WO2019038219A1 - Nouveau procédé de pronostic du cancer du pancréas - Google Patents

Nouveau procédé de pronostic du cancer du pancréas Download PDF

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Publication number
WO2019038219A1
WO2019038219A1 PCT/EP2018/072410 EP2018072410W WO2019038219A1 WO 2019038219 A1 WO2019038219 A1 WO 2019038219A1 EP 2018072410 W EP2018072410 W EP 2018072410W WO 2019038219 A1 WO2019038219 A1 WO 2019038219A1
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Prior art keywords
btn3a
expression level
cells
pancreatic cancer
pancreatic
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PCT/EP2018/072410
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English (en)
Inventor
Daniel Olive
Juan Iovanna
Jean-Charles Dagorn
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Institut Jean Paoli & Irene Calmettes
Centre National De La Recherche Scientifique (Cnrs)
Université D'aix Marseille
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Publication of WO2019038219A1 publication Critical patent/WO2019038219A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of sBTN3A ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.
  • the invention is defined by its claims.
  • Pancreatic Ductal Adenocarcinoma is an aggressive disease with extremely poor prognosis and a 5-year survival rate below 8 %.
  • Conventional treatments such as surgery, chemotherapy and radiotherapy have very limited impact underlying that novel therapeutic strategies are warranted.
  • Immune checkpoint-based immunotherapy strategies have shown their efficacy in the treatment of melanoma and non-small cell lung carcinoma and paved the way to explore immunotherapeutic strategies in poor prognosis solid tumors such as PDAC.
  • recent data support the prognosis value of both systemic and intra-tumoral immune activation including immune-check point molecules expression [Farren MR et al., 2016 and Diana A et al., 2016].
  • TME Tumor Micro-environment
  • ⁇ T cells are anti-tumor immune effector cells that reach to infiltrate the PDAC TME [Kitayama J et al., 1993], in closer proximity to ductal epithelium than what observed in chronic pancreatitis [Helm O et al., 2014].
  • ⁇ T cells encompass two major cytotoxic subtypes: V51 subtype is mainly intra-epithelial while Vy9V52 subtype predominates in blood, accounting for 1-10% of human circulating T lymphocytes.
  • Vy9V52 T cells can migrate to solid tumors and have been isolated among Tumor Infiltrating Lymphocytes (TILs) [Corvaisier M et al, 2005 and Cordova A et al, 2012].
  • TILs Tumor Infiltrating Lymphocytes
  • the Vy9V52 T Cell Receptor senses phosphoAntigens (pAgs) which are intermediary metabolites of the human mevalonate pathway.
  • pAgs phosphoAntigens
  • N-BP Aminobisphosphonates
  • synthetic pAg such as BrHpp and natural microbial pAg HMBPP are also potent TCR agonists.
  • Vy9V52 T cells like NK cells, also recognize malignant epithelial cells through activating receptors, such as DNAM-114 and NKG2D15.
  • NKG2D ligands are up-regulated upon cellular stress and soluble NKG2D Ligand (NKG2DL), such as soluble MICA, can be released by pancreatic tumors.
  • Soluble MICA has been evidenced in patients with pancreatic cancers and alters ⁇ T cell cytotoxicity in vitro.
  • ⁇ T cells isolated from TILs displayed high cytotoxicity against pancreatic tumors ex vivo.
  • the adoptive transfer of N-BP-activated Vy9V52 T cells from Healthy Donors (HD), combined with repeated infusion of IL-2 and N-BP in vivo increased the survival of PDAC line-xenograft mice.
  • BTN3A butyrophilin3A
  • BTN3A1 and BTN3A3 contain a B30.2 cytoplasmic domain.
  • BTN3A members are broadly expressed, notably by most immune cells and various tumors.
  • BTN3A is upregulated under TH1 stimulation and by certain TME factors and cytokines such as VEGF and IL-10.
  • BTN3A molecules are key players in pAg-sensing by Vy9V52 T cells [Harly C et al., 2012; Rhodes DA et al., 2015 and Sebestyen Z et 2016]. This mechanism was shown to involve the three BTN3A isoforms. Of note, B30.2 intracellular domain of BTN3A1 , that binds pAg, is a key determinant in this process.
