WO2019038219A1 - New prognostic method of pancreatic cancer - Google Patents

Abstract

The present invention relates to a prognostic method of pancreatic cancer. In this study, 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. More, they demonstrated a key role of BTN3 A in Vγ9Vδ2 T cells cytolytic functions against PDAC that are conserved under hypoxia. Finally, they found that BTN3A expression in tissues and plasma level of soluble BTN3 A and BTN3 A1 are associated with poor prognosis in patients with PDAC. Thus, the invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising based on the measurement of the expression level of BTN3 A or sBTN3A. More, 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.

Description

NEW PROGNOSTIC METHOD OF PANCREATIC CANCER

FIELD OF THE INVENTION:

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. Particularly, the invention is defined by its claims.

BACKGROUND OF THE INVENTION:

Pancreatic Ductal Adenocarcinoma (PDAC) 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. Moreover, 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]. However, the limited efficacy of immunotherapy has been suggested to arise from the peculiar PDAC Tumor Micro-environment (TME) and highlights the need of combinatorial therapies. Indeed, PDAC is characterized by a preponderant poorly vascularized fibrotic stroma that leads to severe deprivation of nutrients and oxygen. The subsequent hypoxic and metabolic stress induces the selection of aggressive and pro-survival tumor cells together with an immunosuppressive TME.

Though, γδ 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]. γδ 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]. The Vy9V52 T Cell Receptor (TCR) senses phosphoAntigens (pAgs) which are intermediary metabolites of the human mevalonate pathway. In tumors, the blockade of this pathway with pharmacological agents such as Aminobisphosphonates (N-BP), leads to the upstream accumulation of natural pAg that enhances TCR-mediated Vy9V52 T cells activation. In the same line, synthetic pAg such as BrHpp and natural microbial pAg HMBPP are also potent TCR agonists. Once activated, Vy9V52 T cells display TH1 cytokines production and MHC -unrestricted cytotoxicity towards their target cell.

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 γδ T cell cytotoxicity in vitro. However, γδ T cells isolated from TILs displayed high cytotoxicity against pancreatic tumors ex vivo. In addition, 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.

Among the B7-related family, the butyrophilin3A (BTN3A) subfamily is composed of three isoforms (BTN3A1, BTN3A2 and BTN3A3) that contain two extracellular Immunoglobulin (Ig) domains (IgV and IgC) and a transmembrane domain [Compte E et al., 2004]. Only 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 γδ T cell activation via similar extra-cellular conformational changes of BTN3A molecules. Of note, while pAg-activation of Vy9V52 T cells requires the three isoforms, 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.

Together, these data open new perspectives in Vy9V52 T cells based-immunotherapies with anti-BTN3A agonist 20.1 mAb appearing as an interesting tool to enhance Vy9V52 T cells anti-tumor function. However, we currently do not know whether PDAC-associated hypoxic and metabolic stress can regulate BTN3A and Vy9V52 T cells anti-tumor functions.

SUMMARY OF THE INVENTION:

In this study, 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.

Thus, 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. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

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.

In a particular embodiment, 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.

In a particular embodiment, 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.

According to these particular embodiment, 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.

In one embodiment, 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.

In another embodiment, 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.

As used herein, the term "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. The term "sBTN3A" 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. Thus, the term "sBTN3Al" for "soluble BTN3A1" has is general meaning in the art and denotes the soluble form discovered by the inventor of BTN3 Al .

According to the invention, "the expression level of BTN3A" denotes the expression level of the three isoforms of BTN3A that is to say BTN3A1, BTN3A2 and BTN3A3.

According to the invention, "the expression level of sBTN3A" denotes the expression level of the three soluble isoforms of BTN3A that is to say sBTN3Al, sBTN3A2 and SBTN3A3.

As used herein, the term "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.

As used herein and according to the invention, the term "survival time" can regroups the term OS.

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.

As used herein and according to all aspects of the invention, the term "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.

For measuring the expression level of sBTN3A and more particularly sBTN3Al, techniques like ELISA (see below) allowing to measure the level of the soluble proteins are particularly suitable. Typically, 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).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

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.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, 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. Thus, 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 (such as a probe generated by the disclosed methods) 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.

Particular examples of detectable labels include 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). Examples of particular 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. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamme B, sulforhodamme 101 and sulfonyl chloride derivative of sulforhodamme 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable 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. Other 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. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, 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. When semiconductor nanocrystals are illuminated with a primary energy source, 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 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. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Puhlication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition. For example, 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.

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.

Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hyhridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. 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. (See, for example, U.S. Patent Application Puhlication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). 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. (See, for example, U.S. Pat. No. 6,670,113).

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).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with 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.

For example, a biotinylated probe can be detected using 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. For detection by enzyme activity, 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). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al, Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, 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. 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. 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. In other examples, 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). As indicated above, 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.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, 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). For example, 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. Following exposure of the sample to the probes, 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). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

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. Such 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.

In a particular embodiment, 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).

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such 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. To determine the expression level, 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. Typically, 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.

According to the invention, 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. In the present application, the "level of protein" or the "protein level expression" or the "protein concentration" means the quantity or concentration of said protein. In another embodiment, the "level of protein" means the level of BTN3A, BTN3A2, sBTN3A and sBTN3Al proteins fragments. In still another embodiment, the "level of protein" means the quantitative measurement of BTN3A, BTN3A2, sBTN3A and sBTN3Al proteins expression relative to a negative control.

According to the invention, 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).

Typically protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.

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. Such 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.

More particularly, 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. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins BTN3A, BTN3A2, sBTN3A and sBTN3Al . Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the protein BTN3A, BTN3A2, sBTN3A and sBTN3Al . Thus, in a particular embodiment, fragment of BTN3A, BTN3A2, sBTN3A and sBTN3Al protein may also be measured.

In a particular embodiment, the detection of the level of BTN3 A and BTN3 A2 can be performed by flow cytometry. When this method is used, the method consists of determining the amount of BTN3A and/or BTN3A2 expressed on tumor cells. According to the invention and 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.

In another embodiment, 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

a) providing a collection of samples from patients suffering of a pancreatic cancer; b) determining the level of BTN3A, BTN3A2, sBTN3A or sBTN3Alfor each sample contained in the collection provided at step a);

c) ranking the tumor tissue samples according to said level

d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level,

e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient; f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve;

g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets

h) selecting as reference value for the level, the value of level for which the p value is the smallest.

For example 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. According to the information relating to the actual clinical outcome for the corresponding pancreatic cancer patient, 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. In other terms, 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.

In routine work, 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.

The man skilled in the art also understands that the same technique of assessment of the expression level of a protein should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a protein of a patient subjected to the method of the invention.

Such predetermined reference values of expression level may be determined for any protein defined above.

According to the invention, the reference values for sBNT3A and for sBTN3Al may be respectively 8 or 6.92 ng/ml and 6 or 6.98 ng/ml. A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of BTN3A, BTN3A2, sBTN3A and sBTN3Al in the sample obtained from the patient.

The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, 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. Alternatively 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.

The inventors showed that anti-BTN3A more particularly anti-CD277 antibody can activates the cytolytic function, cytokine production and proliferation of T cells (α/β T cells, γ/δ 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).

Thus, in another aspect, 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 α/β 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.

In one embodiment, 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.

In another embodiment, 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. In a particular embodiment, the TCR agonist can be a soluble phosphoantigen like the pAg BrHpp (see for example Rhodes DA et al, 2015).

In one embodiment, 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.

In another embodiment, 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.

In a further embodiment, 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.

In a further embodiment, 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.

In one embodiment, 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.

In another embodiment, 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.

In a further embodiment, 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.

In a further embodiment, 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 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 comprising an anti-CD277 antibody, which activates the cytolytic function, cytokine production and proliferation of α/β 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.

The form of the pharmaceutical compositions, 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.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intrathecal or subcutaneous administration and the like.

Particularly, the pharmaceutical compositions contain 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.

In addition, other pharmaceutically acceptable forms 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 groupe consisting in Gemcitabine, 5-fluorouracil (5-FU), Irinotecan, Oxaliplatin, Albumin-bound paclitaxel, Capecitabine, Cisplatin, Paclitaxel, Docetaxel and Irinotecan liposome. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. 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.

Figure 2: Hypoxia-induced regulation of BTN3A and BTN3A isoforms expression. Transcriptional analysis of the 3 BTN3A isoforms expression in PANC-1 under hypoxia was performed by qRT PCR of total RNA isolated from pancreatic cell lines. Data were normalized using Peptidylprolyl isomerase A (PPIA) as an endogenous control; (ACt = Ct target gene - CtPPIA) and fold change (2- AACt) was established using BTN3A1 expression in normoxia as a calibrator gene. Results were expressed as mean 2-AACt ± SEM and statistical significance was established using the non-parametric Mann Whitney U-test. Non- significant (n. s) : p > 0.05. Cumulative data from 2 independent experiments performed in duplicate.

