bridge between the immunoreaction and DNA amplification.
  • This method combines the versatility and robustness of immunoassays with the exponential signal amplification power of the PCR.
  • IPCR allows a 10-1,000-fold increase in sensitivity over the analogous ELISA. But there is still a need to improve IPCR means to make it suitable to be compatible with the simultaneous detection or quantification of a miRNA biomarker in a single sample.
  • kits comprising said conjugate and suitable components for implementing a multiplex PCR for simultaneously detecting or quantifying a protein target and a nucleic acid target.
  • kit of the invention provides a unique platform to perform diagnostic tests can offer several advantages over using multiple platforms or tests.
  • Standardization A unique platform can offer standardization of testing procedures, which can help reduce variations in results due to differences in testing methods or equipment. This can increase the reliability and accuracy of diagnostic tests, improving patient outcomes.
  • Efficiency Using a single platform for diagnostic tests can increase efficiency in the laboratory by reducing the time and resources required for maintenance, calibration, and training on multiple instruments.
  • a unique platform can also be cost-effective compared to using multiple tests or instruments. This can be particularly important for resource-limited settings, where costs can be a barrier to accessing diagnostic testing.
  • Integration A unique platform can offer integration with electronic medical records and other laboratory systems, which can improve the overall efficiency and quality of patient care. 5. Flexibility: Depending on the specific platform, a unique platform may offer flexibility to perform a wide range of tests, allowing for more comprehensive diagnostic testing from a single instrument. In conclusion, a unique platform for diagnostic testing can offer several advantages, including standardization, efficiency, cost-effectiveness, integration, and flexibility.
  • said probe has a sequence of SEQ ID NO: 4, 9 or 10.
  • the present invention also relates to the use of said immuno-PCR kit for detecting or quantifying the level of a protein target in a sample.
  • the present invention relates to a multiplex immuno-PCR kit for simultaneously detecting or quantifying a protein target and a nucleic acid target, said kit comprising: - a DNA antibody conjugate according to the invention, - a pair of primers for amplifying the oligonucleotide of the DNA-antibody conjugate, and - a pair of primers for amplifying the nucleic acid target.
  • the kit further comprises: - a fluorescent nucleic acid probe that specifically binds to the oligonucleotide of the DNA- antibody conjugate, and - a fluorescent nucleic acid probe that specifically binds to the amplification product of the nucleic acid target.
  • the fluorescent nucleic acid probe that specially binds to the oligonucleotide of the DNA-antibody conjugate may have the sequence of SEQ ID NO: 4, 9 or 10.
  • said multiplex immuno-PCR kit comprises a pair of primer for amplifying a miRNA, in particular hsa-miR-34, more particularly hsa-miR-34a and still more particularly hsa-miR-34a-5p.
  • the DNA-antibody conjugate of the kit comprises an anti-YKL-40 antibody.
  • said multiplex immuno-PCR kit comprises: - a DNA antibody conjugate comprising an anti-YKL-40 antibody and an oligonucleotide of SEQ ID NO: 1, - a pair of primers for amplifying the oligonucleotide of the DNA-antibody conjugate, - a pair of primers for amplifying hsa-miR-34, more particularly hsa-miR-34a and still more particularly hsa-miR-34a-5p
  • the multiplex immuno-PCR kit as described above further comprises: - an internal control for PCR process, - at least one positive control for hsa-miR-34, and/or - at least one positive control for YKL-40.
  • the present invention also relates to the use of the above-described multiplex immuno-PCR for detecting or quantifying the level of a protein target and a nucleic acid target in a sample, in particular for detecting or quantifying the level of YKL-40 and hsa-miR-34 in a sample.
  • the present invention also provides a method for quantifying the levels of a protein target and of a nucleic acid target in a sample, said method comprising the steps of : - contacting the sample with the components of the multiplex immuno-PCR kit of the invention, and - performing a multiplex immuno-PCR to measure the levels of said protein target and of said nucleic acid target.
  • the invention also relates to a method for quantifying the levels of YKL-40 and hsa-miR-34 in a sample, said method comprising the steps of : - contacting the sample with components of the multiplex immuno-PCR kit of the invention, and - performing a multiplex immuno-PCR to measure the levels of YKL-40 and hsa-miR-34.
