WO2020120592A1 - Methods and compositions for predicting and treating melanoma - Google Patents

Abstract

The invention relates to a method for predicting the survival time of a subject suffering from melanoma and treating the subject identified as having a short survival time with an agonist of CELC12B. The inventors have performed transcriptional analysis and several experiments with melanoma cell lines and cells extracted from patients metastases. They have surprisingly identified a new biomarker, CELC12. They have observed that the patients with high expression of CLEC12B have a median survival significantly higher compared to those with a low expression of CLEC12B in their melanomas (3587 vs 1927 days, p=0,0125). Moreover, in order to evaluate the role of CLEC12B in this process, melanoma cell lines (A375 and MeWo) were infected with a lentivirus to overexpress CLEC12B (CLEC12B compare to a control/vector) and to downregulate it (ShCLEC12B compare to a control/vector). Accordingly, inventors have shown that CLEC12B acts as a tumor suppressor gene in melanoma.

Description

METHODS AND COMPOSITIONS FOR PREDICTING AND TREATING

MELANOMA

FIELD OF THE INVENTION:

The present invention is in the field of oncology, more particularly, the invention relates to methods and composition for treating melanoma.

BACKGROUND OF THE INVENTION:

Melanoma is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin, but are also found in other parts of the body, including the bowel, oral cavity and the eye. Melanin has the role of an effective absorber of light; the pigment is able to dissipate over 99.9% of absorbed ultraviolet (UV) radiation. Because of this property, melanin is thought to protect skin cells from UVB radiation damage, reducing the risk of cancer. When people spend time in the sunlight, the melanocytes make more melanin and cause the skin to tan. This also happens when skin is exposed to other forms of ultraviolet light (such as in a tanning booth). If the skin receives too much UV radiation, the melanocytes may begin to grow abnormally and become cancerous, leading to melanoma.

Because of the link to sun exposure, the chance of getting melanoma increases with age, but many young people age also get melanoma. In fact, melanoma is one of the most common cancers in young adults. Each year in the U.S., more than 50,000 people young and old learn that they have melanoma. And, according to a WHO report, about 48,000 melanoma related deaths occur worldwide each year. The treatment includes surgical removal of the tumor, and if melanoma is found early while relatively small and thin, complete removal gives a high cure rate. The chance of the melanoma coming back or spreading depends on how deeply it has invaded into the layers of the skin, and thus, the more progressed the lesion, the great the chancer for recurrence and/or metastasis. For melanomas that recur or spread, treatments include chemo- plus immunotherapy or radiation therapy, but the prognosis for such patients, including those exhibiting metastatic disease (AJCC Stage III and IV) remains poor despite the recent progresses allowed by targeted therapies and immune therapies. As such, news methods and biomarkers are needed to determine the prognosis at early stage of melanoma and to monitor the patients suffering from melanoma. SUMMARY OF THE INVENTION:

The invention relates to a method for predicting the survival time of a subject suffering from melanoma comprising the steps of i) quantifying the expression level of CLEC12B in a biological sample obtained from the subject, ii) comparing the expression level of CLEC12B at step i) with its corresponding predetermined reference value and iii) concluding that the subject will have a short survival time when the expression level of CLEC12B is lower than its corresponding predetermined reference value or concluding that the subject will have a long survival time when the expression level CLEC12B is higher than its corresponding predetermined reference value. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors have performed transcriptional analysis and several experiments with melanoma cell lines and cells extracted from patient metastases. They have surprisingly identified a new biomarker, CELC12. They have observed that the patients with high expression of CLEC12B have a median survival significantly higher compared to those with a low expression of CLEC12B in their melanomas (3587 vs 1927 days, p=0,0125). In order to evaluate the role of CLEC12B in this process, melanoma cell lines (A375 and MeWo) were infected with a lentivirus to overexpress CLEC12B (CLEC12B compare to a control/vector) and to downregulate it (ShCLEC12B compare to a control/vector). These results show that CLEC12B acts as a tumor suppressor gene in melanoma. Thus, the inventors have identified a new biomarker suitable for determining the prognosis and monitoring subjects suffering from melanoma and a target to treat melanoma.

Accordingly, in a first aspect, the present invention relates to a method for predicting the survival time of a subject suffering from melanoma comprising the steps of: i) quantifying the expression level of CLEC12B in a biological sample obtained from the subject, ii) comparing the expression level of CLEC12B at step i) with its corresponding predetermined reference value and iii) concluding that the subject will have a short survival time when the expression level of CLEC12B is lower than its corresponding predetermined reference value or concluding that the subject will have a long survival time when the expression level CLEC12B is higher than its corresponding predetermined reference value.