Figure 3: BTN3 A and MICA/B are shed under nutrient starvation and BTN3 A isoforms exist under soluble form. (A) Transcriptional analysis of the 3 BTN3A isoforms expression under nutrient starvation was performed by qRT PCR of total RNA isolated from PANC-1 cell line. Data were normalized using Peptidylprolyl isomerase A (PPIA) as an endogenous control; (ACt = Ct target gene -CtPPIA) and fold change (2- AACt) was established using BTN3A1 expression in DMEM FCS 10% condition as a calibrator gene. Results were expressed as mean 2-AACt ± SEM and statistical significance was established using paired t-test. *p < 0.05; 0.001 <**p < 0.01. Cumulative data from 2 independent experiments performed in duplicate. (B, C, D) Effect of MMP inhibitor TAPI-1 on BTN3A release as a soluble form and BTN3A surface expression. (B, C) ELISA analysis of BTN3A in PANC-1 supernatants treated or not with TAPI-1 (20 μΜ). Concentrations are expressed in pg/ml. (B) "Pan-BTN3A" dosage. Cumulative data of 3 independent experiments performed in duplicate. Statistical significance was established using paired t-test. *p < 0.05. (C) "BTN3 A 1 -specific" dosage performed in duplicate (n = 1). (D) BTN3A expression assessed with anti-BTN3A 20.1 mAb in PANC-1 cell line treated or not with TAPI-1 (20 μΜ). Data are shown as mean rMFI ±SEM. Cumulative data of 3 independent experiments performed in duplicate. Statistical significance was established using paired t-test. *p < 0.05.

Figure 4: Role of BTN3A and effect of BTN3A triggering on Vj9V52 T cells antitumor function towards PANC-1 in normoxic and hypoxic conditions. (A) Effects of BrHpp or agonist (20.1) mAb or anti-BTN3A antagonist (108.5) mAb on specific lysis of PANC-1 by Vy9V52 T cells from Healthy Donors (HD) (n = 7), assessed in a standard [5 lCr] -release assay. Data are shown for E:T 30:1; 10: 1; 1 : 1. Cumulative data from 3 independent experiments. Results were expressed as mean of specific lysis ± SEM and statistical significance was established using the non-parametric paired Wilcoxon U-test. *p < 0.05. (B) CD107a/b expression of expanded Vy9V62 T cells from HD (n = 8) co-cultured with PANC-1 cell line and treated with anti-BTN3A antagonist (108.5) mAb or agonist (20.1) mAb, and BrHpp in normoxic (20% 02) or hypoxic conditions (0.1% 02). (C) IFNy and (D) TNFa production was assessed by intracellular staining and flow cytometry. Cumulative data from 3 independent experiments. Results were expressed as mean ± SEM and statistical significance was established using the non-parametric paired Wilcoxon U-test. *p < 0.05; 0.001 <**p < 0.01.

Figure 5: BTN3A expression is a marker of PDAC invasiveness. ANOVA test identified differential percentage of BTN3A staining in pancreatic tumor TMA (n = 32). For each TMA, percentage of BTN3A staining in the tumor center and periphery are depicted. BTN3A staining was higher in the group of pancreatic tumors with lymph node involvement ( l) compared with the group devoid of lymph node involvement (NO). Results were expressed as percentage ± SEM. Mann Whitney U test was used to compare differences between groups. 0.001 <**p < 0.01.

Figure 6: Concentration of soluble BTN3A and BTN3A1 (sBTN3Al and sBTN3A) is a prognosis marker in patients with Pancreatic Ductal Adenocarcinoma (PDAC). (A, B) Comparative survival in PDAC patients according to (A) sBTN3A (n = 55) and (B) sBTN3Al dosage (n = 53). 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.

Figure 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).

EXAMPLES:

Example 1

Material & Methods

Patients

Thirty-two PDAC samples were formalin- fixed surgical specimens obtained from the

Pathology Department of Aix-Marseille University.

Plasmas from PDAC patients (n=66), Chronic Calcific Pancreatitis (n=12), Intra Papillary Mucinous Neoplasm (n=8) and Healthy control subjects (n=23) were collected and provided by Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBEREHD), Barcelona.

Tissue microarrays (TMAs)

The procedure for construction of TMAs was as previously described. Briefly, cores were punched from the selected paraffin blocks and distributed in new blocks, with 2 cores of 0.6-mm diameter, one from the periphery (P) and one from the center of each tumor (T). 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-μιη- 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.

Immunohistochemistry

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).

