Methods and compositions for identifying whether subject with cancer will obtain response to immune checkpoint inhibitor

Abstract

The present invention relates to the use of soluble CD27 as a biomarker to predict response to immune checkpoint inhibitor treatment. The inventors have collaborated with two queues of patients and have identified a soluble marker CD27 present in the plasma of renal cell carcinoma patients, the pre-treatment concentration of which can predict a response against PD-1 / PD-L1. The marker is more like a predictive marker of response to anti-PD-1 / PD-L1 therapy, rather than as a prognostic marker. In fact, it is independent of a better survival rate of metastatic renal cell carcinoma patients receiving treatment with anti-angiogenic agents. The markers are not associated with conventional clinical severity markers that classify metastatic kidney cancer patients.

Description

[0001] This application is a divisional application of the patent application with application number 202080011235.4, application date January 29, 2020, and invention name "Methods and compositions for identifying whether a subject with cancer will respond to immune checkpoint inhibitors." Technical Field

[0002] 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

[0003] 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]. 25% of patients responded to this treatment, with a survival advantage of 5.5 months compared with standard treatment.

[0004] 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].

[0005] 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 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 can predict response to immunotherapy [5,6]. Therefore, 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 that co-express PD-1 and Tim-3 are mainly CD28-negative [7].

[0006] In recent decades, the use of immunotherapy, particularly immune checkpoint inhibitors, for cancer has become widespread and is used to treat both solid tumors and hematologic malignancies. However, only some patients respond to treatment with immune checkpoint inhibitors. Therefore, there is a need to identify new biomarkers that can determine whether a subject will respond to treatment with immune checkpoint inhibitors. Summary of the Invention

[0007] 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 (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 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. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION

[0008] 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 from whom blood and tissue samples were collected after obtaining patient consent. 27 patients with renal cell carcinoma from this cohort were included in this study. 22 of these patients had clear cell renal cell 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.

[0009] The inventors 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 therapy. This marker is more of a predictor of 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 who receive anti-angiogenic therapy. This marker does not correlate with conventional clinical severity markers used to stratify patients with metastatic renal cell carcinoma.

[0010] Methods for predicting response to immune checkpoint inhibitor therapy

[0011] Thus, 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 being 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.

[0012] 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.

[0013] 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 an immune checkpoint inhibitor to improve pathological symptoms, so that the subject shows clinical improvement compared to a subject not receiving treatment. The subject is considered to be a "responder" to treatment. The term "no response" refers to a subject who does not show any clinical improvement in response to immune checkpoint inhibitor treatment. 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 Response Evaluation Criteria in Solid Tumors (RECIST) criteria. This standard refers to a set of published rules that define when a cancer subject's tumor improves ("response"), remains unchanged ("stable"), or worsens ("progression") 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 particularly, soluble CD27 is a tool for determining a subject's overall survival (OS) before starting treatment with an immune checkpoint inhibitor.

[0014] As used herein, the term "overall survival (OS)" refers to the percentage of subjects in a study or treatment group who survive for a period of time after being diagnosed with or starting treatment for a disease (eg, cancer) according to the present invention.

[0015] 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.

[0016] Furthermore, the cancer may specifically be of the following histological types, but is not limited thereto: 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; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; bile duct carcinoma; hepatocellular carcinoma; mixed hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenoma in adenomatous polyps; 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 of the breast; adrenal cortex; endometrioid carcinoma; carcinoma of the skin appendages; apocrine gland carcinoma; sebaceous gland carcinoma; cerumenous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous gland carcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma cell tumor, malignant; granulosa cell tumor, malignant; androcyte tumor, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; hematopoietic cell carcinoma; Glomus sarcoma; malignant melanoma; amelanotic melanoma; surface-expanding melanoma; malignant melanoma in giant 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's 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 tumor; 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; neurilemoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma and hairy cell leukemia.

[0017] In a specific embodiment, the cancer is renal cancer. As used herein, the term "kidney cancer," "renal cancer," or "renal cell carcinoma" refers to a cancer that has arisen from the kidney. The term "renal cell carcinoma" or "renal cell carcinoma" (RCC), as used herein, refers to a cancer that originates 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, the cancer is metastatic renal cell carcinoma.

[0018] 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).

[0019] 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 has or is suspected of having renal cell carcinoma (RCC). In a specific embodiment, the subject has or is suspected of having lung cancer.