  • the anti-BTN3A 20.1 agonist monoclonal antibody (mAb) mimics pAg-induced ⁇ T cell activation via similar extra-cellular conformational changes of BTN3A molecules.
  • anti-BTN3A 20.1 mAb can trigger separately each of the three isoforms and sensitize a broad range of tumors to Vy9V52 T cells lysis [Toutirais O et al, 2009 and Benyamine A et al., 2016], including resistant primary Acute Myeloid Leukemia (AML) blasts.
  • the inventors evaluated the effect of hypoxia and metabolic stress on the regulation of BTN3A isoforms using notably qRT PCR and Western Blotting and found an unexpected soluble form. They demonstrated a key role of BTN3A in Vy9V52 T cells cytolytic functions against PDAC that are conserved under hypoxia. Finally, they found that BTN3A expression in tissues and plasma level of soluble BTN3A and BTN3A1 are associated with poor prognosis in patients with PDAC.
  • the present invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of sBTN3A ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.
  • the invention is defined by its claims.
  • a first aspect of the invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of sBTN3A ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.
  • the invention also relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of sBTN3Al ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.
  • a second aspect of the invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTN3A ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.
  • the invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTN3A2 ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.
  • the sample can be a pancreatic tumor sample that is to say a sample obtained from the pancreatic tumor or a biopsy obtained from a pancreatic tumor.
  • a third aspect of the invention relates to a method for predicting the invasiveness of a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTN3A ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.
  • the pancreatic cancer is a stage Tl pancreatic cancer, a stage T2 pancreatic cancer, a stage T3 pancreatic cancer or a stage T4 pancreatic cancer according to the UICC-TNM classification.
  • the pancreatic cancer is a pancreatic ductal adenocarcinoma (PDAC), a pancreatic adenocarcinoma, a pancreatic serous cystadenomas (SCNs), a pancreatic intraepithelial neoplasia, pancreatic mucinous cystic neoplasms (MCNs) or a non resectable pancreatic adenocarcinoma.
  • PDAC pancreatic ductal adenocarcinoma
  • SCNs pancreatic serous cystadenomas
  • MCNs pancreatic intraepithelial neoplasia
  • MCNs pancreatic mucinous cystic neoplasms
  • MCNs pancreatic mucinous cystic neoplasms
  • BTN3A for "butyrophilin3A” (also used as pan-BTN3A in the patent application) has is general meaning in the art and denotes the B7-related family comprising three isoforms: BTN3A1 also called BT3.1, BTF5 or CD277, BTN3A2 also called BT3.2 or BTF4 and BTN3A3 also called BT3.3 or BTF3.
  • BTN3A for "soluble BTN3A” (also used as pan-sBTN3A in the patent application) denotes the soluble form of BTN3A that is to say the soluble forms of the three isoforms of BTN3A.
  • sBTN3Al for "soluble BTN3A1” has is general meaning in the art and denotes the soluble form discovered by the inventor of BTN3 Al .
  • the expression level of BTN3A denotes the expression level of the three isoforms of BTN3A that is to say BTN3A1, BTN3A2 and BTN3A3.
  • the expression level of sBTN3A denotes the expression level of the three soluble isoforms of BTN3A that is to say sBTN3Al, sBTN3A2 and SBTN3A3.
  • survival time denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as pancreatic cancer (according to the invention).
  • the survival time rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.
  • survival time can regroups the term OS.
  • OS Overall survival
  • pancreatic cancer As used herein, the term “Overall survival (OS)” denotes the time from diagnosis of a disease such as pancreatic cancer (according to the invention) until death from any cause.
  • the overall survival rate is often stated as a two-year survival rate, which is the percentage of people in a study or treatment group who are alive two years after their diagnosis or the start of treatment.
  • sample denotes, blood, peripheral-blood, serum, plasma or cancer biopsy and particularly pancreatic cancer biopsy.
