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WO2018046738A1 - Procédés de prédiction du temps de survie de patients souffrant d'un cancer - Google Patents

Procédés de prédiction du temps de survie de patients souffrant d'un cancer Download PDF

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Publication number
WO2018046738A1
WO2018046738A1 PCT/EP2017/072780 EP2017072780W WO2018046738A1 WO 2018046738 A1 WO2018046738 A1 WO 2018046738A1 EP 2017072780 W EP2017072780 W EP 2017072780W WO 2018046738 A1 WO2018046738 A1 WO 2018046738A1
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malignant
carcinoma
cell
tumor
melanoma
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PCT/EP2017/072780
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English (en)
Inventor
Bruno SEGUI
Nicolas Meyer
Thierry Levade
Nathalie ANDRIEU-ABADIE
Florie BERTRAND
Céline COLACIOS VIATGÉ
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paul Sabatier Toulouse Iii
Centre Hospitalier Universitaire De Toulouse
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Publication of WO2018046738A1 publication Critical patent/WO2018046738A1/fr

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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
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/158—Expression markers
    • 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

  • the present invention relates to methods for predicting the survival time of patients suffering from cancer.
  • Sphingolipids act as bioactive molecules in various signaling pathways, modulating cell growth, differentiation, migration and death as well as cancer progression 1 4 .
  • Our group has recently documented some alterations of SL metabolism in cancer, including melanoma 5"8 .
  • Ceramide an anti-oncometabolite can be generated by sphingomyelin (SM) hydrolysis, as a consequence of sphingomyelinase (SMase) activation 9 ' 10 .
  • SMases Several SMases have been described so far including neutral, alkaline and acid SMases u .
  • neutral SMase 2 (nSMase2), which is encoded by SMPD3 12 , is activated by diverse stimuli, including pro-inflammatory cytokines, such as IL- ⁇ 13 and TNFa 14 ⁇ 16 .
  • pro-inflammatory cytokines such as IL- ⁇ 13 and TNFa 14 ⁇ 16 .
  • the role of nSMase2 in oncoimmunology is currently unknown.
  • nSMase2 is involved in exosome budding into multivesicular endosome 17 .
  • NSMase2 expression in breast cancer cell lines enhances the secretion of exosomes, which contain diverse microRNAs (miRNA) including miR-210 18 , miR-lOb 19 and miR-21 20 .
  • miRNA microRNAs
  • NSMase2 is also likely involved in cellular export of miRNAs to HDLs 21 .
  • miR-155 has been identified as a major pro-inflammatory miRNA required for CD8 T cell-dependent immune response 22 ' 23
  • the relationship between miR-155 and nSMase2 remains to be evaluated.
  • the role of nSMase2 in melanoma is currently unknown. Melanoma is a highly immunogenic cancer, the progression of which is likely associated with immune escape mechanisms.
  • Monoclonal antibodies inhibiting immune checkpoints such as CTLA-4 (ipilimumab) and PD-1 (nivolumab, pembrolizumab) have demonstrated significant efficacy in the treatment of metastatic melanoma, promoting high response rate and long-lasting tumor control.
  • CTLA-4 ipilimumab
  • PD-1 nivolumab, pembrolizumab
  • SL metabolism changes in melanoma on the resistance to immune checkpoint inhibitors have not yet been investigated.
  • the biological function of nSMase2 in adaptive immune response towards cancer cells remains unknown.
  • the present invention relates to methods for predicting the survival time of patients suffering from cancer.
  • the present invention is defined by the claims.
  • the first object of the present invention relates to a method for predicting the survival of a patient suffering from cancer comprising i) determining the expression level of miR-155 in a tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) and concluding that the patient will have short survival time when the level determined at step i) is lower than the predetermined reference value or concluding that the patient will have long survival time when the expression level determined at step i) is higher than the predetermined reference value.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • Ewing's sarcoma odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin'
  • the subject suffers from melanoma.
  • melanoma refers to a condition characterized by the growth of a tumor arising from the melanocytic system of the skin and other organs. Most melanocytes occur in the skin, but are also found in the meninges, digestive tract, lymph nodes and eyes. When melanoma occurs in the skin, it is referred to as cutaneous melanoma. Melanoma can also occur in the eyes and is called ocular or intraocular melanoma. Melanoma occurs rarely in the meninges, the digestive tract, lymph nodes or other areas where melanocytes are found.
  • BRAF serine-threonine protein kinase B-RAF
  • OS survival time is generally based on and expressed as the percentage of people who survive a certain type of cancer for a specific amount of time. Cancer statistics often use an overall five-year survival rate. In general, OS rates do not specify whether cancer survivors are still undergoing treatment at five years or if they've become cancer-free (achieved remission). DSF gives more specific information and is the number of people with a particular cancer who achieve remission.
  • progression-free survival (PFS) rates (the number of people who still have cancer, but their disease does not progress) includes people who may have had some success with treatment, but the cancer has not disappeared completely.