Confocal microscopy

Ten-micrometer cryosections of pancreatic tissue were dried and fixed with acetone. After nonspecific binding site blockade with 3% bovine serum albumin, 10% fetal calf serum, and 10% goat serum for 30 minutes, tissue sections were labeled 1 hour at room temperature with primary antibodies BTN3A (clone 103.2) and cytokeratin 19 (abnova (clinisciences), Nanterre, France), followed by incubation for 30 minutes at room temperature with secondary antibodies (Alexa Fluor 488 and 568 (Invitrogen, Cergy Pontoise, France)) and SYTOX Blue when nuclei staining was required. Slides were mounted in ProLong Gold (Invitrogen, Cergy Pontoise, France) and observed with a Zeiss LSM 510 confocal microscope. Images were analyzed using Adobe Photoshop 7.0.

Pancreatic cell lines and culture system

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).

Reagents and antibodies

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).

Vy9V52 T cells expansion and DAUDI cell line culture

Effector γδ T cells were established and maintained as previously described41. Peripheral Blood Mononuclear Cells (PBMCs) from healthy volunteers were provided by the local Blood Bank (EFS) and isolated by density gradient centrifugation. They were stimulated with ZOL (ΙμΜ) or BrHpp (3μΜ) and rhIL-2 (200IU/ml) at Day 0. From Day 5, rhIL-2 was renewed every two days and cells were kept at 1.5 x 106 /ml for 15 days. The purity of γδ T cells was determined and greater than 80%.

HEKShBTN3A

BTN3A Knock-down HEK293FT cells (sh#284; clone#30) were provided by E.Scotet (Inserm U892, Nantes), cultured and transfected with BTN3A1, BTN3A2, BTN3A3 mutated cDNA-containing plasmids, as described [Harly C et al, 2012].

Generation of anti-human BTN3A mAbs and BTN3A-Fc BTN3A1-Fc, BTN3A2-Fc and BTN3A3-Fc and anti-BTN3A mAbs were generated as previously described [Compte E et al, 2004]. Clones 20.1 and 103.2 were further labeled for cytometry using Alexa Fluor® 647 Protein Labeling Kit (Life Technologies).

Determination of BTN3A isoforms Expression by Quantitative RT-PCR (qRT-PCR) 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 μg 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).

Flow cytometry

2 xlO⁵ PBMC were washed in PBS (Cambrex Bio Science) and incubated at 4°C for 20 min with specified mAb. Following incubation and washing, samples were analyzed on LSRFortessa or FACS Canto II (Becton Dickinson) using DIVA software (BD bioscience, Mountain View, CA). For analysis of CD 107 expression, γδ 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

Cytotoxic activity analysis

lxl 0⁶ target cells were incubated with 20 μθ of 51Cr (Perkin-Elmer) for 60 minutes and mixed with effector cells in a Effector: Target (E:T) ratio of 30: 1; 10: 1; 1 : 1. After 4 hours of incubation at 37°C, 50μ1 supernatant of each sample was transferred in LUMA plates and radioactivity was determined by a gamma counter. The percentage of specific lysis was calculated using the formula [(experimental - spontaneous release / total -spontaneous release) x 100] and expressed as the mean of triplicate. Western blot

10 cm-culture dishes were placed on ice, washed in PBS, cells were dissociated and lysed in 250 μΐ of ice-cold HNTG buffer (50 mM HEPES pH 7, 50 mM NaF, 1 mM EGTA, 150 mM NaCl, 1% Triton X- 100, 10% glycerol, and 1.5 mM MgC12) in the presence of protease inhibitors (Roche Applied Science) and 100 μΜ Na3V04. Protein quantification in all cell lysates was performed according to the manufacturer (Biorad quantification kit). Proteins were resolved by SDS-PAGE 10%, followed by western blotting. Primary antibodies used were: anti- BTN3A (20.1) mAb, anti-HIFl (BD Biosciences), anti-γ tubulin, anti-Grb2 (SantaCruz technology). They were detected with peroxidase-conjugated anti-mouse IgGl and anti-rabbit IgG antibody (Jackson Laboratory). Immunoreactive bands were detected using enhanced chemiluminescent reagents (Pierce).

Enzyme Linked Immunosorbent Assay (ELISA)

"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.

Xenograft murine model

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.

Statistical 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 statistical Analysis

To identify differentially expressed biomarkers, ANOVA analysis was performed. Since 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).

Study Approval

Written informed consent was obtained from patients included in this study, in accordance with the Declaration of Helsinki. The study was approved by the local institutional review boards of the Institut-Paoli-Calmettes.