  • Another aspect of the invention relates to a method for diagnosing non-alcoholic steatohepatitis (NASH) and/or for determining the activity, the stage, or the severity of NASH in a subject, and/or for the classification of a subject as a receiver or non receiver of a treatment for NASH, and/or for the evaluation of the efficacy of a medical treatment, and/or for the determination of the progression or the regression of the pathology in NASH patients, and/or for the classification of a patient as a potential responder or non responder to a medical treatment, by measuring the levels of blood, serum or plasma circulating hsa-miR-34 and YKL-40 in a sample of said subject or said patient, said method comprising the steps of : - contacting the sample with the components of the multiplex immuno-PCR kit of the invention, and - performing a multiplex immuno-PCR to measure the levels of YKL-40 and hsa-miR-34.
  • NASH non-alcoholic steato
  • FIG. 1 Fibrosis x NAS spectrum of the training (A) and validation (B) cohorts of patients from screening visit (SV).
  • Figure 2 Corresponding plots of three different miR-34a dosages, i.e. an old dosage data using the usual PCR process (indicated in figures as “oriD”), a new dosage data using the usual PCR process (indicated as “newD”), and the dosage data extracted from the data using the multiplex immuno-PCRprocess (miPCR) of the invention (indicated as “immuPCR”).
  • oriD old dosage data using the usual PCR process
  • newD a new dosage data using the usual PCR process
  • miPCR multiplex immuno-PCRprocess
  • A corresponding plots between the dosage data “oriD” and “newD”
  • B corresponding plots between the dosage data “immuPCR” and “newD”
  • C corresponding plots between the dosage data “immuPCR” and “oriD”.
  • Figure 3 Boxplots of three miR-34a dosage data distributions, i.e. “immuPCR”, “newD” and “oriD”, and ANOVA for repeated measures summary.
  • Figure 4 Corresponding plots of three different YKL-40 dosages, i.e.
  • an old dosage data using the usual ELISA process (indicated in figures as “oriD”), a new dosage data using the usual ELISA process (indicated as “newD”), and the dosage data extracted from the data using the miPCR (indicated as “immuPCR”).
  • C corresponding plots between the dosage data “immuPCR” and “oriD”.
  • Figure 5 Boxplots of different YKL-40, i.e. “immuPCR”, “newD” and “oriD”, and ANOVA for repeated measures summary.
  • FIG. 6 Corresponding plots of miR-34a (A) and YKL-40 (B) dosage values from standard techniques vs miPCR.
  • the corrected data for miR-34a and YKL-40 were extracted from the data using the multiplex immuno-PCR process of the invention.
  • newD the new dosage data using standard techniques
  • ImmuPCR_Corr” the corrected data extracted from miPCR data.
  • Figure 8 Corresponding plots of miR-34a (A) and YKL-40 (B) dosage values from standard techniques vs miPCR.
  • the corrected data for miR-34a and YKL-40 were extracted from the data using the miPCR process.
  • “ImmuPCR” uncorrected data obtained from miPCR process
  • newD the new dosage data using standard techniques
  • ImmuPCR_Corr the corrected data extracted from multiplex immuno-PCR data.
  • A corresponding plots between NIS2+ scores obtained through “NIS2+-newD” and the scores obtained through “NIS2+- immunoPCR”; B: corresponding plots between NIS2+ scores obtained through “NIS2+- immunoPCRCorr” and the scores obtained through “NIS2+-immunoPCR”; C: corresponding plots between NIS2+ scores obtained through “NIS2+-newD” and the scores obtained through “NIS2+-immunoPCRCorr”.
  • nucleic acid molecules which may be used for detecting a target in a sample.
  • nucleic acid molecule refers to a fragment of single-stranded or double-stranded DNA or an analogue of derivative of a nucleic acid molecule which is capable of being amplified by PCR.
  • Said nucleic acid molecule may comprise or consists of an oligonucleotide which is suitable to be amplified during a polymerase chain reaction (PCR).
  • oligonucleotide refers to a single-stranded DNA having from 50 to 200 nucleotides, particularly from 60 to 180 nucleotides, more particularly from 70 to 150 nucleotides, still more particularly from 80 to 120 nucleotides. Said oligonucleotide may comprise natural deoxyribonucleotides and eventually non-natural nucleotides.
  • the sequence of said oligonucleotide has an identity lower than 50% compared to the sequence of any circulating DNA which may present in a sample to be tested, that ensures that, on the one hand, said oligonucleotide will not be amplified by any pair of primers for amplifying a circulating DNA/RNA of the sample, and on the other hand, the pair of primers for amplifying said oligonucleotide will be specific for said oligonucleotide.
  • said oligonucleotide has a sequence with an identity lower than 50%, in particular lower than 40%, more particularly lower than 30%, 20%, 10%, or 5%, compared to the sequence of any circulating human serum DNA/RNA.
  • said oligonucleotide has the sequence set forth by SEQ ID NO: 1, 2 or 3, or a sequence having at least 90% identity to SEQ ID NO: 1, 2 or 3. More particularly, said oligonucleotide has the sequence of SEQ ID NO: 1. Sequence identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences.