  • short survival time indicates that the patient will have a survival time that will be lower than the median (or mean) observed in the general population of patients suffering from said cancer.
  • long survival time indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from said cancer.
  • the patient will have a long survival time it is meant that the patient will have a "good prognosis”.
  • sample refers to any sample obtained from the subject for the purpose of performing the method of the present invention.
  • the sample is a bodily fluid (e.g. a blood sample), a population of cells, or a tissue. Examples of such bodily fluids include, blood, saliva, tears, semen, vaginal discharge, pus, mucous, urine and feces.
  • the sample is a blood sample.
  • blood sample refers to a whole blood sample, serum sample and plasma sample.
  • a blood sample may be obtained by methods known in the art including venipuncture or a finger stick. Serum and plasma samples may be obtained by centrifugation methods known in the art.
  • the sample may be diluted with a suitable buffer before conducting the assay.
  • the sample is a tissue sample.
  • the sample is a tumor tissue sample.
  • tumor tissue sample has its general meaning in the art and encompasses pieces or slices of tissue that have been removed including following a surgical tumor resection.
  • the sample can be subjected to a variety of well-known post- collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.) prior to determining the cell densities.
  • the tumor tissue sample is fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a block which is readily cut.
  • TMA tissue microarrays
  • TMA consists of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis. This technology allows rapid visualization of molecular targets in tissue specimens at a time, either at the DNA, RNA or protein level. TMA technology is described in WO2004000992, US8068988, Olli et al 2001 Human Molecular Genetics, Tzankov et al 2005, Elsevier; Kononen et al 1198; Nature Medicine.
  • miR As used herein, the term "miR” or 'miRNA” has its general meaning in the art and refers to the miRNA sequence publicly available from the data base http://microrna.sanger.ac.uk/sequences/ under the miRBase Accession number. miR-155 pertaining to the invention is thus known per se.
  • measuring the expression level of miR-155 in the sample obtained from the subject can be performed by a variety of techniques.
  • the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • RNA isolation kit (Roche), Trizol (Invitrogen), Guanidinium thiocyanate-phenol-chloroform extraction, PureLinkTM miRNA isolation kit (Invitrogen), PureLink Micro-to- Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol- chloroform extraction, TCA/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, PureYieldTM RNA Midiprep (Promega), PolyATtract System 1000 (Promega), Maxwell® 16 System (Promega), SV Total RNA Isolation (Promega), geneMAG-RNA / DNA kit (Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNATM Kit (Ambion), Poly
  • the expression level of miR-155 in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of miRNA in a sample may be used.
  • miRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms).
  • Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy.
  • RNA is converted to DNA (cDNA) prior to analysis.
  • cDNA can be generated by reverse transcription of isolated miRNA using conventional techniques.
  • miRNA reverse transcription kits are known and commercially available. Examples of suitable kits include, but are not limited to the mirVana TaqMan® miRNA transcription kit (Ambion, Austin, TX), and the TaqMan® miRNA transcription kit (Applied Biosystems, Foster City, CA). Universal primers, or specific primers, including miRNA- specific stem- loop primers, are known and commercially available, for example, from Applied Biosystems.
  • miRNA is amplified prior to measurement. In some embodiments, the expression level of miRNA is measured during the amplification process.
  • the expression level of miRNA is not amplified prior to measurement.
  • Some exemplary methods suitable for determining the expression level of miRNA in a sample are described in greater hereinafter. These methods are provided by way of illustration only, and it will be apparent to a skilled person that other suitable methods may likewise be used.
  • amplification-based methods exist for detecting the expression level of miRNA nucleic acid sequences, including, but not limited to, PCR, RT-PCR, qPCR, and rolling circle amplification.
  • Other amplification-based techniques include, for example, ligase chain reaction (LCR), multiplex ligatable probe amplification, in vitro transcription (IVT), strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art.
  • LCR ligase chain reaction
  • IVTT in vitro transcription
  • SDA strand displacement amplification
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence based amplification
  • RNA (Eberwine) amplification and other methods that are known to persons skilled in the art.
  • a typical PCR reaction includes multiple steps, or cycles, that selectively amplify target nucleic acid species: a denaturing step, in which a target nucleic acid is denatured; an annealing step, in which a set of PCR primers (i.e., forward and reverse primers) anneal to complementary DNA strands, and an elongation step, in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target sequence.
  • Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation.
  • a reverse transcription reaction (which produces a cDNA sequence having complementarity to a miRNA) may be performed prior to PCR amplification.
  • Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.
  • Kits for quantitative real time PCR of miRNA are known, and are commercially available. Examples of suitable kits include, but are not limited to, the TaqMan® miRNA Assay (Applied Biosystems) and the mirVanaTM qRT- PCR miRNA detection kit (Ambion).
  • the miRNA can be ligated to a single stranded oligonucleotide containing universal primer sequences, a polyadenylated sequence, or adaptor sequence prior to reverse transcriptase and amplified using a primer complementary to the universal primer sequence, poly(T) primer, or primer comprising a sequence that is complementary to the adaptor sequence.