[0020] As used herein, the term "biological sample" refers to any sample obtained from a subject, such as a serum sample, plasma sample, urine sample, blood sample, lymph sample, or tissue biopsy. In a specific embodiment, the biological sample used to determine expression levels includes samples such as a blood sample, lymph sample, or tissue biopsy. In a specific embodiment, the biological sample is a blood sample. In another embodiment, the biological sample is a plasma sample.

[0021] 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 its ligand CD70 and plays an important role in regulating B cell activation and immunoglobulin synthesis. The soluble form of CD27 (sCD27), a 32 kD protein, is identical to the extracellular domain of membrane-bound CD27 and can be released from the cell surface after lymphocyte activation through differential splicing of the receptor protein or protease shedding.

[0022] 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 genomic and / or nucleic acid and / or protein level. In a specific embodiment, the expression level of a gene is determined by measuring the amount of nucleic acid transcripts for each gene. In another embodiment, the expression level is determined by measuring the amount of the corresponding protein for 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 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. The extracted mRNA is then 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 nucleic acid sequence-based amplification (NASBA). Nucleic acids having at least 10 nucleotides and showing sequence complementarity or homology with the mRNA of interest 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, 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). Probes typically comprise single-stranded nucleic acids 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. Primers are typically shorter single-stranded nucleic acids, 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, hybridizing to nucleic acids, i.e., they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x SCC. SCC is 0.15 M NaCl, 0.015 M sodium citrate). The nucleic acid primers or probes used in the above-described amplification and detection methods can be assembled into a kit. Such a kit comprises a common primer and a molecular probe. The kit also comprises the components necessary to determine whether amplification has occurred. For example, the kit may also comprise a PCR buffer and enzyme; a positive control sequence, a reaction control primer; and instructions for amplifying and detecting a specific sequence. In a specific embodiment, the method of the present invention comprises the steps of 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, expression levels are determined by DNA chip analysis. Such a DNA chip or nucleic acid microarray consists of different nucleic acid probes chemically attached to a substrate, which can be a microchip, a glass 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 the cDNA or oligonucleotide of about 10 to about 60 base pairs.For determining expression level, biological sample from test subject (optionally first reverse transcription) is labeled and contacts with microarray under hybridization conditions, causes formation complex between target nucleic acid, these complexes are complementary to the probe sequence attached to microarray surface.Then, the hybridization complex of detection mark, 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).

[0023] 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).

[0024] 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 the plasma sample. The amount is compared with a control value. Typically, the log-rank test (Kaplan Meier) or the Cox test is used to associate different parameters with patient survival. Different variables are qualitatively analyzed, a threshold is set at the median to dichotomize them, or quantitatively analyzed (Cox model). Patients are divided into two groups based on 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 the two groups of patients. For the CD27 marker, this correlation can be found regardless of the statistical test used: log-rank (qualitative variable) (p=0.005) ( Figure 1A ) or Cox model (quantitative variables) (Table 1) (p = 0.04).

[0025] 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 experimentally, empirically, or theoretically. The threshold value can also be arbitrarily selected based on 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 appropriately stored historical plasma samples can be used. The threshold value 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, a receiver operating characteristic (ROC) curve based on experimental data can be used to determine the optimal sensitivity and specificity (as well as the threshold value). For example, after determining the expression level of soluble CD27 in the reference group, the expression level determined in the test sample can be statistically processed by algorithmic analysis to obtain a classification standard that is important for sample classification. The ROC curve stands for receiver operator characteristic (ROC) curve, also known as the receiver operating characteristic (ROC) curve. It is primarily used in clinical biochemical diagnostic tests. The ROC curve is a composite indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It uses image synthesis to reveal the relationship between sensitivity and specificity. A series of different cutoff values (thresholds or critical values, the boundary between normal and abnormal diagnostic test results) are set as continuous variables, and a range of sensitivity and specificity values are calculated. The curve is then plotted with sensitivity as the vertical axis and specificity as the horizontal axis. The larger the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the upper left corner of the graph represents the critical point with high sensitivity and high specificity. The AUC value of the ROC curve ranges from 1.0 to 0.5. When the AUC is greater than 0.5, the diagnostic result improves as the AUC approaches 1. When the AUC is between 0.5 and 0.7, the accuracy is low. When the 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 performed using a computer. ROC curves 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.

[0026] 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 respond to treatment with an immune checkpoint inhibitor.

