WO2018134356A1

WO2018134356A1 - Method for predicting the clinical evolution of cancer patients

Info

Publication number: WO2018134356A1
Application number: PCT/EP2018/051315
Authority: WO
Inventors: Laurence Chaperot; Julie CHARLES; Joel Plumas; Caroline Aspord
Original assignee: Etablissement Français Du Sang; Institut National De La Sante Et De La Recherche Medicale (Inserm); Centre Hospitalier Universitaire Grenoble Alpes; Universite Grenoble Alpes
Priority date: 2017-01-20
Filing date: 2018-01-19
Publication date: 2018-07-26

Abstract

The invention pertains to an in vitro method for predicting the clinical evolution of a patient suffering from cancer, said method comprising the steps of: a) obtaining a plasmacytoid dendritic cell (pDC) line pulsed with at least one tumoral antigen, and irradiated; said pDC line sharing at least one major histocompatibility complex (MHC) allele with PBMC from the patient suffering from cancer; b) bringing the pulsed and irradiated pDC line obtained at step a) into contact with PBMC from the patient suffering from cancer, and co-culturing the pulsed and irradiated pDC line and the PBMC; c) bringing the pulsed and irradiated pDC line obtained at step a) again into contact with the co-culture obtained at step b), and co-culturing the pulsed and irradiated pDC line and the co-culture obtained at step b) to obtain stimulated T cells; d) measuring the avidity of the stimulated T cells obtained at step c) for the at least one tumoral antigen; and e) predicting the clinical evolution of the patient suffering from cancer based on the comparison of the avidity measured at step d) to a reference level.

Description

METHOD FOR PREDICTING THE CLINICAL EVOLUTION OF CANCER PATIENTS

The present invention concerns in vitro methods for predicting the clinical evolution of a patient suffering from cancer.

BACKGROUND OF THE INVENTION

Spectacular advances have recently occurred in the field of cancer treatment, and in particular of melanoma treatment, with the emergence of targeted therapies for patients harboring BRAF mutations and the advent of immune-checkpoint blockers as first-line treatments. However, not all patients benefit equally and there is a huge need to identify prognostic biomarkers and predictive factors of evolution at the initial stage of the disease. There is also a lack of response biomarkers that would allow identification of patients with high risk of relapse or who would be candidates for early adjuvant treatment. Such biomarkers allowing for the prediction of response to systemic treatment could avoid inappropriate prescriptions of inefficient or toxic systemic therapies while reducing healthcare cost.

Very little information exists regarding the analysis of links between immune responses, clinical parameters, and response to immunotherapies. High pre-existing numbers of PD-1 and PD-L1 expressing cells and high CD8 T cell infiltration within the tumor bed was identified as being predictive of response to immune-checkpoint blockers. More recently, it was suggested that mutational load, neoantigen load of patient tumor cells, and expression of cytolytic markers in the immune microenvironment were significantly associated with clinical benefit, without predicting response to CTLA4 and/or PD-1 blockade. These methods require tumor samples and/or very complex analysis. In this context, liquid biopsies consisting of blood samples would be interesting for the monitoring of patients.

Some studies suggested that the presence and number of T lymphocyte infiltrates, specifically CD8+ T cells, is an independent and favorable prognostic factor in melanoma (Jacquelot et al., 2016, J Invest Dermatol, 136, 994-1001 ; de Moll et al., 2015, Cancer Immunol Immunother, 64, 1 193-203; Piras et al., 2005, Cancer, 104, 1246-54; Erdag et al., 2012, Cancer Res, 72, 1070-80). However, these studies only focus on tumor- infiltrated lymphocytes (TILs), which are present within the tumor. Ongoing investigations are analyzing the host immune response against cancer to evaluate the prognostic impact of in situ immune-cell infiltrates in tumors and define it as an "immunoscore" (Galon et al., 2012, J Transl Med, 10, 1 ). Based on the enumeration of two lymphocyte populations, both in the tumor and at their invasive margin, this immunohistochemistry analysis is complex, difficult to standardize, and depends upon the availability of tumor biopsies. Thus, there is still a need for a simple, standard and non-invasive prognostic biomarker allowing cancer-patient prognosis assessment.

Functional differences between low- and high-affinity CD8 T cells in the tumor environment were studied by injecting a high-affinity T cell clone and a low-affinity T cell clone in tumor-bearing mice. In such a murine model, it was shown that TCR affinity is a critical factor determining the efficiency of tumor-specific T cells within tumor microenvironments (Bos et al., 2012, Oncoimmunology, 1 , 1239-1247). Indeed, the strength of TCR-ligand interactions can directly affect the infiltration, survival, and cytotoxic potential of tumor-specific T cells. Moreover, high-affinity T cells are less susceptible to suppressive mechanisms, as they exhibit lower expression of inhibitory molecules (Bos et al., 2012, Oncoimmunology, 1 , 1239-1247).

However, the inventors have shown that the avidity value for a tumoral antigen measured from T cells obtained from a patient suffering from cancer is not correlated to the clinical evolution of said patient, when these T cells have not been previously successfully stimulated in vitro with the antigen.

The staining brightness of peptide-HLA-multimer labeling of T cells is a simple approach to quantify functional T cell avidity (Zhang et al., 2016, Sci. Transl. Med., 8(341 ):341 ra77; Corse et al., 201 1 , J. Immunol. ; 186(9):5039-45; Dutoit et al., 2001 , Cancer Res, 61 , 5850-6; Yee et al., 1999, J Immunol, 162, 2227-34).

The inventors recently developed a plasmacytoid dendritic cell (pDC)-based approach to elicit antitumor immunity. PDCs are antigen-presenting cells and play a key role in immunity priming or boosting antigen-specific T cells. Using a pDC line (GEN2.2), the inventors developed a cell-based assay comprising co-cultures of the pDC line loaded with immunogenic peptides together with human leukocyte antigen (HLA)-A^*02:01 peripheral blood mononuclear cells (PBMCs) of melanoma patients.

The inventors observed the induction of an immune response in vitro by the pDC line in all melanoma patients, but the triggered specific T cells displayed a broad range of functional features. In order to understand the basis for such variability in the quality of the response, they investigated whether the features of the antitumor-specific response to the peptide-loaded pDC line were dependent on the disease stage or clinical parameters at diagnosis and if it could represent a prognostic factor of clinical outcome. To address this issue, they performed pDC-line assays in a total of 63 melanoma patients, including 33 patients at diagnosis. The intensity and the quality of the response correlated with the clinical parameters and clinical outcomes of the patients. Interestingly, the inventors found that the peptide-pulsed pDC line elicited tumor-specific T cells with different features depending upon the stage of the disease. Strikingly, their study revealed for the first time the avidity of the cultured tumor-specific T cells triggered by the pDC as a crucial feature in predicting clinical evolution. Indeed, the inventors showed that the avidity of the cultured tumor-specific T cells enables predicting patient relapse time and overall survival.

The inventors have thus identified a non-invasive immune biomarker allowing cancer-patient prognosis assessment. Such prognosis and predictive factor of clinical evolution may help clinicians to identify patients with high risk of relapse and to better orientate therapeutic strategies and schedules for melanoma patients. DESCRIPTION OF THE INVENTION

The invention pertains to an in vitro method for predicting the clinical evolution of a patient suffering from cancer, said method comprising the steps of:

a) obtaining a plasmacytoid dendritic cell (pDC) line pulsed with at least one tumoral antigen, and irradiated; said pDC line sharing at least one major histocompatibility complex (MHC) allele with PBMC from the patient suffering from cancer;

b) bringing the pulsed and irradiated pDC line obtained at step a) into contact with PBMC from the patient suffering from cancer, and co-culturing the pulsed and irradiated pDC line and the PBMC;

c) bringing the pulsed and irradiated pDC line obtained at step a) again into contact with the co-culture obtained at step b), and co-culturing the pulsed and irradiated pDC line and the co-culture obtained at step b) to obtain stimulated T cells;

d) measuring the avidity of the stimulated T cells obtained at step c) for the at least one tumoral antigen; and

e) predicting the clinical evolution of the patient suffering from cancer based on the comparison of the avidity measured at step d) to a reference level.

The invention also relates to an in vitro method for selecting a patient suffering from cancer, suitable to be treated with a non-aggressive therapy comprising the step of:

b) bringing the pulsed and irradiated pDC line obtained at step a) into contact with PBMC from the patient suffering from cancer, and co-culturing the pulsed and irradiated pDC line and the PBMC; c) bringing the pulsed and irradiated pDC line obtained at step a) again into contact with the co-culture obtained at step b), and co-culturing the pulsed and irradiated pDC line and the co-culture obtained at step b) to obtain stimulated T cells;

e) selecting the patient as suitable to be treated with a non-aggressive therapy if the avidity measured at step d) is higher than a reference level.