  • Measuring the expression level of BTN3A, BTN3A2, sBTN3A and sBTN3Al can be done by measuring the gene expression level of BTN3A, BTN3A2, sBTN3A and sBTN3Al or by measuring the level of the protein BTN3A, BTN3A2, sBTN3A and sBTN3Al and can be performed by a variety of techniques well known in the art.
  • the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) 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. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis, in situ hybridization
  • amplification e.g., RT-PCR
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA 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.
  • 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 dif uoride 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 handgap 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 can he produced that are identifiable based on their different spectral characteristics.
  • semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition.
  • quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • radioisotopes such as 3 H
  • metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
  • liposomes include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • enzymes for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can he used in a metallographic detection scheme.
  • SISH silver in situ hyhridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • ISH In situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin,
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR (or q RT-PCR).
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subj ected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • Expression level of a gene may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGKl, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • the level of BTN3A, BTN3A2, sBTN3A and sBTN3Al proteins may also be measured and can be performed by a variety of techniques well known in the art.
  • Detection of protein concentration in the sample may also be performed by measuring the level of BTN3A, BTN3A2, sBTN3A and sBTN3Al proteins.
  • the "level of protein” or the “protein level expression” or the “protein concentration” means the quantity or concentration of said protein.
  • the "level of protein” means the level of BTN3A, BTN3A2, sBTN3A and sBTN3Al proteins fragments.
  • the "level of protein” means the quantitative measurement of BTN3A, BTN3A2, sBTN3A and sBTN3Al proteins expression relative to a negative control.
  • the protein level of BTN3A and BTN3A2 may be measured at the surface of the tumor cells and sBTN3A and sBTN3Al may be measured in an extracellular context (for example in blood or plasma).
  • protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • ELISA ELISA
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS). etc.
  • the reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
  • Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value.
  • concentration of protein refers to an amount or a concentration of a transcription product, for instance the proteins BTN3A, BTN3A2, sBTN3A and sBTN3Al .
  • a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example.
  • relative units can be employed to describe a concentration.
  • concentration of proteins may refer to fragments of the protein BTN3A, BTN3A2, sBTN3A and sBTN3Al .
  • fragment of BTN3A, BTN3A2, sBTN3A and sBTN3Al protein may also be measured.
  • the detection of the level of BTN3 A and BTN3 A2 can be performed by flow cytometry.
  • the method consists of determining the amount of BTN3A and/or BTN3A2 expressed on tumor cells.
  • the flow cytometry method when the florescence intensity is high or bright, the level of BTN3 A and BTN3 A2 express on tumor cells is high and thus the expression level of BTN3 A and BTN3A2 is high and when the florescence intensity is low or dull, the level of BTN3A and BTN3A2 express on tumor cells is low and thus the expression level of BTN3A and BTN3A2 is low.
  • the extracellular part of the BTN3A and BTN3A2 protein is detected.
  • Predetermined reference values used for comparison of the expression levels may comprise "cut-off or "threshold” values that may be determined as described herein.
  • Each reference (“cut-off) value for BTN3A, BTN3A2, sBTN3A or sBTN3Al level may be predetermined by carrying out a method comprising the steps of
  • the expression level of BTN3A, BTN3A2, sBTN3A and sBTN3Al has been assessed for 100 pancreatic cancer samples of 100 patients.
  • the 100 samples are ranked according to their expression level.
  • Sample 1 has the best expression level and sample 100 has the worst expression 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 reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest.
  • the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.
  • the reference value (cut-off value) may be used in the present method to discriminate pancreatic cancer samples and therefore the corresponding patients.
  • Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.
  • Such predetermined reference values of expression level may be determined for any protein defined above.
  • the reference values for sBNT3A and for sBTN3Al may be respectively 8 or 6.92 ng/ml and 6 or 6.98 ng/ml.
  • kits for performing the methods of the invention comprise means for measuring the expression level of BTN3A, BTN3A2, sBTN3A and sBTN3Al in the sample obtained from the patient.
  • kits may include probes, primers macroarrays or microarrays as above described.