Results

BTN3 A molecules are expressed in various pancreatic cell lines including novel Patient-

Derived Xenograft (PDX)-derived cell lines.

We chose a panel of cell lines derived from tumors with various grade of differentiation, harbouring different mutations, and differential potential of invasiveness in patients and in xenograft models.

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).

In order to be more relevant to primary tumors, we next assessed BTN3A expression in ex vivo PDX-derived cell lines established from fresh PDAC tumors by P. Duconseil et al. in our laboratory (n = 12; CRCM-02,-03,-04,-05,-06, -07,-08,-10,-14,-16,-17,-106)30. From a clinical standpoint, these tumors were isolated from patients belonging to two different groups in regards to survival: 1/ short-term survival i.e overall survival less than 8 months and 21 long- term survival i.e overall survival greater than 8 months. These same two groups respectively corresponded to poorly differentiated and middle/well-differentiated tumors [Duconseil P et al, 2015]. 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).

Altogether these data suggest that BTN3A could associate with poor differentiation of PDAC tumors and short-term survival of patients with PDAC.

BTN3A2 is the most abundant isoform in PDAC.

We next decided to tackle which iso forms were expressed in pancreatic tumors. We evaluated BTN3A isoforms expression at transcriptional level by qRT-PCR and protein level by Western Blot in both pancreatic tumor cell lines and PDX-derived cell lines. We observed that the three isoforms were expressed in pancreatic cell lines. At transcriptional level, BTN3 A2 was the most abundant isoform in all tested pancreatic cell lines (data not shown) and PDX- derived cell lines (data not shown). 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).

Collectively, these data show that BTN3A2 expression is a hallmark of PDAC tumors.

BTN3 A2 expression in pancreatic cell lines is enhanced under hypoxia.

Hypoxia is a feature of PDAC TME. Thus, we asked whether 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). To deepen the analysis of BTN3A under hypoxia in vivo, we used a PDX mouse model transplanted with human PDAC (CRCM05) and looked for BTN3A staining in pimonidazole (PDZ)-stained hypoxic regions. We demonstrated that BTN3A is overexpressed in hypoxic region of PDAC tumor in vivo (data not shown).

We next aimed to determine whether BTN3A isoforms expression was modified under hypoxic stress in PANC-1 cell line. We showed that BTN3A2 transcript level increased under hypoxia (Figure 2). Hence, we sought to assess BTN3A isoforms at protein level by Western Blot (data not shown). In parallel, we verified the expression of HIFl used as a marker of hypoxia (Figure 3D). We observed that BTN3A2 protein expression (36 kDa) increased after 24 hours of culture under hypoxia (0.1%). A 30 kDa band appeared after 48H of hypoxia exposure that presumably correspond to non-glycosylated BTN3A231. BTN3A1 and BTN3A3 were not detected at respective expected molecular weights of 57 and 65 kDa (data not shown).

Our data suggest that BTN3 A global expression is stable under hypoxia and that hypoxic conditions tuned BTN3A isoforms expression leading to increased expression of BTN3A2 isoform.

BTN3A is shed under soluble form by MMP with increased shedding under nutrient starvation.

Limited supply of oxygen is associated to limited supply of nutrients that select aggressive pancreatic cancer cell. We examined BTN3A isoforms expression in PANC-1 cell line cultured with nutrient-deprived medium (EBSS). BTN3A1 and BTN3A2 expression increased at transcript level (mean fold increase of 4.56, p = 0.0131 and 2.15, p = 0.0095 respectively) under nutrient starvation compared to standard nutrient supply in PANC-1 cell line (Figure 3 A). Hence, we wondered whether BTN3A global expression increased at the membrane under nutrient starvation. We monitored BTN3A expression by flow cytometry on PANC-1 cell line cultured O/N with standard nutrient supply (DMEM FCS10%) or under nutrient starvation (EBSS). We observed that BTN3A surface expression slightly increased under nutrient starvation (mean rMFI of 14.4 ± 5.4) (data not shown) compared to standard nutrient supply (mean rMFI 17.9 ± 4.2) but to a lesser extent than what observed at transcriptional level (Figure 3 A). In parallel, we investigated the expression of known MMP- cleaved molecules namely MICA/B. We observed a significant decrease of MICA/B expression under nutrient starvation compared to standard nutrient supply (mean rMFI of 1.6 ± 0.1 and 7.13 ± 1.3 respectively; p = 0.0239) (data not shown). We asked whether such cleavage of BTN3 A could occur. This hypothesis led us to test BTN3 A along with MICA/B expression on nutrient-starved cells after treatment with TAPI-1, a broad MMP inhibitor. We showed that TAPI-1 -treated cells exhibited significantly higher level of expression of BTN3A (24.9 ± 4.8) and MICA/B (6.8 ± 0.5) than untreated nutrient-starved cells (data not shown) (17.8 ± 4. land 1.64 ± 0.1; p = 0.0298 and p = 0.0021 respectively). This observation indirectly suggested BTN3A and MICA/B cleavage by MMP under nutrient starvation.