The invention further concerns an in vitro method for selecting a patient suffering from cancer suitable to be treated with an aggressive therapy comprising:

e) selecting the patient as suitable to be treated with an aggressive therapy if the avidity measured at step d) is lower than a reference level. The invention also pertains to an in vitro method for monitoring the response of the patient to a treatment comprising the steps of:

a) measuring the T cells avidity for at least one tumoral antigen by implementing steps a) to steps d) of the method for predicting the clinical evolution of a patient suffering from cancer according to the invention, before onset of said treatment; and

b) measuring the T cells avidity for at least one tumoral antigen by implementing steps a) to steps d) of the method for predicting the clinical evolution of a patient suffering from cancer according to the invention, after onset of said treatment; wherein an increase in the T cells avidity in the course of time indicates that said treatment is efficient for treating said patient. The invention further relates to an in vitro method for monitoring the progression of the cancer comprising the steps of:

a) measuring the T cells avidity for at least one tumoral antigen by implementing steps a) to steps d) of the method for predicting the clinical evolution of a patient suffering from cancer according to the invention, when monitoring is started; and b) measuring the T cells avidity for at least one tumoral antigen by implementing steps a) to steps d) of the method for predicting the clinical evolution of a patient suffering from cancer according to the invention, at a certain point in time;

wherein an increase in the T cells avidity in the course of time indicates a favorable cancer progression.

The invention finally concerns an anti-cancer treatment for use for the treatment of cancer in a patient whose clinical evolution has been predicted by the in vitro method according to the invention.

Diseases and patients

The invention relates to a method for predicting the clinical evolution of a patient suffering from cancer.

As used herein, the term "cancer" refers to any type of malignant (i.e. non benign) tumor. The malignant tumor may correspond to a primary tumor or to a secondary tumor (i.e. a metastasis). The tumor may for instance correspond to a solid malignant tumor.

In a specific embodiment of the invention, the cancer from which the patient suffers is melanoma. The term "melanoma", also known as "malignant melanoma", denotes a type of cancer that develops from the pigment-producing cells known as melanocytes. Melanomas typically occur in the skin, but may also occur in the mouth, intestines, or eye.

In the context of the present invention, a "patient" denotes a human. The subject according to the invention may be in particular a male or a female.

Preferably in the context of the present invention, the patient has been diagnosed as suffering from cancer. In a specific embodiment of the invention, the PBMC are obtained from the patient at the time of cancer diagnosis.

In another particular embodiment of the invention, the patient has already received anti-cancer treatment before the PBMC are obtained from him/her, i.e. the PBMC are obtained from a patient who has already received anti-cancer treatment. Examples of such anti-cancer treatment according to the invention are given in the section "Methods for designing a treatment regimen" hereafter. Cells and antigens

The term "cell line" relates to cells cultured in vitro. Primary cells do not grow in culture or stop being expanded in culture after a limited number of divisions. On the contrary, cell lines are capable of multiplying indefinitely.

In a preferred embodiment of the invention, the "plasmacvtoid dendritic cell" ("pDC") line is obtained from cells of pDC leukemia. The European patent EP 1 572 989 B1 describes a method for obtaining and culturing pDC lines from cells of leukemia. This patent notably discloses a human pDC line called GEN2.2, deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue Du Docteur Roux, F-75015 Paris) on September 24, 2002 under the number CNCM I-2938 according to Rule 6.1 of the Treaty of Budapest, and the human pDC line called GEN3, deposited at the CNCM on October 16, 2003 under the number CNCM 1-31 10 according to Rule 6.1 of the Treaty of Budapest. The phenotype of the pDC lines GEN2.2 and GEN3 is HLA- A^*0201 .

Preferably, the pDC line used in the methods of the invention is the GEN2.2 pDC line or the GEN3 pDC line.

As used herein, a "peripheral blood mononuclear cell" ("PBMC") denotes any cell having a round nucleus that are found in peripheral blood. These cells comprise monocytes and lymphocytes, which include T lymphocytes, B lymphocytes, and NK cells. The PBMC according to the present invention preferably comprise specific effectors. The term "specific effectors" denotes immune cells capable of recognizing a specific antigen or a product of this antigen. In a particular embodiment, the specific effectors are cytotoxic effectors. Preferably, these cytotoxic effectors are antigen-specific T cells, such as CD4+ or CD8+ T lymphocytes. Therefore, the PBMC according to the present invention preferably comprise CD4+ or CD8+ T lymphocytes. Most preferably, the PBMC used in the method of the invention preferably comprise CD8+ T lymphocytes.

Preferably, the "stimulated T cells" obtained at step c) comprise CD8+ T lymphocytes. Also preferably, the "stimulated T cells" obtained at step c) are non-clonal T cells.

The methods according to the invention may optionally comprise a step of obtaining the PBMC from a biological sample from the patient suffering from cancer. As used herein, the term "biological sample" means a substance of biological origin. Preferably, the biological sample is a blood sample, a plasma sample, or a subpopulation of blood cells such as leucocytes or lymphocytes for instance. Also preferably, the biological sample is a biopsy sample, more preferably a tumoral biopsy sample, such as for instance a biopsy sample obtained from a primary tumor or from a metastasis. The biological sample according to the invention may be obtained from the subject by any appropriate means of sampling known from the skilled person.

Primary cells do not grow in culture or stop being expanded in culture after a limited number of divisions. For instance, cells which have been taken from a patient are primary cells. Thus, the PBMC used in the methods of the invention preferably comprise primary PBMC. More preferably, the PBMC used in the methods of the invention comprise primary T cells. Even more preferably, the PBMC used in the methods of the invention comprise primary CD8+ T cells.

The "major histocompatibility complex" (or MHC) is a set of cell surface proteins essential for the adaptive immune system. MHC molecules notably bind to peptide fragments of infectious or tumoral origin and display them on the cell surface allowing recognition by T cells. In humans, the MHC is also called the human leukocyte antigen (HLA).

Dendritic cells are antigen-presenting cells (APC) that may display antigen complexed with major histocompatibility complexes (MHC) on their surfaces. T cells may recognize these complexes using their T cell receptors (TCR): the antigen epitope held in the peptide-binding groove of the MHC molecule may interact with the TCR thus triggering T cell activation. Such antigen presentation is crucial for effective adaptive immune response, as the activity of both cytotoxic and helper T cells dependents on it. Antigen presentation allows for specificity of adaptive immunity and can contribute to immune responses against pathogens but also against tumors.

Antigen presentation may only occur between dendritic cells and effector cells if both cell types share a same major histocompatibility complex (MHC) allele. In the context of the invention, the major histocompatibility complex (MHC) allele shared by the PBMC and the pDC line may be any MHC allele. In a specific embodiment, the major histocompatibility complex (MHC) allele shared by the PBMC and the pDC line may be HLA-A^*02:01 , HLA^*B7:02, or HLA^*B44:02. In a specific embodiment, in particular when the pDC line is GEN 2.2 or GEN3, the major histocompatibility complex (MHC) allele shared by the PBMC and the pDC line is HLA-A^*02:01 .

The pulsed and irradiated pDC line may be co-cultured with the PBMC at any ratio, for instance at a 1 :1 ratio, at a 1 :2 ratio, at a 1 :5 ratio, at a 1 :10 ratio, at a 1 :20 ratio, at a 1 :50 ratio. Preferably, the pulsed and irradiated pDC line is co-cultured with the PBMC at a 1 :10 ratio.

The methods according to the invention comprise a step c) of bringing the pulsed and irradiated pDC line obtained at step a) again into contact with the co-culture obtained at step b), and co-culturing the pulsed and irradiated pDC line and the co-culture obtained at step b) to obtain stimulated T cells. Such repeated stimulations of the PBMC with the pulsed and irradiated pDC line allow the proliferation of the stimulated T cells. For instance, the co-culture may be stimulated again with the pulsed and irradiated pDC line at different time points, for instance after 7 days, 10 days, 14 days, 17 days or 21 days. Preferably, the co-culture is stimulated again weekly with the pulsed and irradiated pDC line. The co-culture may be stimulated again with the pulsed and irradiated pDC line at least once, twice, three times, four times, five times. Preferably, the co-culture is stimulated again with the pulsed and irradiated pDC line at least twice.

Preferably, the methods according to the invention further include a step of verifying that T cells have been stimulated by the pDC at step c). For instance, specific T cell amplification may be measured by multimer labelling of the PBMC initially and/or at different steps of the co-culture.