  • the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled.
  • probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • the kit of the invention may comprise amplification primers that may be pre- labelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • the present invention also relates to sBTN3A and more particularly sBTN3Al as a biomarker for outcome of pancreatic cancer patients.
  • the present invention also relates to BTN3A and more particularly BTN3A2 as a biomarker for pancreatic cancer and more particularly for PDAC.
  • the present invention also relates to BTN3A as a biomarker of invasiveness of pancreatic cancer and more particularly for PDAC.
  • anti-BTN3A more particularly anti-CD277 antibody can activates the cytolytic function, cytokine production and proliferation of T cells ( ⁇ / ⁇ T cells, ⁇ / ⁇ T cells, more particularly Vy9/V52 T cells) and thus can be used to treat patient with pancreatic cancer and with a bad prognosis as described above (see Messa N; et al, 2011).
  • the invention relates to an anti-CD277 antibody, which activates the cytolytic function, cytokine production and proliferation of T cells for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.
  • the invention also relates to an anti-CD277 antibody, which activates the cytolytic function, cytokine production and proliferation of ⁇ / ⁇ T cells and/or Vy9/V52 T cells for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.
  • the invention relates to an anti-CD277 antibody for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.
  • the invention relates to a TCR (T cell receptor) agonist for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.
  • the TCR agonist can be a soluble phosphoantigen like the pAg BrHpp (see for example Rhodes DA et al, 2015).
  • the anti-CD277 antibody of the present invention is an isolated anti- CD277 antibody (mAb 20.1) which is obtainable from the hybridoma accessible under CNCM deposit number 1-4402.
  • the anti-CD277 antibody comprises the 6 CDRs of the antibody obtainable from the hybridoma accessible under CNCM deposit number 1-4402 and derivatives thereof.
  • the anti-CD277 antibody comprises the variable domains (VH and VL) of the antibody obtainable from the hybridoma accessible under CNCM deposit number 1-4402 and derivatives thereof.
  • the anti-CD277 antibody is a derivative of mAb 20.1 which is a monoclonal or a chimeric antibody, which comprises the variable domains of mAb 20.1.
  • the anti-CD277 antibody of the present invention is an isolated anti- CD277 antibody (mAb 7.2) which is obtainable from the hybridoma accessible under CNCM deposit number 1-4401.
  • the anti-CD277 antibody comprises the 6 CDRs of the antibody obtainable from the hybridoma accessible under CNCM deposit number 1-4401 and derivatives thereof.
  • the anti-CD277 antibody comprises the variable domains (VH and VL) of the antibody obtainable from the hybridoma accessible under CNCM deposit number 1-4401 and derivatives thereof.
  • the anti-CD277 antibody is a derivative of mAb 7.2 which is a monoclonal or a chimeric antibody which comprises the variable domains of mAb 7.2.
  • the invention also relates to a method for treating a pancreatic cancer in a patient with a bad prognosis as described above comprising the administration to said patient of an anti- CD277 antibody which activates the cytolytic function, cytokine production and proliferation of T cells.
  • Another aspect of the invention relates to a therapeutic composition
  • a therapeutic composition comprising an anti- CD277 antibody, which activates the cytolytic function, cytokine production and proliferation of T cells for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.
  • the invention also relates to a therapeutic composition
  • a therapeutic composition comprising an anti-CD277 antibody, which activates the cytolytic function, cytokine production and proliferation of ⁇ / ⁇ T cells and/or Vy9/V52 T cells for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.
  • the invention also relates to a therapeutic composition comprising an anti-CD277 antibody for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a 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.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intrathecal or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • Compounds used to already treat pancreatic cancer can be used in combination with an anti-CD277 according to the invention. These compounds can be selected in thr 29, consisting in Gemcitabine, 5-fluorouracil (5-FU), Irinotecan, Oxaliplatin, Albumin-bound paclitaxel, Capecitabine, Cisplatin, Paclitaxel, Docetaxel and Irinotecan liposome.
