Methods and compositions for identifying whether a subject with cancer will respond to immune checkpoint inhibitors

Abstract

The present invention relates to the use of soluble CD27 as a biomarker to predict response to immune checkpoint inhibitor therapy. The inventors have worked with two cohorts of patients and have identified a soluble marker, CD27, present in the plasma of patients with renal cell carcinoma, whose pre-treatment concentrations predict response to anti-PD-1 / PD-L1. This marker is more of a predictive marker for response to anti-PD-1 / PD-L1 therapy than a prognostic marker. In fact, it is not associated with better survival in patients with metastatic renal cell carcinoma treated with anti-angiogenic agents. This marker is not correlated with conventional clinical severity markers used to classify patients with metastatic renal cancer.

Description

Technical Field

[0001] The present invention is in the field of oncology. More specifically, the present invention relates to methods and compositions for determining whether a subject with cancer will respond to treatment with an immune checkpoint inhibitor. Background Art

[0002] The anti-PD-1 antibody (nivolumab) has been approved for second-line treatment of metastatic renal cell carcinoma, and a phase 3 study showed that this treatment was superior to antiangiogenic therapy (everolimus) [1]. Twenty-five percent of patients responded to this treatment, with a survival advantage of 5.5 months compared with standard treatment.

[0003] To date, traditional biomarkers such as PD-L1 or mutational burden or intratumoral CD8+ T cell infiltration have not been able to predict response to this immunotherapy in patients with metastatic renal cell carcinoma [1-3].

[0004] In previous studies by our group, we found that co-expression of PD-1 and Tim-3 cells on CD8+ T cells was associated with a poor prognosis. Functional studies showed that these CD8+ T cells were not functional [4]. Consistent with our results, other preliminary studies have shown that this co-expression predicts response to immunotherapy [5,6]. Thus, in a recent clinical study targeting the PD-1 / PD-L1 axis (programmed death ligand 1), it has been shown that CD8+ T cells expressing PD-1 and CD28 are required for therapeutic effectiveness. Interestingly, CD8+ T cells co-expressing PD-1 and Tim-3 are mainly CD28-negative [7].

[0005] In recent decades, the use of immunotherapy, especially immune checkpoint inhibitors for cancer has become common and is used to treat solid tumors and hematological malignancies. However, only some patients respond to treatment with immune checkpoint inhibitors. Therefore, there is a need to identify new biomarkers to determine whether a subject will respond to treatment with immune checkpoint inhibitors. Summary of the invention

[0006] The present invention relates to a method for determining whether a subject suffering from cancer will obtain a response to an immune checkpoint inhibitor, comprising the steps of: i) quantifying the level of soluble CD27 (sCD27) in a biological sample obtained from the subject, ii) comparing the level of soluble CD27 quantified in step i) with its corresponding predetermined reference value, and iii) concluding that the subject will not respond to the treatment when the level of soluble CD27 is higher than its corresponding predetermined reference value, or concluding that the subject will respond to the treatment when the level of soluble CD27 is lower than its corresponding predetermined reference value. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION

[0007] The inventors have worked with two cohorts of patients: 1) The Checkpoint Cohort (CPP: 2015-08-04-MS2) established at the Georges Pompidou Europea Hospital, which includes all patients treated with immunotherapy (anti-PD-1 / PD-L1) since 2016, and blood and tissue samples were collected from them after obtaining the patient's consent. 27 patients with renal cell carcinoma from this cohort were included in this study. 22 of these patients had clear cell renal carcinoma. 2) Another set of specimens came from the Preinsut clinical study, in which patients with renal cell carcinoma received two cycles of sunitinib before nephrectomy [8]. 27 patients from this cohort were also included in this study.

[0008] The inventors have identified a soluble marker, CD27, present in the plasma of patients with renal cell carcinoma, whose pre-treatment concentrations predict the response to anti-PD-1 / PD-L1. This marker is more of a predictive marker for response to anti-PD1 / PD-L1 therapy than a prognostic marker. In fact, it is not associated with better survival in patients with metastatic renal cell carcinoma treated with anti-angiogenic agents. This marker does not correlate with conventional clinical severity markers for classifying patients with metastatic renal carcinoma.

[0009] Methods for predicting response to immune checkpoint inhibitor therapy

[0010] Therefore, in a first aspect, the present invention relates to a method for determining whether a subject suffering from cancer will respond to an immune checkpoint inhibitor, comprising the steps of: i) quantifying the level of soluble CD27 in a biological sample obtained from the subject treated with an immune checkpoint inhibitor, ii) comparing the level of soluble CD27 quantified in step i) with its corresponding predetermined reference value, and iii) concluding that the subject will not respond to the treatment when the level of soluble CD27 is above its corresponding predetermined reference value, or concluding that the subject will respond to the treatment when the level of soluble CD27 is below its corresponding predetermined reference value.

[0011] In particular, the present invention relates to a method for predicting the overall survival (OS) of a subject with cancer before treatment with an immune checkpoint inhibitor, comprising the steps of i) determining the level of sCD27 in a biological sample obtained from the subject, ii) comparing the level with a predetermined reference value, and iii) providing a better prognosis when the level of sCD27 is lower than the predetermined reference value, and providing a worse prognosis when the level of sCD27 is higher than the predetermined reference value.

[0012] As used herein, the term "will obtain a response" or "response" refers to a response to a treatment of a subject with cancer. Typically, such treatments induce, improve, or otherwise cause an improvement in pathological symptoms, disease progression, or a physiological condition associated with or resistant to cancer. In particular, in the context of the present invention, the term "response" refers to the ability of immune checkpoint inhibitors to improve pathological symptoms, so that the subject shows clinical improvement compared to a subject who has not received treatment. The subject is considered to be a "responder" to treatment. The term "no response" refers to a subject who has not shown any clinical improvement in treatment with immune checkpoint inhibitors. The subject is considered to be a "non-responder" to treatment. Therefore, subjects who are considered to be "non-responders" have specific monitoring in the treatment regimen. In a specific embodiment, the response to treatment is determined by the solid tumor response evaluation criteria (RECIST) criteria. The criteria refer to a set of published rules that define when a cancer subject's tumor improves ("response"), remains unchanged ("stable"), or worsens ("progress") during treatment. In the context of the present invention, when a subject is identified as a responder, it means that the subject has improved overall survival and progression-free survival (OS / PFS). More specifically, soluble CD27 is a tool for determining a subject's overall survival (OS) before starting treatment with an immune checkpoint inhibitor.

