Methods and compositions for preventing and treating cancer

Abstract

We demonstrated that CD70 and CD27 are highly expressed in ccRCC and correlate with poor survival. Multiplex IF demonstrated that CD27+ T cells interact with CD70+ tumor cells in the tumor microenvironment (TME). CD27+ T cells are more apoptotic than CD27- T cells in ccRCC. Elevated plasma sCD27 levels are observed in ccRCC patients and correlate with CD27-CD70 interaction in situ. This study demonstrates that CD27-CD70 interaction contributes to the release of sCD27 in peripheral blood in ccRCC. This indicates that sCD27 is a potential biomarker. Apoptosis of CD27+ T cells suggests a deleterious effect of CD27-CD70 interaction on T cell responses. Therefore, CD27 / CD70 is a promising therapeutic target in ccRCC. Thus, the present invention relates to methods for determining the interaction between CD27 and CD70 by determining the level of soluble CD27 (sCD27) in a biological sample, and methods for targeting the CD27 / CD70 interaction to treat cancer or metastatic cancer.

Description

[Technical Field]

[0001] Field of Invention This invention relates to the field of oncology. In particular, it relates to methods and compositions for the prevention and treatment of cancer or metastatic cancer.

[0002] Background of the Invention Renal cell carcinoma (RCC) accounts for 3-4% of all cancers, with over 300,000 new cases diagnosed worldwide and 140,000 deaths annually (Capitanio et al., 2019). Clear cell renal cell carcinoma (ccRCC) represents 70-75% of RCC histological subtypes and is characterized by inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. This inactivation leads to the stabilization and accumulation of hypoxia-inducible factor-2 (HIF-2). HIF target genes regulate angiogenesis, glycolysis, and apoptosis (Gossage et al., 2015).

[0003] Therefore, uncontrolled activation of HIF explains why ccRCC tumors are lipid- and glycogen-rich and highly vascular. Localized RCC can be treated with partial or radical nephrectomy. However, approximately 30% of patients with localized ccRCC eventually develop metastasis (Hsieh et al., 2017). Given the nature of RCC, anti-angiogenic therapies targeting the vascular endothelial growth factor (VEGF) signaling axis (sunitinib, bevacizumab, axitinib) and mammalian targets of rapamycin (mTOR) inhibitors (everolimus, temsirorumus) are approved for the treatment of metastatic RCC (Hsieh et al., 2017).

[0004] CD70, a member of the tumor necrosis factor superfamily (TNFSF), is typically present on activated memory B and T lymphocytes, natural killer (NK) cells, and mature dendritic cells (DCs) (Borst et al., 2005; Nolte et al., 2009). CD70 expression is essential for effective immune responses, such as T cell activation and proliferation, memory cell generation, and B cell activation and differentiation (Garcia et al., 2004) (Arens et al., 2004). Interestingly, CD70 is abnormally found in hematological malignancies and solid tumors, particularly ccRCCs (approximately 80%) (Law et al., 2006; Ruf et al., 2015). Furthermore, high expression of CD70 in ccRCCs is associated with reduced survival (Jilaveanu et al., 2012). The consequences of this interaction with immune cells in TMEs remain unclear.

[0005] CD27, the ligand for CD70, is a costimulatory molecule belonging to the TNF receptor family (Buchan et al., 2018b; Camerini et al., 1991). CD27 is constitutively expressed on naive T cells and central memory T cells, but is downregulated on effector T cells (Mahnke et al., 2013). The CD27-CD70 axis is important for T cell activation during the priming phase. CD27-CD70 interaction induces a series of additional costimulatory signals, leading to the proliferation and differentiation of memory T cells and effector T cells (Nolte et al., 2009). Studies elucidating CD27-CD70 interaction in solid tumors are rare. One study suggested that RCC-expressed CD70 promotes the terminal differentiation of RCC tumor-infiltrating lymphocytes (TILs) (Wang et al., 2012). Two other in vitro studies suggested that CD27-CD70 interaction induces apoptosis in T cells, which functions as an immune evasion mechanism in CD70-expressing glioblastoma and ccRCC (Diegmann et al., 2006; Wischhusen et al., 2002). Finally, soluble CD27 (sCD27) originates from the proteolytic cleavage of transmembrane molecules on activated T cells after CD27-CD70 interaction (Nolte et al., 2009). The role of this soluble receptor and its prognostic value in solid tumors are unknown.

[0006] Therefore, by identifying the role of soluble CD27 in solid tumors and its interaction with CD70, it becomes possible to understand and discover new tools for treating solid tumors.

[0007] Summary of the Invention The present invention relates to a method for determining the interaction between CD27 and CD70, comprising the steps of: i) determining the level of soluble CD27 (sCD27) in a biological sample; ii) comparing the level of sCD27 quantified in step i) with a corresponding predetermined reference value; and iii) concluding that an interaction exists between CD27 and CD70 if the level of sCD27 is higher than the corresponding predetermined reference value, or concluding that no interaction exists between CD27 and CD70 if the level of sCD27 is lower than the corresponding predetermined reference value. In particular, the present invention is defined by the claims.

[0008] Detailed description of the invention The inventors showed that CD70 and CD27 are highly expressed in ccRCC and correlate with metastatic disease. From multiple IFs, CD27 + T cells in the tumor microenvironment (TME), CD70 + CD27 has been shown to interact with tumor cells. + T cells in ccRCCs - CD27 cells are more apoptotic than T cells. Elevated plasma sCD27 levels were observed in ccRCC patients and correlated with in situ CD27-CD70 interaction. This study demonstrates that CD27-CD70 interaction contributes to the release of sCD27 in peripheral blood in ccRCC. This indicates that sCD27 is a potential biomarker. + T cell apoptosis suggests adverse effects of the CD27-CD70 interaction in T cell responses. Therefore, CD27 / CD70 is a promising therapeutic target in ccRCC.

[0009] A method for determining the interaction between CD27 and CD70 Thus, in a first aspect, the present invention is a method for determining the interaction between CD27 and CD70, comprising: i) determining the level of soluble CD27 (sCD27) in a biological sample; ii) comparing the level of sCD27 quantified in step i) with its corresponding predetermined reference value; and iii) concluding that there is an interaction between CD27 and CD70 if the level of sCD27 is higher than its corresponding predetermined reference value, or concluding that there is no interaction between CD27 and CD70 if the level of sCD27 is lower than its corresponding predetermined reference value.

[0010] In certain embodiments, the method of the present invention is suitable for predicting the dysfunction of intratumoral T lymphocytes (LT).

[0011] In certain embodiments, the method of the present invention is suitable for predicting whether a subject has or is likely to have cancer and / or metastatic cancer.

[0012] As used herein, the term "CD70" is a member of the tumor necrosis factor superfamily (TNFSF) and is typically present on activated memory B lymphocytes and T lymphocytes, natural killer (NK) cells, and mature DCs. The expression of CD70 is essential for an effective immune response, such as T cell activation and proliferation and memory cell generation, B cell activation and differentiation.

[0013] Human CD70 variant 1 has the following nucleotide sequence in the art: SEQ ID NO: 1:

Chemical formula

[0014] Human CD70 variant 2 has the following nucleotide sequence in the art: SEQ ID NO: 2:

Chemical formula

[0015] Human CD70 variant 1 has the following amino acid sequence in the field of this technology: SEQ ID NO: 3: [ka] It has.

[0016] Human CD70 variant 2 has the following amino acid sequence in this technology: SEQ ID NO: 4: [ka] It has.

[0017] As used herein, the term "CD27" refers to a member of the tumor necrosis factor receptor superfamily. It is currently of interest to immunologists as a co-stimulatory immune checkpoint molecule. CD27 binds to ligand CD70 and plays a crucial role in the regulation of B cell activation and immunoglobulin synthesis. A soluble form of CD27 (sCD27), a 32kD protein identical to the extracellular domain of membrane-bound CD27, may be released after lymphocyte activation by differential splicing of the receptor protein or by protease-mediated detachment from the cell surface.