  • Gemcitabine 5-fluorouracil
  • Irinotecan Irinotecan
  • Oxaliplatin Irinotecan
  • Albumin-bound paclitaxel Capecitabine
  • Cisplatin Cisplatin
  • Paclitaxel Docetaxel
  • Irinotecan liposome Irinotecan liposome
  • FIGURES are a diagrammatic representation of FIGURES.
  • BTN3A global surface expression in pancreatic cell lines in vitro and ex vivo BTN3A surface expression assessed in PDX-derived cell lines classified in 2 groups according to survival time (short-term and long-term survival times were respectively defined as survival times ⁇ 8 months and >8 months). Mean expression in each group is shown as mean rMFI ⁇ SEM.
  • FIG. 2 Hypoxia-induced regulation of BTN3A and BTN3A isoforms expression.
  • PPIA Peptidylprolyl isomerase A
  • ACt Ct target gene - CtPPIA
  • fold change (2- AACt) was established using BTN3A1
  • FIG. 3 BTN3 A and MICA/B are shed under nutrient starvation and BTN3 A isoforms exist under soluble form.
  • PPIA Peptidylprolyl isomerase A
  • ACt Ct target gene -CtPPIA
  • fold change (2- AACt) was established using BTN3A1 expression in DMEM FCS 10% condition as a calibrator gene.
  • Figure 4 Role of BTN3A and effect of BTN3A triggering on Vj9V52 T cells antitumor function towards PANC-1 in normoxic and hypoxic conditions.
  • BTN3A expression is a marker of PDAC invasiveness.
  • FIG. 6 Concentration of soluble BTN3A and BTN3A1 (sBTN3Al and sBTN3A) is a prognosis marker in patients with Pancreatic Ductal Adenocarcinoma (PDAC).
  • Kaplan-Meier curves showing overall survival (A) in patients with Low SBTN3A ( ⁇ 8 ng/ml) or High sBTN3A levels (> 8 ng/ml) and (B) in patients Low SBTN3A1 levels ( ⁇ 6 ng/ml) or High sBTN3Al levels (>6 ng/ml).
  • Statistical significance regarding survival curves comparison was established with Log-rank (Mantel-Cox) Test. *p ⁇ 0.05; ***p ⁇ 0.0005.
  • FIG. 7 Receiver operating characteristics (ROC) curve analysis of plasmatic level for, sBTN3Al (A) and pan-sBTN3A (B). For each marker, ROC curves were plotted for sensitivity and specificity of survival classification (left panels). The plasmatic levels of each marker were plotted for STS and LTS patients (right panels). The dashed lines represent the optimal thresholds obtained by ROC analysis. (AUC: area under the curve).
  • Figure 8 Kaplan Meier analysis of overall survival in patients with high and low plasmatic levels of sBTN3Al (A) and pan-sBTN3A (B).
  • Tissue microarrays Tissue microarrays
  • TMA serial tissue sections were prepared 24 hours before immunohistochemical processing and stored at 4°C. The dilution of each antibody was determined by pre-screening on the full 4- ⁇ - thick sections before use on TMA sections.
  • the immunoperoxidase procedures were performed using an automated Ventana BenchMark XT autostainer. Measurements of immunoprecipitate densitometry in cores were made for each marker in an individual core after digitization and "cropping" of microscopic images as previously reported.
  • Pancreatic sections were fixed in 4% paraformaldehyde and paraffin embedded. Immunohistochemistry was performed using standard procedures. Sections were stained with anti-BTN3A mAb (clone 103.2).
  • MiaPACA2, PANC-1, and BxPC-3 cells were obtained from the American Type Culture Collection. Patu8902 and Patu8988t were obtained from the Leibniz Institute DSMZ- German Collection of microorganisms and cell cultures.
  • PDX-derived cell lines were established as previously described30. All cell lines were periodically tested for Mycoplasma contamination and were Mycoplasma-Free. Pancreatic cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% FCS at 37°C with 5% C02. To avoid any supplementary stress, all media were preheated at 37°C before rinsing or changing media. Nutrient starvation was obtained by cultivating cells with Earle's Balanced Salt Solution (EBSS) (Ref# 24010-043). Hypoxia experiments were carried out using C-Shuttle Glove Box coupled hypoxia chamber (BioSpherix).