  • custom qRT-PCR assays can be developed for determination of miRNA levels. Custom qRT-PCR assays to measure miRNAs in a sample can be developed using, for example, methods that involve an extended reverse transcription primer and locked nucleic acid modified PCR.
  • Custom miRNA assays can be tested by running the assay on a dilution series of chemically synthesized miRNA corresponding to the target sequence. This permits determination of the limit of detection and linear range of quantitation of each assay. Furthermore, when used as a standard curve, these data permit an estimate of the absolute abundance of miRNAs measured in the samples. Amplification curves may optionally be checked to verify that Ct values are assessed in the linear range of each amplification plot. Typically, the linear range spans several orders of magnitude. For each candidate miRNA assayed, a chemically synthesized version of the miRNA can be obtained and analyzed in a dilution series to determine the limit of sensitivity of the assay, and the linear range of quantitation.
  • Relative expression levels may be determined, for example, according to the 2(- ⁇ C(T)) Method, as described by Livak et ah, Analysis of relative gene expression data using real-time quantitative PCR and the 2(- ⁇ C(T)) Method. Methods (2001) Dec;25(4):402-8.
  • Rolling circle amplification is a DNA-polymerase driven reaction that can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (see, for example, Lizardi et al, Nat. Gen. (1998) 19(3):225-232; Gusev et al, Am. J. Pathol. (2001) 159(l):63-69; Nallur et al, Nucleic Acids Res. (2001) 29(23):E118).
  • a complex pattern of strand displacement results in the generation of over 109 copies of each DNA molecule in 90 minutes or less.
  • Tandemly linked copies of a closed circle DNA molecule may be formed by using a single primer. The process can also be performed using a matrix- associated DNA. The template used for rolling circle amplification may be reverse transcribed. This method can be used as a highly sensitive indicator of miRNA sequence and expression level at very low miRNA concentrations (see, for example, Cheng et al, Angew Chem. Int. Ed. Engl. (2009) 48(18):3268-72; Neubacher et al, Chembiochem. (2009) 10(8): 1289-91).
  • miRNA quantification may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe.
  • said method comprises a first step wherein the stem-loop primers are annealed to miRNA targets and extended in the presence of reverse transcriptase. Then miRNA-specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of miRNAs is estimated based on measured CT values. Many miRNA quantification assays are commercially available from Qiagen (S. A. Courtaboeuf, France), Exiqon (Vedbaek, Denmark) or Applied Biosystems (Foster City, USA).
  • nucleic acids exhibiting sequence complementarity or homology to the miRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin). The probes and primers are "specific" to the miRNAs they hybridize to, i.e.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • miRNA may be detected using hybridization-based methods, including but not limited to hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, and in situ hybridization.
  • hybridization arrays e.g., microarrays
  • NanoString analysis e.g., NanoString analysis
  • Northern Blot analysis e.g., Northern Blot analysis
  • branched DNA (bDNA) signal amplification e.g., branched DNA (bDNA) signal amplification
  • in situ hybridization e.g., in situ hybridization.
  • Microarrays can be used to measure the expression levels of large numbers of miRNAs simultaneously.
  • Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre- made masks, photolithography using dynamic micromirror devices, inkjet printing, or electrochemistry on microelectrode arrays.
  • micro fluidic TaqMan Low-Density Arrays which are based on an array of microfluidic qRT-PCR reactions, as well as related micro fluidic qRT-PCR based methods.
  • oligonucleotides e.g., 200+ 5'- amino- modified-C6 oligos
  • human sense miRNA sequences are spotted on three- dimensional CodeLink slides (GE Health/ Amersham Biosciences) at a final concentration of about 20 ⁇ and processed according to manufacturer's recommendations.
  • First strand cDNA synthesized from 20 ⁇ g TRIzol-purified total RNA is labeled with biotinylated ddUTP using the Enzo BioArray end labeling kit (Enzo Life Sciences Inc.).
  • Hybridization, staining, and washing can be performed according to a modified Affymetrix Antisense genome array protocol.
  • Axon B-4000 scanner and Gene-Pix Pro 4.0 software or other suitable software can be used to scan images. Non-positive spots after background subtraction, and outliers detected by the ESD procedure, are removed. The resulting signal intensity values are normalized to per-chip median values and then used to obtain geometric means and standard errors for each miRNA. Each miRNA signal can be transformed to log base 2, and a one-sample t test can be conducted. Independent hybridizations for each sample can be performed on chips with each miRNA spotted multiple times to increase the robustness of the data.
  • Microarrays can be used for the expression profiling of miRNAs.
  • RNA can be extracted from the sample and, optionally, the miRNAs are size- selected from total RNA.
  • Oligonucleotide linkers can be attached to the 5' and 3' ends of the miRNAs and the resulting ligation products are used as templates for an RT-PCR reaction.
  • the sense strand PCR primer can have a fluorophore attached to its 5' end, thereby labeling the sense strand of the PCR product.