[0018] Human CD27 has the following nucleotide sequence in this technology: Sequence ID: 5: [ka] It has.

[0019] Human CD27 has the following amino acid sequence in this technology: SEQ ID NO: 6: [ka] It has.

[0020] As used herein, the term “CD27-CD70 interaction” refers to the interaction between CD27 and its ligand, CD70. Such interactions induce a series of additional costimulatory signals, leading to the proliferation and differentiation of memory T cells and effector T cells. Despite the importance of the CD27-CD70 interaction in initiating the immune response, this sequential interaction can lead to immunodysregulation and immunopathology. Studies elucidating the CD27-CD70 interaction in solid tumors are rare.

[0021] 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, tumor sample, or tissue biopsy. In certain embodiments, the biological sample for determining expression levels includes samples such as blood samples, lymph samples, or biopsies.

[0022] In certain embodiments, the biological sample is a blood sample. In other embodiments, the biological sample is a plasma sample. Typically, cancer patients and healthy donors were used to analyze sCD27 concentrations using a CD27 (soluble) human instant ELISA kit (ThermoFisher Scientific, Massachusetts, United States) according to the manufacturer's instructions. Data were acquired using an MRX Revelation microplate reader (DYNEX Technologies, Virginia, United States).

[0023] In a further embodiment, the biological sample is a tumor sample.

[0024] Typically, flow cytometry is performed on tumor samples as described in the examples. In certain embodiments, the interaction between CD27 and CD70 is analyzed by immunofluorescence analysis. In particular, interaction multiplex immunofluorescence (mIF) multiplex stained slides are imaged using Vectra® Polaris® Automated Quantitative Pathology Imaging system version 2 (Akoya). Using multispectral images obtained from single-stained slides for each marker, a spectral library containing spectrally peak-emitting fluorophores was created using inForm (version 2.4.6) image analysis software (PerkinElmer). Such software analysis enables detection and segmentation of specific tissues by powerful pattern recognition algorithms, and machine learning algorithms are trained to segment tumors from the stroma and identify labeled cells.

[0025] As used herein, the term “level of soluble CD27” refers to the concentration of soluble CD27. Typically, 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 levels. In further embodiments, soluble CD27 is characterized by Luminex, electrochemiluminescence, or ultra-sensitive immunoassays (Simoa...). Typically, apoptosis, exhaustion, proliferation, and cytotoxicity of T lymphocytes are measured by flow cytometry, single-cell RNA-seq, in situ multiplex immunofluorescence, and / or immunohistochemistry. In another embodiment, cytokine and / or chemokine production by T lymphocytes is measured by Luminex.

[0026] In certain embodiments, the gene expression level is determined by measuring the amount of nucleic acid transcript for each gene. In other embodiments, this expression level is determined by measuring the amount of protein corresponding to each gene. The amount of nucleic acid transcript can be measured by any technique known to those skilled in the art. In particular, this 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 well known in the art. The amount of nucleic acid transcript from mRNA or cDNA samples 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 certain embodiments, this 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 requires no precise explanation. Methods for determining the amount of mRNA are well known in the art. For example, nucleic acids contained in a biological sample are first extracted according to standard methods, for example, using lytic enzymes or chemical solutions, or extracted with nucleic acid-binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and / or amplification (e.g., 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 substitution amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest as described herein are found to be useful as hybridization probes or amplification primers. Such nucleic acids do not need to be identical, but are typically understood to be at least about 80% identical, more preferably 85%, and even more preferably 90-95% identical to a homologous region of comparable size.In certain embodiments, it would be advantageous to use nucleic acids in combination with appropriate means, e.g., detectable labels, to detect hybridization. A wide variety of suitable indicators, including fluorescent, radioactive, enzymatic, or other ligands (e.g., avidin / biotin), are known in the art. Probes typically consist of single-stranded nucleic acids 10 to 1000 nucleotides long, e.g., 10 to 800, more preferably 15 to 700, typically 20 to 500. Primers are typically shorter single-stranded nucleic acids 10 to 25 nucleotides long, designed to perfectly or nearly perfectly match the nucleic acid of interest to be amplified. The probes and primers are "specific" to the nucleic acid they hybridize with. That is, they are preferably hybridized under high stringency hybridization conditions (the highest melting temperature Tm, e.g., 50% formamide, 5× or 6× SCC, where SCC is 0.15M NaCl, 0.015M sodium citrate). The nucleic acid primers or probes used in the amplification and detection method described above can be assembled as a kit. Such a kit includes consensus primers and molecular probes. The kit also includes components necessary to determine whether amplification has occurred. The kit may also include, for example, PCR buffer and enzymes, a positive control sequence, reaction control primers, and instructions for amplifying and detecting specific sequences. In certain embodiments, the method of the present invention includes the steps of providing total RNA extracted from a biological sample and subjecting this RNA to amplification and hybridization to specific probes, particularly by quantitative or semi-quantitative RT-PCR. In another embodiment, the expression level is determined by DNA chip analysis. Such a DNA chip or nucleic acid microarray consists of various nucleic acid probes chemically bound to a substrate. This substrate can be a microchip, a glass slide, or a microsphere-sized bead. The microchip can be made of polymer, plastic, resin, polysaccharide, silica or silica-based material, carbon, metal, inorganic glass, or nitrocellulose. The probe contains nucleic acids, such as cDNA or oligonucleotides.This can be approximately 10 to 60 base pairs. To determine the expression level, a biological sample from the test subject initially subjected to reverse transcription is sometimes labeled and brought into contact with a microarray under hybridization conditions. This forms a complex between the target nucleic acid and the probe sequence attached to the microarray surface. The labeled hybridization complex can then be detected and quantified or semi-quantified. Labeling can be achieved by various methods, for example, by using radioactive or fluorescent labels. Many variations of microarray hybridization techniques are available to those skilled in the art (see, for example, the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

[0027] In some embodiments, the amount of sCD27 present in the plasma sample is detected by mass spectrometry.

[0028] In some embodiments, a score, which is a composite of the levels of soluble CD27, is determined and compared to a reference value. If the concentration of soluble CD27 is determined to be higher than the reference value, this indicates that an interaction exists between CD27 and CD70. If the concentration of soluble CD27 is determined to be lower than the reference value, this indicates that no interaction exists between CD27 and CD70. In certain embodiments, the predetermined reference value is 48.2 UI / mL. In further embodiments, the standard deviation is 11.46 ± 2. The predetermined reference value is determined in healthy subjects. Typically, the predetermined reference value is a threshold or cutoff value that can be determined experimentally, empirically, or theoretically. The threshold may also be arbitrarily selected based on existing experimental and / or clinical conditions, as will be recognized by those skilled in the art. For example, retrospective measurements of levels of soluble CD27 in appropriately banked historical plasma samples can be used to establish the predetermined reference value. The threshold must be determined to obtain optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical outcomes of false positives and false negatives). Typically, the optimal sensitivity and specificity (and therefore the threshold) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the expression level of soluble CD27 in a reference group, algorithmic analysis can be used for the statistical processing of the determined expression level in the sample under test, thereby obtaining a classification criterion with significance for sample classification. The full name of the ROC curve is the receiver operating characteristic curve, also known as the receiver operating characteristic curve. It is primarily used in clinical biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). This reveals the relationship between sensitivity and specificity by image synthesis. To calculate a set of sensitivity and specificity values, a set of different cutoff values ​​(thresholds or critical values, boundary values ​​between normal and abnormal results of a diagnostic test) are set as continuous variables.Next, sensitivity is used as the vertical coordinate for drawing the curve, and specificity is used as the horizontal coordinate for drawing the curve. 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 coordinate diagram is the critical point that has both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When the AUC is greater than 0.5, the diagnostic results get better and better 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 the AUC is greater than 0.9, the accuracy is high. This algorithm is preferably performed by computer. Existing software or systems in this technical field, 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., can be used to plot ROC curves.