  • a nucleic acid molecule of the present invention may further comprise a linker. Said linker is attached to the oligonucleotide described above. Typically, the linker may comprise a carbon chain or a PEG chain as a spacer and a reactive group which allows to conjugate said nucleic acid molecule to a protein or a peptide.
  • Said reactive group may be any reactive group which can directly react with a residue of a protein or indirectly react with a protein via a compatible reactive group carried by said protein.
  • Suitable reactive groups include, but are not limited to, thiol, carboxyl, amine, hydroxyl, aldehyde, biotin, streptavidin and chemical groups suitable for click chemistry, e.g. an alkyne group, an azide group.
  • a linker comprised in a nucleic acid molecule of the invention may be any linker described in the art suitable for oligo-antibody conjugation. Examples of linkers include, but are not limited to, a linker comprising a biotin group, an amino C6 to C18 linker, e.g.
  • said nucleic acid molecule comprises or consists of an oligonucleotide of sequence SEQ ID NO: 1, which is attached to a biotin group.
  • a nucleic acid molecule of the invention may be produced by any conventional method. More particularly, the method for producing a biotinylated oligonucleotide is well known in the art.
  • DNA-antibody conjugates comprising (i) a nucleic acid molecule as described above and (ii) an antibody directed against a protein of interest.
  • DNA-antibody conjugate refers to a complex formed by an oligonucleotide and an antibody, wherein the oligonucleotide is attached to the antibody by a linker.
  • the oligonucleotide of the DNA-antibody conjugate has a sequence of SEQ ID NO: 1, 2 or 3, or a sequence having at least 90% identity to SEQ ID NO: 1, 2 or 3.
  • the oligonucleotide of the DNA-antibody conjugate has a sequence of SEQ ID NO: 1.
  • the antibody of the DNA-antibody conjugate of the invention may be an antibody directed to any protein of interest.
  • proteins of interest include, without being limited to, alpha 2 macroglobulin (A2M), glycated haemoglobin (HbA1c), insulin, C-peptide, PIIINP N- terminal pro-peptide of collagen type III, CK18-M30(cytokeratin 18 fragment 30), CK18-M65, HSCRP High Sensitivity-Reactive Protein HSCRP, TSP-2 Thrombospondin 2 and Soluble Vascular Cell Adhesion Molecule (sVCAM).
  • A2M alpha 2 macroglobulin
  • HbA1c glycated haemoglobin
  • insulin C-peptide
  • CK18-M30(cytokeratin 18 fragment 30) CK18-M65
  • HSCRP High Sensitivity-Reactive Protein HSCRP TSP-2 Thro
  • Said antibodies may be monoclonal or polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, nano- antibodies, full length or fragments thereof including Fab, Fab' or F(ab')2, scFV, aptamers or diabody.
  • said antibody is an antibody directed against the protein YKL-40 (also named chitinase 3-like protein 1, CHI3L1).
  • a suitable anti-YKL-40 antibody may be any anti-YKL-40 antibody described in the prior art or produced according to conventional methods.
  • a DNA-antibody conjugate may be produced by any conventional method described in the prior art (Wiener. J., Kokotek. D., Rosowski. S., Lickert. H.
  • a DNA-antibody conjugate may be produced by reacting an antibody with a nucleic acid molecule bearing a linker or by reacting together an antibody and an oligonucleotide with a linker.
  • the antibody, the oligonucleotide and the linker may bear suitable reactive groups.
  • an oligonucleotide bearing a biotin group or an alkyne group may react with a protein bearing a streptavidin group or an azide group, respectively.
  • the DNA-antibody conjugate comprises an oligonucleotide of sequence SEQ ID NO: 1 and an anti-YKL-40 antibody, wherein the oligonucleotide is attached to the antibody via biotin-streptavidin-biotin linker.
  • Kits The present invention also provides kits for detecting or quantifying a protein target by immuno-PCR. More particularly, the present invention concerns a kit for detecting or quantifying by multiplex immuno-PCR a protein target and a nucleic acid target.
  • immuno-PCR refers to the use of polymerase chain reaction to detect or quantify a protein by specifically amplifying an oligonucleotide attached to said protein.
  • An immuno-PCR allows to detect or quantify a protein target by PCR.
  • the term “multiplex immuno-PCR” refers to the use of polymerase chain reaction to amplify simultaneously at least an oligonucleotide of a DNA-antibody conjugate and at least one other DNA. Therefore, a multiplex immuno-PCR allows to simultaneously detect or quantify more than one target by PCR.