[0013] As used herein, the term "overall survival (OS)" refers to the percentage of subjects in a study or treatment group who survive a certain period of time after being diagnosed with or starting treatment for a disease (such as cancer) (according to the present invention).

[0014] As used herein, the term "cancer" refers to a malignant growth or tumor caused by uncontrolled cell division. The term "cancer" includes primary tumors and metastatic tumors.

[0015] Furthermore, the cancer may specifically be of the following histological types, but is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branching alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic cell carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosis carcinoma, adrenocortical carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine carcinoma; sebaceous carcinoma; cerumen carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant neoplasm; ovarian stromal tumor, malignant neoplasm; thecoma cell tumor, malignant neoplasm; granulosa cell tumor, malignant neoplasm; male cell tumor, malignant neoplasm; Sertoli cell carcinoma; Leydig cell tumor, malignant neoplasm; lipid cell tumor, malignant neoplasm; paraganglioma, malignant neoplasm; extramammary paraganglioma, malignant neoplasm; pheochromocytoma; hematopoietic cell carcinoma; Glomerular sarcoma; malignant melanoma; amelanotic melanoma; superficially expanding melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant tumor; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant tumor; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant tumor; mixed Müllerian tumor; Wilms tumor; hepatoblastoma; carcinosarcoma; stromal tumor, malignant tumor; Brenner tumor, malignant tumor; phyllodes tumor, malignant tumor; synovial sarcoma; mesothelioma, malignant tumor; dysgerminoma; embryonal carcinoma; teratoma, malignant tumor; goiter-like ovarian tumor, malignant tumor; choriocarcinoma; mesonephroblastoma, malignant tumors; angiosarcoma; hemangioendothelioma, malignant tumor; Kaposi's sarcoma; hemangiopericytoma, malignant tumor; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant tumor; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumors, malignant tumors; ameloblastic odontosarcoma; ameloblastoma, malignant tumor; ameloblastic fibrosarcoma; pinealoma, malignant tumor; chordoma; glioma, malignant tumor; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioblastoma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma;Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Lymphoma, malignant; Hodgkin's disease; Paragranuloma; Lymphoma, malignant, small lymphocytic; Lymphoma, malignant, large cell, diffuse; Lymphoma, malignant, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphoid leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryocyte leukemia; Myeloid sarcoma and hairy cell leukemia. ;

[0016] In a specific embodiment, cancer is renal cancer. As used herein, the term "kidney cancer", "renal cancer" or "renal cell carcinoma" refers to cancer that has arisen from the kidney. The term "renal cell carcinoma" or "renal cell carcinoma" (RCC), as used herein, refers to a cancer originating from the inner wall of the proximal convoluted tubule. More specifically, RCC includes several relatively common histological subtypes: clear cell renal cell carcinoma, papillary (pheochromocytic) carcinoma, chromophobe cell carcinoma, collecting duct carcinoma and medullary carcinoma. Clear cell renal cell carcinoma (ccRCC) is the most common RCC subtype. In a specific embodiment, cancer is metastatic renal cell carcinoma.

[0017] In another embodiment, the cancer is lung cancer. As used herein, the term "lung cancer" includes, but is not limited to, all similar lung cancers in all stages of progression, such as lung cancer, metastatic lung cancer, non-small cell lung cancer (NSCLC), such as lung adenocarcinoma, squamous cell carcinoma, or small cell lung cancer (SCLC). In some embodiments, the subject suffers from non-small cell lung cancer (NSCLC).

[0018] As used herein, the term "subject" refers to a mammal, such as a rodent, a feline, a canine, and a primate. In particular, the subject according to the present invention is a human. More particularly, the subject according to the present invention suffers from or is suspected of suffering from renal cell carcinoma (RCC). In a specific embodiment, the subject suffers from or is suspected of suffering from lung cancer.

[0019] 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 tissue biopsy. In a specific embodiment, the biological sample used to determine the expression level includes a sample such as a blood sample, a lymph sample, or a tissue biopsy. In a specific embodiment, the biological sample is a blood sample. In another embodiment, the biological sample is a plasma sample.

[0020] As used herein, the term "CD27" is a member of the tumor necrosis factor receptor superfamily. Immunologists are currently interested in it as a co-stimulatory immune checkpoint molecule. CD27 binds to the ligand CD70 and plays an important role in regulating B cell activation and immunoglobulin synthesis. The soluble form of CD27 (sCD27), a 32kD protein, is identical to the extracellular domain of membrane-bound CD27 and can be released from the cell surface after lymphocyte activation by differential splicing of the receptor protein or protease shedding.