[0029] In certain embodiments, the method of the present invention further includes the steps of classifying objects using an algorithm and determining the interaction between CD27 and CD70.

[0030] Typically, the method of the present invention includes a) quantifying the level of soluble CD27 in a biological sample, b) running a classification algorithm on the data including the quantified sCD27 level to obtain an algorithmic output, and c) determining the probability of interaction between CD27 and CD70 from the algorithmic output of step b).

[0031] In some embodiments of the present invention, the algorithm in the method is selected from linear discriminant analysis (LDA), phase data analysis (TDA), neural networks, support vector machine (SVM) algorithms and random forest algorithms (RF).

[0032] In some embodiments, the method of the present invention includes the step of determining a target response using a classification algorithm. As used herein, the term “classification algorithm” has its general meaning in the art and refers to classification and regression tree methods and multivariate classification methods well known in the art, such as those described in US Patent No. 8,126,690; WO 2008 / 156617. As used herein, the term “support vector machine (SVM)” refers to a general-purpose learning machine useful for pattern recognition, in which the discrimination boundary is parameterized by a set of support vectors and a corresponding set of weights, and which processes multiple variables simultaneously rather than separately. For this reason, support vector machines are useful as a statistical tool for classification. A support vector machine nonlinearly maps its n-dimensional input space to a higher-dimensional feature space and presents optimal interfaces (optimal partitioning surfaces) between features. A support vector machine consists of two stages: a training stage and a testing stage. In the training stage, support vectors are generated, while estimation is performed in the testing stage according to specific rules. Generally, SVM provides a model for classifying each of n subjects into two or more disease categories based on a single k-dimensional vector (called a k-tuple) of biomarker measurements for each subject. SVM first uses a kernel function to transform the k-tuples into a space of equal or higher dimension. The kernel function projects the data into a space where categories can be better separated using a hyperplane that would be possible in the original data space. To determine the hyperplane for distinguishing between categories, a set of support vectors closest to the boundary between disease categories can be selected. Then, the hyperplane is selected by known SVM techniques such that the distance between the support vectors and the hyperplane is maximized within the boundary of a cost function that penalizes incorrect predictions. This hyperplane optimally separates the data with respect to predictions (Vapnik, 1998 Statistical Learning Theory. New York: Wiley). Then, any new observation is classified as belonging to one of the desired categories based on how the observation exists relative to the hyperplane.If three or more categories are considered, this process is performed in pairs for all categories, and their results are combined to create rules for distinguishing between all categories. As used herein, the terms “random forest algorithm” or “RF” have their general meaning in the art and refer to classification algorithms such as those described in US No. 8,126,690; WO No. 2008 / 156617. A random forest is a decision tree-based classifier built using an algorithm first 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 a class by selecting a mode of class determined by the individual trees. Each tree is 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 M; (2) Select the number of input variables that will be used to determine the decisions at the nodes of the tree (this number m should be much smaller than M); (3) Select the training set by selecting N samples from the training set with permutations; (4) For each node of the tree, randomly select m of the M variables based on the decision at that node; (5) Calculate the best partition based on these m variables in the training set. In some embodiments, the score is generated by a computer program.

[0033] The algorithm of the present invention can be implemented by one or more programmable processors that execute one or more computer programs to perform a function by operating on input data and generating an output. Alternatively, this algorithm can be implemented by dedicated logic circuits, such as FPGAs (Field-Programmable Gate Arrays) or ASICs (Application-Specific Integrated Circuits), and devices can implement these. Processors suitable for executing computer programs include, for example, both general-purpose and dedicated microprocessors, as well as any one or more processors of any type of digital computer. Generally, a processor will receive instructions and data from read-only memory or random-access memory, or both. Essential elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include or be operably connected to one or more high-capacity memory devices for storing data, such as magnetic disks, magneto-optical disks, or optical disks, to receive data from and transfer data to or both. However, a computer is not required to have such devices. Furthermore, a computer can be incorporated into another device. Computer-readable media suitable for storing computer program instructions and data include, 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, and all forms of non-volatile memory, media, and memory devices. Processors and memory may be supplemented by or incorporated into dedicated logic circuits. To provide user interaction, embodiments of the present invention can be implemented on a computer having a display device for displaying information to the user, for example, in a non-limiting example, a CRT (cathode ray tube) or LCD (liquid crystal display) monitor, and a keyboard and pointing device, for example, a mouse or trackball, on which the user can provide input to the computer.Other types of devices can also be used to provide interaction with the user. For example, the feedback provided to the user can be any form of sensory feedback, such as visual, auditory, or tactile feedback, and input from the user can be received in any form, including acoustic, voice, or tactile input. Therefore, in some embodiments, the algorithm can be implemented in a computer system that includes a backend component (e.g., a data server), or a middleware component (e.g., an application server), or a frontend component (e.g., a client computer having a graphical user interface or web browser that allows the user to interact with the implementation of the invention), or any combination of one or more such backend, middleware, or frontend components. The components of the system can be interconnected by digital data communication in any form or medium, such as a communication network. Examples of communication networks include local area networks ("LANs") and wide area networks ("WANs"), such as the Internet. The computer system can include clients and servers. Clients and servers are generally geographically separated from each other and typically interact via a communication network. The client-server relationship arises from computer programs running on each computer that have a client-server relationship with each other.

[0034] In the context of the present invention, the inventors have shown that interaction between CD70 and CD27 in ccRCC induces TIL apoptosis, resulting in an increase in sCD27 in the patient's peripheral blood. When the concentration of soluble CD27 is determined to be lower than the reference value, it indicates that there is no interaction between CD27 and CD70. This means that a series of additional and sustained co-stimulatory signals do not occur, and the risk of T cell dysfunction is avoided.

[0035] Therefore, the method of the present invention is suitable for predicting tumor-mediated T lymphocyte (LT) dysfunction.

[0036] Typically, the present invention relates to a method for predicting intratumor lymphocyte (LT) dysfunction, comprising: i) determining an interaction between CD27 and CD70 according to the method described above; and ii) concluding that intratumor T lymphocyte dysfunction is present if an interaction between CD27 and CD70 is present, or concluding that intratumor T lymphocyte dysfunction is not present if an interaction between CD27 and CD70 is not present.

[0037] Typically, the present invention relates to a method for predicting intratumor lymphocyte (LT) dysfunction, comprising: i) determining the level of soluble CD27 (sCD27) in a biological sample; ii) comparing the level of sCD27 quantified in step i) with a corresponding predetermined reference value; and iii) concluding that intratumor lymphocyte (LT) dysfunction is present if the level of sCD27 is higher than the corresponding predetermined reference value, or concluding that intratumor lymphocyte (LT) dysfunction is not present if the level of sCD27 is lower than the corresponding predetermined reference value.

[0038] As used herein, the term “intratumor T lymphocytes,” also known as tumor-infiltrating lymphocytes (TILs), refers to white blood cells that have left the bloodstream and migrated to a tumor. They are mononuclear immune cells and can consist of a mixture of various cell types (e.g., T cells, B cells, NK cells, macrophages) in varying proportions, with T cells usually being the most abundant. In certain embodiments, in the context of the present invention, TILs are T lymphocyte cells.

[0039] As used herein, the term “intratumor T lymphocyte dysfunction” refers to lymphocytes (T cells) that are unable to proliferate and differentiate. Such lymphocytes become exhausted within the tumor and are therefore unable to defend against tumor cells.

[0040] In certain embodiments, the method of the present invention is suitable for predicting whether a subject will develop or is susceptible to CD70-expressing cancer and / or metastatic cancer.

[0041] Accordingly, the present invention relates to a method for predicting whether a subject will develop or is susceptible to cancer and / or metastatic cancer that expresses CD70, the method comprising: i) determining the interaction between CD27 and CD70 as described above; and ii) concluding that the subject will develop or is affected by cancer if the interaction between CD27 and CD70 is present, or concluding that the subject will not develop or is not affected by cancer if the interaction between CD27 and CD70 is not present.