Within the scope of the present invention the term "antigen" defines a molecule recognized by cells of the immune system and capable of triggering a specific immune response. This specific immune response may be a cell-mediated immune response.

Antigens of the present invention are tumor antigens of any kind, such as peptides, proteins, glycopeptides, glycoproteins, phosphorylated proteins. Preferably, the antigens are peptides obtained from antigenic proteins of tumoral origin.

In the context of the invention, the at least one antigen may for instance be a peptide comprised in the sequence of CEA, NY-BR1 , Her-2/Neu, PSA, RAGE -1 , PRAME, TRP-2, MAGE-A1 , MAGE-A2, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C2, MUC -1 , p53, hTERT, survivin, melan-A/MART-1 (also noted Mel A), GP100, tyrosinase, MAGE- A3 or NY-ES01 . In a specific embodiment, the at least one tumoral antigen is a panel comprising peptides comprised in the sequence of the antigens MelA, GP100, tyrosinase and MAGE- A3.

The at least one antigen may also be a panel of two or more peptides selected from the group consisting of the peptides comprised in the sequence of CEA, NY-BR1 , Her- 2/Neu, PSA, RAGE -1 , PRAME, TRP-2, MAGE-A1 , MAGE-A2, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C2, MUC -1 , p53, hTERT, survivin, melan-A/MART-1 (also noted MelA), GP100, tyrosinase, MAGE- A3 or NY-ES01 .

In a preferred embodiment, the at least one tumoral antigen is a peptide derived from an antigen selected from the group consisting of MelA, GP100, tyrosinase and MAGE-A3. In another embodiment, the at least one tumoral antigen is a panel of two or more peptides derived from an antigen selected from the group consisting of MelA, GP100, tyrosinase and MAGE-A3. In a specific embodiment, the at least one tumoral antigen is a panel comprising peptides derived from the antigens MelA, GP100, tyrosinase and MAGE-A3. In another specific embodiment, the at least one tumoral antigen is a peptide derived from the antigen MelA. Preferably, the at least one tumoral antigen is one the following peptides: MelA₂₆-35

(SEQ NO: 1 ), GPI OO209-217 (SEQ NO: 2), tyrosinase₃₆9-377 (SEQ NO: 3), or MAGE-A3₂₇i- ₂₇₉ (SEQ NO: 4). Also preferably, the at least one tumoral antigen is a panel of two or more of the following peptides: MelA₂₆-35 (SEQ NO: 1 ), GPI OO₂09-217 (SEQ NO: 2), tyrosinase₃₆9-377 (SEQ NO: 3), MAGE-A3₂₇i-279 (SEQ NO: 4). Most preferably, the at least one tumoral antigen is a panel comprising the following peptides: MelA₂₆-35 (SEQ NO: 1 ), GPI OO209-217 (SEQ NO: 2), tyrosinase₃₆9-377 (SEQ NO: 3), and MAGE-A3₂₇i -279 (SEQ NO: 4). In a specific embodiment, the at least one tumoral antigen is the peptide MelA₂₆-35 (SEQ NO: 1 ).

The at least one antigen of the invention may comprise a peptide which sequence is derived from the sequence of CEA, NY-BR1 , Her-2/Neu, PSA, RAGE -1 , PRAME, TRP-2, MAGE-A1 , MAGE-A2, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C2, MUC -1 , p53, hTERT, survivin, melan-A/MART-1 (also noted MelA), GP100, tyrosinase, MAGE-A3 or NY-ES01 .

An amino acid sequence derived from a reference sequence is understood to mean an amino acid sequence which differs from the reference sequence by substitution, deletion and/or insertion of an amino acid or a plurality of amino acids, preferably a reduced number of amino acids, particularly by substitution of natural amino acids by non-natural amino acids or pseudo-amino acids at positions such that these modifications do not significantly undermine the antigenicity of the peptide or polypeptide.

The derivative peptide or polypeptide may for instance have or comprise a sequence identical to at least 70%, preferably at least 80%, more preferably at least 90%, even at least 95%, of the reference sequence.

By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. Methods for comparing the identity and homology of two or more sequences are well known in the art. The "needle" program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk web site. The percentage of identity in accordance with the invention is preferably calculated using the EMBOSS::needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

Peptides or polypeptides comprising or consisting of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a reference sequence may comprise one or more mutations such as deletions, insertions and/or substitutions compared to the reference sequence.

In a preferred embodiment, the mutation corresponds to a conservative substitution, which is a substitution of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (such as asparagine, glutamine, serine, threonine, and tyrosine), amino acids with basic side chains (such as lysine, arginine and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid); amino acids with apolar side chains (such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and cysteine) as indicated in the table below:

Preferably, the antigen used in the methods according to the invention is a tumoral antigen expressed on the cell surface of the patient cancer cells. In other words, if the patient suffers from melanoma, the pDC line is pulsed with one or more melanoma tumor antigens. Alternatively, the "at least one antigen" used in the methods according to the invention may be a panel of tumoral antigens likely to be expressed on the surface of the tumor cells of the patient. Preferably, such panel of tumoral antigens comprise one or more antigens that are altogether expressed on the cell surface of most cancer patients or, more preferably, of all cancer patients. For instance, 100% of melanoma patients express at least one tumor antigen within the group consisting of MelA, GP100, tyrosinase and MAGE- A3.

In order to pulse the pDC line, i.e. to obtain an antigen-loaded pDC line, the antigen may for instance be supplied into the culture medium of the pDC line. Alternatively, the antigen may be expressed by the pDC line following the transfection of said pDC line with a vector allowing the expression of said antigen.

According to a specific embodiment, several pools of pDC lines may be loaded with different antigens and then single antigen-loaded pDC lines may be mixed together. Measurement of T cells avidity

The term "avidity" refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, in particular between an antigen-specific cell and said antigen. In the context of non-covalent binding interactions, avidity is inversely correlated with the dissociation rate. Avidity is commonly referred to as functional affinity (affinity describes the strength of a single interaction). Individually, each binding interaction may be readily broken, however, when many binding interactions are present at the same time, transient unbinding of a single site does not allow the molecule to diffuse away, and binding of that weak interaction is likely to be restored.

In a specific embodiment of the invention, the avidity of tumor-specific T cells, in particular T CD8+ cells, is measured by MHC-antigen multimer staining. For instance, the avidity of tumor-specific T cells is measured by MHC-antigen dimer staining, tetramer staining, or pentamer staining. Preferably, the avidity is measured by MHC-antigen tetramer staining. More preferably, the avidity is measured by MHC-peptide tetramer staining. For instance, the avidity of tumor-specific T cells may also be determined from the mean fluorescence intensity (MFI) of the MHC-antigen multimer labelling. The cells may for instance be labeled with an iTAg™ HLA-A^*02:01 multimer, for instance an iTAg™ HLA-A^*02:01 tetramer, (for example for 30 min at 2°C to 8°C), and then with anti-CD3 and anti-CD8 antibodies (for example for 15 min at room temperature). Typically, cells are incubated at 37°C, fixed and analyzed by flow cytometry. A FACSCalibur and CellQuest software (BD Biosciences) may for instance be used.

In another embodiment, the avidity of tumor-specific T cells, in particular T CD8+ cells, is assessed by measuring MHC-antigen multimer dissociation from T cell receptor (TCR) over time after staining. Preferably, the avidity is determined by measuring MHC- antigen tetramer dissociation from T cell receptor (TCR) over time after staining. More preferably, the avidity is determined by measuring MHC-peptide tetramer dissociation from T cell receptor (TCR) over time after staining. T cells are typically labeled with an iTAg™ ΗΙ_Α-Α^*02:0Γ multimer, for instance an iTAg™ ΗΙ_Α-Α^*02:0Γ tetramer, (for example for 30 min at 2°C to 8°C), and then with anti-CD3 and anti-CD8 antibodies (for example for 15 min at room temperature). Typically, cells are incubated at 37°C, fixed at different time points (for instance 0, 0.5, 1 , 2, 4, and 7 h), and analyzed by flow cytometry. A FACSCalibur and CellQuest software (BD Biosciences) may for instance be used.