  • the PCR product is denatured and then hybridized to the microarray.
  • a PCR product referred to as the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed.
  • the spot will then fluoresce when excited using a microarray laser scanner.
  • the fiuorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA.
  • Total RNA containing the miRNA extracted from the sample can also be used directly without size-selection of the miRNAs.
  • the RNA can be 3' end labeled using T4 RNA ligase and a fluorophore-labeled short RNA linker.
  • Fluorophore-labeled miRNAs complementary to the corresponding miRNA capture probe sequences on the array hybridize, via base pairing, to the spot at which the capture probes are affixed.
  • the fiuorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA.
  • microarrays can be employed including, but not limited to, spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays or spotted long oligonucleotide arrays.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a "detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook- A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315- 22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016- 2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos.
  • quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, Calif).
  • thermocyclers typically involve a format of glass capillaries, plastics tubes, 96-well plates or 384-well plates.
  • the thermocylcer also involves software analysis.
  • miRNAs can also be detected without amplification using the nCounter Analysis
  • nCounter miRNA assay kits are available from NanoString Technologies, which are capable of distinguishing between highly similar miRNAs with great specificity. The basis of the nCounter® Analysis system is the unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO 08/124847, U.S. Patent No. 8,415,102 and Geiss et al. Nature Biotechnology. 2008.
  • the code is composed of an ordered series of colored fluorescent spots which create a unique barcode for each target to be assayed.
  • a pair of probes is designed for each oligonucleotide target, a biotinylated capture probe and a reporter probe carrying the fluorescent barcode. This system is also referred to, herein, as the nanoreporter code system. Specific reporter and capture probes are synthesized for each target.
  • the reporter probe can comprise at a least a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; at least a second label attachment region, which is non-over-lapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and a first target- specific sequence.
  • each sequence specific reporter probe comprises a target specific sequence capable of hybridizing to no more than one gene and optionally comprises at least three, or at least four label attachment regions, said attachment regions comprising one or more label monomers that emit light, constituting at least a third signal, or at least a fourth signal, respectively.
  • the capture probe can comprise a second target-specific sequence; and a first affinity tag.
  • the capture probe can also comprise one or more label attachment regions.
  • the first target-specific sequence of the reporter probe and the second target- specific sequence of the capture probe hybridize to different regions of the same gene to be detected.
  • Reporter and capture probes are all pooled into a single hybridization mixture, the "probe library".
  • the relative abundance of each target is measured in a single multiplexed hybridization reaction.
  • the method comprises contacting the sample with a probe library, such that the presence of the target in the sample creates a probe pair - target complex.
  • the complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution.
  • the tripartite hybridized complexes (probe pairs and target) are purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to universal sequences present on the capture and reporter probes.
  • This dual purification process allows the hybridization reaction to be driven to completion with a large excess of target-specific probes, as they are ultimately removed, and, thus, do not interfere with binding and imaging of the sample. All post hybridization steps are handled robotically on a custom liquid-handling robot (Prep Station, NanoString Technologies). Purified reactions are typically deposited by the Prep Station into individual flow cells of a sample cartridge, bound to a streptavidin-coated surface via the capture probe, electrophoresed to elongate the reporter probes, and immobilized.
  • the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technologies).
  • the expression level of a target is measured by imaging each sample and counting the number of times the code for that target is detected. For each sample, typically 600 fields-of-view (FOV) are imaged (1376 X 1024 pixels) representing approximately 10 mm2 of the binding surface.
  • Typical imaging density is 100- 1200 counted reporters per field of view depending on the degree of multiplexing, the amount of sample input, and overall target abundance.
  • Data is output in simple spreadsheet format listing the number of counts per target, per sample. This system can be used along with nanoreporters. Additional disclosure regarding nanoreporters can be found in International Publication No.
  • nucleic acid probes and nanoreporters can include the rationally designed (e.g. synthetic sequences) described in International Publication No. WO 2010/019826 and US Patent Publication No. 2010/0047924, incorporated herein by reference in its entirety.
  • RNA endonucleases RNases
  • MS/MS tandem MS
  • the first approach developed utilized the on-line chromatographic separation of endonuclease digests by reversed phase HPLC coupled directly to ESTMS. The presence of post-transcriptional modifications can be revealed by mass shifts from those expected based upon the RNA sequence.
  • MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry
  • MALDI-MS has also been used as an analytical approach for obtaining information about post- transcriptionally modified nucleosides.
  • MALDI-based approaches can be differentiated from EST-based approaches by the separation step.
  • the mass spectrometer is used to separate the miRNA.
  • a system of capillary LC coupled with nanoESI-MS can be employed, by using a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) or a tandem-quadrupole time-of- flight mass spectrometer (QSTAR® XL, Applied Biosystems) equipped with a custom-made nanospray ion source, a Nanovolume Valve (Valco Instruments), and a splitless nano HPLC system (DiNa, KYA Technologies). Analyte/TEAA is loaded onto a nano-LC trap column, desalted, and then concentrated.