[0042] Typically, the present invention relates to a method for predicting whether a subject will develop or is susceptible to CD70-expressing cancer and / or metastatic cancer, comprising: i) determining the level of soluble CD27 (sCD27) in a biological sample; ii) comparing the level of sCD27 quantified in step i) with a corresponding predetermined reference value; and iii) concluding that the subject will develop or is diagnosed with CD70-expressing cancer and / or metastatic cancer if the level of sCD27 is higher than the corresponding predetermined reference value, or concluding that the subject will not develop or is not diagnosed with CD70-expressing cancer and / or metastatic cancer if the level of sCD27 is lower than the corresponding predetermined reference value.

[0043] In another embodiment, the present invention is suited to a method for predicting whether a subject with cancer will achieve a response to immunotherapy treatment.

[0044] Typically, the present invention relates to a method for predicting whether a subject with CD70-expressing cancer and / or metastatic cancer will achieve a response to immunotherapy treatment, comprising: i) determining the interaction between CD27 and CD70 as described above; and ii) concluding that if there is no interaction between CD27 and CD70, the subject will achieve a response to immunotherapy treatment, or if there is an interaction between CD27 and CD70, the subject will not achieve a response to immunotherapy treatment.

[0045] In a further embodiment, the present invention relates to a method for predicting whether a subject suffering from CD70-expressing cancer and / or metastatic cancer will achieve a response to immunotherapy treatment, comprising the steps of: i) quantifying the level of soluble CD27 in a biological sample; ii) comparing the level of soluble CD27 quantified in step i) with a corresponding predetermined reference value; and iii) concluding that if the level of soluble CD27 is higher than the corresponding predetermined reference value, the subject will not respond to immunotherapy treatment, or if the level of soluble CD27 is lower than the corresponding predetermined reference value, the subject will respond to immunotherapy treatment.

[0046] As used herein, the term “predict” means that the subjects analyzed by the method of the present invention are assigned to either a group of subjects who will develop or are susceptible to cancer and / or metastatic cancer, or a group of subjects who will not develop or are not affected by cancer.

[0047] As used herein, the term “cancer” refers to malignant proliferation or tumor resulting from uncontrolled cell division. The term “cancer” includes primary tumors and metastatic tumors. In certain embodiments, cancer is cancer expressing CD70. In addition, cancer specifically refers to the following tissue types: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; pilomatal carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; combination of hepatocellular carcinoma and cholangiocarcinomas; cord-like adenocarcinomas; adenoid cystic carcinomas; adenocarcinomas in adenomatous polyps; familial colon polyposis adenocarcinomas; solid cancers; malignant carcinoid tumors; branch alveolar adenocarcinomas; papillary adenocarcinomas; Pigment-averse carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granulocytic carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; unencapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; squamous dysplasia adenocarcinoma; Malignant thymoma; malignant ovarian stromal tumor; malignant follicular cell tumor; malignant granulosa cell tumor, malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; chromaffin cell tumor; angioglobulosarcoma; malignant melanoma; chromosomal melanoma; chromosomal melanoma; chromosomal melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma; malignant blue nevi; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma Tumor; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Interstitial sarcoma; Malignant mixed tumor; Müllerian duct mixed tumor; Nephroblastoma; Hepatoblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Brenner tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Undifferentiated germ cell tumor; Fetal carcinoma; Malignant teratoma; Malignant ovarian goiter; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangioendothelioma; Lymphangiosarcoma; Osteosarcoma; Paraosteal osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor;Ameloblastoma; Malignant ameloblastoma; Ameloblastoma fibrosarcoma; Malignant pineal glandoma; Chordoma; Malignant glioma; Ependymoma; Asterocyte; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioblastoma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Gangioblastoma; Neuroblastoma; Retinoblastoma; Olfactory neuron tumor; Malignant meningioma; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; Lateral granuloma; Microlymphocyte Malignant lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other certain non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma and hairy cell leukemia may, but are not limited to, these.

[0048] In certain embodiments, the cancer is renal cancer. As used herein, the terms “renal cancer,” “renal carcinoma,” or “renal cell carcinoma” refer to cancer originating from the kidney. As used herein, the terms “renal cell carcinoma (cancer)” or “renal cell carcinoma (RCC)” refer to cancer originating from the inner lining of the proximal tubule. More specifically, RCC encompasses several relatively common histological subtypes: clear cell renal cell carcinoma, papillary (chromophilic) chromophilic collecting tubular carcinoma, and medullary carcinoma. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC. In certain embodiments, the cancer is metastatic renal cell carcinoma.

[0049] In another embodiment, the cancer is lung cancer. As used herein, the term “lung cancer” includes, but is not limited to, all types of lung cancer at all stages of progression, e.g., lung cancer, metastatic lung cancer, non-small cell lung cancer (NSCLC), e.g., lung adenocarcinoma, squamous cell carcinoma, or small cell lung cancer (SCLC). In some embodiments, the subject suffers from non-small cell lung cancer (NSCLC).

[0050] As used herein, the term “metastatic melanoma” refers to cancer that does not respond to classical treatments. Cancer may be resistant at the start of treatment or may become resistant during treatment. Resistance to drugs leads to the rapid progression of metastasis. Cancer resistance to drugs arises from mutations in various genes involved in cell proliferation, division, or differentiation.

[0051] As used herein, the term “classical treatment” refers to any natural or synthetic compound used to treat cancer and / or metastatic cancer. In certain embodiments, classical treatment refers to radiotherapy, immunotherapy, antibody therapy, or chemotherapy.

[0052] As used herein, the term “subject” means mammals, such as rodents, cats, dogs, and primates. In particular, the subject of the present invention is humans. In particular, the subject of the present invention has or is prone to having cancer that expresses CD70. In certain embodiments, the subject of the present invention has or is prone to having the above-described cancer. In other embodiments, the subject of the present invention has or is prone to having metastatic cancer.

[0053] In another embodiment, the subject of the present invention has or is prone to having renal cell carcinoma. In another embodiment, the subject of the present invention has or is prone to having clear cell renal cell carcinoma (ccRCC). In another embodiment, the subject of the present invention has or is prone to having lung cancer. In another embodiment, the subject of the present invention has or is prone to having NSCLC or SCLC. In another embodiment, the subject of the present invention has or is prone to having melanoma.

[0054] Methods for treating cancer In a second aspect, the present invention relates to a method for treating cancer and / or metastatic cancer in a subject requiring treatment for cancer and / or metastatic cancer, the method comprising the step of administering a therapeutically effective amount of a CD27 / CD70 interaction inhibitor to the subject.

[0055] In certain embodiments, the present invention relates to a method for treating cancer or metastatic cancer in subjects identified as having no interaction between CD27 and CD70, according to the method of the present invention.

[0056] In a particular embodiment, in the method of the present invention, the cancer and / or metastatic cancer is a cancer that expresses CD70.

[0057] Accordingly, the present invention relates to a method for treating cancer or metastatic cancer in a subject requiring treatment for cancer or metastatic cancer, the method comprising: i) determining the interaction between CD27 and CD70 according to the method described above; and ii) if there is no interaction between CD27 and CD70, treating the subject with a therapeutically effective amount of a CD27 / CD70 interaction inhibitor.

[0058] Typically, the present invention relates to a method for treating cancer or metastatic cancer in a subject requiring treatment for cancer or metastatic cancer, comprising: i) determining the level of soluble CD27 (sCD27) in a biological sample; ii) comparing the level of sCD27 quantified in step i) with a corresponding predetermined reference value; iii) concluding that if the level of sCD27 is lower than the corresponding predetermined reference value, there is no interaction between CD27 and CD70; and iv) treating the subject with a therapeutically effective amount of a CD27 / CD70 interaction inhibitor.