We decided to confirm whether BTN3A could be released by PANC-1 cell line under standard nutrient supply (DMEM FCS10%). Hence, we analyzed BTN3A concentrations in PANC-1 cell lines supernatants by ELISA. BTN3A molecules and BTN3A1 were detected in PANC-1 supernatants (Figure 3B and Figure 3C). The whole pool of soluble BTN3A (sBTN3A) molecules was significantly higher in the supernatants of TAPI-1 -treated cells (99.3 ± 27.11 pg/ml versus 81.83 ± 22.1 pg/ml, p = 0.0355) (Figure 3B). Similar soluble BTN3A1 (sBTN3Al) levels were observed in both conditions (82.50 ± 1.50 pg/ml versus 80.50 ± 1.71 pg/ml) (Figure 3C). Concomitantly, treatment with TAPI-1 was associated with significantly increased BTN3A expression in PANC-1 cell line assessed by flow cytometry (7.11 ± 0.8 versus 9.7 ± 1.3, p = 0.0123) (Figure 3D). Collectively, our data suggest that BTN3A molecules are released from PANC-1 cell line and that MMP are involved in this process.

BTN3A triggering with TCR agonist or agonist 20.1 mAb enhances Vy9V52 T cells anti-tumor functions under normoxic and hypoxic conditions.

We next aimed to take advantage of BTN3A expression to improve Vy9V52 T cells recognition and lysis of pancreatic cell lines. In parallel, we assessed whether hypoxia could affect anti-tumor functions of expanded Vy9V52 T cells from Healthy Donors.

We showed that treatment with the synthetic pAg BrHpp or anti-BTN3A agonist 20.1 mAb enhanced Vy9V52 T cells killing of PANC-1 cell line (p = 0.0156) (Figure 4 A) and of all tested PDAC cell lines (BxPC3, Patu8902, Patu8988t) and PDX-derived cell line (CRCM04) excepting MiaPACA2 (data not shown). Enhanced PDAC lysis was associated with a greater degranulation ability assessed with CD107a/b expression (p = 0.0313) (Figure 4B) (data not shown) and a greater cytokine production of TNFa (p = 0.0313) (Figure 4C) and IFNy (p = 0.0313) (Figure 4D). In addition, BTN3A- and BrHpp-stimulated Vy9V52 T cells conserved their ability to degranulate and to produce TH1 cytokine towards PANC-1 cells under hypoxia. Hypoxia was associated with higher production of TNFa by Vy9V52 T cells towards Panc-1 (p =0.0313).

Finally, we demonstrated that BrHpp induced a greater enhancement of Vy9V52T cells cytokine production than anti-BTN3 A 20.1 mAb (p=0.0313) (Figure 4C and 4D). Collectively, these data demonstrate that BTN3A can be triggered to enhance Vy9V52 T cells abilities to degranulate and produce TH1 cytokines including under hypoxic conditions. They underscore the heterogeneity of sensitivity of pancreatic tumors, confirming MiaPACA2 intrinsic resistance to Vy9V52 T cells killing despite BTN3A-mediated enhancement of Vy9V52 T cells degranulation.

BTN3 A plays a key role in BrHpp-mediated enhancement of Vy9V52 T cells lysis of

PDAC cell lines.

To investigate the role played by BTN3A in pAg-mediated enhancement of Vy9V52 T cells, we combined BrHpp and antagonist anti-BTN3A 108.5 mAb and assessed their effect upon Vy9V52 T cells functions. The BrHpp-mediated enhancement of lysis, degranulation and cytokine production of Vy9V52T cells towards PANC-1 and all tested PDAC cell lines was significantly abrogated by anti-BTN3A 108.5 antagonist mAb (p = 0.0313) (Figure 4A-4D).

This highlighted a key role of BTN3A triggering by BrHpp in the enhancement of Vy9V52T cells lysis of poorly-sensitive PANC-1 cell line.