As used herein, the term“CLEC12B” has its general meaning in the art and refers to C-Type Lectin Domain Family 12 Member B. Its Entrez Gene reference is 387837 and its Uniprot number is Q2HXU8. As used herein, the term“method for predicting survival time” refers to the method for predicting the duration of the overall survival (OS), progression-free survival (PFS) and/or the disease-free survival (DFS) of the cancer subject. Those of skill in the art will recognize that OS survival time is generally based on and expressed as the percentage of people who survive a certain type of cancer for a specific amount of time. Cancer statistics often use an overall five-year survival rate. In general, OS rates do not specify whether cancer survivors are still undergoing treatment at five years or if they have become cancer-free (achieved remission). DSF gives more specific information and is the number of people with a particular cancer who achieve remission. Also, progression-free survival (PFS) rates (the number of people who still have cancer, but their disease does not progress) include people who may have had some success with treatment, but the cancer has not disappeared completely. As used herein, the expression“short survival time” indicates that the subject will have a survival time that will be lower than the median (or mean) observed in the general population of subjects suffering from said cancer. When the subject will have a short survival time, it is meant that the subject will have a“poor prognosis”. Inversely, the expression“long survival time” indicates that the subject will have a survival time that will be higher than the median (or mean) observed in the general population of subjects suffering from said cancer. When the subject will have a long survival time, it is meant that the subject will have a“good prognosis”.

As used herein, the term“subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject is a human is suffering or is susceptible to suffer from melanoma.

As used herein, the term“melanoma” also known as malignant melanoma refers to, is a type of cancer that develops from the pigment-containing cells known as melanocytes. Melanomas are usually caused by DNA damage resulting from exposure to ultraviolet (UV) light from the sun or other sources (such as tanning booth). The American Joint Committee on Cancer (AJCC) TNM classification is the most widely accepted staging system for cutaneous melanoma. It is based on the combination of three factors: 1) tumor depth (T), as described by Breslow's thickness (expressed in millimeters), 2) lymph node status (N), including in-transit metastasis, and 3) distant metastasis (M), including plasma levels of lactate dehydrogenase (LDH). Their combination defines four stages and nine substages. In a particular embodiment, the subject suffers from early stage of melanoma. As used herein, the term“early stage of melanoma” refers to melanomas stages 0 and I which are localized: stage 0 tumors are in situ, meaning that they are noninvasive and have not penetrated below the surface of the skin, while stage I tumors have invaded the skin but are small, nonulcerated, and are growing at a slow mitotic rate. In a particular embodiment, the subject suffers from metastatic melanoma. As used herein, the term“metastatic melanoma”, also known as Stage IV melanoma, is used when melanoma cells of any kind (cutaneous, mucosal or ocular) have spread through the lymph nodes to distant sites in the body and/or to the body's organs. The liver, lungs, bones and brain are most often affected by these metastases.

As used herein, the term“expression level” refers to the expression of CLEC12B at genomic and/or nucleic and/or protein level.

As used herein, the term“biological sample” refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy. In a particular embodiment, the biological samples for the determination of an expression level include samples such as a blood sample, a lymph sample, or a biopsy. In a particular embodiment, the biological sample is a tumor tissue sample obtained from a subject suffering from melanoma. As used herein, the term“tumor tissue sample” has its general meaning in the art and encompasses pieces or slices of tissue that have been removed including following a surgical tumor resection. The tumor tissue sample can be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.) prior to determining the cell densities.

In a further embodiment, the invention is suitable to determining whether a subject suffering from melanoma will achieve a response with an immune-checkpoint inhibitor. Accordingly, in a further embodiment, the present invention relates to a method for determining whether a subject suffering from melanoma will achieve a response with an immune-checkpoint inhibitor.

In particular, the present invention relates to a method for determining whether a subject suffering from melanoma will achieve a response with an immune-checkpoint inhibitor comprising the steps of i) quantifying the expression level of CLEC12B in a biological sample obtained from the subject treated with an immune-checkpoint inhibitor, ii) comparing the expression level of CLEC12B at step i) with its corresponding predetermined reference values and iii) concluding that the subject will not respond to the treatment when the expression level of CLEC12B is lower than its corresponding predetermined reference value or concluding that the subject will respond to the treatment when expression level of CLEC12B is higher than its corresponding predetermined reference value. As used herein, the term“respond” refers when the survival time of the subject is increased with a treatment. In particular, in the context of the invention, the term“respond” refers to the ability of the immune system to decrease tumour masse, thus, the subject presents a clinical improvement compared to the subject who does not receive the treatment. The said subject is considered as a“responder” to the treatment. The term“not respond” refers to a subject who does not present any clinical improvement to the treatment with an immune checkpoint inhibitor treatment. This subject is considered as a “non-responder” to the treatment.

As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA- 4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T- Lymphocyte-Associated protein 4 and also called CD 152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2, 3 -dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.

In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.

In a particular embodiment, the immune checkpoint inhibitor is an antibody.

Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.

In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002,

WO2010117057 and W02013006490.

In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.