[0021] As used herein, the term "level of soluble CD27" refers to the concentration of soluble CD27. Generally, the level or concentration of the soluble CD27 gene can be determined by any technique known to those skilled in the art. In particular, the concentration can be measured at the genome and / or nucleic acid and / or protein level. In a specific embodiment, the expression level of the gene is determined by measuring the amount of each gene nucleic acid transcript. In another embodiment, the expression level is determined by measuring the amount of the corresponding protein of each gene. The amount of nucleic acid transcripts can be measured by any technique known to those skilled in the art. In particular, the measurement can be performed directly on an extracted messenger RNA (mRNA) sample, or on a reverse transcribed complementary DNA (cDNA) prepared from the extracted mRNA by techniques known in the art. From an mRNA or cDNA sample, the amount of nucleic acid transcripts can be measured using any technique known to those skilled in the art, including nucleic acid microarrays, quantitative PCR, microfluidic cards, and hybridization with labeled probes. In a specific embodiment, the expression level is determined by using quantitative PCR. Quantitative or real-time PCR is a technique well known and readily available to those skilled in the art and does not require precise description. Methods for determining the amount of mRNA are well known in the art. For example, first, the nucleic acid contained in the biological sample is extracted according to standard methods, for example, using a lyase or chemical solution or extracting by a nucleic acid binding resin according to the instructions of the manufacturer. Then the extracted mRNA is detected by hybridization (for example, Northern blot analysis) and / or amplification (for example, RT-PCR). Preferably, quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and amplification based on nucleic acid sequence (NASBA). Nucleic acids having at least 10 nucleotides and showing sequence complementarity or homology with the mRNA concerned herein can be used as hybridization probes or amplification primers. It should be understood that such nucleic acids do not need to be identical, but are generally at least about 80% identical to homologous regions of comparable size, more preferably 85% identical, and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with suitable means such as detectable labels to detect hybridization. A variety of suitable indicators are well known in the art, including fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin). The probe typically comprises a single-stranded nucleic acid between 10 and 1000 nucleotides in length, e.g., between 10 and 800, more preferably between 15 and 700, and typically between 20 and 500. The primer is typically a shorter single-stranded nucleic acid, between 10 and 25 nucleotides in length, intended to perfectly or nearly perfectly match the target nucleic acid to be amplified.Probes and primers are "specific" for nucleic acids, they hybridize to nucleic acids, that is, they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, for example, 50% formamide, 5x or 6x SCC. SCC is 0.15M NaCl, 0.015M sodium citrate). The nucleic acid primers or probes used in the above-mentioned amplification and detection methods can be assembled into a kit. Such a kit contains a common primer and a molecular probe. The kit also contains the components necessary to determine whether amplification has occurred. For example, the kit can also contain PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting specific sequences. In a specific embodiment, the method of the present invention includes the following steps: providing total RNA extracted from a biological sample, and amplifying the RNA and hybridizing it with a specific probe, more specifically by quantitative or semi-quantitative RT-PCR. In another embodiment, the expression level is determined by DNA chip analysis. Such DNA chips or nucleic acid microarrays consist of different nucleic acid probes chemically attached to a substrate, which can be a microchip, a slide, or microbead-sized beads. Microchip can be made of polymer, plastic, resin, polysaccharide, silicon dioxide or silicon dioxide based material, carbon, metal, inorganic glass or nitrocellulose.Probe comprises nucleic acid, as can be about 10 to about 60 base pairs of cDNA or oligonucleotide.To determine expression level, biological sample from test subject (optionally first reverse transcription) is labeled and contacts with microarray under hybridization conditions, causes formation of complex between target nucleic acid, these complexes are complementary to the probe sequence attached to microarray surface.Then, detect the hybridization complex of labeling, and can be quantitative or semi-quantitative to it.Can be labeled by various methods, for example, by using radioactivity or fluorescent labeling.Those skilled in the art can obtain many variants of microarray hybridization technology (referring to, for example, the review of Hoheisel, Nature Reviews, Genetics, 2006,7:200-210).

[0022] In a specific embodiment, the level of soluble CD27 (sCD27) present in a plasma sample obtained from a subject is determined, for example, by detecting the amount of sCD27 present in the plasma sample. In some embodiments, to determine the amount of sCD27, the sample is contacted with a specific binding agent (such as an antibody, e.g., a monoclonal antibody anti-CD27 or protein, such as CD7) that specifically binds to sCD27, and the amount of the specific binding agent bound to sCD27 is detected. In the context of the present invention, the Procartaplex kit (Thermofischer) is used to detect a group of soluble inhibitors or activator receptors (BTLA, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, Tim-3, CD28, CD80, 4-1BB, CD27, and CTLA-4).

[0023] In some embodiments, the amount of sCD27 present in a plasma sample is detected by mass spectrometry. In some embodiments, the amount of sCD27 present in a plasma sample is detected by measuring the activity of sCD27 in a plasma sample. The amount is compared with a control value. Typically, a log-rank test (Kaplan Meier) or a Cox test is used to associate different parameters with patient survival. Different variables are qualitatively analyzed, a threshold is set at the median to divide it into two, or a quantitative analysis (Cox model). Patients are divided into two groups according to plasma CD27 concentration (above or below the median). The patient's survival is determined from the start of treatment. A log-rank test is performed to compare the survival of 2 groups of patients. For the CD27 marker, no matter what statistical test is used, this correlation can be found: log-rank (qualitative variable) (p=0.005) (Figure 1A) or Cox model (quantitative variable) (Table 1) (p=0.04).

[0024] In some embodiments, a score consisting of soluble CD27 levels is determined and compared with a reference value. When the concentration of soluble CD27 is determined to be higher than the reference value, it indicates that the subject will not respond to the immune checkpoint inhibitor. When the concentration of soluble CD27 is lower than the reference value, it indicates that the subject will respond to the immune checkpoint inhibitor. Typically, the predetermined reference value is a threshold or cutoff value, which can be determined by experiment, experience or theory. The threshold value can also be arbitrarily selected according to existing experimental and / or clinical conditions, as will be recognized by those of ordinary skill in the art. For example, when establishing a predetermined reference value, a retrospective measurement of soluble CD27 levels in historical plasma samples that have been properly stored can be used. The threshold must be determined based on the function of the test and the benefit / risk balance (clinical consequences of false positives and false negatives) to obtain optimal sensitivity and specificity. Typically, the receiver operating characteristic (ROC) curve based on experimental data can be used to determine the optimal sensitivity and specificity (and threshold). For example, after determining the expression level of soluble CD27 in the reference group, the expression level determined in the sample to be tested can be statistically processed by algorithmic analysis to obtain a classification standard that is of great significance to sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operating characteristic curve. It is mainly used for clinical biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of the true positive rate (sensitivity) and the false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity through image synthesis methods. A series of different cutoff values ​​(thresholds or critical values, the boundary values ​​between normal and abnormal results of diagnostic tests) are set as continuous variables, and a series of sensitivity and specificity values ​​are calculated. Then, the curve is drawn with sensitivity as the ordinate and specificity as the abscissa. The larger the area under the curve (AUC), the higher the accuracy of the diagnosis. On the ROC curve, the point closest to the upper left corner of the coordinate graph is the critical point with high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, as AUC approaches 1, the diagnostic results are getting better and better. 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 greater than 0.9, the accuracy is high. Preferably, this algorithm is completed by computer. The ROC curve can be drawn using existing software or systems in the art, such as: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0025] In a specific embodiment, the method according to the present invention further comprises the step of classifying the subject by an algorithm and determining whether the subject will obtain a response to immune checkpoint inhibitor therapy.

[0026] Generally, the methods of the present invention comprise a) quantifying the level of soluble CD27 in a biological sample; b) performing a classification algorithm on data comprising the quantified sCD27 levels to obtain an algorithm output; and c) determining from the algorithm output of step b) the probability that the subject will or will not respond to an immune checkpoint inhibitor.

[0027] In some embodiments, according to the method of the present invention, the algorithm is selected from linear discriminant analysis (LDA), topological data analysis (TDA), neural network, support vector machine (SVM) algorithm and random forest algorithm (RF), selected from linear discriminant analysis (LDA), topological data analysis (TDA), neural network, support vector machine (SVM) algorithm and random forest algorithm (RF).