[0027] 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 the data comprising the quantified sCD27 levels to obtain an algorithm output; and c) determining, from the algorithm output of step b), a probability that the subject will or will not respond to an immune checkpoint inhibitor.

[0028] 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).

[0029] In some embodiments, the methods of the present invention include the step of determining the subject's response using a classification algorithm. As used herein, the term "classification algorithm" has its ordinary meaning in the art and refers to classification and regression tree algorithms and multivariate classification algorithms well known in the art, such as those described in US Pat. No. 8,126,690 and 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 corresponding set of weights, referring to a method that processes multiple variables simultaneously rather than individually. 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 the features. Support vector machines comprise two phases: a training phase and a testing phase. Support vectors are generated in the training phase, while they are estimated according to specific rules in the testing phase. In general, an 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 the k-tuples into an equal-dimensional or higher-dimensional space. The kernel function projects the data into a space in which a 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 vectors 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, 1998 Statistical Learning Theory. New York: Wiley). Any new observation is then classified as belonging to any one of the categories of interest based on the position of the observation relative to the hyperplane. 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 determines the class by selecting the pattern of classes determined by the individual trees.Individual trees are constructed using the following algorithm: (1) Assume 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 to be used as the basis for the decision at that node; (5) Compute the best split based on these m variables in the training set. In some embodiments, the score is generated by a computer program.

[0030] The algorithms of the present invention can be executed by one or more programmable processors to execute one or more computer programs to perform functions by operating on input data and generating output. The algorithms can also be executed by, and the apparatus can also be implemented as, dedicated logic circuitry, such as an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit). Processors suitable for executing computer programs include, for example, general-purpose and special-purpose microprocessors, as well as any one or more processors of any type of digital computer. Typically, a processor will receive instructions and data from read-only memory or random access memory, or both. The basic elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Typically, a computer will also include, or be operatively coupled to receive data from, or transfer data to, one or more mass storage devices (e.g., magnetic disks, magneto-optical disks, or optical disks) for storing data. 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, for example, semiconductor memory 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 memory may be supplemented by or incorporated into dedicated logic circuitry. To provide interaction with a user, embodiments of the present invention may be implemented on a computer having a display device, such as, in non-limiting examples, a CRT (cathode ray tube) or LCD (liquid crystal display) monitor, for displaying information to the user, as well as a keyboard and pointing device, such as a mouse or trackball, through which the user can provide input to the computer. Other types of devices may also be used to provide interaction with the user; for example, the feedback provided to the user may be any form of sensory feedback, such as visual feedback, auditory feedback, or tactile feedback; and input from the user may be received in any form, including acoustic, voice, or tactile input. Thus, in some embodiments, the algorithm may be implemented in a computing system that includes a back-end component, such as a data server, or a middleware component, such as an application server, or a front-end component, such as a client computer with a graphical user interface or a web browser, through which a user can interact with the present invention, or any combination of one or more such back-end, middleware, or front-end components. The components of the system may be interconnected by any form or medium of digital data communication, such as a communications network. Examples of communication networks include local area networks ("LANs") and wide area networks ("WANs"), such as the Internet. A computer system may include clients and servers. The clients and servers are generally 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.

[0031] 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.

[0032] 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 signal (stimulatory checkpoint molecules) or weaken signal (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). The example of stimulatory checkpoints includes CD27, CD28, CD40, CD122, CD137, OX40, GITR and ICOS. The example of inhibitory checkpoint molecules includes A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The adenosine A2A receptor (A2AR) is considered a key checkpoint in cancer therapy because adenosine in the immune microenvironment leads to A2a receptor activation, creating a negative immune feedback loop. Adenosine concentrations are relatively high in the tumor microenvironment. 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. BTLA surface expression is gradually downregulated during the differentiation of human CD8+ T cells from naive to effector cell phenotypes. Subsequently, tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, also known as cytotoxic T lymphocyte-associated protein 4, is also known as CD152. CTLA-4 expression on Treg cells controls T cell proliferation. IDO, indoleamine 2,3-dioxygenase, is a tryptophan catabolizing enzyme. A related immunosuppressive enzyme. Another important molecule is TDO, or 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, or killer cell immunoglobulin-like receptor, is a receptor for MHC class I molecules on natural killer cells. LAG3, or lymphocyte activation gene 3, inhibits immune responses through effects on Tregs and direct effects on CD8+ T cells. PD-1, or 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, short for T cell immunoglobulin domain-containing and mucin domain-containing 3, 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, short for V-domain Ig inhibitor of T cell activation, is primarily expressed on hematopoietic cells. Consistent VISTA expression on tumor-infected leukocytes may make VISTA blockers effective across a wide range of solid tumors. Tumor cells often exploit these checkpoints to evade detection by the immune system. Therefore, inhibiting checkpoint proteins on the immune system can enhance anti-tumor T cell responses.