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. Immunohistochemical analysis of Tissue Micro Arrays (TMA) of PDAC showed BTN3A expression in 93.75 % of the tumors tested (n=32) (data not shown). Immunofluorescence analysis revealed a strong prominent epithelial staining assessed by a co- localization of BTN3A and Keratinl9 stainings (data not shown). ANOVA analysis demonstrated a differential expression of BTN3A among tumors (p = 0.013). Using Tukey Kramer post-hoc test and Mann Whitney test, we showed a significantly higher percentage of BTN3A staining (6.99 ± 2.49) in the group of patients with pancreatic cancer with lymph node involvement (Nl) (n=23) compared with patients devoid of lymph node involvement (NO) (n=9) (3.13 ± 0.60) (p = 0.036 and p = 0.003 respectively for each test) (Figure 5).

Soluble BTN3A and BTN3A1 concentration is a prognosis marker in PDAC patients. As BTN3A was found as a released soluble form in pancreatic tumor cell line supernatant, we investigated whether sBTN3A was present in PDAC patients' plasmas. We first evaluated whether sBTN3Al concentrations obtained with "BTN3 A 1 -specific" dosage correlated with concentrations obtained with "pan-BTN3A" dosage. We found a significant positive correlation between sBTN3A and sBTN3Al concentrations (rS = 0.88; ***p<0.0001). We next compared sBTN3Al concentrations obtained in PDAC patients with concentrations observed in patients with Chronic Calcificating Pancreatitis (CCP) and Intraductal Papillary Mucinous Neoplasm (IPMN). The highest mean concentration was observed in PDAC patients (3.48 ± 0.8 ng/ml) and was significantly higher than in Healthy Donors (1.5 ± 0.3 ng/ml). With regards to overall survival of patients with PDAC, we found that sBTN3A concentrations greater than 8 ng/ml (Figure 6 A) and sBTN3Al concentrations greater than 6 ng/ml were associated with decreased survival (Figure 6B). Altogether these data show that sBTN3A and sBTN3Al could constitute biomarkers reflecting the progression and prognosis of PDAC. Example 2: Prognostic significance of circulating pan-BTN3As and BTN3A1 in patients with non resectable pancreatic adenocarcinoma

Material & Methods

Patients

Three expert clinical centers (Institut Paoli Calmettes, Hospital Nord and Hospital La

Timone) from Marseille participated in this study after receiving ethics review board approval. Thirty-two diagnosed patients with non-resectable PDAC (locally advanced n=12 and already metastatic at diagnosis n=20) were included in this project led by the Paoli-Calmettes Institute (clinical trial NCTO 1692873). Consent forms of informed patients were collected and registered in a central database. All patients were followed from diagnosis until their death or the last follow-up date. Full annotated clinical characteristics were listed in a central database.

Determination of soluble pan-BTN3A and BTN3A1 concentrations in plasma

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.

All 6 ELISAs follow the same schedule: All steps are run at room temperature. Plates are coated overnight with the antibody selected for capturethen washed. Remaining binding sites are blocked to minimize background. All next steps end with plate washing. For PD-L1 assay, all steps are conducted under shaking. Samples to be tested are incubated for 3 h. Then, the biotinylated antibody selected for detection is incubated for 30 min, followed by incubation for 15 min with the avidine -peroxidase conjugate. Finally, the substrate TMB is incubated for 15 min, the reaction stopped with H2S04 and the O.D. read at 450 nm. Concentrations are established by comparison with a range obtained with known concentrations of the recombinant antigen. All recombinant antigens except PD-L1 (obtained from R&D, cat# 156 B7) were synthesized in the laboratory

Studies comparing concentrations of all 6 markers measured in serum and plasma from the same blood collection showed that apparent concentrations in serum were at least ten times less than in plasma. This observation shows that clotting results in the apparent loss of a large part of the assayed proteins. Because the mechanism of such loss is unknown, determination of protein concentrations in serum might be affected by factors other than the clinical status of the patient. As consequence, use of serum samples could be misleading and should be avoided. All samples assayed in this study were plasmas. We also observed in all 6 ELISAs an interference of the plasma matrix, which becomes negligible when plasma samples are diluted at least 1/5. In the present study, all plasma samples were at least diluted 1/5 before assay.

For the ELISA, three isoforms of BTN3A are identified (Al, A2, A3). Among available monoclonal antibodies to BTN3A, one is specific of Al (α-ΒΤΝ3Α1 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 ».

Blood samples

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.