Clinical evolution of cancer

The inventors showed that the avidity of antigen-specific T cells induced upon stimulation of PBMC with a pulsed and irradiated pDC line is a predictive marker of clinical evolution. Therefore a first aspect of the invention pertains to an in vitro method for predicting the clinical evolution of a patient suffering from cancer, said method comprising the steps of:

Following diagnosis of a cancer in a patient, the symptoms of such disease and the clinical state of the patient may progress in various manners. For instance, the cancer may develop slowly or even regress, or it may develop and spread, possibly with metastases appearing. The patient may respond well to the treatment and recover while the symptoms alleviate or, on the contrary, the patient may poorly respond to the treatment, or relapse after a period of time, and have a short life-expectancy. In the context of the invention, "predicting the clinical evolution of a patient suffering from cancer" can therefore mean predicting the risk of the patient to relapse, or to develop metastasis, or to have a short life-expectancy or a poor long-term survival, or to poorly respond to treatments. As used herein, a "relapse" or "recurrence" of a cancer means the return of said cancer, or of the signs and symptoms of said cancer, after a period of improvement or after a period of time when the cancer couldn't be detected after treatment. In the case of cancer relapse, the cancer can be detected again weeks, months, or even many years after the primary or original cancer was treated. In a particular embodiment of the invention, predicting the clinical evolution of the patient comprises predicting the risk of relapse of the patient.

According to another embodiment, predicting the clinical evolution of the patient comprises predicting the long-term survival of the patient. The "long-term survival" of a patient may for instance be evaluated by measuring the Overall Survival (OS) or the Progression-Free Survival (PFS). The Overall Survival (OS) denotes the time from a certain date (for instance the date of diagnosis, the date of sampling, the first day of treatment...) until death from any cause. The Progression-Free Survival (PFS) denotes the time from a certain date (for instance the date of diagnosis, the date of sampling, the first day of treatment...) until disease progression or death from any cause.

Strikingly, the inventors showed that the avidity of antigen-specific T cells induced upon stimulation of patient's PBMC with a pDC line pulsed with tumor antigen and irradiated predicted both PFS and OS after the sampling date. In contrast, measurement of the avidity of T cells obtained from PBMC of a cancer patient, which have not been co- cultured with a pDC line pulsed with tumor antigen and irradiated, does not allow predicting the clinical evolution of the patient, nor the patient's PFS or OS.

In the present application, the term "treatment" refers to the kind of therapeutical means used to treat a patient. Examples of anti-cancer treatments according to the invention are given in the section "Methods for designing a treatment regimen" hereafter.

Methods for predicting the clinical evolution

The last step of the methods of the invention is a step of predicting the clinical evolution of the patient suffering from cancer based on the comparison of the measured T cells avidity to a reference level.

As used herein, the term "reference level" may refer to the median value or the mean value of the T cells avidity measured in a population of patients suffering from cancer. Preferably, the term "reference level" refers to the median value of the T cells avidity measured in a population of patients suffering from cancer.

The median value or the mean value of the T cells avidity in a population of patients suffering from cancer may be calculated from the T cells avidity values obtained in a certain number of individuals suffering from cancer. For each individual suffering from cancer, said T cells avidity may be determined by:

a) obtaining a plasmacytoid dendritic cell (pDC) line pulsed with at least one tumoral antigen, and irradiated; said pDC line sharing at least one major histocompatibility complex (MHC) allele with PBMC from an individual suffering from cancer;

b) bringing the pulsed and irradiated pDC line obtained at step a) into contact with PBMC from the individual, and co-culturing the pulsed and irradiated pDC line and the PBMC;

d) measuring the avidity of the stimulated T cells obtained at step c) for the at least one tumoral antigen.

As used throughout the present specification, the expression "a patient with good clinical evolution" or "a patient with good prognosis" refers to a patient that is likely to present a long life-expectancy, not to develop metastases, not to relapse, and/or to well respond to treatments.

Similarly, the expression "a patient with poor clinical evolution" or "a patient with poor prognosis" refers to a patient that is likely to present a short life-expectancy, to develop metastases, to relapse, and/or not to respond, or poorly respond, to treatments.

Preferably, in the methods of the invention, it is further determined whether the measured T cells avidity is increased or decreased compared to the reference level according to the invention. Still preferably, in the methods of the invention, it is further determined the level of increase or decrease of the measured T cells avidity compared to the reference level according to the invention.

As used herein, the expression "level of increase" means the percentage of increase of the measured T cells avidity, compared to the reference level according to the invention or the number of fold of increase of the measured T cells avidity, compared to the reference level according to the invention.

Preferably, when the measured T cells avidity is increased compared to the reference level, its value is significantly higher than the threshold value.

Also preferably, when the measured T cells avidity is decreased compared to the reference level, its value is significantly lower than the threshold value.

The inventors specifically demonstrated that the increase of the measured T cells avidity for a subject suffering from cancer compared to the reference level enabled predicting a good clinical evolution. Accordingly, in the methods for predicting the clinical evolution of a patient suffering from cancer according to the invention, a T cells avidity measured at step d) which is higher than the reference level is predictive of a good clinical evolution, of a low risk of relapse, of a good long-term survival for the patient, or of a good response of the patient to an anti-cancer treatment.

Also, in the methods for predicting the clinical evolution of a patient suffering from cancer according to the invention, a T cells avidity measured at step d) which is lower than the reference level is predictive of a poor clinical evolution, of a high risk of relapse, of a poor long-term survival for the patient, or of a poor response of the patient to an anti- cancer treatment.

Preferably, in the methods for predicting the clinical evolution of a patient suffering from cancer according to the invention, a T cells avidity measured at step d) which is at least 40%, 50%, 60%, 70%, 80% or 90% higher than the reference level is predictive of a good clinical evolution.

Also preferably, in the methods for predicting the clinical evolution of a patient suffering from cancer according to the invention, a T cells avidity measured at step d) which is at least 40%, 50%, 60%, 70%, 80% or 90% lower than the reference level is predictive of a poor clinical evolution.

More preferably, in the methods for predicting the clinical evolution of a patient suffering from cancer according to the invention, a T cells avidity measured at step d) which is at least 2-fold, 3-fold, 4-fold, or 5-fold higher than the reference level is predictive of a good clinical evolution.

Also more preferably, in the methods for predicting the clinical evolution of a patient suffering from cancer according to the invention, a T cells avidity measured at step d) which is at least 2-fold, 3-fold, 4-fold, or 5-fold lower than the reference level is predictive of a poor clinical evolution.

Methods for designing a treatment regimen

The inventors showed that the avidity of antigen-specific PBMC upon stimulation with a pulsed and irradiated pDC line could be used as a marker for selecting the treatment regimen of a patient.

Therefore the above methods for predicting the clinical evolution of a patient suffering from cancer may also be used for designing a treatment regimen.

When the above methods are used to design a treatment regimen, they further comprise the step of designing a treatment regimen based on the result of step e). In the present application, the expression "treatment regimen" or "treatment" refers to the kind of therapeutical means used to treat a patient. The treatment regimen, or treatment, of a patient suffering from cancer may for instance include chemotherapy, biological therapy or radiation therapy, performed alone or in combination.

Anti-cancer treatment options are related to a number of factors such as the stage of the cancer, the grade of the cancer, the invasiveness of the cancer, but also the overall prognosis of the cancer, the patient life-expectancy, the risk of metastasis, and the risk of relapses. Therefore, determining the prognostic of a patient may help selecting the treatment regimen of said patient.

Patients suffering from early-stage or low-grade cancer with good prognostic generally receive a light therapy. Such light therapy may only include careful watching, and behaviour modifications such as e.g. exercise and dietary changes, usually together with a light surgery, such as e.g. local excision of the tumor.

Typically, the patient is given a non-aggressive treatment regimen if the clinical evolution is found to be good.

The above methods can be used to decide how to monitor a patient. Indeed, as shown herein, a high avidity measured at step d) is indicative of a good cancer prognosis or clinical evolution. Therefore, patients for who high avidity in measured need to be treated by a non-aggressive therapy.

The invention is thus directed to an in vitro method for selecting a patient suffering from cancer, suitable to be treated with a non-aggressive therapy comprising the step of: a) obtaining a plasmacytoid dendritic cell (pDC) line pulsed with at least one tumoral antigen, and irradiated; said pDC line sharing at least one major histocompatibility complex (MHC) allele with PBMC from the patient suffering from cancer;

b) bringing the pulsed and irradiated pDC line obtained at step a) into contact with

PBMC from the patient suffering from cancer, and co-culturing the pulsed and irradiated pDC line and the PBMC;

In the field of the invention, a "non-aggressive therapy" may refer to surgery, and/or to radiotherapy. In another embodiment, the "non-aggressive therapy" may refer to low doses of a systemic therapy. By systemic therapy is meant a therapy that is given thought the bloodstream, such as e.g. hormone therapy, chemotherapy and/or immunotherapy. Hormone therapy refers to the use of hormones and/or hormone antagonists, such as e.g. tamoxifen or raloxifene, in medical treatment. Chemotherapy refers to the treatment by chemicals such as antineoplastic drugs or a combination of these drugs. Antineoplastic drugs include e.g. cyclophosphamide, methotrexate, and 5- Fluorouracil. Immunotherapy refers to the treatment by induction, enhancement, or suppression of an immune response, using immuno-modulators such as e.g. trastuzumab.