  • Said target may be a protein target or a nucleic acid target.
  • protein targets include, without being limited to, human serum circulating proteins, especially serum circulating proteins expressed in patients suffering from NASH.
  • nucleic acid targets include, without being limited to, human serum circulating nucleic acids, e.g. microRNAs, especially serum circulating microRNAs expressed in patients suffering from NASH.
  • the protein target is circulating YKL-40 protein and said nucleic acid target is miR-34, in particular hsa-miR-34, more particularly hsa-miR-34a and still more particularly hsa-miR-34a-5p.
  • the kit comprises a DNA-antibody conjugate as described herein.
  • the invention relates to an immuno-PCR kit for detecting a protein target, said kit comprising: - a DNA-antibody conjugate as described in the present invention, and - a pair of primers for amplifying the oligonucleotide of said DNA-antibody conjugate.
  • said kit comprises a DNA-antibody conjugate comprising an anti-YKL-40 antibody and an oligonucleotide of sequence SEQ ID NO: 1, 2 or 3.
  • the sequences of the primers for amplifying an oligonucleotide of a DNA-antibody conjugate may be designed according to any conventional method for designing PCR primers and on the basis of the sequence of the oligonucleotide of the DNA-antibody conjugate.
  • the forward and the reverse primers for amplifying the oligonucleotide of sequence SEQ ID NO: 1 have the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the forward and the reverse primers for amplifying the oligonucleotide of sequence SEQ ID NO: 2 have the sequences of SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the forward and the reverse primers for amplifying the oligonucleotide of sequence SEQ ID NO: 3 have the sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
  • the immuno-PCR kit of the invention further comprises a fluorescent nucleic acid probe that specifically binds to the oligonucleotide of the DNA- antibody conjugate.
  • Said fluorescent nucleic acid probe can be used to indicate in real time the quantity of the amplification product and is suitable to be used in a quantitative PCR.
  • Said fluorescent nucleic acid probe is in particular a TaqMan probe, i.e. a nucleic acid probe consisting of a fluorescent dye covalently attached to the 5’ of the probe and a quencher covalently attached to the 3’ and of the probe.
  • Suitable couple of fluorescent dye and quencher can be selected according to the rules well-known in the art, i.e. the fluorescence emission of the fluorescent dye should be efficiently inhibited by the quencher before the probe being degraded by a DNA polymerase during a PCR cycle.
  • the sequence of said fluorescent nucleic acid probe may be designed according to conventional methods and on the basis of the sequence of the oligonucleotide of the DNA-antibody conjugate. Said probe binds to the oligonucleotide within the region that will be amplified.
  • said fluorescent nucleic acid probe has the sequence of SEQ ID NO: 4, 9 or 10.
  • the probe of sequence SEQ ID NO: 4 can specifically binds to the oligonucleotide of sequence SEQ ID NO: 1.
  • the probe of sequence SEQ ID NO: 9 can specifically binds to the oligonucleotide of sequence SEQ ID NO: 2.
  • the probe of sequence SEQ ID NO: 10 can specifically binds to the oligonucleotide of sequence SEQ ID NO: 3. More particularly, said probe has the sequence of SEQ ID NO: 4 and bears at 5’ end a Cy5 fluorescent dye and at 3’ end BHQ-2 as a quencher.
  • An immuno-PCR kit comprising an above described fluorescent nucleic acid probe is particularly suitable to be used in a quantitative PCR.
  • the invention relates to a multiplex immuno-PCR kit for detecting a protein target and a nucleic acid target, said kit comprising: - a DNA-antibody conjugate as described in the present invention, - a pair of primers for amplifying the oligonucleotide of the DNA-antibody conjugate, and - a pair of primers for amplifying the nucleic acid target.
  • said kit comprises a DNA-antibody conjugate comprising an anti-YKL-40 antibody and an oligonucleotide of sequence SEQ ID NO: 1, 2 or 3.
  • sequences of the primers for amplifying the oligonucleotide of said DNA-antibody conjugate may be designed according to conventional methods for designing PCR primers and on the basis of the sequence of said oligonucleotide.
  • the forward and the reverse primers for amplifying the oligonucleotide of sequence SEQ ID NO: 1 have the sequences SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the forward and the reverse primers for amplifying the oligonucleotide of sequence SEQ ID NO: 2 have the sequences SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the forward and the reverse primers for amplifying the oligonucleotide of sequence SEQ ID NO: 3 have the sequences SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
  • the sequence of the primers for amplifying the nucleic acid target may be designed according to any conventional method and on the basis of the sequence of the nucleic acid target.