[0028] In some embodiments, the method of the present invention includes a step of determining the subject's response using a classification algorithm. As used herein, the term "classification algorithm" has the usual meaning in the art and refers to classification and regression tree algorithms and multivariate classification well known in the art, such as described in US8,126,690; WO2008 / 156617. As used herein, the term "support vector machine (SVM)" is a general learning machine for pattern recognition, whose decision surface is parameterized by a set of support vectors and a set of corresponding weights, referring to a method of processing multiple variables simultaneously instead of processing them separately. Therefore, support vector machines can be used as statistical tools for classification. Support vector machines nonlinearly map their n-dimensional input space to a high-dimensional feature space and present an optimal interface (optimal classification surface) between features. Support vector machines include two stages: a training stage and a testing stage. Support vectors are generated in the training stage, and are estimated according to specific rules in the testing stage. In general, SVM provides a model for classifying each of n subjects into two or more disease categories based on a k-dimensional vector (called a k-tuple) of biomarker measurements for each subject. SVM first uses a kernel function to convert k-tuples into an equal-dimensional or higher-dimensional space. The kernel function projects the data into a space in which the hyperplane can be used to better separate the categories than in the original data space. In order to determine the hyperplane used to distinguish the categories, a set of support vectors closest to the boundary between the disease categories can be selected. The hyperplane is then selected by known SVM techniques so that the distance between the support vector and the hyperplane is the largest within the range of the cost function that penalizes incorrect predictions. The hyperplane is the hyperplane that best separates the data in terms of prediction (Vapnik, 1998Statistical Learning Theory. New York: Wiley). Then, according to the position of the observation relative to the hyperplane, any new observation is classified as belonging to any one of the categories of interest. When considering more than two categories, the process is performed in pairs for all categories, and these results are combined to create rules that distinguish all categories. As used herein, the term "random forest algorithm" or "RF" has its general meaning in the art and refers to a classification algorithm, such as described in US 8,126,690; WO2008 / 156617. Random forest is a decision tree-based classifier built using an algorithm originally developed by Leo Breiman (Breiman L, "Random forests," Machine Learning 2001, 45: 5-32). The classifier uses a large number of individual decision trees and decides on the class by selecting the pattern of classes decided by the individual trees.Individual trees are constructed using the following algorithm: (1) Assume that the number of cases in the training set is N and the number of variables in the classifier is N; (2) Select the number of input variables that will be used to determine the decision at the tree node; this number, m, should be much smaller than M; (3) Select the training set by selecting N samples from the training set with replacement; (4) For each node of the tree, randomly select m of the M variables as the basis for the decision at that node; (5) Calculate the best split based on these m variables in the training set. In some embodiments, the score is generated by a computer program.

[0029] The algorithms of the present invention can be executed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. The algorithms can also be executed by dedicated logic circuits, and the apparatus can also be implemented as dedicated logic circuits, e.g., FPGA (Field Programmable Gate Array) or ASIC (Application Specific Integrated Circuit). Processors suitable for executing computer programs include, for example, general and special purpose microprocessors, and any one or more processors of any type of digital computer. Generally, a processor will receive instructions and data from a read only memory or a random access memory or both. The basic elements of a computer are a processor for executing instructions and one or more storage devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from and / or transfer data to one or more mass storage devices for storing data (e.g., magnetic disks, magneto-optical disks, or optical disks). However, a computer need not have such devices. Computer readable media suitable for storing computer program instructions and data include all forms of non-volatile memory, media, and storage devices, including semiconductor storage devices, such as, EPROM, EEPROM, and flash memory devices; magnetic disks, such as internal hard disks or removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, dedicated logic circuits. To provide for interaction with a user, embodiments of the present invention can be implemented on a computer having a display device, e.g., in a non-limiting example, a CRT (Cathode Ray Tube) or LCD (Liquid Crystal Display) monitor for displaying information to the user and a keyboard and a pointing device, such as a mouse or a trackball, by which the user can provide input to the computer. Other types of devices can also be used to provide for interaction with the user; for example, the feedback provided to the user can be any form of sensory feedback, such as visual feedback, auditory feedback, or tactile feedback; and input from the user can be received in any form, including acoustic, speech, or tactile input. Thus, in some embodiments, the algorithms can be implemented in a computing system that includes backend components, such as a data server, or includes middleware components, such as an application server, or includes frontend components, such as a client computer having a graphical user interface or a web browser through which the user can interact with the present invention, or any combination of one or more such backend, middleware, or frontend components. The components of the system can be interconnected by any form or medium of digital data communication, e.g., a communication network. Examples of communication networks include local area networks (“LAN”) and wide area networks (“WAN”), such as the Internet. A computer system can include clients and servers. Clients and servers are typically remote from each other and typically interact through a communication network.The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0030] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that fully or partially reduces, inhibits, interferes with, or modulates one or more immune checkpoint proteins.

[0031] As used herein, the term "immune checkpoint protein" has its general meaning in the art, and refers to molecules expressed by T cells, wherein or open signals (stimulatory checkpoint molecules) or weaken signals (inhibitory checkpoint molecules). Immune checkpoint molecules are well known in the art, to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, for example, Pardoll, 2012.Nature Rev Cancer 12: 252-264; Mellman et al., 2011.Nature 480: 480-489). Examples of stimulatory checkpoints include CD27, CD28, CD40, CD122, CD137, OX40, 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. Adenosine A2A receptor (A2AR) is considered an important checkpoint for cancer treatment because adenosine in the immune microenvironment leads to the activation of A2a receptors, which is an immune negative feedback loop, and the concentration of adenosine in the tumor microenvironment is relatively high. B7-H3, also known as CD276, was originally considered a co-stimulatory molecule, but is now considered a co-inhibitory molecule. B7-H4, also known as VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape. B and T lymphocyte attenuator (BTLA), also known as CD272, has HVEM (herpes virus entry mediator) as its ligand. The surface expression of BTLA is gradually downregulated during the differentiation of human CD8+T cells from naive to effector cell phenotypes, and then, tumor-specific human CD8+T cells express high levels of BTLA. CTLA-4 is cytotoxic T lymphocyte-associated protein 4, and is also known as CD152. The expression of CTLA-4 on Treg cells is used to control T cell proliferation. IDO, indoleamine 2,3-dioxygenase, is a tryptophan decomposition enzyme. A related immunosuppressive enzyme. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to inhibit T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. KIR, killer cell immunoglobulin-like receptor, is a receptor for MHC class I molecules on natural killer cells. LAG3, lymphocyte activation gene 3, inhibits immune responses through effects on 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 was approved by the FDA in September 2014. One advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.TIM-3 is the abbreviation of T cell immunoglobulin domain and mucin domain 3, which is expressed on activated human CD4+T cells and regulates Th1 and Th17 cytokines. TIM-3 triggers cell death by interacting with its ligand galectin-9, acting as a negative regulator of Th1 / Tc1 function. VISTA is the abbreviation of V-domain Ig inhibitor of T cell activation. VISTA is mainly expressed on hematopoietic cells, so that the consistent expression of VISTA on intratumoral leukocytes may make VISTA blockers effective in a wide range of solid tumors. Tumor cells often use these checkpoints to evade detection by the immune system. Therefore, inhibiting checkpoint proteins on the immune system can enhance anti-tumor T cell responses.