  • LTQ Orbitrap XL linear ion trap-orbitrap hybrid mass spectrometer
  • QSTAR® XL tandem-quadrupole time-of- flight mass spectrometer
  • Analyte/TEAA is loaded onto a nano-LC trap column, desalted, and then concentrated.
  • Intact miRNAs are eluted from the trap column and directly injected into a CI 8 capillary column, and chromatographed by RP-HPLC using a gradient of solvents of increasing polarity.
  • the chromatographic eluent is sprayed from a sprayer tip attached to the capillary column, using an ionization voltage that allows ions to be scanned in the negative polarity mode.
  • miRNA detection and measurement include, for example, strand invasion assay (Third Wave Technologies, Inc.), surface plasmon resonance (SPR), cDNA, MTDNA (metallic DNA; Advance Technologies, Saskatoon, SK), and single- molecule methods such as the one developed by US Genomics.
  • Multiple miRNAs can be detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle- amplified SPR imaging (SPRI).
  • SPRI nanoparticle- amplified SPR imaging
  • the surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI.
  • This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attamole levels.
  • miRNAs can also be detected using branched DNA (bDNA) signal amplification (see, for example, Urdea, Nature Biotechnology (1994), 12:926- 928).
  • miRNA assays based on bDNA signal amplification are commercially available.
  • One such assay is the QuantiGene® 2.0 miRNA Assay (Affymetrix, Santa Clara, CA).
  • Northern Blot and in situ hybridization may also be used to detect miRNAs. Suitable methods for performing Northern Blot and in situ hybridization are known in the art. Advanced sequencing methods can likewise be used as available.
  • miRNAs can be detected using Illumina ® Next Generation Sequencing (e.g.
  • the expression level of miR-155 may be expressed as absolute expression levels or normalized expression levels. Typically, expression levels are normalized by correcting the absolute expression level of miRNAs by comparing its expression to the expression of a mRNA that is not a relevant marker for determining whether a subject suffering from acute severe colitis (ASC) will be a responder or a non-responder to a corticosteroid, infliximab and cyclosporine, e.g., a housekeeping mRNA that is constitutively expressed. Suitable mRNAs for normalization include housekeeping mRNAs such as the U6, U24, U48 and SI 8.
  • expression levels are normalized by correcting the absolute expression level of miRNAs by comparing its expression to the expression of a reference mRNA.
  • the predetermined reference value is a threshold value or a cutoff value.
  • a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of expression level of the gene in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • receiver operator characteristic curve which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate.
  • the predetermined reference value is determined by carrying out a method comprising the steps of a) providing a collection of samples; b) providing, for each ample provided at step a), information relating to the actual clinical outcome for the corresponding subject (i.e.
  • the expression level of the gene has been assessed for 100 samples of 100 subjects.
  • the 100 samples are ranked according to the expression level of the gene.
  • Sample 1 has the highest level and sample 100 has the lowest level.
  • a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
  • the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
  • Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
  • the predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest.
  • the expression level of the gene corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value.
  • the predetermined reference value is not necessarily the median value of expression levels of the gene.
  • the predetermined reference value thus allows discrimination between a poor and a good prognosis for a subject.
  • high statistical significance values e.g. low P values
  • a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g.
  • a range of quantification values includes a "cut-off value as described above.
  • the outcome can be determined by comparing the expression level of the gene with the range of values which are identified.
  • a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found).
  • a suitable (exemplary) range may be from 4-6.
  • a subject may be assessed by comparing values obtained by measuring the expression level of the gene, where values higher than 5 reveal a poor prognosis and values less than 5 reveal a good prognosis.
  • a subject may be assessed by comparing values obtained by measuring the expression level of the gene and comparing the values on a scale, where values above the range of 4-6 indicate a poor prognosis and values below the range of 4-6 indicate a good prognosis, with values falling within the range of 4-6 indicating an intermediate occurrence (or prognosis).
  • the method of the present invention is also suitable for determining whether a patient is eligible or not to a treatment with an immune checkpoint inhibitor.
  • the second object of the present invention relates to a method of treating cancer in patient in need thereof comprising i) determining the expression level of miR-155 in a tumor sample obtained from the patient ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering to the patient a therapeutically effective amount of an immune checkpoint inhibitor.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • immune checkpoint protein has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).
  • Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489).
  • inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA.
  • Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because the tumor microenvironment has relatively high levels of adenosine, which lead to a negative immune feedback loop through the activation of A2AR.
  • B7-H4, also called VTCN1 is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape.
  • B and T Lymphocyte Attenuator (BTLA), also called CD272 is a ligand of HVEM (Herpesvirus Entry Mediator) .
  • BTLA tumor-specific human CD8+ T cells express high levels of BTLA.
  • CTLA-4 Cytotoxic T -Lymphocyte- Associated protein 4 and also called CD 152 is overexpressed on Treg cells serves to control T cell proliferation.
  • IDO Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme, a related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis.