  • EBSS Earle's Balanced Salt Solution
  • BrHpp was from Innate Pharma (Marseilles, France). Zoledronate (ZOL) was from Novartis (United Kingdom). Recombinant human (Rh) IL2 was from BD Biosciences (San Jose, CA, USA). TAPI-1 was from Peptides International (Louisville, KY, USA).
  • PBMCs Peripheral Blood Mononuclear Cells
  • EFS local Blood Bank
  • rhIL-2 rhIL-2
  • BTN3A Knock-down HEK293FT cells (sh#284; clone#30) were provided by E.Scotet (Inserm U892, France), cultured and transfected with BTN3A1, BTN3A2, BTN3A3 mutated cDNA-containing plasmids, as described [Harly C et al, 2012].
  • RNA from cells was prepared using Trizol (Invitrogen, Cergy Pontoise, France) according to the manufacturer's instructions. RNA concentration was determined by absorption and RNA integrity was checked on RNA Nano chips (Agilent, Santa Clara, CA). Reverse transcription (RT) reactions were performed on 1 ⁇ g of total RNA using Go Script (Promega, Madison, WI) according to the manufacturer's protocol.
  • qPCR reactions were run in duplicate on two independent cDNA preparations. qPCR was performed in Stratagene MX3005P machine (Agilent, Santa Clara, CA) using TaqMan® Universal Master Mix II, with UNG (Applied biosystem (Invitrogen), Cergy Pontoise, France). The crossing point (Cp), defined as the point at which the fluorescence rises appreciably above the background fluorescence, was determined for each transcript. The 2AACp method was used to analyze the relative gene expression. The Peptidylprolyl Isomerase A (PPIA) gene (ref 4331182) was chosen as control. Three BTN3A isoforms are measured: BTN3A1 (Hs01063368_ml), BTN3A2 (Hs00389328_ml) and BTN3A3 (Hs00757230_ml).
  • PPIA Peptidylprolyl Isomerase A
  • ⁇ T cells were incubated at 37°C in the presence of anti-CD 107a/b and Golgi stop with or without BrHpp, anti-BTN3A 20.1 mAb or anti-BTN3A 108.5 mAb in normoxia or hypoxia. After 4 hours, cells were collected, washed in PBS and analyzed by flow cytometry. To study cytokine production, cells were further permeabilized with Permwash (BD bioscience) to allow intracellular staining with labeled antibodies
  • Pan-BTN3A and BTN3 A 1 -specific sandwich ELISAs were conceived by Dynabio®. Were used as capture antibodies: l/anti-BTN3A mAb clone that recognizes the 3 BTN3A-Fc recombinant proteins i.e BTN3A1-Fc, BTN3A2-Fc and BTN3A3-Fc for "Pan- BTN3A” ELISA and 2/anti-BTN3A mAb clone that only recognizes BTN3A1-Fc for "BTN3 A 1 -specific" ELISA.
  • Anti-BTN3A 103.2 mAb that recognizes the 3 BTN3A isoforms was biotinylated for detection of BTN3A isoforms.
  • the efficiency of biotinylation was validated comparing to detection mAb used for Pancreatitis-Associated Protein (PAP) ELISA test (Dynabio®) and using recombinant BTN3A-Fc proteins. After blockade of the plate, supernatants of pancreatic cell lines, patients' plasmas or BTN3A1-Fc, BTN3A2-Fc and BTN3A3-Fc recombinant proteins used as standards were added. After repeated washes, biotinylated detection-anti-BTN3A mAb were added. Revelation was achieved with avidin- HRP. The optical density of each well was determined using a microplate reader set to 450 nm. The concentration of each BTN3 A isoform was assessed following the standard curve obtained with BTN3A1-Fc protein.
  • mice were home-bred and maintained under pathogen-free conditions. All animal procedures were in accordance with protocols approved by the local Committee for Animal Experiments.