  • the multiplex immuno-PCR kit comprises a pair of primers for amplifying a miRNA, in particular a miRNA selected from hsa-miR-34, hsa-miR-132, hsa- miR-125, hsa-miR-505, hsa-miR-365, hsa-miR-22, hsa-miR-378, hsa-miR-320, hsa-miR-885, hsa-miR-483, hsa-miR-30, hsa-miR-422a, hsa-miR-100, hsa-miR-4324, hsa-miR-193 and hsa- miR-452.
  • a miRNA in particular a miRNA selected from hsa-miR-34, hsa-miR-132, hsa- miR-125, hsa
  • the multiplex immuno-PCR kit comprises a pair of primers for amplifying hsa-miR-34a and still more particularly hsa-miR-34a-5p (SEQ ID NO: 7).
  • the multiplex immuno-PCR kit comprises a forward primer and a reverse primer for amplifying hsa-miR-34a-5p.
  • Said primers are those comprised in the Taqman MicroRNA assay ((Applied Biosystems, reference 4440886)).
  • said kit further comprises: - a fluorescent nucleic acid probe that specifically binds to the oligonucleotide of the DNA-antibody conjugate as described above, and - a fluorescent nucleic acid probe that specifically binds to the amplification product of the nucleic acid target.
  • the fluorescent nucleic acid probe for the oligonucleotide and the fluorescent nucleic acid probe for the nucleic acid target comprise respectively fluorescent dyes which can be distinguished.
  • said fluorescent nucleic acid probe has the sequence of SEQ ID NO: 4, 9 or 10.
  • the fluorescent nucleic acid probe binding with the amplification product of the nucleic acid target may be designed on the basis of the sequence of the nucleic acid target.
  • a multiplex immuno-PCR kit of the invention may further comprise: - an internal control for PCR process, - at least one positive control for hsa-miR-34, and - at least one positive control for YKL-40.
  • an internal control and/or at least one positive control of the targets to be detected or quantified may be used for normalizing the amplification data of the target(s).
  • An appropriate internal control may be selected according to the general knowledge in the art.
  • said internal control may be an exogenous DNA for a sample, for example a miRNA molecule found in a different species than human, such as a miRNA molecule from Caenorhabditis elegans.
  • said internal control is cel-miR- 40-3p (SEQ ID NO:8: 5’-UCACCGGGUGUACAUCAGCUAA-3’) from Caenorhabditis elegans.
  • said kit further comprises a pair of primers for amplifying the internal control and a fluorescent nucleic acid probe that specifically binds to the amplification product of the internal control.
  • said fluorescent nucleic acid probe for the internal control cel-miR-40-3p is TaqMan MicroRNA Assay (Applied Biosystems, reference CCU001L).
  • a positive control with a known hsa-miR-34 value can be used.
  • three positive controls with known different hsa-miR-34 value can be used. These positive controls can cover the range of hsa- miR-34 level in NASH population. In case three positive control are used, one corresponds to a low hsa-miR-34 level, one to a medium level and another to a high level.
  • the medium level can also be referred to as the calibrator, serving in the assay to calculate the fold change value.
  • the kit comprises three positive controls for hsa-miR-34 with the concentrations: 24.8fM , 2.48fM , 0.99fM , respectively.
  • YKL-40 To quantify the level of YKL-40 during a quantitative PCR, at least one positive control with a known YKL-40 value can be used. Particularly, three positive controls with known different YKL-40 value can be used.
  • the kit comprises three positive controls for YKL-40 with the concentrations: 30000 pg/mL, 85000 pg/mL, 200000 pg/mL respectively.
  • the kits of the invention may further comprise a DNA polymerase, e.g. a TaqMan® polymerase, several deoxynucleotides, and one or more reaction buffer(s).
  • the reaction buffer(s) may contain salt and any conventional reagents to optimize the DNA polymerase activity and/or the PCR assay.
  • the immuno-PCR kit of the present invention can be used for detecting or quantifying the level of a protein target in a sample.
  • the multiplex immuno-PCR kit of the present invention is particularly advantageous, because it can be used for simultaneously detecting or quantifying the levels of a protein target and a nucleic acid target in a sample.
  • the kit described above can be used for simultaneously detecting or quantifying the levels of YKL-40 and miR-34, two biomarkers used in the diagnostic method as described in WO2017167934. Therefore, the multiplex immuno-PCR kit of the invention allows to simultaneously detect and/or quantify the levels of circulating biomarkers which are used in the diagnosis of NASH.
  • the multiplex immuno-PCR kit of the invention allows to implement a more rapid and easier method for measuring these biomarkers.