  • KIR Killer-cell Immunoglobulin- like Receptor
  • LAG3, Lymphocyte Activation Gene-3 works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells.
  • PD-1 Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014.
  • An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.
  • TIM-3 short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines.
  • TIM-3 acts as a negative regulator of Thl /Tel function by triggering cell death upon interaction with its ligand, galectin-9.
  • VISTA Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors.
  • immune checkpoint inhibitor has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade.
  • Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future.
  • the immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules.
  • CD8+ T cells has its general meaning in the art and refers to a subset of T cells, which express CD8 on their surface. They are MHC class I-restricted, and function as cytotoxic T cells. "CD8+ T cells” are also called called cytotoxic T lymphocytes (CTL), T-killer cells, cytolytic T cells, or killer T cells.
  • CTL cytotoxic T lymphocytes
  • CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.
  • the ability of the immune checkpoint inhibitor to enhance T CD8 cell killing activity may be determined by any assay well known in the art.
  • said assay is an in vitro assay wherein CD8+ T cells are brought into contact with target cells (e.g. target cells that are recognized and/or lysed by CD8+ T cells).
  • the immune checkpoint inhibitor of the present invention can be selected for the ability to increase specific lysis by CD8+ T cells by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%, or more of the specific lysis obtained at the same effector: target cell ratio with CD8+ T cells or CD8 T cell lines that are contacted by the immune checkpoint inhibitor of the present invention.
  • Examples of protocols for classical cytotoxicity assays are conventional.
  • the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
  • the antibody is a humanized antibody. In some embodiments, the antibody is a fully human antibody.In some embodiments, the antibody comprises human heavy chain constant regions sequences but will not deplete CD8+ T cells to which they are bound and preferably do not comprise an Fc portion that induces antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody of the present invention does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD 16) polypeptide. In some embodiments, the antibody of the present invention lacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the antibody of the present invention is not linked to a toxic moiety.
  • ADCC antibody dependent cellular cytotoxicity
  • anti-CTLA-4 antibodies examples include anti-CTLA-4 antibodies.
  • One anti- CTLA-4 antibody is tremelimumab, (ticilimumab, CP-675,206).
  • the anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonal IgG antibody that binds to CTLA-4.
  • PD-1 and PD-L1 antibodies are described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.
  • the PD-1 blockers include anti-PD-Ll antibodies.
  • the PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1 ; CT-011 a humanized antibody that binds PD-1 ; AMP -224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX- 1105-01) for PD-L1 (B7-H1) blockade.
  • nivolumab MDX 1106, BMS 936558, ONO 4538
  • a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2
  • immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al, 2007, J. Immunol. 179:4202-4211).
  • Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors.
  • the anti-B7-H3 antibody MGA271 (Loo et al, 2012, Clin. Cancer Res. July 15 (18) 3834).
  • TIM3 T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al, 2010, J.
  • TIM-3 has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3.
  • the natural ligand of TIM-3 is galectin 9 (Gal9).
  • TIM-3 inhibitor refers to a compound, substance or composition that can inhibit the function of TIM-3.
  • the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and/or block the binding of TIM-3 to galectin-9.
  • Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO2011155607, WO2013006490 and WO2010117057.
  • the immune checkpoint inhibitor is an IDO inhibitor.
  • IDO inhibitors are described in WO 2014150677.
  • IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), ⁇ - (3-benzofuranyl)-alanine, ⁇ -(3- benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 - methyl tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3- diacetate, 9- vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine
  • the IDO inhibitor is selected from 1-methyl-tryptophan, ⁇ -(3- benzofuranyl)-alanine, 6-nitro-L- tryptophan, 3-Amino-naphtoic acid and ⁇ -[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.
  • a “therapeutically effective amount” is meant a sufficient amount of the immune checkpoint inhibitor for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the immune checkpoint inhibitor is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as pharmaceutically acceptable polymers
  • sustained-release matrices such as biodegradable polymers
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 nSMase2 enhances exosomal secretion of miR-155 from melanoma cells, increasing melanoma cell-derived exosome immunogenicity.
  • A Correlation analysis of SMPD3 and MIR155HG expression in human melanoma samples.
  • B Analysis of overall survival in metastatic melanoma patients from the TCGA melanoma cohort, exhibiting high (80th percentile) and low (20th percentile) MIR155HG expression in melanoma samples.
  • R A was isolated from B16K1 cells transduced with a retroviral vector encoding wild-type (WT) or catalytically-inactive (C.I.) nSMase2 as well as from secreted exosomes produced by each cell line (Exo.), and miR As were analysed by using RT-qPCR. Data are means ⁇ sem of 4 independent experiments carried out on 4 independent exosome preparations.
  • D Bone marrow-derived DC were incubated with or without 10 ⁇ g/mL exosomes from B16K1 expressing either WT or C.I nSMase2. After 24h, CXCL9, IL-12 and SOCS1 transcripts were analyzed by using RT-qPCR.