[0059] As used herein, the terms “to treat” or “treatment” include both prophylactic or preventive treatments and curative or disease-modifying treatments, and include treatments for subjects at risk of or suspected of having the disease, and subjects who have or have been diagnosed with the disease or condition, and include suppression of clinical relapses. Treatments may be administered to subjects who have or are at risk of acquiring a medical disability to prevent, cure, delay the onset of, reduce the severity of, or improve one or more of the disability or recurrent disability, or to extend the survival of the subject beyond the expected survival in the absence of such treatment. “Treatment plan” means a pattern of treatment for the disease, e.g., a pattern of administration used during treatment. A treatment plan may include an induction plan and a maintenance plan. The terms “induction plan” or “induction period” mean a treatment plan (or part of a treatment plan) used for the initial treatment of the disease. The general goal of an induction plan is to provide the subject with a high level of medication during the initial period of the treatment plan. The initiation plan may utilize a “loading plan” (in part or in whole). A loading plan may include administering a higher dose of medication than the physician would use during the maintenance plan, administering medication more frequently than the physician would use during the maintenance plan, or both. The terms “maintenance plan” or “maintenance period” refer to a treatment plan (or part of a treatment plan) used to maintain a subject during treatment for a disease, for example, to keep the subject in remission for an extended period (several months or several years). The maintenance plan may utilize continuous therapy (e.g., administering medication at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interruption treatment, intermittent treatment, treatment on relapse, or treatment when certain predetermined criteria [e.g., pain, disease symptoms, etc.] are reached).

[0060] As used herein, the term “subject” means mammals, such as rodents, cats, dogs, and primates. In particular, the subject of the present invention is human. In particular, the subject of the present invention has or is prone to cancer. In certain embodiments, the subject of the present invention has or is prone to metastatic cancer. In certain embodiments, the subject of the present invention has or is prone to CD70-expressing cancer. In certain embodiments, the subject of the present invention has or is prone to renal cell carcinoma (RCC). In certain embodiments, the subject of the present invention has or is prone to clear cell renal cell carcinoma (ccRCC). In certain embodiments, the subject has or is prone to lung cancer. In certain embodiments, the subject has or is prone to melanoma.

[0061] As used herein, the term “cancer” means malignant proliferation or tumor resulting from uncontrolled cell division, as defined above. As used herein, the term “metastatic cancer” means cancer that does not respond to classical treatment, as defined above. In certain embodiments, cancer is cancer that expresses CD70. The cancers of the present invention are as described above.

[0062] As used herein, the term “CD27 / CD70 interaction inhibitor,” also known as “antagonist,” refers to a natural or synthetic compound that has a biological effect of inhibiting the interaction between CD27 and CD70. Such inhibitors are antagonists of either CD27 or CD70. Such inhibition blocks the interaction between CD27 and CD70.

[0063] In certain embodiments, the CD27 / CD7 interaction inhibitor is a peptide, peptide mime, polypeptide, decoy, small organic molecule, antibody, aptamer, siRNA, or antisense oligonucleotide.

[0064] In certain embodiments, the CD27 / CD70 interaction inhibitor is an inhibitor of CD27.

[0065] In certain embodiments, the CD27 / CD70 interaction inhibitor is an inhibitor of CD70.

[0066] In certain embodiments, the CD27 / CD70 interaction inhibitor is a polypeptide. The term "polypeptide" refers to both short peptides, oligopeptides (11 to 100 amino acid residues) having lengths of at least two amino acid residues and up to 10 amino acid residues, as well as longer peptides (the usual interpretation of "polypeptide," i.e., having lengths of more than 100 amino acid residues) and proteins (functional entities comprising at least one peptide, oligopeptide, or polypeptide that can be chemically modified by being glycosylated, lipidized, or containing a prosthetic group).

[0067] In certain embodiments, the polypeptide is a decoy peptide or peptide mime that can bind to CD27 or CD70.

[0068] In certain embodiments, the inhibitor of the CD27 / CD70 interaction is a peptide mime. The term "peptide mime" refers to a small protein-like chain designed to mimic a peptide.

[0069] In certain embodiments, a peptide mime is, in the context of the present invention, a small protein-like chain designed to mimic a peptide, such as CD27 or CD70. As used herein, the term “peptide mime” or “PM” means the non-peptide chemical moiety. A peptide is a short chain of amino acid monomers linked by a peptide (amide) bond. A peptide (amide) bond is a covalent chemical bond formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid. The shortest peptide is a dipeptide, consisting of two amino acids linked by a single peptide bond, followed by tripeptides, tetrapeptides, and so on. The peptide mime chemical moiety includes a non-amino acid chemical moiety. The peptide mime chemical moiety may also include one or more amino acids separated by one or more non-amino acid chemical units. The peptide mime chemical moiety does not contain two or more adjacent amino acids linked by peptide bonds in any part of its chemical structure. As used herein, the term “amino acid” means glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, or citrulline.

[0070] In certain embodiments, the peptide mime is a functionally equivalent fragment of CD27 or CD70.

[0071] As used herein, a “functional equivalent,” also known as a “sink” or “trap” decoy, is a compound capable of binding to CD27 or CD70, thereby interfering with their interaction. Such peptide mimes of CD27 or CD70 are in an inactive form.

[0072] As used herein, a “functional equivalent,” also known as a “decoy” or “decoy receptor,” is a compound capable of binding to CD27 or CD70 and thereby interfering with their interaction. In particular, it is a compound that binds to a ligand but is structurally incapable of signaling or presenting an agonist to the signaling receptor complex. A decoy functions as a molecular trap for a ligand, thereby preventing the ligand from binding to its functional receptor. A decoy can be a peptide mime or fragment thereof of CD27 or CD70. Therefore, the term “functional equivalent fragment” includes any equivalent of CD27 or CD70 obtained, for example, by the deletion, substitution, or addition of one or more amino acids, by altering the amino acid sequence so that the protein analogue retains its ability to bind to soluble CD70. Amino acid substitutions can be performed, for example, by point mutations in the DNA encoding the amino acid sequence. Functional equivalents include molecules that bind to CD27 or CD70.

[0073] The term "mutant" in relation to a peptidomic mimite should be understood as a peptide mimite that differs from the peptide mimite from which it is derived due to one or more changes in the amino acid sequence. The peptide mimite from which a protein mutant is derived is also known as the parent polypeptide. Typically, mutants are constructed artificially, preferably by genetic means. Typically, the parent polypeptide is the wild-type protein or wild-type protein domain. The mutants usable in this invention may also be derived from homologs, orthologs, or paralogs of the parent polypeptide. Changes in the amino acid sequence can be amino acid exchanges, insertions, deletions, N-terminal or C-terminal cleavage, or any combination of these changes, and these may occur at one or more sites. In certain embodiments, the mutants usable in the present invention exhibit up to 200 total (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) changes (i.e., exchanges, insertions, deletions, N-terminal cleavage, and / or C-terminal cleavage) in the amino acid sequence. Amino acid exchanges may be conservative and / or non-conservative. In preferred embodiments, the variants usable in the present invention differ from the protein or domain from which they are derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid exchanges, preferably conservative amino acid changes. Alternatively or additionally, as used herein, a “variant” may be characterized by some degree of sequence identity with respect to the parent polypeptide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 80% sequence identity with respect to its parent polypeptide. In particular, the sequence identity of the protein variant extends over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 600 or more amino acids, and more preferably, over the entire length of the reference polypeptide (parent polypeptide).The term “at least 80% sequence identity” is used throughout this specification with respect to polypeptide sequence comparisons. This expression specifically refers to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity for each reference polypeptide. In particular, the polypeptide of interest and the reference polypeptide exhibit the indicated sequence identity over a continuous stretch as identified above.