In a particular embodiment, the small organic molecules interfere with Indoleamine- pyrrole 2, 3 -dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1 -methyl-tryptophan (IMT), b- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- m ethoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5-bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a b- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1 -methyl-tryptophan, b-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and b-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof. In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4- fluorophenyl)-N'-hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide :

In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-1, 2, 4-Triazole-3, 5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:

In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA- 170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

In some embodiments, the immune checkpoint inhibitor is an aptamer.

Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015. In a particular embodiment, the expression level of CLEC12B is determined by Immunohistochemistry (IHC). Immunohistochemistry typically includes the following steps i) fixing the tumor tissue sample with formalin, ii) embedding said tumor tissue sample in paraffin, iii) cutting said tumor tissue sample into sections for staining, iv) incubating said sections with the binding partner specific for the marker, v) rinsing said sections, vi) incubating said section with a secondary antibody typically biotinylated and vii) revealing the antigen-antibody complex typically with avidin-biotin-peroxidase complex. Accordingly, the tumor tissue sample is firstly incubated the binding partners. After washing, the labeled antibodies that are bound to marker of interest are revealed by the appropriate technique, depending of the kind of label is borne by the labeled antibody, e.g. radioactive, fluorescent or enzyme label. Multiple labelling can be performed simultaneously. Alternatively, the method of the present invention may use a secondary antibody coupled to an amplification system (to intensify staining signal) and enzymatic molecules. Such coupled secondary antibodies are commercially available, e.g. from Dako, EnVision system. Counterstaining may be used, e.g. Hematoxylin & Eosin, DAPI, Hoechst. Other staining methods may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems. For example, one or more labels can be attached to the antibody, thereby permitting detection of the target protein (i.e the marker). Exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof. In some embodiments, the label is a quantum dot. Non-limiting examples of labels that can be conjugated to primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bioluminescent proteins (e.g. luciferin, luciferase), haptens (e.g. biotin). A variety of other useful fluorescers and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke J R (1972) Annu. Rev. Biochem. 41 :843-868. Affinity ligands can also be labeled with enzymes (e.g. horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g. ³H, ¹⁴C, ³²P, ³⁵S or ¹²⁵I) and particles (e.g. gold). The different types of labels can be conjugated to an affinity ligand using various chemistries, e.g. the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can be used, e.g. aldehydes, carboxylic acids and glutamine. Various enzymatic staining methods are known in the art for detecting a protein of interest. For example, enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC or Fast Red. In other examples, the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody. In an indirect IHC assay, a secondary antibody or second binding partner is necessary to detect the binding of the first binding partner, as it is not labeled. The resulting stained specimens are each imaged using a system for viewing the detectable signal and acquiring an image, such as a digital image of the staining. Methods for image acquisition are well known to one of skill in the art. For example, once the sample has been stained, any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as, for example, upright or inverted optical microscopes, scanning confocal microscopes, cameras, scanning or tunneling electron microscopes, canning probe microscopes and imaging infrared detectors. In some examples, the image can be captured digitally. The obtained images can then be used for quantitatively or semi-quantitatively determining the amount of the marker in the sample, or the absolute number of cells positive for the maker of interest, or the surface of cells positive for the maker of interest. Various automated sample processing, scanning and analysis systems suitable for use with IHC are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS-200 system (Becton, Dickinson & Co.). In particular, detection can be made manually or by image processing techniques involving computer processors and software. Using such software, for example, the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see e.g., published U.S. Patent Publication No. US20100136549). The image can be quantitatively or semi-quantitatively analyzed and scored based on staining intensity of the sample. Quantitative or semi-quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry, to identify and quantitate the presence of the specified biomarker (i.e. the marker). Quantitative or semi-quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically. For example, images can be quantitatively analyzed using a pixel count algorithms and tissue recognition pattern (e.g. Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), or Tribvn with Ilastic and Calopix software), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat. No. 7,646,905; published U.S. Patent Publication No. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8: 1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328). A ratio of strong positive stain (such as brown stain) to the sum of total stained area can be calculated and scored. The amount of the detected biomarker (i.e. the marker) is quantified and given as a percentage of positive pixels and/or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels. For example, a sample can have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels as compared to the total staining area. For example, the amount can be quantified as an absolute number of cells positive for the maker of interest. In some embodiments, a score is given to the sample that is a numerical representation of the intensity or amount of the histochemical staining of the sample, and represents the amount of target biomarker (e.g., the marker) present in the sample. Optical density or percentage area values can be given a scaled score, for example on an integer scale. Thus, in some embodiments, the method of the present invention comprises the steps consisting in i) providing one or more immunostained slices of tissue section obtained by an automated slide-staining system by using a binding partner capable of selectively interacting with the marker (e.g. an antibody as above described), ii) proceeding to digitalisation of the slides of step i).by high resolution scan capture, iii) detecting the slice of tissue section on the digital picture iv) providing a size reference grid with uniformly distributed units having a same surface, said grid being adapted to the size of the tissue section to be analyzed, and v) detecting, quantifying and measuring intensity or the absolute number of stained cells in each unit whereby the number or the density of cells stained of each unit is assessed.