  • the present invention relates to the use of an immuno-PCR kit of the present invention for detecting or quantifying the level of a protein target in a sample.
  • the invention also relates to the use of a multiplex immuno-PCR kit of the present invention for detecting or quantifying the levels of a protein target and a nucleic acid target in a sample. More particularly, the invention relates to the use of the kit as described above for detecting or quantifying the level of YKL-40 and hsa-miR-34 in a sample.
  • the invention relates to the use of the kit as described above for diagnosing non-alcoholic steatohepatitis (NASH) and/or for determining the activity, the stage, or the severity of NASH in a subject, and/or for the classification of a subject as a receiver or non receiver of a treatment for NASH, and/or for the evaluation of the efficacy of a medical treatment, and/or for the determination of the progression or the regression of the pathology in NASH patients, and/or for the classification of a patient as a potential responder or non responder to a medical treatment.
  • NASH non-alcoholic steatohepatitis
  • the present invention also provides a method for quantifying the levels of a protein target and of a nucleic acid target in a sample, said method comprising the steps of: - contacting the sample with the components of the multiplex immuno-PCR kit of the invention, - performing a multiplex immuno-PCR to measure the levels of said protein target and of said nucleic acid target.
  • the present invention provides a method for quantifying the levels of YKL- 40 and hsa-miR-34 in a sample, said method comprising the steps of: - contacting the sample with the components of a multiplex immuno-PCR kit comprising: (i) a DNA-antibody conjugate comprising an anti-YKL-40 antibody and an oligonucleotide of sequence SEQ ID NO: 1, 2 or 3, (ii) a pair of primers for amplifying the oligonucleotide of the DNA-antibody conjugate, (iii) a pair of primers for amplifying hsa-miR-34, - performing a multiplex immuno-PCR to measure the levels of YKL-40 and hsa-miR-34.
  • a multiplex immuno-PCR kit comprising: (i) a DNA-antibody conjugate comprising an anti-YKL-40 antibody and an oligonucleotide of sequence SEQ ID NO: 1, 2 or 3, (ii
  • the multiplex PCR may be performed according to any conventional method, for example using “TaqMan” probes.
  • the present invention also provides a method for diagnosing non-alcoholic steatohepatitis (NASH) and/or for determining the activity, the stage, or the severity of NASH in a subject, and/or for the classification of a subject as a receiver or non receiver of a treatment for NASH, and/or for the evaluation of the efficacy of a medical treatment, and/or for the determination of the progression or the regression of the pathology in NASH patients, and/or for the classification of a patient as a potential responder or non responder to a medical treatment, by measuring the levels of blood, serum or plasma circulating hsa-miR-34 and YKL-40 in a sample of said subject or said patient, said method comprising the steps of: - contacting the sample with the reagents of a multiplex immuno-PCR kit comprising: (i) a DNA-antibody conjugate comprising an ani-Y
  • ⁇ 1 is comprised between 1 and 5, in particular between 2 and 4.
  • ⁇ 2 is comprised between 0 and 4.5, in particular between 0.5 and 3.
  • ⁇ 3 is comprised between -2 and 2, in particular between -1 and 1.
  • ⁇ 4 is comprised between -1 and 2, in particular between 0 and 2.
  • ⁇ 0 is comprised between -3 and 3
  • ⁇ 1 is comprised between 1 and 5
  • ⁇ 2 is comprised between 0 and 4.5
  • ⁇ 3 is comprised between -2 and 2
  • ⁇ 4 is comprised between -1 and 2.
  • ⁇ 0 is comprised between - 2 and 2
  • ⁇ 1 is comprised between 2 and 4
  • ⁇ 2 is comprised between 0.5 and 3
  • ⁇ 3 is comprised between -1 and 1
  • ⁇ 4 is comprised between 0 and 2.
  • the score calculated from the mathematical function can then be compared to predetermined cutoff values, such as low and high cutoff values.
  • predetermined cutoff values such as low and high cutoff values.
  • a calculated S value lower that the low cutoff is indicative of a subject not having at-risk NASH
  • a calculated S value greater or equal to the high cutoff value is indicative of a subject having at-risk NASH.
  • the low cutoff is comprised between 0.24 and 0.5, in particular between 0.41 and 0.49.
  • the high cutoff is comprised between 0.6 and 0.95, in particular between 0.62 and 0.74.
  • the low cutoff is equal to 0.4564.
  • the high cutoff is equal to 0.6815.
  • the low cutoff is equal to 0.4564 and the high cutoff is equal to 0.6815.
  • V3 Visit3 samples was obtained for validation of the potential mathematical correction of biomarkers (BMs) concentration and thus NIS2+ scores.