  • DC were transfected with (TSB) or without (Veh) a target site blocker that specifically interacts with the 3' untranslated region of SOCS1 mRNA, preventing miR-155 binding. 24 hours later, DC were incubated with the indicated exosomes and IL-12 and CXCL9 mRNA were analysed as described in D. Data are expressed as percentage of maximal response measured in mock-transfected DC incubated with exosomes from B16K1 expressing nSMase2 WT. Values are means ⁇ sem of three independent experiments.
  • Figure 4 Melanoma nSMase2, which triggers miR-155 exosomal secretion, enhances the response to anti-PD-1 therapy.
  • NSMase2 expression enhances CD8+ Tumor-Infiltrating Lymphocytes in melanoma.
  • nSMase2 is expressed at low levels in human metastatic melanoma as compared to primary tumors, suggesting that nSMase2 downregulation is likely associated with melanoma progression.
  • the clinical outcome in metastatic melanoma patients exhibiting high (80th percentile) and low (20th percentile) SMPD3 expression was next analysed.
  • Low SMPD3 expression was statistically associated with shortened overall survival, further argues that SMPD3 downregulation is associated with a bad prognosis in melanoma.
  • CD8A, CD8B and CD4 were enriched in melanoma samples expressing SMPD3 at high levels.
  • various Thl-related genes such as IFNG, TNF, CXCL9, CXCL10 and CCL5 as well as cell-mediated cytotoxicity genes were highly expressed in melanoma samples exhibiting high SMPD3 expression. Accordingly, SMPD3 expression was significantly correlated with the expression of diverse genes, which likely reflect CD8 T cell infiltration.
  • SMPD2 the expression of which poorly, yet significantly, correlated with that of CD8B.
  • SMPD4 was anti-correlated with T cell-related genes.
  • SMPD3 expression is associated with a signature of CD8+ T cell tumor infiltration in human melanoma samples, and this cannot be extended to the other sphingomyelinase isoforms.
  • SMPD3 downregulation contributes to melanoma immune escape and nSMase2 expression level is critical for the CD8+ T cell-dependent immune response towards melanoma.
  • B16K1 MHC-I hlgh
  • B16K1 melanoma cell lines overexpressing or not nSMase2. Overexpressed enzyme was mainly located at the plasma membrane and led to robust increase in intracellular neutral sphingomyelinase activity and ceramide level without affecting two- and three-dimensional cell growth in vitro.
  • mice grafted with B16K1 melanoma cells expressing nSMase2 at low or high levels Twelve days after B16K1 cell injection, the tumor content of leukocytes (CD45+) and T lymphocytes (Thyl+) was significantly enhanced in tumors that expressed nSMase2 at high levels.
  • the proportion of CD8+ TILs was 3- fold higher in tumors expressing nSMase2 at high levels.
  • nSMase2 overexpression as evaluated by western blot triggered an intra- tumor increase (i) in the nSMase activity, C16 and C24-ceramides and sphingosine and (ii) a reduction of B16K1 tumor growth in WT mice.
  • tumor sphingomyelin and SIP content were noticed for tumor sphingomyelin and SIP content in nSMase2 overexpressing tumors.
  • a similar trend was observed in B16F10 melanoma cells in which nSMase2 overexpression significantly reduced tumorigenesis in WT mice without affecting their proliferation rate in vitro.
  • nSMase2 overexpression failed to impair B16K1 melanoma growth in CD8- deficient mice.
  • nSMase2 enhances the exosomal secretion of miR-155 by melanoma cells.
  • NSMase2 has been shown to facilitate the budding of exosomes 11 , which likely contribute to the modulation of the anti-melanoma immune response 25 .
  • nSMase2 has been involved in the exosomal secretion of microRNA (miRNA) 18 ⁇ 20 .
  • miRNA microRNA
  • the expression of SMPD3 in human melanoma samples was correlated with that of miR155HG, which encodes miR-155 (Fig. 1A), a major pro-inflammatory miRNA required for CD8 T cell-dependent immune response 22 ' 23 , and the expression of which translates into improved survival in metastatic melanoma (Fig. IB).
  • nSMase2 overexpression did not increase the intracellular content of miR-155 in B16K1 cells (Fig. 1C).
  • WT WT
  • catalytically-inactive nSMase2 expressing cells The total amount of secreted exosomes, their ultra-structural morphology and content in exosome markers (CD63, TSG101) and melanoma antigens (MC1R, TRP2), were similar for both exosome types.
  • exosomes from B16K1 expressing WT nSMase2 greatly enhanced the intracellular levels of mRNA encoding CXCL9, a chemokine facilitating T cell tumor infiltration, and IL-12, both of which being induced by IFNy (Fig. ID).
  • This phenomenon was anti-correlated with the expression of mRNA encoding SOCSl, which is a major cytokine signaling repressor and a well-established miR-155 target 27 (Fig. ID).
  • TAB SOCSl target site blocker
  • TSB SOCSl transfection prevented the increase of mRNA encoding IL-12 and CXCL9 mRNA in response to exosomes derived from B16K1 expressing WT nSMase2 (Fig. IE).