As used herein, the term“corresponding predetermined reference value” refers to a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of cell densities in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the cell density in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured densities in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER. S AS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE- ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

Inventors have shown that CLEC12B, a new gene which is implicated in the melanomagenesis process in acting as a tumor suppressor gene. Loss of CLEC12B promotes melanomagenesis in vitro and in vivo and overexpression induces the opposite. CLEC12B is a transmembrane receptors, thus its identification as a player in melanomagenesis is of great interest and could provide a novel target for anti-melanoma agents. According, in a second aspect, the invention relates to a method for treating melanoma in a subject in need thereof, comprising a step of administering to said subject a therapeutically effective amount of an agonist of CLEC12B.

In a particular embodiment, the method according to the invention, wherein the method comprising i) a first step consisting in determining the survival time of the subject by the method of invention as described above and ii) administering to said subject a therapeutically amount of agonist of CLEC12B. In a particular embodiment, the conventional treatment or a combination of thereof can be combined with an agonist of CLEC12B when the expression level of CLEC12B is lower than its corresponding predetermined reference value.

In a particular embodiment, the invention relates to use of an agonist of CLEC12B in the treatment of melanoma.

As used herein, the terms“treating” or“treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term“agonist of CLEC12B” refers to a natural or synthetic compound which binds and activates CLEC12B for initiating a pathway signalling and further biological processes. Typically, the agonist of CLEC12B through a dephosphorylation of SHP2 (Y542) negatively regulates S6K, STAT 1, 3 and 5 and promotes p38 activation. In the context of the invention, the agonist of CLEC12B inhibits proliferation and migration of melanoma cells in a subject. Typically, the agonist of CLEC12B is CLEC12B, an aptamer, a small molecule, an antibody, a peptide, a polypeptide peptidomimetic or glycomimetic.

In a particular embodiment, the agonist of CLEC12B is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In a particular embodiment, the agonist of CLEC12B is a small molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In a particular embodiment, the agonist of CLEC12B is a peptide. In a particular embodiment, the CLEC12B antagonist is a polypeptide. The term“polypeptide” refers both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups). In a particular embodiment, the polypeptide is a functional equivalent fragment of CELC12B. As used herein, a“functional equivalent” also known as a decoy or "decoy receptor", as "sink" or "trap" is a compound which is capable of binding to soluble CLEC12B, thereby stimulating its interaction with CLEC12B receptor. More particularly, it is a compound that binds to a ligand, and is structurally capable of signaling or presenting the agonist to signaling receptor complexes. In a particular embodiment, the polypeptide is a peptidomimetic. As used herein, the term“peptidomimetic” refers to a polypeptide designed to mimic a peptide. The polypeptide may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of CLEC12B or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

In some embodiments, the agonist of CLEC12B is a glycomimetic molecule. As used herein, the term“glycomimectic molecule” refers to compounds that mimic the bioactive function of carbohydrates and address the drawbacks of carbohydrate leads, namely their low activity and insufficient drug-like properties. Typically, the glycomimectic molecule is selected from the group consisting of: Cylexin (CY-1503), Bimosiamose (TBC-1269), OJ- R9188, GMI-1070, PSI-697, GSC-150, Efomycin M, as described in Ernst et al 2009, Nature Reviews Drug Discovery.

In a particular embodiment, the agonist of CLEC12B is an antibody. The term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs or VHH), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. In some embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In some embodiments, the antibody is non-internalizing. As used herein the term “non-internalizing antibody” refers to an antibody, respectively, that has the property of to bind to a target antigen present on a cell surface, and that, when bound to its target antigen, does not enter the cell and become degraded in the lysosome.

Particularly, in the context of the invention, the antibody is a single domain antibody. The term“single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also called VHH or“nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends Biotechnol, 2003, 21(11):484-490; and WO 06/030220, WO 06/003388. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/). Particularly, in the context of the invention, the antibody is a single chain variable fragment. The term "single chain variable fragment" or "scFv fragment" refers to a single folded polypeptide comprising the VH and VL domains of an antibody linked through a linker molecule. In such a scFv fragment, the VH and VL domains can be either in the VH - linker - VL or VL - linker - VH order. In addition to facilitate its production, a scFv fragment may contain a tag molecule linked to the scFv via a spacer. A scFv fragment thus comprises the VH and VL domains implicated into antigen recognizing but not the immunogenic constant domains of corresponding antibody.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an agonist of CLEC12B) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

A“therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In a second aspect, the invention relates to a pharmaceutical compositions comprising an agonist of CLEC12B for use in the treatment or the prevention of melanoma.

The agonists of CLEC12B as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer 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 pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral- route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, 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 pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. In fourth aspect, the invention relates to i) an agonist of CLEC12B as described above and ii) a conventional treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of melanoma.