  • BMs biomarkers
  • YKL- 40 has not been re-dosed using ELISA since it was already observed a high correlation between initial values (those in GFT database) and re-dosed ones based on SV results.
  • the fibrosis versus NAS histological spectrum of both cohorts are graphically reported in Figure 1.
  • the training cohort was enriched with F2 patients (30.7%), while in the validation cohort F1 and F3 were the most represented (28.1% and 32.4%, resp).
  • Positive controls and internal control Positive controls for hsa-miR-34a-5p named C1mir, C2mir and C3mir were prepared from the biological matrix of voluntary blood donor patients, in which synthetic hsa-miR-34a-5p was added. These positive controls mimic respectively the high, medium and low expression levels of this microRNA in the NASH population.
  • the concentrations of hsa-miR-34a-5p in the positive controls are respectively 24.8fM , 2.48fM , 0.99fM .
  • the concentrations of YKL-40 in the positive controls are 30 000 pg/ml, 85000 pg/ml, 200 000 pg/ml, respectively
  • An internal control (Internal Process Control (IPC)) was used.
  • the internal control servs as a control for the entire process for hsa-miR-34a-5p assay ranging from extraction to PCR and was prepared from the biological matrix of volunteer blood donor patients and Cel-miR-40-3p (microRNA of C. elegans).
  • PCR for measuring hsa-miR-34a level A quantitative PCR was performed for separately measuring hsamiR-34a level in serum samples obtained from the patient cohorts.
  • RNA is extracted from a patient serum samples, using Promega magnetic bead-based extraction Maxwell® Plasma and Serum Kit (AS1680, Promega) and RCS48 Instrument (AS8500, Promega) according to the manufacturer’s instructions.
  • Promega magnetic bead-based extraction Maxwell® Plasma and Serum Kit AS1680, Promega
  • RCS48 Instrument AS8500, Promega
  • synthetic vesicles containing Caenorhabditis elegans Cel-miR-40-3p (Mature miRNA sequence UCACCGGGUGUACAUCAGCUAA-3’ (SEQ ID NO: 8), Integrated DNA Technologies, purification RNAse free HPLC) are used as IPC and are added to each sample prior to RNA extraction.
  • RNA from serum samples, containing IPC and total RNA from positive controls containing IPC as well are concomitantly reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems, Thermo Fisher Scientific).
  • Reverse transcription reaction is carried out in a final mixture of 24 ⁇ L containing 3 ⁇ L of TaqMan MicroRNA Assay 5X and incubated in a Thermal Cycler T100 (Biorad). cDNAs are stored in low binding tubes at -20°C until further use. Expression of mature miRNAs is quantified according to the manufacturer’s instructions using the TaqMan miRNA RT-qPCR Assay 20X and TaqMan Universal Master Mix II, no Uracil-N-Glycosilase (UNG) (4440040, Applied Biosystems, ThermoFisher Scientific). A fixed volume of 5 ⁇ L of total cDNA is used as a template for the qPCR assay using a CFX96 Real-Time PCR detection System.
  • UNG Uracil-N-Glycosilase
  • ELISA was performed by using manufacturer’s procedure (Quantikine® ELISA Human Chitinase 3-like 1 Immunoassay Kit (RUO), DC3L10, R&D Systems, Minneapolis, USA) and with automation. Washing steps were performed with the Tecan HydroSpeedTM plate washer (Tecan, Gurnnedorf, ref.30054550 Switzerland), the reading was determined using Thermo ScientificTM MultiskanTM GO Microplate Spectrophotometer (ThermoFisher, ref. 51119200 Massachusetts, USA) set to 450nm. Positive controls were processed similarly to serum samples and assayed in duplicate.
  • manufacturer’s procedure Quantikine® ELISA Human Chitinase 3-like 1 Immunoassay Kit (RUO), DC3L10, R&D Systems, Minneapolis, USA
  • Washing steps were performed with the Tecan HydroSpeedTM plate washer (Tecan, Switzerland, Switzerland), the reading was determined using Thermo ScientificTM MultiskanTM GO Microplate Spectrophotometer (Ther
  • Multiplex immuno-PCR A multiplex immuno-PCR was performed according to the following protocol for measuring hsa-miR-34a and YKL-40 levels in serum samples obtained from the patient cohorts.
  • Sample preparation for measuring hsa-miR-34a The method for extracting total RNA from a serum sample and the method of reverse transcription are the same as that described above in “PCR for measuring hsa-miR-34a level”.
  • Sample preparation for measuring YKL-40 protein Coating of capture antibody: the capture antibody is fixed on the plate by overnight non-specific adsorption at room temperature. The capture antibody is a rat monoclonal antibody specific for the human YKL-40 protein.