[0032] In some embodiments, an immune checkpoint inhibitor refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduced function and complete blockade. In some embodiments, an immune checkpoint inhibitor can be an antibody, synthetic or natural sequence peptide, small molecule, or aptamer that binds to an immune checkpoint protein and its ligand.

[0033] In a specific embodiment, the immune checkpoint inhibitor is an antibody.

[0034] Typically, the antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, or VISTA.

[0035] In a specific embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody, such as described in WO2011082400, WO2006121168, WO2015035606, WO2004056875, WO2010036959, WO2009114335, WO2010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of commercialized anti-PD-1 antibodies are: nivolumab ( BMS), pembrolizumab (also known as lambrolizumab, or MK-3475, MERCK).

[0036] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody, such as described in WO2013079174, WO2010077634, WO2004004771, WO2014195852, WO2010036959, WO2011066389, WO2007005874, WO2015048520, US8617546, and WO2014055897. Examples of anti-PD-L1 antibodies in clinical trials are: atezolizumab (MPDL3280A, Genentech / Roche), durvalumab (AZD9291, AstraZeneca), avelumab (also known as MSB0010718C, Merck), and BMS-936559 (BMS).

[0037] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody as described in US7709214, US7432059 and US8552154.

[0038] In the context of the present invention, immune checkpoint inhibitor Tim-3 or its ligand.

[0039] In a specific embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and WO2013006490.

[0040] In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

[0041] As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules commonly used in medicine. The term does not include biomacromolecules (e.g., proteins, nucleic acids, etc.). Typically, small organic molecules range in size from up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0042] Typically, organic small molecules interfere with the transduction pathways of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, or VISTA.

[0043] In a specific embodiment, the small organic molecule interferes with the transduction pathways of PD-1 and Tim-3. For example, it can interfere with molecules, receptors or enzymes involved in the PD-1 and Tim-3 pathways.

[0044] In a specific embodiment, the organic small molecule interferes with an indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor. IDO is involved in tryptophan catabolism (Liu et al., 2010, Vacchelli et al., 2014, Zhai et al., 2015). Examples of IDO inhibitors are described in WO2014150677. Examples of IDO inhibitors include, but are not limited to, 1-methyl-tryptophan (IMT), β-(3-benzofuranyl)-alanine, β-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyltryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-methanol, 3,3'-diindolylmethane, epigallocatechingallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemet, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-aminonaphthoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole, a brassinolide derivative, a thiohydantoin derivative, a β-carboline derivative, or a brassinolide derivative. In a specific embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphthoic acid and β-[3-benzo(b)thienyl]-alanine or a derivative or prodrug thereof.

[0045] In a specific embodiment, the IDO inhibitor is Epacadostat (INCB24360, INCB024360), which has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-1,2,5-oxadiazole-3-carboxamidine:

[0046]

[0047] In a specific embodiment, the inhibitor is BGB324, also known as R428, as described in WO2009054864, refers to 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(1-pyrrolidinyl)-5H-benzocyclohepten-2-yl]-1H-1,2,4-triazole-3,5-diamine, and has the following chemical formula in the art:

[0048]

[0049] In a specific 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 inhibitor of T-cell activation (VISTA) (Liu et al. 2015). Preclinical data for CA-170 were presented by the Curis Collaborator and Aurigene at the ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics in November.

[0050] In some embodiments, the immune checkpoint inhibitor is an aptamer.

[0051] Typically, aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, or VISTA.

[0052] In a specific embodiment, the aptamer is a DNA aptamer, such as described in Prodeus et al., 2015, etc. A major disadvantage of aptamers as therapeutic entities is that their pharmacokinetic characteristics are poor, because these short DNA chains are rapidly removed from the circulation due to renal filtration. Therefore, the aptamer according to the present invention is conjugated to a high molecular weight polymer such as polyethylene glycol (PEG). In a specific embodiment, the aptamer is an anti-PD-1 aptamer. In particular, the anti-PD-1 aptamer is a PEGylated MP7 as described in Prodeus et al., 2015.

[0053] Methods for treating subjects who have been identified as responders to immune checkpoint inhibitor therapy

[0054] In a second aspect, the present invention relates to a method for treating cancer in a subject who has been identified as a responder to immune checkpoint inhibitor treatment, which results in i) quantifying the level of soluble CD27 in a biological sample obtained from the subject before treatment with an immune checkpoint inhibitor; ii) providing an assessment based on the amount of said soluble CD27 in said biological sample of said subject; and iii) if in step ii) said subject is identified as a responder to immune checkpoint inhibitor treatment, treating said subject with an immune checkpoint inhibitor.

[0055] In a specific embodiment, the present invention relates to a method for treating renal cancer in a subject who has been identified as a responder to immune checkpoint inhibitor treatment, which results in i) quantifying the level of soluble CD27 in a biological sample obtained from the subject prior to treatment with an immune checkpoint inhibitor; ii) providing an assessment based on the amount of said soluble CD27 in said biological sample of said subject; iii) communicating said assessment to said subject and iii) if in step ii) said subject is identified as a responder to immune checkpoint inhibitor treatment, treating said subject with an immune checkpoint inhibitor.

[0056] In a further embodiment, the present invention relates to a method for treating renal cancer in a subject who has been identified as a responder to immune checkpoint inhibitor treatment, comprising the steps of: i) quantifying the level of soluble CD27 in a biological sample obtained from a subject treated with an immune checkpoint inhibitor, ii) comparing the level of soluble CD27 quantified in step i) with its corresponding predetermined reference value, iii) concluding that the subject will not respond to the treatment when the level of soluble CD27 is higher than its corresponding predetermined reference value, or concluding that the subject will respond to the treatment when the level of soluble CD27 is lower than its corresponding predetermined reference value, and iv) treating the subject with an immune checkpoint inhibitor.