As used herein, the term “simultaneous” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “separate” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “sequential” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

As used herein, the term“conventional treatment” refers to the treatment that is widely accepted and used by most healthcare professionals. Examples of conventional treatment for cancer include immunotherapy, chemotherapy, radiation therapy, and surgery.

In a particular embodiment, the conventional treatment is a treatment with the inhibitors of BRAF mutations. BRAF is a member of the Raf kinase family of serine/threonine-specific protein kinases. This protein plays a role in regulating the MAP kinase / ERKs signaling pathway, which affects cell division, differentiation, and secretion. A number of mutations in BRAF are known. In particular, the V600E mutation is prominent. Other mutations which have been found are R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, K600E, A727V, and most of these mutations are clustered to two regions: the glycine-rich P loop of the N lobe and the activation segment and flanking regions. In a particular embodiment, the BRAF mutation is V600E.

In a particular embodiment, the conventional treatment is the treatment with the inhibitors of BRAF mutations are well known in the art. In a particular embodiment, the melanoma is resistant to a treatment with vemurafenib. Vemurafenib also known as PLX4032, RG7204 ou R05185426 and commercialized by Roche as Zelboraf. In a particular embodiment, the melanoma is resistant to a treatment with dacarbazine. Dacarbazine also known as imidazole carboxamide is commercialized as DTIC-Dome by Bayer. In a particular embodiment, the melanoma is resistant to a treatment with dabrafenib also known as tafmlar which is commercialized by Novartis.

In a further embodiment, the conventional treatment is the treatment with the inhibitors of MEK. MEK refers to Mitogen-activated protein kinase kinase, also known as MAP2K, MEK, MAPKK. It is a kinase enzyme which phosphorylates mitogen-activated protein kinase (MAPK). MEK is activated in melanoma. The inhibitors of MEK are well known in the art. In a particular embodiment, the melanoma is resistant to a treatment with trametinib also known as mekinist which is commercialized by GSK. In a particular embodiment, the melanoma is resistant to a treatment with cobimetinib also known as cotellic commercialized by Genentech. In a particular embodiment, the melanoma is resistant to a treatment with Binimetinib also knowns as MEK162, ARRY-162 is developed by Array Biopharma.

In a particular embodiment, the conventional treatment is the treatment with the inhibitors of NRAS. The NRAS gene is in the Ras family of oncogene and involved in regulating cell division. NRAS mutations in codons 12, 13, and 61 arise in 15-20 % of all melanomas. The inhibitors of BRAF mutation or MEK are used to treat the melanoma with NRAS mutations. In a particular embodiment, the melanoma is resistant in which double negative BRAF and NRAS mutant melanoma.

In a particular embodiment, the conventional treatment is the treatment with an immune checkpoint inhibitor as described above.

A further object of the present invention relates to a method of screening a drug suitable for the treatment of melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to activate the activity of CLEC12B.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to activate the activity of CLEC12B. In some embodiments, the assay first comprises determining the ability of the test compound to bind to CLEC12B. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of CLEC12B. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of activating the activity of CLEC12B, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, glycomimemtic, antibody, small organic molecules, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

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: Lower CLEC12B mRNA shortens melanoma patient survival. Kaplan- Meier survival curves for CLEC12B“high” tumors (n=321) versus CLEC12B“low” tumors (n=137). Data were extracted from Tumor Cancer Genome Atlas (TCGA) database (n=458 samples from primary and metastatic melanoma). A high level expression of CLEC12B was defined as a level expression upper or equal to 1 Transcripts Per Kilobase Million (TPM) and low level lower than 1 TPM. Survival analysis was performed according to the Kaplan- Meier method and the log-rank test was used to assess the significance. Significance defined as * p<0,05; ** p<0,01.

Figure 2: CLEC12B inhibits A375 melanoma cells xenograft tumor growth in vivo. The tumor growth and tumor volume is significantly (A) slower and weaker in Ov- CLEC12B (Ov) compared to empty vector (EV) and (B) is higher and faster in Sh-CLEC12B (Sh) compared to empty vector’ (EV’). Twenty- four nude mice were injected (on 7^th March) with A375 melanoma cells (4 groups and 6 mice per group; CLEC12B/vector and ShCLEC12B/vector). A total of 4x l0⁶ (100 pi) were subcutaneously implanted in right flank using a 27-gauge needle. Tumor size was measured with calipers. Tumor volume (V) was determined by the equation V = (L xw x h )p/6 , in which L is the length, w is the width and h is the height. When tumors reached volumes of -200 mm3, the mice were sacrificed.