  • Said antibody was diluted beforehand in phosphate-buffered saline (PBS) and then deposited at a concentration of 360 ⁇ g/ml. Washing the plate: washing was repeated 3 times with one minute interval between each wash.
  • the washing buffer was prepared with a concentrated wash buffer solution, which containing PBS and 1% bis(trimethylsilyl)acetamide (BSA). The solution was diluted in 1L of water before adding 1ml of Tween 20.
  • Plate saturation a saturation buffer containing 1% BSA, 1 g/L of salmon sperm DNA and 0.1mM of EDTA was used for plate saturation. The incubation was performed at 37°C for 1 hour. It was then followed by 3 washes by using the same washing buffer of the previous step.
  • Standard preparation in order to prepare a series of standards of YKL-40 protein, a recombinant YKL-40 was diluted. This series of standards was made up of 8 points (identical to the ELISA range) with a decrease in the YKL-40 concentrations, obtained by successive cascade dilutions of the YKL-40 protein in the saturation buffer (500 ⁇ L of standard + 500 ⁇ L of saturation buffer).
  • the first range point (std 1) was at a concentration of 8000 pg/ml of YKL- 40.
  • the second to the eighth range points have respectively a concentration of 4000 (std 2), 2000 (std 3), 1000 (std 4), 500 (std 5), 250 (std 6), 125 (std 7) and 62.5 pg/ml (std 8).
  • the standards and the positive controls were then diluted in the saturation buffer. Positive controls were diluted 100 fold in the saturation buffer. Addition of samples: 25 ⁇ L of serum samples were added to each plate wells of the plate. The standards and the positive controls were also added to different wells respectively. The incubation lasts 1 hour at 37°C.
  • biotinylated detection antibody (goat polyclonal antibody directed against human YKL-40 protein) was added to the wells.
  • the detection antibody was used at a concentration of 0.05 ⁇ g/ml.
  • the incubation of the plate was carried out during 1 hour at 37°C.
  • Stock solution of streptavidin (1 nM) was diluted 50,000 fold in the saturation buffer.
  • Biotinylated oligonucleotide of sequence SEQ ID NO: 1 was prepared at a concentration of 0.35M. The streptavidin and the biotinylated oligonucleotide were then mixed in equal volumes and then incubated for 45 min at 4°C.
  • PCR mix contains the three pairs of PCR primers and three specific “TaqMan” probes for each target: oligonucleotide of sequence SEQ ID NO: 1, miR-34a and miR-40 (internal process control), the master mix and the sterile water. 15 ⁇ l of this PCR mix and 5 ⁇ L of cDNA were added to each well. Each positive control was also respectively added to a well.
  • the PCR program was then launched on the CFX96TM IVD Real-Time PCR Systems thermocycler (Bio-rad, reference 185-5095 IVD) using the following program: 1 cycle of 10 minutes at 95°C, 50 cycles of 15 seconds at 95°C and 1 minute at 60°C. Calculation of NASH score The NASH score was calculated by using the method NIS2+ described above. Results 1. Comparison between conventional method and the method of the invention In order to verify the accuracy of the method of the invention for measuring the levels of hsa- miR-34a-5p and YKL-40, three different sources of data for these biomarkers were compared between each two of them.
  • Results are summarized in Table 2 and graphically shown in Figure 3.
  • ANOVA modelling was performed by hypothesizing that all three sets of measures have been obtained from the same serum sample, at the same time. In that way, ANOVA modelling was performed by hypothesizing that the only source of difference would be the method used, which is not exactly true and could explain why significance was reached here while we can observe that distributions were quite similar. Besides of this consideration, we observed that all three distributions were similar with means values of -0.138, -0.162 and - 0.214.
  • NIS2+ score calculations In order to further evaluate the accuracy of miPCR values, in particular the corrected miPCR values, NIS2+ scores calculated respectively from corrected miPCR BMs values, uncorrected miPCR BMs values and the BMs values obtained by the standard techniques were compared. The corresponding plots are graphically shown in Figure 10.
  • an ANOVA modelling on repeated measures was performed.
  • Results are summarized in Table 6 and graphically shown on Figure 11.

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Abstract

La présente invention concerne un kit pour effectuer une immuno-PCR multiplex. Les produits, compositions, kits et procédés mettant en oeuvre lesdits kits peuvent être avantageusement utilisés pour le diagnostic de la stéatohépatite non alcoolique (NASH).
PCT/EP2024/065375 2023-06-06 2024-06-05 Kits et procédés pour immuno-pcr multiplex Pending WO2024251754A1 (fr)

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