[0033] In some embodiments, an immune checkpoint inhibitor refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes both 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.

[0034] In a specific embodiment, the immune checkpoint inhibitor is an antibody.

[0035] 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.

[0036] 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).

[0037] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody, such as those 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).

[0038] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody, as described in US7709214, US7432059, and US8552154.

[0039] In the context of the present invention, immune checkpoint inhibitor Tim-3 or its ligand.

[0040] In a specific embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody, as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and WO2013006490.

[0041] In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

[0042] As used herein, the term "small organic molecule" refers to a molecule of a size comparable to those commonly used in pharmaceuticals. The term does not include biological macromolecules (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.

[0043] 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.

[0044] 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.

[0045] In a specific embodiment, the small organic 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, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemet, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-aminonaphthoic acid, pyrrolidinedithiocarbamate, 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.

[0046] 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:

[0047]

[0048] 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:

[0049]

[0050] In a specific embodiment, the inhibitor is CA-170 (or AUPM-170), an oral small molecule immune checkpoint antagonist that targets 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.

[0051] In some embodiments, the immune checkpoint inhibitor is an aptamer.

[0052] Typically, aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, or VISTA.

[0053] In a specific embodiment, the aptamer is a DNA aptamer, such as described in Prodeus et al., 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic properties, as these short DNA chains are rapidly removed from the circulation by renal filtration. Therefore, the aptamers according to the present invention are conjugated to high molecular weight polymers, 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 PEGylated MP7, such as described in Prodeus et al., 2015.

[0054] Methods for treating subjects who have been identified as responders to immune checkpoint inhibitor therapy

[0055] 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 treatment with an immune checkpoint inhibitor, resulting 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 soluble CD27 in the biological sample of the subject; and iii) if in step ii) the subject is identified as a responder to treatment with an immune checkpoint inhibitor, treating the subject with an immune checkpoint inhibitor.

[0056] 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 treatment with an immune checkpoint inhibitor, resulting 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 from said subject; iii) communicating said assessment to said subject and iii) if in step ii) said subject is identified as a responder to treatment with an immune checkpoint inhibitor, treating said subject with an immune checkpoint inhibitor.

[0057] 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 treatment with an immune checkpoint inhibitor, 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 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, and iv) treating the subject with an immune checkpoint inhibitor.

[0058] As used herein, the term "treating" or "treatment" refers to prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of subjects at risk for 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 administered to subjects who have a medical condition or may eventually acquire the condition to prevent, cure, delay the onset of, reduce the severity of, or improve one or more symptoms of the condition or recurrent condition, or to prolong the subject's survival beyond the expected survival time in the absence of such treatment. A "treatment regimen" refers to a treatment pattern for a disease, for example, the mode of administration used during treatment. A treatment regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a treatment regimen (or part of a treatment 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 stages of the treatment regimen. An induction regimen can use (in part or in whole) a "loading regimen," which may include administering a larger dose of the drug than the doctor uses during the maintenance regimen, administering the drug more frequently than the doctor uses during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a subject during treatment for 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, yearly, etc.) or intermittent therapy (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment after reaching specific predetermined criteria [e.g., pain, disease manifestations, etc.]).

[0059] 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 has or is suspected of having renal cell carcinoma (RCC). In a specific embodiment, the subject has or is suspected of having lung cancer.

[0060] As used herein, the term "cancer" refers to a malignant growth or tumor resulting from the uncontrolled division of cells as defined above.

[0061] 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.

[0062] 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.

[0063] In a further embodiment, the method according to the present invention, wherein the anti-PD-1 antibody and the 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, the method according to the present invention, wherein 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.

[0065] 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.

[0066] As used herein, the term "angiogenesis" refers to the physiological process involved in the growth of new blood vessels from pre-existing blood vessels. Angiogenesis is a combinatorial process that is regulated by a balance between pro-angiogenic and anti-angiogenic molecules. Angiogenic stimuli (e.g., hypoxia or inflammatory cytokines) lead to the induction of expression and release of angiogenic growth factors such as vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF).