[0074] The sequence identity of the “mutant” peptide mimetics of the present invention can be determined over a specified range of the amino acid sequence of CD27 or CD70 by referring to the entire amino acid sequence of a specified CD27 or CD70 decoy peptide mimetics. When determining the % sequence identity of mutant peptide mimetics, the sequence alignment may omit any specifically excluded amino acid residues. For example, for mutant peptide mimetics of CD27 or CD70 decoy peptide mimetics that show at least 80% sequence identity with respect to the amino acids of SEQ ID NO: 3; SEQ ID NO: 4 or SEQ ID NO: 6, the sequence alignment for comparison may be performed over only amino acids or may take into account two or more specified ranges of the amino acid sequence within the full-length CD27 or CD70 decoy peptide mimetics.

[0075] In certain embodiments, the inhibitor of the CD27 / CD70 interaction is an aptamer. An aptamer is a class of molecules that represent an antibody substitute in terms of molecular recognition. An aptamer is an oligonucleotide or oligopeptide sequence that has the ability to recognize substantially any class of target molecules with high affinity and specificity.

[0076] In certain embodiments, the CD27 / CD70 interaction inhibitor is an organic small molecule. The term “organic small molecule” refers to a molecule of a size comparable to organic molecules commonly used in pharmaceuticals. This term excludes biomacromolecules (e.g., proteins, nucleic acids, etc.). Preferred organic small molecules are in the size range of up to about 5000 Da, more preferably up to about 2000 Da, and most preferably up to about 1000 Da.

[0077] In certain embodiments, the inhibitor of the CD27 / CD70 interaction is an antibody. Such an antibody blocks the CD27 / CD70 interaction. In certain embodiments, the antibody is a CD70 neutralizing monoclonal antibody. In other embodiments, the antibody is a CD27 neutralizing monoclonal antibody.

[0078] As used herein, the term “antibody” is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments (insofar as they exhibit the desired biological activity). This term includes antibody fragments containing antigen-binding domains, such as Fab', Fab, F(ab')2, single-domain antibodies (DAB), TandAbs dimers, Fv, scFv (single-stranded Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, biobodies, tribodies (scFv-Fab fusions, each bispecific or trispecific); sc-diabodies; kappa (lambda) antibodies (scFv-CL fusions); BiTE (bispecific T-cell activator that induces T cells, scFv-scFv tandem); DVD-Ig (bivariable domain antibody, bispecific format); SIP (small immunoprotein, a type of minibody); SMIP ("small modular immunopharmaceutical"); scFv-Fc dimer; DART (ds-stabilized diabodies "biaffinity reTargeting"); small antibody mimics containing one or more CDRs, etc. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, which is specifically incorporated herein by reference). In particular, diabodies are further described in EP 404,097 and WO 93 / 11161. Linear antibodies, on the other hand, are further described in Zepata et al., 1995. Antibodies can be fragmented using conventional techniques. For example, an F(ab')2 fragment can be produced by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce the disulfide crosslinks to produce a Fab' fragment. Papain digestion can lead to the formation of a Fab fragment.Furthermore, Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAb, TandAb, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques or chemically. Techniques for producing antibody fragments are well known and described in the art. For example, (Beckman et al., 2007; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; Young et al., 1995) each further describe and demonstrate the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody, as described in US Patent No. 4,816,567. In some embodiments, the antibody is a humanized antibody, for example, those described in US Patent Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody, for example, the "human antibody" described in US Patent Nos. 6,075,181 and 6,150,584. In some embodiments, the antibody is a single-domain antibody, for example, those described in EP No. 0368684, WO No. 06 / 030220 and 06 / 003388.

[0079] In certain embodiments, the CD27 / CD70 interaction inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides the production of antibody molecules by a serial cell line in culture. Techniques for production and isolation include, but are not limited to, hybridoma techniques, human B-cell hybridoma techniques, and EBV hybridoma techniques.

[0080] In certain embodiments, the inhibitor is an intrabody specific to CD27 or CD70. As used herein, the term “intrabody” generally refers to an intracellular antibody or antibody fragment. Antibodies, particularly single-chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modifications may involve, for example, fusion to a stable intracellular protein, such as a maltose-binding protein, or the addition of an intracellular transport / localization peptide sequence, such as endoplasmic reticulum retention. In some embodiments, the intrabody is a single-domain antibody. In some embodiments, the antibody of the present invention is a single-domain antibody. The terms “single-domain antibody” (sdAb) or “VHH” refer to a single heavy-chain variable domain of an antibody of a type that can be found in camelid mammals that essentially lack a light chain. Such VHHs are also called “nanobody®”. According to the present invention, the sdAb can be, in particular, a llama sdAb.

[0081] In certain embodiments, the inhibitor of the CD27 / CD70 interaction is an anti-CD27 antibody.

[0082] In certain embodiments, the inhibitor of the CD27 / Cd70 interaction is an anti-Cd70 antibody.

[0083] In certain embodiments, the inhibitor of the CD27 / Cd70 interaction is an anti-CD27 antibody, where the anti-CD27 antibody is HPAB-0095-LSX; human anti-CD70 recombinant antibody (clone 2H5); HPAB-469-WJ-F(E); human anti-CD70 recombinant antibody (clone Ab2); HPAB-0095-LSX-F(E); human anti-CD70 recombinant antibody (clone 2H5) );HPAB-0468-WJ-F(E);Human anti-CD70 recombinant antibody (clone Ab1);HPAB-AP586-YC;Human anti-CD70 recombinant antibody (clone h1F6-HHLA);HPAB-AP589-YC-F(E);Human anti-CD70 recombinant antibody (clone h1F6-HMLA);TAB-336LC-F(E);Selected from, but not limited to, the group consisting of anti-human CD70 therapeutic antibody Fab fragment (Mb VLVH) or rat anti-Cd70 recombinant antibody (clone FR70).

[0084] In certain embodiments, the CD27 / CD70 interaction inhibitor is ARGX-110. ARGX-110, also known as xatuzumab, was developed by Argenx BVBA and is described in WO 2012 / 123586 and Aftimos et al 2017.

[0085] In certain embodiments, the CD27 / CD70 interaction inhibitor is borsetuzumab mahodotin (SGN-75). SGN-75 is an antibody-drug conjugate (ADC) in which borsetuzumab, a humanized monoclonal antibody, is conjugated with the cytotoxic agent non-cleaving monomethyl auristatin F (MMAF).

[0086] As used herein, the terms “administer” or “dosage” mean the act of injecting or otherwise physically delivering a substance present outside the body (e.g., an inhibitor of CD27 / CD70 interaction) to a target, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When a disease or its symptoms are treated, the administration of the substance is typically performed after the onset of the disease or its symptoms. When a disease or its symptoms are prevented, the administration of the substance is typically performed before the onset of the disease or its symptoms.

[0087] In certain embodiments, CD27 / CD70 interaction inhibitors are administered orally.

[0088] In another aspect, the present invention relates to a CD27 / CD70 interaction inhibitor and ii) classical treatment as a combination preparation for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0089] In certain embodiments, the present invention relates to i) inhibitors of CD27 / CD70 interaction and ii) classical treatments for use by simultaneous, separate, or sequential administration in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0090] As used herein, the terms “combination treatment,” “combination therapy,” or “combination therapy” refer to a treatment that uses two or more drugs.

[0091] As used herein, the term "simultaneous administration" refers to the administration of two active ingredients simultaneously or substantially simultaneously via the same route. The term "separate administration" refers to the administration of two active ingredients simultaneously or substantially simultaneously via different routes. The term "sequential administration" refers to the administration of two active ingredients at different times, with the same or different routes of administration.

[0092] As used herein, the term “classic treatment” refers to treatments that are well known in the art and used to treat cancer. In the context of the present invention, classic treatment refers to targeted therapy, radiotherapy, immunotherapy, or chemotherapy.