In another embodiment, the "non-aggressive therapy" may be a combination of surgery, optionally followed by radiotherapy, and of low doses of systemic therapy.

Preferably, the treatment regimen of a patient having a T cells avidity higher than the reference level should include means of treatment other than chemotherapy, alone or in combination with chemotherapy. Such means of treatment may for instance include surgery or radiation therapy. Also preferably, the treatment regimen of a patient having a T cells avidity lower than the reference level should include chemotherapy.

Typically, the patient is given an aggressive treatment regimen, if the clinical evolution is found to be poor.

The above methods can be used to decide how to monitor a patient. Indeed, as shown herein, a low avidity measured at step d) is indicative of a poor cancer prognosis or clinical evolution. Therefore, patients for who low avidity in measured should be under very tight observation by their oncologist and need to be treated by an aggressive therapy.

The invention also pertains to an in vitro method for selecting a patient suffering from cancer suitable to be treated with an aggressive therapy comprising:

d) measuring the avidity of the stimulated T cells obtained at step c) for the at least one tumoral antigen; and e) selecting the patient as suitable to be treated with an aggressive therapy if the avidity measured at step d) is lower than a reference level.

By "aggressive therapy" is meant a therapy adapted for treating aggressive cancers. Specifically, such aggressive therapies may induce side effects and do therefore not constitute the preferred treatment regimen in the case of a non-aggressive cancer. An aggressive therapy typically corresponds to a combination chemotherapy carried out with high doses of drugs. The combination chemotherapy may for example comprise the administration of high doses of at least one compound selected from the group consisting of an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, a hormonal therapy drug, a signaling inhibitor, an aromatase inhibitor, a differentiating agent, a monoclonal antibody, a biologic response modifier and an antiangiogenic agent. Thus combination chemotherapy may for example comprise the administration of at least one of the following anti-cancer agents (simultaneously or sequentially):

- an alkylating agent such as Cyclophosphamide, Chlorambucil and Melphalan; - an antimetabolite such as Methotrexate, Cytarabine, Fludarabine, 6-

Mercaptopurine and 5- Fluorouracil;

- an antimitotic such as Vincristine, Paclitaxel (Taxol), Vinorelbine, Docetal and Abraxane;

- a topoisomerase inhibitor such as Doxorubicin, Irinotecan, Platinum derivatives, Cisplatin, Carboplatin, Oxaliplatin;

- a hormonal therapy drug such as Tamoxifen;

- an aromatase inhibitor such as Bicalutamide, Anastrozole, Examestane and Letrozole;

- a signaling inhibitor such as Imatinib (Gleevec), Gefitinib and Erlotinib;

- a monoclonal antibody such as Rituximab, Trastuzumab (Herceptin) and

Gemtuzumab ozogamicin;

- a biologic response modifier such as Interferon-alpha;

- a differentiating agent such as Tretinoin and Arsenic trioxide; and/or

- an agent that block blood vessel formation (antiangiogenic agents) such as Bevicizumab, Serafinib and Sunitinib.

- an agent that block osteoclast maturation and/or function such as bisphosphonate or denosumab

- a checkpoint blocker, in particular an anti-CTLA-4 monoclonal antibody such as Ipilimumab, Tremelimumab; a PD-1 blocker such as MEDI0680, Nivolumab, Pembrolizumab, Pidilizumab; a PD-L1 blocker such as BMS-936559, MEDI4736, MPDL3280A, MSB0010718C; an anti-LAG-3 blocking monoclonal antibody such as NCT01968109; an anti-KIR monoclonal antibody such as Lirilumab; an anti-B7-H3 monoclonal antibody such as NCT01391 143; an anti-TIM-3 blocking antibody; an anti- VISTA blocking antibody; an anti-TIGIT blocking antibody; an IDO pathway inhibitor such as D-IMT (Indoximod) and a small molecule enzymatic inhibitor of IDOI such as INCB024360, NLG919.

The aggressive therapy may also correspond to radiation therapy and/or surgery, or to a combination of chemotherapy with a radiation therapy and/or surgery.

Methods for monitoring cancer progression, or for monitoring a patient response to a treatment

The above methods for predicting the clinical evolution of a patient suffering from cancer may also be used for monitoring the progression of the cancer, and/or for monitoring the response of the patient to a treatment.

When the above methods are used to monitor the progression of a disorder or to monitor the response to a treatment, it is repeated at least at two different points in time (e.g. before and after onset of a treatment).

Accordingly, the invention also relates to an in vitro method for monitoring the response of the patient to a treatment comprising the steps of:

b) measuring the T cells avidity for at least one tumoral antigen by implementing steps a) to steps d) of the method for predicting the clinical evolution of a patient suffering from cancer according to the invention, after onset of said treatment; wherein an increase in the T cells avidity in the course of time indicates that said treatment is efficient for treating said patient.

The invention also relates to an in vitro method for monitoring the progression of the cancer comprising the steps of:

The monitoring of disease progression or treatment efficiency is typically performed by determining the T cells avidity at different points in time, for instance at 2-week, 1 - month, 2-month, 3-month intervals, etc.

An "increase in the T cells avidity" is evaluated by comparing the T cells avidity when monitoring is started with the T cells avidity at any point in time. Said increase is preferably statistically significant. A statistically significant increase can for example correspond to an increase of at least 5, 10, 25 or 50%.

Methods of treating a patient suffering from cancer

Another aspect of the invention pertains to an anti-cancer treatment for use for the treatment of cancer in a patient whose clinical evolution has been predicted by the method according to the invention.

The invention further pertains to an in vitro method of treating a patient suffering from cancer, said method comprising the steps of:

a) predicting the clinical evolution of the patient suffering from cancer by the method for predicting the clinical evolution of the patient suffering from cancer according to the invention, and

b) administering an anti-cancer treatment to the patient if the T cells avidity for the at least one tumoral antigen measured at step d) of the method for predicting the clinical evolution of the patient suffering from cancer according to the invention is lower than the reference level.

Thus, the invention also relates to an in vitro method of treating a patient suffering from cancer, said method comprising the steps of:

e) predicting the clinical evolution of the patient suffering from cancer based on the comparison of the avidity measured at step d) to a reference level; and

f) administering an anti-cancer treatment to the patient if a poor clinical evolution has been predicted at step e).

The invention further relates to an in vitro method of treating a patient suffering from cancer, said method comprising the steps of:

e) administering an anti-cancer treatment to the patient if the avidity measured at step d) is lower than a reference level.

In the present application, the term "treatment" is understood to mean treatment for a curative purpose (aimed at curing or reducing the symptoms, or aimed at alleviating or stopping the development of the pathology) or for a prophylactic purpose (aimed at reducing the risk of appearance of the pathology).

The anti-cancer treatment of the invention can correspond to any one of the anticancer treatment described above in the paragraph entitled "Methods for designing a treatment regimen, for monitoring cancer progression, or for monitoring a patient response to a treatment", such as surgery, radiotherapy or a systemic therapy (such as hormone therapy, chemotherapy and/or immunotherapy). Preferably, the anti-cancer treatment is a combination chemotherapy which may for example comprise the administration of high doses of at least one compound selected from the group consisting of an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, a hormonal therapy drug, a signaling inhibitor, an aromatase inhibitor, a differentiating agent, a monoclonal antibody, a biologic response modifier and an antiangiogenic agent.

Thus combination chemotherapy may for example comprise the administration of at least one of the following anti-cancer agents (simultaneously or sequentially):

- an alkylating agent such as Cyclophosphamide, Chlorambucil and Melphalan;

- an antimetabolite such as Methotrexate, Cytarabine, Fludarabine, 6- Mercaptopurine and 5- Fluorouracil;

- a hormonal therapy drug such as Tamoxifen;

- a signaling inhibitor such as Imatinib (Gleevec), Gefitinib and Erlotinib;

- a monoclonal antibody such as Rituximab, Trastuzumab (Herceptin) and Gemtuzumab ozogamicin;

- a biologic response modifier such as Interferon-alpha;

- a differentiating agent such as Tretinoin and Arsenic trioxide; and/or

- a checkpoint blocker, in particular an anti-CTLA-4 monoclonal antibody such as

Ipilimumab, Tremelimumab; a PD-1 blocker such as MEDI0680, Nivolumab, Pembrolizumab, Pidilizumab; a PD-L1 blocker such as BMS-936559, MEDI4736, MPDL3280A, MSB0010718C; an anti-LAG-3 blocking monoclonal antibody such as NCT01968109; an anti-KIR monoclonal antibody such as Lirilumab; an anti-B7-H3 monoclonal antibody such as NCT01391 143; an anti-TIM-3 blocking antibody; an anti- VISTA blocking antibody; an anti-TIGIT blocking antibody; an IDO pathway inhibitor such as D-IMT (Indoximod) and a small molecule enzymatic inhibitor of IDOI such as INCB024360, NLG919. When the anti-cancer treatment is a drug, for instance a chemotherapeutic drug, it may be administered by any route that achieves the intended purpose. For example, administration may be achieved by a number of different routes including, but not limited to subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intracerebral, intrathecal, intranasal, oral, rectal, transdermal, buccal, topical, local, inhalant or subcutaneous use. Parenteral route is particularly preferred.