EXAMPLE:

Material & Methods :

Melanoma cells (A375 and MeWo) were cotransfected with a puromycin resistance vector and either a lentivirus overexpressing CLEC12B (Ov CLEC12B) or silencing CLEC12B (Sh CLEC12B), or control (vector) and protein lysate was analyzed by western blot. The molecular weight in kDa is indicated. Actin was the loading control. Western blot analysis of CLEC12B, SHP2, STAT1, STAT3, STAT5 and their phosphorylated forms (PTyr542SHP2 = P-SHP2, PSer727-STATl = P-STAT1 PTyr705-STAT3 = P-STAT3, PTyr694-STAT5 = P-STAT5). CLEC12B inhibits proliferation and colony formation in melanoma cell lines

Melanoma cells (A375 (a-c) and MeWo (b-d)) were cotransfected with a puromycin resistance vector and either a lentivirus overexpressing CLEC12B (Ov CLEC12B), silencing CLEC12B (Sh CLEC12B) or control (vector) and were grown in medium, supplemented with 10% fetal bovine serum, containing puromycin for several days, according to the type of experiments and cell lines. For each experiment, (a-b) cell number and (c-d) number of colony, results are expressed in percent of control (100%). Data are mean ± S.D. of three independent experiments performed in triplicate. Significantly different from the corresponding control *P<0.01; **P<0.001; ***P<0.0001.

Cell cultures - transfection

Melanoma cells lines were from; MeWo (ATCC #HTB-65, LGC Standards S.a.r.l. 6, Molsheim, France), and A375. A375 (BRAV600E mutated, NRAS wild-type) and MeWo (BRAV600E and NRAS wild-type, deletion exon 2 of CDKN2A) cells were cultured at 37°C and 5% C02 in RPMI supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 IU; 50 g mil). To generate stably-transfected Sh CLEC12B and Over CLEC12B cell lines, A375 and MeWo cells were cotransfected with a puromycin resistance vector and either a lentivirus overexpressing CLEC12B (Ov CLEC12B) or silencing CLEC12B (Sh CLEC12B), or control (vector).

Cell proliferation assay

Cells were plated in six-well tissue culture plates at a density of 20000 cells per well for A375 melanoma cells and 60000 for MeWo melanoma cells. Then the cell number was counted with Malassez chamber at 72hours and 48 hours for A375 and MeWo cells respectively.

Colony formation assay

Cells were plated in six-well tissue culture plates at a density of 500 cells per well. After 12 days for A375 melanoma cells and 14 days for MeWo melanoma cells, colonies were fixed with ethanol 70% and stained with 2% crystal violet, washed with water to remove the excess dye, and imaged by a scanner. Quantitative changes in clonogenicity were determined by counting the colonies, using Bio-Rad Vision-Capt software.

In vivo experiments - tumor xenograft experiments

Swiss nude mice were purchased from Charles River Laboratories. Animal experiments were carried out in accordance with the Declaration of Helsinki Principles and were approved by a local ethics committee. All mice were housed under pathogen-free conditions in the animal facility and received autoclaved water and food. 9 weeks old Swiss nude mice were used in the study. Thirty nude mice were injected with A375 melanoma cells (4 groups and 6 mice per group; Over CLEC12B/vector and Sh CLEC12B/vector). A375 cells were suspended in phosphate-buffered saline (PBS) at 1 x 106 and 4 ^c 106 x l06 cells/ml. A total of 1 x 106 and 4x 106 cells (100 pi) were subcutaneously implanted into both left and right flank using a 27-gauge needle. Tumor size was measured with calipers. Tumor volume (V) was determined by the equation V = (L xw x h )p/6 , in which L is the length, w is the width of the tumor and h is the height. The tumor growth (with the weight of mice) were monitored 3 days by weeks. When tumors reached volumes of -200 mm3, the mice were sacrificed or before in case of signs of discomfort (weight loss, signs of pain...).

Immunofluorescence of tumor samples

Paraffine-embedded tumors were sectioned into 7-pm-thick transverse sections. After dewax and rehydratation, they are rinsed in Tween Buffer saline (TBS; Tris (Sigma: T-1503) 20mM pH 7.6), 150mM NaCl (OSI, A 4321152) and Tween-20 (VWR 8221840500) 0.1% [v/v]), boiled for 20 min in lOmM sodium citrate and block-end with TBST (TBS/0.1% Tween-20) containing 3% Bovine serum albumin (BSA, Sigma A9418). Sections were incubated overnight at 4°C in TBST (TBS/0.1% Tween-20) containing 3% BSA with antibodies against p-STAT3 (Cell signaling, #9131), or CLEC12B (Proteintech, #26077-1- AP). Donkey anti-Rabbit IgG (H&L), Affinity Pure DyLight® 488 (Diagomics, ref DkxRb- 003-F488NHSX) and Alexa fluor 594 donkey anti-rabbit IgG (Life technologies - Thermo Fisher ref A-21207) secondary antibodies were used for 25 minutes at room temperature (RT). All sections were mounted with Fluoroshield with DAPI mounting medium (Sigma, ref F6057). Images were taken using the ZEISS Axio Imager 2 with Axiocam 506 color cameras. Image analysis was performed in ZEISS ZEN, Adobe Photoshop, and ImageJ software”.