  • exosomal miR-155 which is enriched upon WT nSMase2 overexpression, is biologically active and facilitates the expression of Thl cytokines by DC.
  • nSMase2 enhances the exosomal secretion of miR- 155, thereby increasing the immunogenicity of exosomes produced by melanoma cells, which likely contributes to the establishment of a beneficial CD8+ T cell-dependent immune response.
  • MIR155HG expression in human melanoma samples was correlated with various Thl -related genes such as IFNG, CXCL9, CXCLIO and CCL5 as well as cell-mediated cytotoxicity genes (Fig. 2A).
  • MIR155HG expression was significantly correlated with CD8A and CD8B, which reflect CD8 T cell infiltration (Fig. 2B).
  • Pre-existing CD8+ TILs that are negatively regulated by the immune checkpoint PD-1 are predictive of the response to anti-PD-1 in melanoma patients 28 .
  • SMPD3 and miR-155 levels were also correlated with immunosuppressive genes, such as PDCD1, which encodes the immune checkpoint PD-1, and PDCD1LG1 as well as PDCD1LG2, encoding the PD-1 ligands PD-L1 and PD-L2, respectively (Fig. 3).
  • PDCD1 which encodes the immune checkpoint PD-1
  • PDCD1LG1 encoding the PD-1 ligands PD-L1 and PD-L2
  • nSMase2 significantly delayed nSMase2 low and melanoma growth
  • all tumors relapsed presumably due to immune escape mechanisms and, consequently, all mice died within 40 days post-B16Kl injection.
  • nSMase2 overexpression delayed melanoma growth and slightly, yet significantly, increased the overall survival.
  • the therapeutic efficacy of anti-PD-1 was dramatically enhanced by nSMase2 overexpression (Fig. 4).
  • mice injected with B16K1 nSMase2 hlgh and anti-PD-1 all mice survived and none of them developed melanoma upon a second B16K1 cell injection, indicating that they were fully vaccinated against melanoma cells.
  • Our observation was unlikely restricted to anti-PD-1 since WT nSMase2 expression greatly enhanced the therapeutic effect of anti-CTLA-4 blocking antibodies against melanoma.
  • melanoma nSMase2 enhanced immunogenicity of melanoma cell-derived exosomes. This tenet is supported by the following findings: (i) WT nSMase2 increased the exosomal secretion of miR-155, which is required for CD8+ T cell-dependent immune response 23 ; (ii) the uptake of exosomes derived from WT nSMase2 expressing cells by DC was accompanied by the silencing of SOCS1 mRNA (a miR-155 target) 27 , and, consequently, a concomitant augmentation of IL-12 and CXCL9 transcripts, and (iii) the increased CD8+ TIL content in melanoma tumors upon injection of exosomes derived from B16K1 expressing WT nSMase2.
  • SMPD3 and MIR155HG expression in melanoma samples from metastatic patients was correlated not only with a CD8 T cell-dependent immune gene signature but also with genes encoding immune checkpoints such as PD-1, presumably leading to melanoma immune escape.
  • PD-1 expression which reflects T cell activation, constitutes a key negative immune checkpoint, inducing T cell apoptosis and/or exhaustion, thus promoting tumor progression 29 .
  • CD4+PD-1+ and CD8+PD-1+ TILs in mouse melanoma tumors, which overexpressed nSMase2.
  • targeting SL metabolism may constitute an original therapeutic strategy to enhance the exosomal secretion of miR-155 and overcome resistance of melanoma to anti- PD-1.
  • SMPD3 and MIR155HG expression in melanoma samples as well as circulating exosomes containing miR-155 may serve as novel biomarkers to predict response to immunotherapy.
  • Neutral sphingomyelinase 2 (nSMase2) is the primary neutral sphingomyelinase isoform activated by tumour necrosis factor-alpha in MCF-7 cells. Biochem J 435, 381-390 (2011).

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Abstract

La présente invention concerne des procédés permettant de prédire le temps de survie de patients souffrant d'un cancer. La sphingomyélinase 2 neutre (nSMase2), codée par SMPD3, catalyse la dégradation de la sphingomyéline en céramide. Les inventeurs ont montré que SMPD3 est fréquemment régulé négativement dans le mélanome métastatique humain, ce qui contribue probablement à des mécanismes d'échappement immunitaires et à une survie globale raccourcie. De manière mécanistique, ils ont découvert que nSMase2 augmente l'immunogénicité des exosomes dérivés de cellules de mélanome, en augmentant leur association avec miR-155, un ARNmi pro-inflammatoire majeur. En particulier, la présente invention concerne un procédé de prédiction de la survie d'un patient souffrant d'un cancer, comprenant la détermination du niveau d'expression de miR-155 dans un échantillon de tumeur d'un patient.
PCT/EP2017/072780 2016-09-12 2017-09-11 Procédés de prédiction du temps de survie de patients souffrant d'un cancer WO2018046738A1 (fr)

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