[0067] As used herein, the term "anti-angiogenic" refers to any molecule that can inhibit (anti-angiogenic) 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 antagonists, angiostatic steroids + heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, angiostatin, endostatin, 2-methoxyestradiol, teicogalactan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin, αVβ3 inhibitors, linomide, ramucirumab, taquimod, ranibizumab, sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), everolimus (Afinitor), cabozantinib.

[0068] 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.

[0069] 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 bitherapy.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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, whether by the same or different routes of administration.

[0074] 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; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (particularly bletabsin and bletabsinone); camptothecins (including the synthetic analogue topotecan); bryostatin; caustatin; CC-1065 (including its synthetic analogues adolesin, carzelesin, biszelesin); candida albicans (particularly candida albicans 1 and candida albicans 8); Aplysia; Dukatomycin (including the synthetic analogs KW-2189 and CBI-TMI); Acanthopanax; Cytodoxine; Cytodoxol; Spongistatin; 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, and lanimustine; Antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin 11 and calicheamicin 211, see, e.g., Agnew Chem. Intl. Ed. Engl.33:183-186 (1994); danamicin, including danamicin A; espamicin; and the neocanstatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, calicheamicin, carrubicin, carmomycin, 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, streptomycin, oligomycin, peclomycin, pantothecin, puromycin, quinamycin, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folinic acid, 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 captestosterone, drostanolone propionate, cyclothiocarbamate, melastane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; acetaminophen Ester; aldophosphamide glycoside; aminoacetoacetic acid; amacrine; bibirimide; bisantrene; edatrexate; difaramine; descarboxycolchicine; diazocone; eflornithine; eliplironium acetate; epothilone; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and antomycin; mitoguanidine; mitoxantrone; mopidarol; nitropropane; pentostatin; beremate; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; Razoxane; risoxine; cizofilan; spirogermanium; tenuzolic acid; triazinon; 2,2',2"-trichlorotriethylamine; mucormycins (particularly T-2 toxin, veracrucin A, rhodanin A, and guanidinidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; garcitosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and docetaxel Chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; laceration inhibitor C; mitoxantrone; vincristine; vinorelbine; naviridine, novarone; teniposide; daunorubicin; aminopterin; xeloda; ibandronic acid; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof. Also included within this definition are antihormonal agents 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 (Faloxifene); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0075] As used herein, the term "radiotherapy" has its ordinary meaning in the art and refers to the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy, injuring or destroying cells by destroying the genetic material of the treated area (target tissue), making it impossible for these cells to continue to grow. A commonly used type of radiotherapy involves photons, for example, X-rays. Depending on the amount of energy they possess, 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 penetrate into 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 radiotherapy. Gamma rays are another form of photons used for radiotherapy. Certain elements (such as radium, uranium and cobalt 60) release radiation when they decompose or decay, and gamma rays are produced spontaneously. In some embodiments, radiotherapy is external beam radiotherapy. 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), such as 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 doses of radiation to small tumor areas in a single treatment session; hyperfractionated radiation therapy, such as continuous hyperfractionated accelerated radiation therapy (CHART), in which the 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 in each fraction, but in fewer fractions.

[0076] In some embodiments, the methods of the present invention are particularly suitable for use in 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 delivered less than once a day.

[0077] Kit or device of the present invention

[0078] A third aspect of the invention relates to a kit or device for performing the method of the invention comprising means for determining the level of soluble CD27 in a biological sample.

[0079] In some embodiments, the kit or device comprises at least one binding partner (e.g., an 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., an 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 paper.

[0080] In some embodiments, the kit or device of the present invention further comprises a microprocessor to execute an algorithm on data comprising the level of soluble CD27 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 audible signal indicating the probability determined by the microprocessor.

[0081] In some embodiments, the kit or device of the present invention comprises:

[0082] -Mass spectrometer;

[0083] - 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;

[0084] 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;

[0085] - A visual display and / or an audible signal indicating the probability determined by the microprocessor.

[0086] The present invention will be further illustrated by the following figures and examples, which however should not be interpreted as limiting the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1: Plasma levels of CD27 and Tim-3 correlate with survival in patients with renal cell carcinoma treated with anti-PD-1 / PD-L1 therapy. Plasma concentrations of soluble CD27 (A) and soluble Tim-3 (B) were measured in 27 patients with metastatic renal cell carcinoma before anti-PD-1 / PD-L1 therapy. Patients were divided into two groups based on their plasma CD27 concentration (above or below the median). Patient survival was determined from the start of treatment. A log-rank test was performed to compare survival between the two groups.