[0057] As used herein, the term "treating" or "treatment" refers to preventive or prophylactic treatment and curative or disease-modifying treatment, including treatment of subjects at risk of or suspected of contracting a disease and subjects who are ill or diagnosed with a disease or medical condition, including suppression of clinical relapse. Treatment can be applied to subjects with a medical condition or who may eventually acquire the condition to prevent, cure, delay onset, reduce its severity, or improve one or more symptoms of a condition or recurrent condition, or to prolong the survival of the subject beyond the expected survival time in the absence of such treatment. "Therapeutic regimen" refers to the treatment mode of the disease, for example, the mode of administration used during treatment. The therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a therapeutic regimen (or part of a therapeutic regimen) for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the subject in the initial stage of the therapeutic regimen. The induction regimen may (partially or entirely) use a "loading regimen," which may include a larger dose of the drug than the doctor uses during the maintenance regimen, a higher frequency of drug administration than the doctor during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a subject during treatment of a disease, e.g., to keep the subject in remission for a long period of time (months or years). A maintenance regimen can use continuous therapy (e.g., administration at regular intervals, e.g., weekly, monthly, annually, etc.) or intermittent therapy (e.g., interruption of treatment, intermittent treatment, treatment at relapse, or treatment after reaching a specific predetermined criterion [e.g., pain, disease manifestation, etc.]).

[0058] As used herein, the term "subject" refers to mammals, such as rodents, felines, canines and primates. In particular, the subject according to the present invention is a human. More particularly, the subject according to the present invention suffers from or is suspected of suffering from renal cell carcinoma (RCC). In a specific embodiment, the subject suffers from or is suspected of suffering from lung cancer.

[0059] As used herein, the term "cancer" refers to a malignant growth or tumor resulting from the uncontrolled division of cells as defined above.

[0060] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more immune checkpoint proteins. Such immune checkpoint inhibitors are as defined above.

[0061] In a specific embodiment, the method according to the present invention, wherein at least two immune checkpoint inhibitors are used as a combined preparation for treating a subject identified as a responder to immune checkpoint inhibitor treatment.

[0062] In a further embodiment, according to the method of the present invention, an anti-PD-1 antibody and an anti-CTLA-4 antibody are used as a combined preparation for treating a subject identified as a responder to immune checkpoint inhibitor therapy.

[0063] In a specific embodiment, according to the method of the present invention, an anti-PD-L1 antibody and an anti-CTLA-4 antibody are used as a combined preparation for treating a subject identified as a responder to immune checkpoint inhibitor therapy.

[0064] In a specific embodiment, i) an immune checkpoint inhibitor and ii) an anti-angiogenic compound are used as a combined preparation for treating a subject identified as a responder to immune checkpoint inhibitor therapy.

[0065] As used herein, the term "angiogenesis" refers to the physiological process involving the growth of new blood vessels from pre-existing blood vessels. Angiogenesis is a combinatorial process regulated by the balance between pro-angiogenic and anti-angiogenic molecules. Angiogenic stimuli (e.g., hypoxia or inflammatory cytokines) lead to the induced expression and release of angiogenic growth factors such as vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF).

[0066] As used herein, the term "anti-angiogenesis" refers to any molecule that can inhibit (anti-angiogenesis) the formation of new blood vessels. Generally, anti-angiogenic compounds are well known in the art and refer to the following compounds but are not limited to bevacizumab (Avastin, anti-VEGF), itraconazole (anti-VGFR), carboxyamidotriazole, TNP-470 (fumagillin analog), CM101, IFN-α, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonist, angiostatic steroid + heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, angiostatin, endostatin, 2-methoxyestradiol, teicogalactan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin, αVβ3 inhibitor, linomide, ramucirumab, taquimod, ranibizumab, sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), everolimus (Afinitor), cabozantinib.

[0067] In a specific embodiment, the method according to the present invention, wherein i) an immune checkpoint inhibitor and ii) a chemotherapeutic agent or radiation therapy are used as a combined preparation for treating a subject identified as a responder to immune checkpoint inhibitor treatment.

[0068] As used herein, the term "combination therapy," "combination therapy," or "therapy combination" refers to a treatment using more than one drug. A combination therapy can be a dual therapy or a bitherapy.

[0069] In a specific embodiment, at least two immune checkpoint inhibitors are used as a combined preparation according to the present invention for simultaneous, separate or sequential use in a method for treating cancer.

[0070] In a specific embodiment, i) an immune checkpoint inhibitor and ii) an anti-angiogenic compound are used as a combined preparation according to the present invention for simultaneous, separate or sequential use in a method for treating cancer.

[0071] In a specific embodiment, i) an immune checkpoint inhibitor and a chemotherapeutic agent or a radiotherapeutic agent are used as a combined preparation according to the present invention for simultaneous, separate or sequential use in a method for treating cancer.

[0072] As used herein, the term "simultaneous use" refers to the simultaneous or substantially simultaneous administration of two active ingredients by the same route. The term "separate use" refers to the simultaneous or substantially simultaneous administration of two active ingredients by different routes. The term "sequential use" refers to the administration of two active ingredients at different times, with the same or different administration routes.