  • PDX murine models were established as previously described42. Briefly, patient- derived pancreatic tumor pieces (lmm3) were embedded in Matrigel before to be s.c implanted into flank of adult male Swiss nude Mice (Charles River laboratories) under isoflurane anesthesia. Tumors were measured weekly with a caliper until tumor volume reached lmm3. At 4h after intratumoral injection of PDZ hydrochloride, pieces of tumor were removed fixed in 4% (wt/vol) formaldehyde or frozen in cold isopentane for further analysis.
  • Results are expressed as median ⁇ SEM. Statistical analysis was performed using paired t-test, Wilcoxon test, Mann-Whitney t test and Spearman correlation. P values ⁇ 0.05 were considered significant. Survival curves were compared using LogRank Test. Analyses were performed using GraphPad Prism program.
  • TMA comprises 2 groups of PDAC patients NO and Nl
  • ANOVA analysis was followed by post hoc analysis (Tukey-Kramer, and Mann-Whitney t test) to perform pairwise comparisons and determine which pairs were significantly different from one another.
  • the analysis was performed using NCSS software (Kay sville, Utah).
  • BTN3 A molecules are expressed in various pancreatic cell lines including novel Patient-related
  • PANC-1, MiaPACA2 and BxPc3 were extensively used as PDAC models in the literature. These cell lines differ in their KRAS, P53, SMAD4 mutational status. Patu8902 and Patu8988t originate from primary and liver-metastatic PDAC and are respectively highly metastatic and poorly metastatic in mice. We observed that BTN3A was expressed at the surface of all the tested pancreatic tumor cell lines irrespective of their origin, mutational status or differentiation state (data not shown).
  • BTN3A was expressed in all tested PDX-derived cell lines including liver-metastasis derived one (CRCM-14) (data not shown) and PDX-derived cell lines belonging to both survival groups (data not shown).
  • the mean level of BTN3A surface expression was higher in the short-term survival group (313.6 ⁇ 61.4) than in the long-term survival group (226 ⁇ 27.38) ( Figure 1).
  • BTN3A2 is the most abundant isoform in PDAC.
  • BTN3A2 was also the most abundant isoform at protein level (mean density quantification relative to loading control: 8.2 ⁇ 6.2) compared with BTN3A1 (1.3 ⁇ 0.75) and BTN3A3 (3.7 ⁇ 3.4) in MiaPACA2, BxPC3, Patu8902, Patu8988t (data not shown), PANC-1 (data not shown) and PDX-derived cell line CRCM04 (supplementary Figure 1).
  • BTN3 A2 expression in pancreatic cell lines is enhanced under hypoxia.
  • hypoxia is a feature of PDAC TME.
  • BTN3A surface expression was influenced by hypoxia.
  • Flow cytometry analysis revealed that BTN3A global surface expression in Panc-1 cell line remained stable under hypoxia in vitro (data not shown).
  • PDZ pimonidazole
  • BTN3A is shed under soluble form by MMP with increased shedding under nutrient starvation.
  • BTN3A isoforms expression in PANC-1 cell line cultured with nutrient-deprived medium (EBSS).
  • DMEM FCS10% or nutrient starvation EBSS
  • BTN3A triggering with TCR agonist or agonist 20.1 mAb enhances Vy9V52 T cells anti-tumor functions under normoxic and hypoxic conditions.
  • BTN3 A plays a key role in BrHpp-mediated enhancement of Vy9V52 T cells lysis of
  • BTN3A expression in Human primary pancreatic tumors is associated with invasiveness.
  • the BTN3A subfamily is a critical determinant of Vy9V52T cells recognition and lysis of primary tumor and has been shown to be expressed in many solid tumors but its expression in pancreatic tumors remains unknown. We thus decided to address its expression on primary pancreatic tumors.
  • Immunofluorescence analysis revealed a strong prominent epithelial staining assessed by a co- localization of BTN3A and Keratinl9 stainings (data not shown).
  • Soluble BTN3A and BTN3A1 concentration is a prognosis marker in PDAC patients.
  • BTN3A was found as a released soluble form in pancreatic tumor cell line supernatant, we investigated whether sBTN3A was present in PDAC patients' plasmas.