Dosages to be administered depend on individual needs, on the desired effect and the chosen route of administration. It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The total dose required for each treatment may be administered by multiple doses or in a single dose.

Depending on the intended route of delivery, the drug may be formulated as liquid (e.g., solutions, suspensions), solid (e.g., pills, tablets, suppositories) or semisolid (e.g., creams, gels) forms.

The invention also pertains to a method of treating a patient suffering from cancer comprising the step of administering an effective amount of a chemotherapeutic drug as defined herein to a patient having a T cells avidity that is lower than a reference level.

By "effective amount" is meant an amount sufficient to achieve a concentration of drug which is capable of preventing, treating or slowing down the disease to be treated. Such concentrations can be routinely determined by those of skilled in the art. The amount of the compound actually administered will typically be determined by a physician or a veterinarian, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the subject, the severity of the subject's symptoms, and the like. It will also be appreciated by those of skilled in the art that the dosage may be dependent on the stability of the administered drug.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 shows the sequence of MelA₂₆-35 (ELAGIGILTV).

SEQ ID NO: 2 shows the sequence of GP1 OO209-217 (IMDQVPFSV).

SEQ ID NO: 3 shows the sequence of tyrosinase₃₆9-377 (YMDGTMSQV).

SEQ ID NO: 4 shows the sequence of MAGE-A3₂₇i-279 (FLWGPRALV). DESCRIPTION OF THE FIGURES

Figure 1 : Features of tumor-specific T cells triggered by pDC-line assay.

PBMCs and TILs from stage I— IV HLA-A^*02:01 ⁺ melanoma patients (n=67) were co- cultured with pDC loaded with HLA-A^*02:01 -restricted peptides. The amplification and functionality of tumor-specific T cells was evaluated at culture day 20. (A) Percentage of MelA-specific CD8 T cells. (B) Proportions of patients responding to one to four antigens. (C) Percentage of IFN-y⁺ tumor-specific T cells upon restimulation with T2 cells pulsed with MelA or control peptide. (D) Cytotoxic activity of tumor-specific T cells on peptide- loaded T2 cells (ratio E:T = 60:1 ). (E) Tetramer-dissociation rate of tumor-specific T cells expressed as a percentage of the initial tetramer labeling (each line and symbol referred to one patient). (F) MFI of MelA-multimer labelling on tumor-specific T cells. P-values were determined using Wilcoxon matched-pairs test.

Figure 2: The features of tumor-specific T cells triggered by the pDC-line assay depend upon disease stage.

PBMCs from all HLA-A^*02:01 ⁺ melanoma patients (obtained from stage l-ll (n=38) and stage lll-IV (n=12) were co-cultured with irradiated pDC loaded with HLA-A^*02:01 - restricted peptides derived from MelA, GP100, tyrosinase, and MAGE-A3. Cultures were restimulated weekly in the presence of IL-2. The amplification and functional properties of tumor-specific T cells were evaluated at culture day 20. (A) Percentage of MelA-specific T cells (left panel), cytotoxic activity of T cells toward peptide-loaded T2 cells (middle panel), and cytotoxic activity of T cells toward melanoma tumor cells (right panel). (B) Percentage of IFNy+ MelA-specific T cells (left panel) and MFI of the tetramer labelling (right panel). (C) Number of antigens for which a specific T cell amplification could be obtained. P- values were determined using Mann-Whitney f test.

Figure 3: The affinity of tumor-specific T cells elicited by the pDC-line assay from PBMCs predicted time to relapse.

PBMCs from stage I— IV HLA-A^*02:01 ⁺ melanoma patients were co-cultured with irradiated pDC loaded with HLA-A^*02:01 -restricted peptides derived from MelA, GP100, tyrosinase, and MAGE-A3, and restimulated weekly in the presence of IL-2. The features of tumor-specific T cells were evaluated at day 20. (A) Correlation between the progression-free survival (PFS) and the percentage of MelA-specific T cells, (B) the proportion of IFNy-secreting tumor-specific T cells, (C) the cytotoxic activity on peptide- loaded T2 cells and (D) melanoma lines, and (E) the affinity of MelA-specific T cells. Groups were separated according to the median value of each parameter (A, median=8.2; B, median=14.8; C, median=47; D, median=19; E, median=223). (A-D) n=44-50; (E) n=34. PFS was calculated from time of diagnosis. P-values were determined using the log-rank test.

Figure 4: The affinity of tumor-specific T cells elicited by the pDC-line assay from PBMCs from stage I— IV melanoma patients predicted the clinical outcome after sampling.

PBMCs obtained from stage I— IV HLA-A^*02:01 ⁺ melanoma patients were co- cultured with irradiated pDC loaded with HLA-A^*02:01 -restricted peptides derived from MelA, GP100, tyrosinase, and MAGE-A3. Cultures were restimulated weekly in the presence of IL-2. The affinity of tumor-specific T cells was evaluated at culture day 20. Correlation between (A) the overall survival (OS) or (B) progression-free survival (PFS) and the affinity of MelA-specific T cells upon stimulation with the pDC (d20; n = 34 patients). OS and PFS were calculated from sampling time. Groups were separated according to the median value (223). P-values were determined using the log-rank test.

Figure 5. The basal affinity of circulating tumor-specific T cells does not predict time to relapse.

The affinity of tumor-specific T cells was evaluated from PBMCs obtained from stage I— IV HLA-A^*02:01 + melanoma patients. Correlation between the progression-free survival (PFS) and the basal affinity of circulating MelA-specific T cells. Groups were separated according to the median value (144; n = 34). PFS was calculated from time of diagnosis. P-values were determined using the log-rank test.

Figure 6. The basal affinity of circulating tumor-specific T cells does not predict the clinical outcome after sampling.

The affinity of tumor-specific T cells was evaluated from PBMCs obtained from stage I— IV HLA-A^*02:01 + melanoma patients. Correlation between the overall survival (OS) or progression-free survival (PFS) and the basal affinity of MelA-specific T cells (n=34 patients). OS and PFS were calculated from sampling time. Groups were separated according to the median value (144). P-values were determined using the log-rank test.

Figure 7. The affinity of tumor-specific T cells elicited by the pDC-line assay from PBMCs from stage l-ll melanoma patients predicted the clinical outcome after sampling.

PBMCs obtained from stage l-ll HLA-A^*02:01 + melanoma patients were co- cultured with irradiated pDC loaded with H LA- A^*02:01 -restricted peptides derived from MelA, GP100, tyrosinase, and MAGEA3. Cultures were restimulated weekly in the presence of IL-2. The affinity of tumor-specific T cells was evaluated at culture day 20. Correlation between the overall survival (OS) or progression-free survival (PFS) and the affinity of MelA-specific T cells upon stimulation with the pDC (d20; n = 24 patients). OS and PFS were calculated from sampling time. Groups were separated according to the median value (223). P-values were determined using the logrank test.

Figure 8. Correlations between the features of tumor-specific T cells elicited by the pDC-line assay from TILs and clinical evolution.

TILs obtained from HLA-A^*02:01 + melanoma patients were co-cultured with irradiated pDC loaded with HLAA^* 02:01 -restricted peptides derived from MelA, GP100, tyrosinase, and MAGE-A3. Cultures were restimulated weekly in the presence of IL-2. The features of tumor-specific T cells were evaluated at culture day 20. Correlation between OS or PFS and (A) the percentage of MelAspecific T cells, (B) the proportion of IFNv- secreting tumor-specific T cells, (C) the cytotoxic activity of tumor-specific T cells on melanoma lines, and (D) the affinity of MelA-specific T cells upon stimulation with the pDC. Groups were separated according to the median value of each parameter (A, median = 15.5; B, median = 12.4; C, median = 24.3; D, median = 174) (n = 1 1 -17 patients). OS and PFS were calculated from sampling time. P-values were determined using the log-rank test.

Figure 9. Correlations between the proportion of tumor-infiltrating T cells and clinical evolution.