Statistical analysis

Correlation analysis was performed using the Pearson correlation test according to the Gaussian distribution of data. Overall survival analysis was performed using the Kaplan-Meier method and log-rank test. Univariate analysis of survival was performed using the Cox- regression model. The receiver operating characteristic (ROC) curves were generated for each marker. The areas under the curves (AUC) were assessed to evaluate each marker performance for discriminating short term from long term survivors. SPSS PASW 23.0 (SPSS Inc. Chicago, IL, USA) software was used for regression analysis. ROC curves were generated using the MedCalc software for Windows, version 18.2.1 (MedCalc Software, Ostend, Belgium). GraphPad Prism 5.01 (GraphPad software Inc. La Jolla, CA, USA) was used for correlation and Kaplan-Meier survival analysis. P values <0.05 were considerate significant.

Results

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

05 median between 6-8 months for this type of non-operable patients under palliative chemotherapy (Heinemann et al., 2012). We split the cohort in two subgroups according to the

6 months OS cut-off. Patients who died of disease before six months were named short term survival patients (STS, n=16) and those who died after six months were named long term survival patients (LTS, n=16). All the patients characteristics are provided in Table 1. Ninety percent of patients (29/32) received palliative treatments consisting of gemcitabine (n=8), gemcitabine-capecitabine (GEMZAR®/XELODA®) combination treatments (n=2), FOLFIRINOX combination (n=19).

The levels of pan-sBTN3 A are highly correlated in non-operable PDAC patients

In this study, two specific ELISA tests were utilized to measure plasma levels of Pan- sBTN3A and sBTN3Al . Median values were 7.56 ng/ml for sBTN3Al (range 0 to 20.41 ng/ml) and 8.39 ng/ml for pan-sBTN3A (range 0 to 24.33 ng/ml). There is a close correlation in the levels of all immune checkpoint soluble forms. The best correlation (rp=0.93, p=3.2 10-14) is observed between sBTN3Al and the pan-sBTN3A levels.

Plasma levels of sBTN3Al and pan-sBTN3A negatively correlate with overall survival in non resectable PDAC patients

In order to correlate the levels of soluble forms of the immune checkpoints investigated in this study with overall patient's survival, we first used the linear Pearson correlation model. We observed that pan-sBTN3A family and sBTN3Al negatively correlate with OS in advanced pancreatic cancer patients (data not shown) (rp=-0.57, pval=0.0006; rp=-0.6, pval=0.0003, respectively).

Determination of the optimal cut-off for each marker to classify short term versus long term PDAC survivors

We used ROC curves analysis (Hajian-Tilaki, 2013) to determine for each marker the optimal cut-off that discriminates short- versus long-term survivors, As shown in Figure 7 the optimal cut-off for are 6.98 ng/ml for sBTN3Al (AUC = 0.918, pvalO.001) and 6.92 ng/ml for pan-sBTN3A (AUC = 0.922, pvaKO.OOl). The plasma levels of each marker are plotted in the right panels for long term and short term survivors, the dashed lines indicate the threshold level of each marker. Note that soluble forms of BTN3A1 show the most important predictive power.

Clinical characteristics of patients with high plasma concentrations of immune checkpoints.

We classified, for each marker tested, the patients with low and high plasma levels for each immune checkpoint, using cut-offs determined beforehand with ROC curves. We plotted the overall survival for these patients by Kaplan Meier curves (Figure 8). Patients with high level of sBTN3Al have a median survival of 2.8 months compared to 20.0 months for patients with low level of sBTN3Al (log rank pval<0.0001) and patients with high level of pan- sBTN3A have a median survival of 2.5 months compared to 20.0 months for patients with low level of pan-sBTN3A (log rank pvalO.0001).

Conclusion

In conclusion, our study reveals that monitoring the concentration of soluble forms of inhibitory immune checkpoints in plasma like sBTN3Al and pan-sBTN3A can help predict survival in advanced and unresectable PDAC patients.

REFERENCES:

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Claims

CLAIMS:

1. 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 sBTN3 A 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.

2. A method according to claim 1 wherein sBTN3A is sBTN3Al .

3. 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.

4. A method according to claim 3 wherein BTN3A is BTN3A2.

5. A method for predicting the invasiveness of a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTN3 A 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.

6. A method according to claims 1 to 4 wherein 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..

7. 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 according to claims 1 to 4.

8. An anti-CD277 antibody which activates the cytolytic function, cytokine production and proliferation of α/β T cells and/or Vy9/V52 T cells for use in the treatment of pancreatic cancer in a patient with a bad prognosis according to claims 1 to 4.

9. A method for treating a pancreatic cancer in a patient with a bad prognosis according to claim 1 to 4 comprising the administration to said patient of an anti-CD277 antibody which activates the cytolytic function, cytokine production and proliferation of T cells.