[0088] 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.

[0089] Different clinical parameters whose prognostic value has been identified (A. tumor histological type, B. ECOG, C. anterior nephrectomy, D. number of metastatic sites, E. MSKCC criteria) are associated with survival in patients treated with anti-PD-1 / PD-L1 therapy.

[0090] Figure 3 : Lack of correlation between biological parameters related to inflammation and survival in patients with renal cell carcinoma receiving anti-PD-1 / PD-L1 therapy. In a similar series of patients with metastatic renal cell carcinoma, two parameters related to inflammation (CRP and 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 patients were divided into 2 groups using the median value or a CRP threshold of 5 mg / L.

[0091] Figure 4: Correlation analysis between plasma levels of CD27 and Tim-3 and survival in patients with renal cell carcinoma treated with sunitinib (an anti-angiogenic molecule). A. Soluble CD27 and B. Soluble Tim-3 plasma concentrations were measured in 27 patients with metastatic renal cell carcinoma before treatment with the anti-angiogenic molecule sunitinib. Patients were divided into two groups based on plasma concentrations above or below the previously determined median (see Figure 1). Patient survival was determined from the start of treatment. A log-rank test was performed to compare survival between the two groups.

[0092] This application contains the following items:

[0093] 1. A method for determining whether a subject having 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 being 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.

[0094] 2. A method for treating cancer in a subject who has been identified as a responder to treatment with an immune checkpoint inhibitor, comprising: i) quantifying the level of soluble CD27 in a biological sample obtained from the subject being treated with an immune checkpoint inhibitor; ii) providing an assessment based on the amount of soluble CD27 in the biological sample of the subject; and iii) if the subject is identified as a responder to treatment with an immune checkpoint inhibitor in step ii), treating the subject with an immune checkpoint inhibitor.

[0095] 3. The method according to item 1, wherein the biological sample is a plasma sample.

[0096] 4. 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 treatment with an immune checkpoint inhibitor.

[0097] 5. The method according to item 4, wherein 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).

[0098] 6. The method of claim 1, wherein the immune checkpoint inhibitor is an antibody.

[0099] 7. The method according to claim 1, wherein the immune checkpoint inhibitor is a monoclonal antibody.

[0100] 8. The method according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0101] 9. The method according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0102] 10. The method according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-L2 antibody.

[0103] 11. The method according to item 2, wherein the cancer is renal cancer or lung cancer.

[0104] 12. The method of claim 2, wherein at least two immune checkpoint inhibitors are used as a combination preparation to treat the subject who has been identified as a responder to immune checkpoint inhibitor treatment.

[0105] 13. The method of claim 2, wherein i) the immune checkpoint inhibitor and ii) a chemotherapeutic agent or a radiotherapeutic agent are used as a combination preparation to treat the subject who has been identified as a responder to immune checkpoint inhibitor treatment.

[0106] 14. The method of claim 2, wherein i) an immune checkpoint inhibitor and ii) an anti-angiogenic compound are used as a combination preparation to treat the subject who has been identified as a responder to immune checkpoint therapy.

[0107] 15. A kit or device for performing the method of item 1, comprising means for determining the level of said soluble CD27 in a biological sample.

[0108] Example

[0109] Materials and methods

[0110] Patients: We selected two cohorts of patients for this project:

[0111] 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, from whom routine blood and tissue samples were collected with patient consent. This study included 27 patients with renal cell carcinoma from this cohort, 22 of whom had clear cell renal cell carcinoma.

[0112] - Another cohort 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

[0113] plasma

[0114] The plasma of both groups of patients was obtained from the Bioresource Platform of the Georges Pompidou European Hospital (Claudia de Toma)

[0115] Clinical and biological data

[0116] 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 patient's inflammatory state.

[0117] Measurement of soluble receptors

[0118] 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).

[0119] Statistical analysis

[0120] The log-rank (Kaplan Meier) test or the Cox test was used to correlate the different parameters with patient survival.

[0121] Different variables were analyzed qualitatively, with a threshold set at the median to dichotomize them, or quantitatively (Cox model).

[0122] result

[0123] 1) Correlation between plasma concentrations of soluble CD27 and soluble Tim-3 and survival in patients with renal cell carcinoma receiving anti-PD-1 / PD-L1 therapy.