[0073] As used herein, the term "chemotherapeutic agent" refers to a chemical compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, methyldopa and uredopa; ethylimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (particularly bletasin and bletasinone); camptothecins (including synthetic analogs topotecan); bryostatin; callusstatin; CC-1065 (including its synthetic analogs adolesin, carzelesin, biszelesin); scutellarin (particularly scutellarin 1 and scutellarin 8); tail Aplysia; Dukamycin (including synthetic analogs KW-2189 and CBI-TMI); Acanthopanax; Bananadine; Cyclocorinol; Spongestatin; Nitrogen mustards, such as chlorambucil, chlorphenazine, clofosamide, estramustine, ifosfamide, mechlorethamine, methoquinone hydrochloride, melphalan, neomycin, phenacetin, prednisone, trofosamide, uramustine; Nitrosoureas, such as carmustine, chlorzoxazone, fermustine, lomustine, nimustine, lanimustine; Antibiotics, such as enediyne antibiotics (e.g., calicheamicin, in particular calicheamicin 11 and calicheamicin 211, see, e.g., Agnew Chem. Intl. Ed. Engl.33:183-186 (1994); danimicin, including danimicin A; espamicicin; and the neocanstatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, calicheamicin, carubicin, carminomycin, carmophorin, chromomycin, dactinomycin, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin) , cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexicomycin, mitomycin, mycophenolic acid, linemycin, oligomycin, pelocycin, panmycin, puromycin, quinamycin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, zoloft, doxycycline; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylfolate, methotrexate , pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as caprotestosterone, drostanolone propionate, cyclothiodine, melastosane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; acetoglucuronide esters; aldophosphamide glycosides; aminoacetoacetic acid; amacrine; bitribitin; bisantrene; edatrexate; difazodone; decarboxylcolchicine; diazocone; eflornithine; elitrilonium acetate; epothilones; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and antomycin; mitoguanidine; mitoxantrone; mopidarol; nitropropanidine; pentostatin; belemet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine;. Razoxane; lisoxine; cizofilan; spirogermanium; tenuzolic acid; triazoline; 2,2',2"-trichlorotriethylamine; mucormycins (particularly T-2 toxin, veracrulin A, rhodanin A, and guanidinidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; garcitosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, for example, paclitaxel and docetaxel Chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; tearing mycin C; mitoxantrone; vincristine; vinorelbine; navirin, novarone; teniposide; daunomycin; aminopterin; xeloda; ibandronic acid; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof. Also included in this definition are antihormonal drugs used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, the aromatase inhibitors 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, ketoprofen, LY117018, onapristone and toremifene (Falenton); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0074] As used herein, the term "radiation therapy" has its general meaning in the art and refers to the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy to injure or destroy cells by destroying the genetic material of the treated area (target tissue), making it impossible for these cells to continue to grow. A type of radiation therapy commonly used involves photons, such as X-rays. Depending on the amount of energy they have, these rays can be used to destroy cancer cells on the surface of the body or deeper. The higher the energy of the X-ray beam, the deeper the X-rays enter the target tissue. Linear accelerators and electron accelerators produce X-rays with increasing energy. Using a machine to focus radiation (such as X-rays) on the cancer site is called external beam radiation therapy. Gamma rays are another form of photons used for radiation therapy. Certain elements (such as radium, uranium, and cobalt 60) release radiation when they decompose or decay, and gamma rays are spontaneously generated. In some embodiments, radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams from different directions to closely follow the shape of the tumor; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beam to closely follow the shape of the tumor and varies the radiation dose based on the shape of the tumor; conformal proton beam radiation therapy; image guided radiation therapy (IGRT), which combines scanning and radiation techniques to provide real-time images of the tumor to guide radiation therapy; intraoperative radiation therapy (IORT), which delivers radiation directly to the tumor during surgery; stereotactic radiosurgery, which can deliver large, precise radiation doses to small tumor areas in a single treatment session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which a subject is given more than one radiation therapy treatment (fraction) per day; and hypofractionated radiation therapy, in which a larger dose of radiation therapy is given per fraction, but in fewer fractions.

[0075] In some embodiments, the methods of the present invention are particularly applicable to the context of hypofractionated radiotherapy. As used herein, the term "hypofractionated radiotherapy" has its ordinary meaning in the art and refers to radiotherapy in which the total radiation dose is divided into larger doses and the treatment is less than once a day.

[0076] Kit or device of the present invention

[0077] A third aspect of the invention relates to a kit or device for carrying out the method of the invention, comprising means for determining the level of soluble CD27 in a biological sample.

[0078] In some embodiments, the kit or device comprises at least one binding partner (e.g., antibody or aptamer) specific for soluble CD27 (immobilized or not immobilized on a solid support as described above). In some embodiments, the kit or device may comprise a second binding partner (e.g., antibody or aptamer) of the invention that produces a detectable signal. Examples of kits include, but are not limited to, ELISA assay kits, and kits comprising test strips and test papers.

[0079] In some embodiments, the kit or device of the present invention further comprises a microprocessor to perform an algorithm on data comprising soluble CD27 levels in a sample to determine the probability of responding to an immune checkpoint inhibitor. In some embodiments, the kit or device of the present invention further comprises a visual display and / or an auditory signal indicating the probability determined by the microprocessor.

[0080] In some embodiments, the kit or device of the present invention comprises:

[0081] -Mass spectrometer;

[0082] - a container for holding a biological sample, the container being connectable to a mass spectrometer so that the mass spectrometer can quantify the level of soluble CD27 in the sample;

[0083] a microprocessor for executing an algorithm on data comprising the level of soluble CD27 in a sample to determine the probability of response to an immune checkpoint inhibitor;

[0084] - A visual display and / or an audible signal indicating the probability determined by the microprocessor.

[0085] The present invention will be further described by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1: Plasma levels of CD27 and Tim-3 are associated with survival in patients with renal cell carcinoma treated with anti-PD-1 / PD-L1. Plasma concentrations of soluble CD27 (A.) and soluble Tim-3 (B.) measured before anti-PD-1 / PD-L1 therapy in 27 patients with metastatic renal cell carcinoma. Patients were divided into two groups according to their plasma CD27 concentration (above or below the median). Patient survival was determined from the start of treatment. Log-rank test was performed to compare the survival of the two groups of patients.

[0087] Figure 2: Lack of correlation between prognostic clinical parameters related to renal cell carcinoma and survival in patients treated with anti-PD-1 / PD-L1 therapy.

[0088] Different clinical parameters whose prognostic value has been identified (A. histological type of tumor, B. ECOG, C. anterior nephrectomy, D. number of metastatic sites, E. MSKCC criteria) were associated with the survival of patients treated with anti-PD-1 / PD-L1.

[0089] Figure 3 : Lack of correlation between biological parameters related to inflammation and survival in patients with renal cell carcinoma treated with anti-PD-1 / PD-L1 therapy. In the same series of patients with metastatic renal cell carcinoma, two parameters related to inflammation (CRP, neutrophil / lymphocyte ratio) were measured before treatment with anti-PD-1 / PD-L1. The variables were dichotomized according to defined thresholds to consider values ​​as pathological. Similar results were observed when the median values ​​were used to divide patients into 2 groups or when a CRP threshold of 5 mg / L was used.

[0090] Figure 4: Correlation analysis between plasma levels of CD27 and Tim-3 and survival of patients with renal cell carcinoma treated with sunitinib (antiangiogenic molecule). A. Soluble CD27 and B. Soluble Tim-3 plasma concentrations measured before treatment with the antiangiogenic molecule sunitinib in 27 patients with metastatic renal cell carcinoma. The patients were divided into two groups according to values ​​above or below the median plasma concentration previously determined (see Figure 1). The survival of the patients was determined from the start of treatment. The log-rank test was performed to compare the survival of the two groups of patients.