Western blotting, antibodies and detection

Cells were grown in six-well dishes at the time indicated in the corresponding figure. Whole-cell lysate was prepared from human melanoma cell lines using RIPA buffer supplemented with Complete inhibitor (Roche, #11873580001) and PhosStop (Roche, #04906837001) followed by centrifugation at 15,000g for 20 min at 4°C. Total protein amounts in cell lysates were quantified. SDS-PAGE was carried out on homemade 10% or 12% polyacrylamide protein gels. Membranes were blocked in TBST with 5% non-fat dry milk (for 1 hour at RT, afterwards probed with antibodies against CLEC12B (Proteintech, #26077- 1-AP), b-actin (Sigma, A5441), STAT3 (BD, #610189) phospho-STAT3 (Tyr 705, BD, #612356), Primary antibodies were applied in TBST 5% non-fat dry milk overnight at 4°C and visualized using secondary antibodies (goat anti-rabbit IgG, HRP conjugated, Jackson, 111-035-003 and goat anti-mouse IgG, HRP conjugated, Jackson, 115-035-003) in TBST 5% non-fat dry milk for 1 hour at RT. Blots were incubated in ECL (Pierce, #34075) and revealed in the dark chamber using hyperfilm ECL (GE Healthcare, RPN3103K). All primary antibodies were used at a dilution of 1/1,000, except b-actin (1/10,000) and phospho- STAT3 (1/5000). All secondary antibodies were used at dilution of (1/20,000). Molecular marker (Pageruler, ThermoFisher, #26616).

Results

CLEC12B shows clinical significance in melanoma.

To explore the clinical significance of CLEC12B in melanoma, we investigated its expression in human melanoma samples. We used the human melanoma dataset generated by The Cancer Genome Atlas (TCGA) and analyzed its mRNA level. Interestingly, the Kaplan- Meier survival analysis for patients in this dataset revealed that those with high CLEC12B expression had a significantly higher median survival than those with low expression (Log- rank Test, p=0,006, Figure 1).

CLEC12B inhibits proliferation and colony formation in melanoma cell lines

In order to evaluate the biological function of CLEC12B in melanoma, we firstly examined the effect of its modulation on melanoma cell proliferation and colony formation. To this end, we established stably transfected melanoma cell lines, by infecting them with a lentivirus to overexpress CLEC12B (Ov CLEC12B compare to a vector) or to downregulate it (Sh CLEC12B compare to a vector). The cell proliferation was measured by Malassez chambers and CLEC12B overexpression inhibits cell proliferation by -60% and conversely CLEC12B knockdown promotes that by -100% (data not shown). We investigated the effect of CLEC12B on its impact on the ability of melanoma cells to form colonies using colony formation assay. Compared to vector control, -55% less colonies were observed in CLEC12B overexpressing melanoma cells. The opposite effect was observed after CLEC12B knockout, with more -75% colonies observed compared to vector control (data not shown). The same results were observed in MeWo melanoma cells (data not shown).

Collectively, the above results indicate that CLEC12B inhibits proliferation and colony formation in melanoma cell lines.

CLEC12B inhibits the activation of STAT1, STAT3, and STAT5 and increases the expression of p21/p53 and p27.

The inhibitory function of CLEC12B is mediated by the recruitment of the tyrosine phosphatases SHP1 and SHP2 to its ITIM domain upon receptor phosphorylation in human epithelial embryonic-kidney 293T cells f The recruitment and activation of SHP2 induces its own rapid dephosphorylation and, thus, after being activated, SHP2 becomes dephosphorylated ². CLEC12B overexpression induced dephosphorylation of SHP2 (Y542) (data not shown) and pSHPY542 levels were significantly higher in CLEC12B silenced cells (data not shown). We further generated a point mutant of CLEC12B by exchanging the ITIM tyrosine with phenylalanine (Ov-mut). Co-immunoprecipitation assays showed an interaction between CLEC12B and SHP2 in melanoma cells. Moreover, the recruitment of SHP2 no longer occurred when using the Ov-mut (data not shown). As expected, the effect on pSHP2Y542 observed after CLEC12B overexpression no longer occurred with the Ov-mut, either in A375 or MeWo cells (data not shown). Overexpression of CLEC12B with an altered ITIM domain had a significantly lower antiproliferative effect than overexpression of the wildtype form (data not shown).