[0093] In certain embodiments, the present invention relates to 1) an inhibitor of the CD27 / CD70 interaction and ii) radiotherapy as combination preparations for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0094] As used herein, the terms “radiation therapy” or “radiotherapy” have their general meanings in the art and refer to the treatment of cancer with ionizing radiation. Ionizing radiation provides energy to damage or destroy cells in the treated area (target tissue) by damaging their genetic material, making it impossible for these cells to continue to proliferate. One type of radiotherapy commonly used involves photons, such as X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of the body or deeper inside the body. The higher the energy of the X-ray beam, the deeper the X-rays can reach the target tissue. Linear accelerators and betatrons emit X-rays with increasingly higher energy. The use of machines that concentrate radiation (e.g., X-rays) on the site of cancer is called extracorporeal beam radiotherapy. Gamma rays are another form of photons used in radiotherapy. Gamma rays are emitted spontaneously as certain elements (e.g., radium, uranium, and cobalt-60) decompose or decay, emitting radiation. In some embodiments, radiotherapy is extracorporeal radiotherapy. Examples of extracorporeal radiotherapy include, but are not limited to, conventional extracorporeal beam radiotherapy; three-dimensional conformal radiotherapy (3D-CRT) which delivers a shaped beam from different directions to closely conform to the shape of the tumor; intensity-modulation radiotherapy (IMRT), such as helical tomotherapy which shapes the radiation beam to closely conform to the shape of the tumor and also changes the radiation dose according to the shape of the tumor; conformal proton beam radiotherapy; image-guided radiotherapy (IGRT) which combines scanning and radiotherapy techniques to provide real-time images of the tumor to guide radiotherapy; intraoperative radiotherapy (IORT) which delivers radiation directly to the tumor during surgery; stereotactic radiosurgery which delivers a large, precise radiation dose to a small tumor area in a single session; multifractionated radiotherapy, such as sequential multifractionated accelerated radiotherapy (CHART), in which the patient is given two or more treatments (fractions) per day of radiotherapy; and low-fractionated radiotherapy, in which a higher dose of radiotherapy is given per fraction but fewer fractions.

[0095] In certain embodiments, the present invention relates to 1) an inhibitor of the CD27 / CD70 interaction and ii) a chemotherapy, which are used as combination preparations for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0096] As used herein, the term "chemotherapy" refers to the use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to a compound that is effective in inhibiting tumor growth.

[0097] Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and piposulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa and uredopa; ethyleneimines and methylamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomellamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); briostatin; carstatin; CC-1065 (including its synthetic analogs adzeresin, carzeresin and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); and drasta Tin; Duocalmycin (including synthetic analogs KW-2189 and CBI-TMI); Eleuterobin; Pancratistatin; Sarcodicin; Spongistatin; Nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorphosphamide, estralnustine, ifosphamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, uracil mustard; Nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics, e.g., enegyoin antibiotics (e.g., calicheamicin, in particular calicheamicin 11 and calicheamicin 211, e.g., Agnew) See Chem Intl. Ed. Engl. 33: 183-186 (1994); Dynemycin containing Dynemycin A;Esperamycin and neocardinostatin chromophores and related pigment proteins (endiin antibiotic chromophores), acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kactinomycin, carabicin, canninomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin) Epirubicin, esorubicin, idanrubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptomagrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., flud Rabin, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergic agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofsphamide glycosides; a Minolevulinic acid; Amsacrine; Bestrabusil; Bisanthren; Edatraxate; Defofamine; Demecolsin; Diadicone; Elfornithine; Erliptinium acetate; Epotilon; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamine; Mytansinoids, e.g., Maytansine and Ansamitosine; Mitoguazone; Mitoxanthrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK®; Lazoxane; Rhizoxin;Schizophyllan; Spirogenanium; Tenuazonic acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Triclothecene (especially T-2 toxin, bellacrin A, loridine A and angidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobromitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.) and Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeroda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine and any of the above pharmaceutically acceptable salts, acids, or derivatives. Furthermore, this definition also includes anti-hormone agents that act to regulate or inhibit hormonal activity in tumors, such as anti-estrogens and anti-androgens including tamoxifen, raloxifene, 4(5)-imidazole (which inhibits aromatase), 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston), such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0098] In certain embodiments, the present invention relates to 1) an inhibitor of the CD27 / CD70 interaction and ii) an immune checkpoint inhibitor, as combination preparations for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of such cancer and / or metastatic cancer.

[0099] 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.

[0100] As used herein, the term “immune checkpoint protein” has its general meaning in the art and refers to a molecule expressed by T cells that either turn up a signal (stimulative checkpoint molecule) or turn down a signal (inhibitory checkpoint molecule). It is recognized in the art that immune checkpoint molecules constitute immune checkpoint pathways similar to the CTLA-4 and PD-1-dependent pathways (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. 2011. Nature 480:480-489). Examples of stimulative 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. The adenosine A2A receptor (A2AR) is considered a key checkpoint in cancer treatment. This is because adenosine in the immune microenvironment, which leads to A2a receptor activation, forms a negative immune feedback loop, and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also known as CD276, was originally understood to be a co-stimulatory molecule, but is now considered a co-inhibitor. B7-H4, also known as VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor evasion. B lymphocyte and T lymphocyte attenuators (BTLA), also known as CD272, have HVEM (herpesvirus entry mediator) as their ligand. Surface expression of BTLA is gradually downregulated during the differentiation of human CD8+ T cells from naive cell phenotype to effector cell phenotype, but tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, also known as cytotoxic T lymphocyte-associated protein 4 (CD152), is expressed on Treg cells, where CTLA-4 expression helps regulate T cell proliferation. IDO, indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme.Related immunosuppressive enzymes. Another important molecule is TDO tryptophan 2,3-dioxygenase. IDO is known to suppress T cells and NK cells, generate and activate Treg and bone marrow-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 activator gene-3 suppresses the immune response by acting on Treg and gives a direct effect 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 received FDA approval in September 2014. The advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3 stands for T cell immunoglobulin domain and mucin domain 3. It is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 acts as a negative regulator of Th1 / Tc1 function by inducing cell death through interaction with its ligand, galectin-9. VISTA stands for V-domain Ig suppressor of T cell activation. VISTA is primarily expressed on hematopoietic cells, and consistent expression of VISTA on leukocytes within tumors allows VISTA blockade to be effective across a wide range of solid tumors. Tumor cells often utilize these checkpoints to evade detection by the immune system. Therefore, inhibiting checkpoint proteins in the immune system may enhance the anti-tumor T cell response.

[0101] 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 may be an antibody, synthetic or naturally occurring sequence peptide, small molecule, or aptamer that binds to an immune checkpoint protein and its ligands.

[0102] In certain embodiments, immune checkpoint inhibitors are antibodies.

[0103] Typically, the antibodies are against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, or VISTA.

[0104] In certain embodiments, the immune checkpoint inhibitor is, for example, an anti-PD-1 antibody described in WO Nos. 2011082400, 2006121168, 2015035606, 2004056875, 2010036959, 2009114335, 2010089411, 2008156712, 2011110621, 2014055648, and 2014194302. Examples of commercially available anti-PD-1 antibodies include nivolumab (Opdivo®, BMS) and pembrolizumab (also known as lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

[0105] In some embodiments, the immune checkpoint inhibitor is, for example, an anti-PD-L1 antibody as described in WO Nos. 2013079174, 2010077634, 2004004771, 2014195852, 2010036959, 2011066389, 2007005874, 2015048520, US No. 8617546, and WO No. 2014055897. Examples of anti-PD-L1 antibodies in clinical trials include: atezolizumab (MPDL3280A, Genentech / Roche), durvalumab (AZD9291, AstraZeneca), avelumab (also known as MSB0010718C, Merck), and BMS-936559 (BMS).

[0106] In some embodiments, the immune checkpoint inhibitor is, for example, an anti-PD-L2 antibody as described in US Patent Nos. 7709214, 7432059, and 8552154.