[0091] Example

[0092] Materials and methods

[0093] Patients: We selected two cohorts of patients in this project:

[0094] - The Checkpoint Cohort (CPP: 2015-08-04-MS2) established at the Georges Pompidou European Hospital includes all patients treated with immunotherapy (anti-PD-1 / PD-L1) since 2016 and from whom routine blood and tissue samples were collected with the patient's consent. Twenty-seven patients with renal cell carcinoma from this cohort were included in this study. Twenty-two of these patients had clear cell renal carcinoma.

[0095] - Another cohort of patients was from the Preinsut clinical study, in which patients with renal cell carcinoma received two cycles of sunitinib before nephrectomy [8]. Twenty-seven patients from this cohort were also included in this study.

[0096] plasma

[0097] The plasma of both groups of patients came from the Bioresource Platform of the Georges Pompidou European Hospital (Claudia de Toma)

[0098] Clinical and biological data

[0099] Different clinical data (histological type of tumor, ECOG status, number of metastatic sites, MSKCC criteria, patient survival) and biological data (CRP, NLR) were collected. These latter parameters (CRP, NLR...) were chosen because they are most commonly associated with the inflammatory state of the patient.

[0100] Measurement of soluble receptors

[0101] The Procartaplex kit (Thermofischer) was used to measure a panel of soluble inhibitor or activator receptors (BTLA, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, Tim-3, CD28, CD80, 4-1BB, CD27, and CTLA-4)

[0102] Statistical analysis

[0103] The log-rank (Kaplan Meier) test or the Cox test was used to correlate the different parameters with patient survival.

[0104] Different variables were analyzed qualitatively, with a threshold set at the median to dichotomize them, or quantitatively (Cox model).

[0105] result

[0106] 1) Correlation between plasma concentrations of soluble CD27 and soluble Tim-3 and survival in patients with renal cancer treated with anti-PD-1 / PD-L1 therapy.

[0107] In the cohort of checkpoint patients treated with immunotherapy, only the pretreatment plasma concentrations of 2 parameters measured before the start of treatment (Tim-3 and CD27) were associated with patient survival in a statistically significant manner (Figure 1A and B).

[0108] For the CD27 marker, this correlation was found regardless of the statistical test used: log-rank (qualitative variable) (p=0.005) (Figure 1A) or Cox model (quantitative variable) (Table 1) (p=0.04).

[0109] RR IC inf IC sup p-value CD27 1 1 1.001 0.041 TIM3 1 1 1.001 0.1

[0110] Table 1: Correlation between plasma concentrations of soluble CD27 and soluble Tim-3 and survival determined by Cox model.

[0111] 2) There is no correlation between clinical and conventional biological prognostic criteria and response to anti-PD-1 / PD-L1 immunotherapy.

[0112] Interestingly, in the same series of patients, none of the clinical criteria (histological type, ECOG, anterior nephrectomy, number of metastatic sites, MSKCC criteria) ( Figure 2A-E ) or biological criteria (CRP, neutrophil / lymphocyte ratio (NLR)) ( Figure 3 ) was associated with survival in these patients treated with immunotherapy.

[0113] Different clinical parameters whose prognostic value has been identified (tumor histology, ECOG, anterior nephrectomy, number of metastatic sites, MSKCC criteria) are associated with survival in patients treated with anti-PD-1 / PD-L1 therapy.

[0114] 3) Plasma concentrations of CD27 were not correlated with survival in patients treated with antiangiogenic drugs.

[0115] To verify whether these 2 parameters can predict the response to immunotherapy or serve as prognostic markers for clinical outcomes in patients with metastatic renal cell carcinoma, we measured them in a group of patients treated with the antiangiogenic drug sunitinib. We found a correlation between the plasma concentration of Tim-3 before treatment and the patient's survival (p = 0.03) (Figure 4A), indicating that this marker is more likely to be a prognostic factor for metastatic renal cell carcinoma and therefore is not truly specific for the prediction of immunotherapy response.

[0116] In contrast, the concentration of plasma CD27 was not associated with patient survival ( Figure 4B ), indicating that this marker is more relevant and predictive of anti-PD-1 / PD-L1 response.

[0117] 4) CD27 marker has no correlation with clinical prognostic criteria for classification of metastatic renal cell carcinoma

[0118] We have found that the plasma concentration of soluble CD27 is not associated with different clinical parameters used to categorize patients with metastatic renal cell carcinoma for their prognostic role (ECOG, previous nephrectomy, number of metastatic sites, MSKCC criteria, etc.) (Table 2).

[0119]

[0120]

[0121] Table 2: Correlation between plasma soluble CD27 concentrations and prognostic clinical criteria in patients with metastatic renal cell carcinoma treated with anti-PD-1 / PD-L1 therapy.

[0122] in conclusion

[0123] We have identified a soluble marker, CD27, that is present in the plasma of patients with renal-cell carcinoma and whose pretreatment concentration predicts response to PD-1 / PD-L1.

[0124] This marker is more of a predictive marker for response to anti-PD1 / PD-L1 therapy than a prognostic marker. In fact, it was not associated with better survival in patients with metastatic renal cell carcinoma treated with antiangiogenic agents.

[0125] This marker does not correlate with conventional clinical severity markers used to stratify patients with metastatic renal cancer.

[0126] References:

[0127] Throughout this application, various references describe prior art related to the present invention. The disclosures of these references are hereby incorporated into the present disclosure by reference.

Claims

1. Use of a reagent for quantifying the level of soluble CD27 in the preparation of a detection reagent for determining whether a subject with renal cancer will obtain a response to an anti-PD-1 or anti-PD-L1 antibody, wherein the determination The following steps are involved: i) quantifying the level of soluble CD27 in a biological sample obtained from the subject before treatment with an anti-PD-1 or anti-PD-L1 antibody, ii) comparing the level of soluble CD27 quantified in step i) with its corresponding predetermined reference value, and iii) concluding that the subject will not respond to the treatment when the level of soluble CD27 is above its corresponding predetermined reference value, or concluding that the subject will respond to the treatment when the level of soluble CD27 is below its corresponding predetermined reference value.

2. The use according to claim 1, wherein the biological sample is a plasma sample.

3. The method according to claim 1, further comprising the step of classifying the subject by an algorithm for determining whether the subject will respond to anti-PD-1 or anti-PD-L1 antibody treatment.

4. The method according to claim 3, wherein the algorithm is selected from the group consisting of linear discriminant analysis, topological data analysis, neural network algorithm, support vector machine algorithm and random forest algorithm.

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