  • Example 2 Prognostic significance of circulating pan-BTN3As and BTN3A1 in patients with non resectable pancreatic adenocarcinoma
  • ELISAs for pan-BTN3A and BTN3A1 are not commercially available. Because some discrepancies were observed in monitoring the three other proteins when using commercial kits obtained from different sources, we decided to have ELISAs of the 6 markers produced by DYNABIO S.A. (Pare de Luminy, Marseille France) according to our specifications. These specifications included i/ verification by tandem mass spectrometry of the sequence of the antigen ii/ optimization of the assay by testing all combinations of available monoclonal antibodies in capture and detection, targeting maximal signal/background ratio and sensitivity. Combinations of two or more antibodies in coating and/or detection were also tested to improve performances iii/ checking sample compatibility (serum vs plasma, interference of the matrix), iv/ ensure that assay can be run at room temperature for easy handling and robustness.
  • BTN3A For the ELISA, three isoforms of BTN3A are identified (Al, A2, A3). Among available monoclonal antibodies to BTN3A, one is specific of Al ( ⁇ - ⁇ 3 ⁇ 1 S240). Coating with a- BTN3A1 S240 allows specific assay of the Al isoform, whereas the couple of antibodies a- BTN3A S148 and a-BTN3A 103.2 allows simultaneous detection of all 3 forms (Pan-BTN3A assay). It is however noteworthy that BTN3A concentrations obtained with the Pan-BTN3A assay are only indicative since the range used in the assay is pure BTN3A1. BTN3A concentrations should therefore be expressed as pg/ml « equivalent BTN3A1 Colour
  • the samples were obtained, under consent, at the time of the EUS-FNA biopsy procedure. According to inclusion criterias, all patients were naive of any chemotherapeutic treatment during blood sampling. Total blood fractions were processes within 4 hours from the sampling and centrifuged at 2,200g during 15 min at 4°C in presence of EDTA. The supernatants (plasma fraction) were aliquoted in cryotubes and stored a -80°C until processing.
  • Non resectable PDAC patients Between 2012 and 2016, EUS-FNA tumor biopsies and blood samples of 32 non resectable PDAC patients were collected. All patients were recruited under the Institut Paoli Calmette clinical trial NCT01692873 (https://clinicaltrials.gov/show/NCT01692873) exclusively in case of pancreatic ductal adenocarcinoma diagnosis. The overall survival median of this cohort is 6.9 months (95% CI: (4.4-10.19)) that is very close to the worldwide reference
  • pan-sBTN3 A The levels of pan-sBTN3 A are highly correlated in non-operable PDAC patients
  • TIL tumour-infiltrating lymphocytes

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Abstract

La présente invention concerne un procédé de pronostic du cancer du pancréas. Dans cette étude, les inventeurs ont évalué l'effet de l'hypoxie et du stress métabolique sur la régulation des isoformes de BTN3A en utilisant notamment la PCR qRT et le transfert Western et ont découvert une forme soluble inattendue. De plus, ils ont démontré un rôle clé du BTN3 A dans des lymphocytes T Vγ9Vδ2 T cytolytiques contre le PDAC conservés sous hypoxie. Enfin, ils ont découvert que l'expression de BTN3A dans les tissus et le niveau de plasma de BTN3 A et de BTN3 A1 solubles sont associés à un pronostic médiocre chez des patients atteints de PDAC. Ainsi, l'invention concerne un procédé de prédiction du temps de survie d'un patient souffrant d'un cancer du pancréas notamment en fonction de la mesure du niveau d'expression de BTN3 A ou de sBTN3A. En outre, l'invention concerne un anticorps anti-CD277, qui active la fonction cytolytique, la production de cytokine et la prolifération de lymphocytes T à utiliser dans le traitement du cancer du pancréas chez un patient présentant un mauvais pronostic.
PCT/EP2018/072410 2017-08-21 2018-08-20 Nouveau procédé de pronostic du cancer du pancréas WO2019038219A1 (fr)

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