Correlation between OS or PFS and the proportion of tumor-infiltrating CD8 T cells. Groups were separated according to the median value (20; n = 17 patients). OS and PFS were calculated from time of diagnosis time. P-values were determined using the log- rank test.

Table 1 : Clinical features of atients enrolled in the stud .

Table 2: Correlations between the features of the tumor-specific T cells triggered by the pDC-line assay and the clinical parameters for patients at diagnosis.

EXAMPLES

Example 1 : Materials and Methods

1.1. Melanoma-patient samples

Samples were obtained from stage I to IV HLA-A^*02:01 ⁺ melanoma patients (Table

1 ). Detailed clinical parameters, treatments and follow-up time are described in Table 1 . Censoring was performed adequately for survival curves. Approval to conduct the study was given by the local ethics committee of Grenoble University Hospital. The Declaration of Helsinki principles were followed, and all participants gave their written consent. Blood samples were obtained from 50 patients, and PBMCs were purified by Ficoll-Hypaque density gradient centrifugation (Eurobio, Les Ulis, France). Fresh tumor samples were obtained from 13 patients who underwent surgery for in-transit metastasis. Samples were mechanically dilacerated and digested in 2 mg/mL collagenase D (Roche Diagnostics, Basel, Switzerland) and 20 U/mL DNase (Sigma-Aldrich, St. Louis, MO, USA). TILs were separated from tumor cells by adherence. Pleural effusions were obtained from four patients with lung metastasis and submitted to density gradient centrifugation.

1.2. Cell lines

Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 Glutamax supplemented with non-essential amino acids, 1 mM sodium pyruvate (Sigma-Aldrich), 20 μg mL gentamycin, and 10% FCS (Invitrogen, Carlsbad, CA, USA). Melanoma line Me275 was provided by Pr J-C Cerottini (Ludwig Institute for Cancer Research, Epalinges, Switzerland). Melanoma lines COL0829, A375, T2, and K562 were purchased from ATCC (LGC Standards, Molsheim, France). Melanoma line Mel89 was generated in our laboratory. The human pDC line GEN2.2 was cultured as previously described in Chaperot et al. 2006, J. Immunol., 176, 248-55. 1.3. Peptides and tetramers

The inventors used the following tumor-derived HLA-A^*02:01 -restricted peptides (NeoMPS) and the corresponding iTAg™ HLA-A^*02:01 tetramers (Beckman Immunomics, Brea, CA, USA) or dextramers (Immudex, Kobenhavn, Denmark): MelA₂₆-35 (ELAGIGILTV, SEQ NO: 1 ), GP100₂₀₉-217 (IMDQVPFSV, SEQ NO: 2), tyrosinase₃₆9-377 (YMDGTMSQV, SEQ NO: 3), MAGE-A3₂₇i-₂79 (FLWGPRALV, SEQ NO: 4).

1.4. pDC-line assay for induction of antitumor T cell responses

The GEN2.2 pDC line was first loaded with the four peptides of interest. Briefly, cells were washed with serum-free RPMI and 32-microglobulin (0.1 μg mL; Sigma-Aldrich), and peptide(s) (10 μΜ; NeoMPS) were added. After 3 h at 37°C, single peptide-loaded pDC were mixed together. All peptide-loaded pDC were then washed, irradiated (30 Gy), and co-cultured with ΗΙ_Α-Α^*02:0 PBMCs or TILs at a 1 :10 ratio in RPMI supplemented with 10% FCS. Cultures were restimulated weekly with peptide-loaded pDC and 200 U/mL IL-2 (Proleukine; Chiron, Emeryville, CA, USA). Specific CD8 T cell amplification was measured by tetramer labelling of PBMCs initially and at different steps of the culture. Cells were resuspended in phosphate-buffered saline with 2% FCS and stained with CD45, iTAg™ HLA-A^*02:01 tetramers (BD Biosciences, San Jose, CA, USA) or dextramers (Immudex), CD3 or CD8 antibodies (Beckman Coulter), and analyzed by flow cytometry using a FACSCalibur and CellQuest software (BD Biosciences).

1.5. Functional assays

Affinity of specific T cells

The affinity of tumor-specific T cells was assessed by measuring tetramer dissociation over time. T cells were labeled with an iTAg™ HLA-A^*02:01 ⁺ tetramer for 30 min at 2°C to 8°C, and then with anti-CD3 and anti-CD8 antibodies for 15 min at room temperature. Cells were incubated at 37°C, fixed at different time points (0, 0.5, 1 , 2, 4, and 7 h), and analyzed by flow cytometry using a FACSCalibur and CellQuest software (BD Biosciences). The affinity of tumor-specific T cells was also determined from the mean fluorescence intensity (MFI) of the iTAg™ HLA-A^*02:01 multimer labelling.

IFN-y secretion by tumor-specific CD8 T cells

T cells were first labelled with iTAg™ HLA-A^*02:01 -multimers, washed, and re- stimulated with peptide-pulsed T2 cells (10:1 ratio) for 5 h and 30 min. BrefeldinA (1 μΙ/mL; BD Biosciences) was added for the last 3 h. Cells were then surface-stained with anti-CD3 and anti-CD8 antibodies and submitted to IFN-γ intracellular staining (BD Biosciences). Cytotoxic activity of tumor-specific T cells

Antigen-specific cytotoxicity was measured by performing a standard ⁵¹Cr-release assay. Effector T cells were sorted from the co-culture using an EasySep human T cell enrichment kit (StemCell, Vancouver, Canada). Target cells (MelA or MUC1 (control) peptide-pulsed HLA-A^*02:01 ⁺ T2 cells, allogeneic tumor cells) were loaded with 50 μθί for 1 h, washed, and plated with effector T cells at the indicated E:T ratio in round-bottom 96- well plates. After 4 h of incubation, radioactivity was measured in 30 μΙ_ of supernatant using a scintillation counter (TopCount NXT; PerkinElmer, Waltham, MA, USA). The mean of triplicate measurements was expressed as a percentage of specific lysis using the formula: (sample release - spontaneous release) / (maximal release - spontaneous release) ^χ 100.

1.6. Statistical analysis

Statistical analysis was performed using the Kruskal-Wallis test, Mann-Whitney non- parametric U test, Wilcoxon matched-pairs f test, Spearman correlation, and Log-rank test using Prism software (GraphPad Software, La Jolla, CA, USA).

Example 2: pDC line triggers tumor-specific T cells exhibiting a broad diversity of functional features from PBMCs and TILs

The inventors designed an assay involving co-culture of the HLA-A^*02:01 peptide- pulsed pDC line (GEN2.2) with PBMCs or TILs sampled from stage I— IV HLA-A^*02:01 ⁺ melanoma patients in order to trigger in vitro expansion of tumor-specific T cells (Table 1 ). Four H LA- A^*02:01 -restricted immunodominant peptides derived from melanoma-tumor antigens (MelA, GP100, tyrosinase, and MAGE-A3) were used. As shown in Figure 1 A, the peptide-loaded GEN2.2 cell line elicited tumor-specific CD8 T cells from PBMCs and TILs in all patients and displayed a wide range of functional properties (positive response defined as % tetramer⁺ CD8 T cell multiplied by at least three between dO and d20). The percentages of MelA-specific tetramer⁺ CD8 T cell responses induced at day 20 varied between 0.1 1 % and 62.2%. The diversity of the antitumor responses was also variable, as some patients responded to only one antigen, while others responded to four antigens (Figure 1 B). Upon assessment of the functionality of the tumor-specific T cells, the inventors observed variations in the proportions of tumor-specific CD8⁺ T cells secreting IFN-γ upon specific restimulation, ranging from 1 .6% to 75.1 % (Figure 1 C), and with cytotoxic properties ranging from 1 % to 100% (Figure 1 D). The inventors also evaluated the avidity of the tumor-specific T cells by measuring tetramer dissociation at different time points after staining, with avidity being inversely correlated with the tetramer dissociation rate. Interestingly, tumor-specific T cells elicited by the peptide-pulsed pDC line displayed very different avidity levels (Figure 1 E), with dissociation rates of the MelA-tetramer at 7 h ranging from 0% (100% of the initial tetramer label) to 68% (32% of the initial tetramer label). The avidity of tumor-specific T cells was also addressed by analyzing the mean fluorescence intensity (MFI) of MelA-multimer labeling, with the results varying depending on the patient (Figure 1 F). Therefore, this pDC-line assay showed that it was possible to expand antitumor-specific CD8+ T cells presenting different functional features both from PBMCs and TILs, even if the latter are found in a potential immune-suppression context induced by the tumor cells. Example 3: The features of tumor-specific T cells triggered by the pDC line from PBMCs at diagnosis are independent of the clinical parameters at diagnosis

The inventors next determined whether the characteristics of the expanded antitumor T cells were dependent upon tumor development. Therefore, the inventors looked for potential correlations between each immune feature and the histological parameters at diagnosis (Breslow index, Clark score, ulceration, and AJCC staging) in a cohort of 33 stage l-ll HLA-A^*02:01 ⁺ melanoma patients who were sampled at the time of diagnosis. The level of response toward MelA, the proportion of IFN-y-secreting T cells, the cytotoxic activity, and the affinity of the triggered T cells were independent of the clinical parameters tested, with no correlation observed between the parameters (Table 2). These data revealed that at an early stage, the evolution of the disease did not impact the response of the antitumor T cells from the inventors' pDC-line assay.