[0124] In the cohort of checkpoint patients treated with immunotherapy, only pre-treatment plasma concentrations of 2 parameters (Tim-3 and CD27) measured before treatment initiation were associated with patient survival ( Figure 1A and B) were statistically significantly correlated.

[0125] 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 variables) (Table 1) (p = 0.04).

[0126] RR IC inf IC sup p-value CD27 1 1 1.001 0.041 TIM3 1 1 1.001 0.1

[0127] Table 1: Correlation between plasma concentrations of soluble CD27 and soluble Tim-3 and survival determined by Cox model.

[0128] 2) There is no correlation between clinical and conventional biological prognostic criteria and response to anti-PD-1 / PD-L1 immunotherapy.

[0129] 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.

[0130] Different clinical parameters of identified prognostic value (tumor histology, ECOG, anterior nephrectomy, number of metastatic sites, MSKCC criteria) were associated with survival in patients treated with anti-PD-1 / PD-L1 therapy.

[0131] 3) Plasma concentrations of CD27 were not correlated with the survival of patients treated with anti-angiogenic drugs.

[0132] To test whether these two parameters can predict response to immunotherapy or serve as prognostic markers for clinical outcome 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 plasma concentrations of Tim-3 before treatment and patient survival (p = 0.03) ( Figure 4A ), suggesting that this marker is more likely a prognostic factor for metastatic renal cell carcinoma and therefore not truly specific for the prediction of response to immunotherapy.

[0133] In contrast, plasma CD27 concentrations were not associated with patient survival ( Figure 4B ), indicating that this marker has a stronger correlation and predictive power with anti-PD-1 / PD-L1 response.

[0134] 4) CD27 markers are not correlated with clinical prognostic criteria for the classification of metastatic renal cell carcinoma

[0135] We have found that plasma concentrations of soluble CD27 are not associated with different clinical parameters used to stratify patients with metastatic renal cell carcinoma for their prognostic role (ECOG, previous nephrectomy, number of metastatic sites, MSKCC criteria, etc.) (Table 2).

[0136]

[0137]

[0138] 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.

[0139] in conclusion

[0140] 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.

[0141] 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 who received antiangiogenic agents.

[0142] This marker did not correlate with conventional clinical severity markers used to stratify patients with metastatic renal cancer.

[0143] References:

[0144] 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. A method for determining whether a subject with 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 being 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.

2. A method for treating cancer in a subject who has been identified as a responder to treatment with an immune checkpoint inhibitor, comprising: i) quantifying the level of soluble CD27 in a biological sample obtained from the subject being treated with an immune checkpoint inhibitor; ii) providing an assessment based on the amount of soluble CD27 in the biological sample of the subject; and iii) if the subject is identified as a responder to treatment with an immune checkpoint inhibitor in step ii), treating the subject with an immune checkpoint inhibitor. The method of claim 1 , wherein the biological sample is a plasma sample.

4. The method of claim 1, further comprising the step of classifying the subject by an algorithm for determining whether the subject will respond to treatment with an immune checkpoint inhibitor.

5. The method according to claim 4, wherein the algorithm is selected from the group consisting of linear discriminant analysis (LDA), topological data analysis (TDA), neural network, support vector machine (SVM) algorithm and random forest algorithm (RF).

6. The method of claim 1, wherein the immune checkpoint inhibitor is an antibody.

7. The method of claim 1, wherein the immune checkpoint inhibitor is a monoclonal antibody.

8. The method of claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

9. The method of claim 1, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

10. The method of claim 1, wherein the immune checkpoint inhibitor is an anti-PD-L2 antibody.

11. The method of claim 2, wherein the cancer is renal cancer or lung cancer.

12. The method of claim 2, wherein at least two immune checkpoint inhibitors are used as a combination preparation to treat the subject who has been identified as a responder to immune checkpoint inhibitor therapy.

13. The method according to claim 2, wherein: i) the immune checkpoint inhibitor and ii) a chemotherapeutic agent or a radiotherapeutic agent as a combined preparation for treating the subject who has been identified as a responder to immune checkpoint inhibitor treatment.

14. The method of claim 2, wherein i) an immune checkpoint inhibitor and ii) an anti-angiogenic compound are used as a combined preparation to treat the subject who has been identified as a responder to immune checkpoint therapy.

15. A kit or device for performing the method of claim 1, comprising means for determining the level of said soluble CD27 in a biological sample.

Images