Overall, these data demonstrate that CLEC12B directly recruits and activates SHP2 through its ITIM domain in melanoma cells. SHP2 plays a dual role in cancer, as it stimulates the MAPK pathway but also inhibits the STATs ^{3 5}. The JAK/STAT signaling pathway plays a crucial role in melanoma and is generally constitutively activated during melanoma progression ⁶’⁷. SHP2 appears to play a critical role in regulating the JAK/STAT pathway in cancer, particularly in melanoma ^{8 u}. We thus assessed whether modulating CLEC12B levels can affect the JAK/STAT pathway. STAT1, STAT3, and STAT5 phosphorylation was significantly lower in CLEC12B-overexpressing A375 cells than those carrying the control vector (data not shown). In contrast, Sh-mediated CLEC12B downmodulation promoted phosphorylation of STAT1, STAT3, and STAT5 (data not shown). We observed the same effects on the activation of STATs in MeWo cells (data not shown). SHP2 also appears to regulate the MAPK pathway ^{12 14}, especially in melanoma cells not harboring mutations in BRAF or NRAS. However, we did not observe any modification of ERK or pERK when we modulated CLEC12B expression in A375 (BRAFV600E, NRASWT) or MeWo cells (BRAFWT, NRASWT) (data not shown). We further aimed to assess whether inhibition of the STATs was due to the recruitment of SHP2 by CLEC12B. Use of the altered-ITIM mutant of CLEC12B showed that the decrease of pSHP2 observed after CLEC12B overexpression was accompanied by lower phosphorylation of STAT3 (data not shown) and that these effects were lost when the Ov-mut was expressed.

Overall, our results indicate that the recruitment of SHP2 by CLEC12B may activate SHP2, which in turn leads to the dephosphorylation of STATs and lower proliferative capacity of the cells. We studied the role of CLEC12B in the cell cycle to better understand the mechanisms involved in its regulation of melanoma-cell proliferation. The percentage of cells in the G0-G1 phase of the cell cycle increased significantly in CLEC12B-overexpressing A375 cells (data not shown). In contrast, the percentage of cells in S phase decreased significantly in CLEC12B-silenced cells (data not shown). Moreover, the modulation of CLEC12B expression did not significantly modify the number of cells in the SubGl phase, showing that the observed effects were not due to apoptotic cell death. In accordance with these results, western-blot analysis showed elevated levels of p21, p27, and p53 in CLEC12B- overexpressing A375 cells (data not shown). Conversely, the level of these proteins was reduced when CLEC12B was knocked down (data not shown).

CLEC12B suppresses tumor development in vivo

Given the observed promising in vitro role of CLEC12B in blocking melanomagenesis process, we wanted to provide further proof-of-concept studying its role in vivo. Tumorigenic properties of CLEC12B were analyzed in swiss nude mice. Tumor size was monitored for 42 days after inoculation of A375 melanoma cell lines. In accordance with in vitro results, the tumor growth and tumor volume in the CELC12B group were significantly smaller than those in vehicle control group and opposite observation was done with ShCLEC12B compare to vector (Figure 2). These data suggest that CLEC12B could be unfavorable for tumor invasion.

Moreover, consistent with the observations in cultured A375 cells, CLEC12B overexpression leads to decreased pSTAT3 level in xenograft tumor samples and opposite effect was observed after turning off CLEC12B. Otherwise, the immunofluorescence analyses of tumor samples revealed that CLEC12B represses the level of pSTAT3 level, indicating that CLEC12B attenuated tumor cell proliferation (data not shown).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method for predicting the survival time of a subject suffering from melanoma comprising the steps of: i) quantifying the expression level of CLEC12B (C-Type Lectin Domain Family 12 Member B) in a biological sample obtained from the subject, ii) comparing the expression level of CLEC12B at step i) with its corresponding predetermined reference value and iii) concluding that the subject will have a short survival time when the expression level of CLEC12B is lower than its corresponding predetermined reference value or concluding that the subject will have a long survival time when the expression level CLEC12B is higher than its corresponding predetermined reference value.

2. A method for determining whether a subject suffering from melanoma will achieve a response with an immune-checkpoint inhibitor comprising the steps of i) quantifying the expression level of CLEC12B in a biological sample obtained from the subject treated with an immune-checkpoint inhibitor, ii) comparing the expression level of CLEC12B at step i) with its corresponding predetermined reference values and iii) concluding that the subject will not respond to the treatment when the expression level of CLEC12B is lower than its corresponding predetermined reference value or concluding that the subject will respond to the treatment when expression level of CLEC12B is higher than its corresponding predetermined reference value.

3. The method according to claims 1 to 2, wherein the biological sample is a tumor tissue sample.

4. The method according to claims 1, to 2 wherein the expression level of CLEC12B is determined by Immunohistochemistry (IHC).

5. A method for treating melanoma in a subject in need thereof, comprising a step of administering to said subject a therapeutically effective amount of an agonist of CLEC12B.

6. The method according to claim 5, wherein the method comprising i) a first step consisting in determining the survival time of the subject by the method of claim 1 and ii) administering to said subject a therapeutically amount of agonist of CLEC12B, when the expression level of CLEC12B is lower than its corresponding predetermined reference value.

7. The method according to claim 5, wherein the method further comprises i) an agonist of CLEC12B and ii) a conventional treatment, as a combined preparation for simultaneous, separate or sequential use in the treatment of melanoma..

8. The method according to claims 5 to 7, wherein the agonist of CLEC12B is CLEC12B, an antibody or a peptide.

9. A method of screening a drug as being useful for the treatment of melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to activate the activity of CLEC12B.