Example 4: The features of tumor-specific T cells depend upon disease stage at sampling time

The inventors then determined whether the stage of disease could influence the parameters of the immune responses studied by comparing results obtained with the blood of stage l-ll versus stage lll-IV patients (n = 38 and n = 12, respectively). Surprisingly, a better amplification of tumor-specific T cells displaying higher cytotoxic activity was obtained from advanced-stage patients as compared with patients at earlier stages (Figure 2A). However, the tumor-specific T cells displayed a lower ability to secrete IFN-γ and a lower avidity (identified by a lower intensity of tetramer staining) when elicited from the blood of stage lll-IV patients as compared with stage l-ll patients (Figure 2B). No difference depending on disease stage was observed regarding the diversity of the immune response elicited (Figure 2C). Therefore, tumor T cells elicited from patients using the inventors' pDC-line assay display differential properties depending on the stage of the disease. Example 5: The avidity of expanded tumor-specific T cells can predict patient clinical outcome

The inventors investigated whether the features of the tumor-specific T cells elicited by the pDC line could predict progression-free survival (PFS) and/or overall survival (OS). The inventors observed that the percentage of MelA-specific T cells, the proportion of IFNy-secreting tumor-specific T cells, the cytotoxic activity of tumor-specific T cells, and the initial affinity of MelA-specific T cells were not correlated with the PFS (Figure 3A-D, Figure 5) or OS (not shown) from the time of diagnosis. Strikingly, the avidity of MelA- specific T cells elicited from PBMCs predicted the PFS when calculated from diagnosis date (Figure 3E). Although the basal avidity of MelA-specific T cells was not informative of PFS and OS after sampling (Figure 6), the avidity of MelA-specific T cells upon GEN2.2 stimulation predicted both PFS and OS after the sampling date (Figure 4), even when considering only stage l-ll patients (Figure 7). These data revealed that the avidity of the expanded tumor-specific T cells could be a predictive marker of clinical evolution. No link was noticed between the features of tumor-specific T cells elicited from TILs and the clinical evolution of patients (Figure 8). As expected, the proportion of tumor-infiltrating total CD8 T cells appeared as a predictive marker of clinical evolution (Figure 9). Example 6: Discussion

The identification of predictive factors of clinical evolution is needed to allow clinicians to better orientate therapeutic strategies and schedules for patients. Here, the inventors revealed that 1 ) the quality of the response to in vitro stimulation with a peptide- loaded pDC line was independent of the histological parameters at diagnosis, but depended upon the stage of the disease, and 2) this peptide-loaded pDC assay represents an innovative, non-invasive immune tool for melanoma-patient prognosis assessment.

The inventors observed that the features of MelA-specific T cells elicited by a pDC line from PBMCs from stage l-ll patients were independent of the histological parameters at diagnosis (Breslow index, Clark level, ulceration and AJCC staging). However, the quality of this response was influenced by the disease stage. Indeed, in vitro stimulation triggered similar diverse responses, but elicited tumor-specific T cells with lower IFNv- secretion potential, lower avidity, and higher cytotoxic activity in advanced-stage as compared with early stage PBMCs. These results suggested that patients with more advanced stages of disease displayed tumor-specific T cells that had been shaped by the disease, which could be related to the immunosuppressive tumor environment. Very little information is available regarding the predictive factors associated with response to therapy in melanoma. The presence of primary melanoma ulceration could be a marker of good response to IFN-a adjuvant therapy in stage III melanoma patients, given that this parameter is positively associated with an increase in relapse-free survival, distant metastasis-free survival, and OS. Elsewhere, vitiligo, reflecting anti-melanoma immunity, is associated with a better PFS and OS for melanoma patients, especially for stage lll-IV patients receiving immunotherapy. These clinical observations and the demonstration of spontaneous regression suggest that patient immunity can control tumor growth and prevent relapses. This is reinforced by the effectiveness of immunotherapies based on immune-checkpoint blockers that allow for reversal of melanoma-induced immunosuppression correlated with the presence of neo-antigens expressed by the tumor.

The inventors' results highlighted for the first time the avidity of tumor-specific T cells as a critical feature for predicting patient time to relapse and OS in melanoma. T lymphocyte activity critically depends upon the strength of the binding of T cell receptors (TCR) to cognate peptide-HLAs, directing TCR affinity and avidity. TCR affinity contributes to functional T cell avidity, and complex methods have been developed to precisely evaluate this affinity and allow demonstration of the wide range of anti-pathogen na^'ive T cell TCR affinities. Conversely, there is a bias in the frequency of high-avidity lymphocytes directed toward self-derived tumor-associated Ag, which is very low, because these cells are deleted by central and peripheral deletion mechanisms.

The staining brightness of peptide-HLA-multimer labeling of T cells is a simple approach to quantify functional T cell avidity. The assay presented here is able to reveal, quantify, and qualify the presence of antitumor T cells exhibiting high avidity from circulating PBMCs, with the presence of such T cells being a good prognostic factor for melanoma patients, regardless of initial disease staging. Therefore, the use of this peptide-loaded pDC assay could represent a non-invasive immunological tool for cancer- patient prognosis assessment.

Claims

1. An in vitro method for predicting the clinical evolution of a patient suffering from cancer, said method comprising the steps of:

2. The in vitro method according to claim 1 , wherein predicting the clinical evolution of the patient comprises predicting the risk of relapse of the patient, predicting the long- term survival of the patient, or predicting the response of the patient to an anti-cancer treatment.

3. The in vitro method according to claim 1 or 2, wherein an avidity lower than the reference level is predictive of a high risk of relapse of the patient, of a poor long-term survival for the patient, or of a poor response of the patient to the anti-cancer treatment.

4. The in vitro method according to any one of claims 1 to 3, wherein an avidity higher than the reference level is predictive of a low risk of relapse of the patient, of a good long-term survival for the patient, or of a good response of the patient to the anticancer treatment.

5. An in vitro method for selecting a patient suffering from cancer, suitable to be treated with a non-aggressive therapy comprising the step of: a) obtaining a plasmacytoid dendritic cell (pDC) line pulsed with at least one tumoral antigen, and irradiated; said pDC line sharing at least one major histocompatibility complex (MHC) allele with PBMC from the patient suffering from cancer;

6. An in vitro method for selecting a patient suffering from cancer suitable to be treated with an aggressive therapy comprising:

e) selecting the patient as suitable to be treated with an aggressive therapy if the avidity measured at step d) is lower than a reference level.

7. The in vitro method according to any one of claims 1 to 6, wherein the PBMC are obtained from a patient at the time of cancer diagnosis, or from a patient who has already received anti-cancer treatment.

8. An in vitro method for monitoring the response of the patient to a treatment comprising the steps of: a) measuring the T cells avidity for at least one tumoral antigen by implementing steps a) to steps d) of the method for predicting the clinical evolution of a patient suffering from cancer according to the invention, before onset of said treatment; and

9. An in vitro method for monitoring the progression of the cancer comprising the steps of:

10. The in vitro method according to any one of claims 1 to 9, wherein the at least one major histocompatibility complex (MHC) allele shared by the PBMC and the pDC line is HLA-A^*02:01 .

11. The in vitro method according to any one of claims 1 to 10, wherein the pDC line is the GEN2.2 pDC line or the GEN3 pDC line.

12. The in vitro method according to any one of claims 1 to 1 1 , wherein the cancer is melanoma.

13. The in vitro method according to any one of claims 1 to 12, wherein the at least one tumoral antigen is a peptide derived from an antigen selected from the group consisting of MelA, GP100, tyrosinase and MAGE- A3.

14. The in vitro method according to any one of claims 1 to 13, wherein the avidity is measured by MHC-antigen multimer staining.

15. An anti-cancer treatment for use for the treatment of cancer in a patient whose clinical evolution has been predicted by the in vitro method according to any one of claims 1 to 4, 7 and 10 to 14.