[0107] In the context of the present invention, immune checkpoint inhibitors inhibit Tim-3 or its ligand.

[0108] In certain embodiments, the immune checkpoint inhibitor is, for example, an anti-Tim-3 antibody as described in WO No. 03063792, No. 2011155607, No. 2015117002, No. 2010117057 and No. 2013006490.

[0109] In some embodiments, immune checkpoint inhibitors are small organic molecules.

[0110] As used herein, the term “small organic molecule” refers to a molecule of a size comparable to organic molecules commonly used in pharmaceuticals. This term excludes biomacromolecules (e.g., proteins, nucleic acids, etc.). Typically, small organic molecules range in size from about 5000 Da to about 2000 Da, more preferably to about 1000 Da.

[0111] Typically, small organic 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.

[0112] In certain embodiments, small organic molecules interfere with the transduction pathways of PD-1 and Tim-3. For example, they can interfere with molecules, receptors, or enzymes involved in the PD-1 and Tim-3 pathways.

[0113] In certain embodiments, small organic molecules interfere with indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitors. 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 WO No. 2014150677. 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-methyl-tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diaacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-amino-naphthoic acid, pyrrolidinedithiocarbamate, 4-phenylimidazole, brassinin derivatives, thiohydantoin derivatives, β-carbolin derivatives, or brassirexin derivatives. In certain embodiments, the IDO inhibitor is selected from 1-methyltryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-aminonaphthoic acid, and β-[3-benzo(b)thienyl]-alanine or their derivatives or prodrugs.

[0114] In certain embodiments, the inhibitor of IDO is, in the art, of the following chemical formula: [ka] It contains -N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-1,2,5-oxadiazole-3-carboxymidoamide, which is epacadostat (INCB24360, INCB024360).

[0115] In certain embodiments, the inhibitor is BGB324, also known as R428, as described in WO No. 2009054864, for example, 1H-1,2,4-triazole-3,5-diamine,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]-, which in the art is defined as follows: [ka] It has.

[0116] In certain embodiments, the inhibitor is CA-170 (or AUPM-170): an oral small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and the V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data for CA-170 were presented at the Curis Collaborator and Aurigene conference on November at the ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

[0117] In some embodiments, immune checkpoint inhibitors are aptamers.

[0118] Typically, aptamers are for A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, or VISTA.

[0119] In certain embodiments, the aptamer is a DNA aptamer, for example, as described in Prodeus et al 2015. The main drawback of aptamers as therapeutic entities is their poor pharmacokinetic profile, as these short DNA strands are rapidly removed from circulation for renal filtration. For this reason, the aptamers of the present invention are conjugated with a high molecular weight polymer, such as polyethylene glycol (PEG). In certain embodiments, the aptamer is an anti-PD-1 aptamer. In particular, the anti-PD-1 aptamer is MP7 pegylated, as described in Prodeus et al 2015.

[0120] In certain embodiments, the present invention relates to i) an inhibitor of the CD27 / CD70 interaction and ii) an anti-PD1 antibody as a combination preparation for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0121] In certain embodiments, the present invention relates to i) an inhibitor of the CD27 / CD70 interaction and ii) an anti-PDL1 antibody as a combination preparation for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of such cancer and / or metastatic cancer.

[0122] In certain embodiments, the present invention relates to i) an inhibitor of the CD27 / CD70 interaction and ii) an anti-PD2 antibody as a combination preparation for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0123] In certain embodiments, the present invention relates to i) an inhibitor of the CD27 / CD70 interaction and ii) an anti-CTLA4 antibody as a combination preparation for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of such cancer and / or metastatic cancer.

[0124] In certain embodiments, the present invention relates to i) an inhibitor of the CD27 / CD70 interaction and ii) an anti-angiogenic compound, as combination preparations for use in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0125] As used herein, the term “angiogenesis” refers to the physiological process involved in the growth of new blood vessels from existing ones. Angiogenesis is a combinatorial process regulated by a 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).

[0126] As used herein, the term “anti-angiogenic” refers to any molecule that can inhibit the formation of new blood vessels (anti-angiogenic). Typically, anti-angiogenic compounds are well known in the art and include the following compounds: bevacizumab (avastin, anti-VEGF), itraconazole (anti-VGFR), carboxamide triazole, TNP-470 (an analogue of fumagiline), CM101, IFN-α, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, anti-angiogenic steroids + heparin, cartilage-derived angiogenesis inhibitors, matrix metallo This includes, but is not limited to, proteinase inhibitors such as angiostatin, endostatin, 2-methoxyestradiol, tecogalane, tetrathiomolybdate, thalidomide, thrombospondin, prolactin, αVβ3 inhibitors such as linomi, ramucirumab, tascinimod, ranibizumab, sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), everolimus (Afinitor), and cabozantinib.

[0127] "Therapeutic dose" means a sufficient amount of CD27 / CD70 interaction inhibitor to be used in a method for treating cancer, with a reasonable benefit / risk ratio applicable to any medical treatment. It should be understood that the total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the bounds of appropriate medical judgment. A specific therapeutically effective dose level for any particular subject will depend on various factors, including the subject's age, weight, general health, sex, and diet; timing of administration, route of administration, and excretion rate of the particular compound being used; duration of treatment; drugs used in combination with or concurrently with the particular polypeptide being used; and similar factors well known in the medical field. For example, it is well known to those skilled in the art to start with a dose of the compound at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product can vary over a wide range of 0.01 to 1,000 mg per adult per day. Typically, compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient for indicational adjustment of the dose to the subject being treated. The drug typically contains about 0.01 mg to about 500 mg of the active ingredient, and typically 1 mg to about 100 mg of the active ingredient. The effective dose of the drug is usually supplied at dose levels of 0.0002 mg / kg body weight / day to about 20 mg / kg body weight / day, and particularly about 0.001 mg / kg body weight / day to 7 mg / kg body weight / day.

[0128] Pharmaceutical composition CD27 / CD70 interaction inhibitors can be used alone or in combination with the classical treatments described above to form pharmaceutical compositions with pharmaceutically acceptable excipients and optionally with sustained-release matrices, such as biodegradable polymers.

[0129] Therefore, in another embodiment, the present invention relates to a pharmaceutical composition comprising a CD27 / CD70 interaction inhibitor and a pharmaceutically acceptable excipient.

[0130] In certain embodiments, the pharmaceutical composition of the present invention comprises i) an inhibitor of CD27 / CD70 interaction and ii) a classic treatment for use by simultaneous, separate, or sequential administration in the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer in subjects requiring the prevention and / or treatment of CD70-expressing cancer and / or metastatic cancer.

[0131] As used herein, the terms “pharmaceutically acceptable” or “pharmaceutically acceptable” mean, as necessary, molecular entities and compositions that do not produce harmful, allergic, or other inappropriate reactions when administered to mammals, in particular humans. A pharmaceutically acceptable carrier or excipient means any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration may be administered to animals and humans in unit dose forms, with the active ingredient alone or in combination with another active ingredient, as a mixture with conventional pharmaceutical carriers. Suitable unit dose forms include oral route forms, e.g., tablets, gel capsules, powders, granules, and oral suspensions or solvents; sublingual and buccal administration forms; aerosols; implants; subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal, and intranasal administration forms; and rectal administration forms. Typically, pharmaceutical compositions contain a pharmaceutically acceptable medium for injectable formulations. These can be, in particular, isotonic and sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts) or dry, especially lyophilized compositions. This can be configured for injection by, as applicable, the addition of sterile water or saline. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or suspensions. In all cases, the form must be sterile and fluid enough to be easily injected. The form must be stable under manufacturing and storage conditions and must be resistant to microbial contamination, such as bacteria and fungi. Solutions containing the compounds of the present invention as free bases or pharmaceutically acceptable salts can be prepared in water appropriately mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.Polypeptides (or nucleic acids encoding them) can be formulated into neutral or salt compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins). Acid addition salts are formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can be obtained from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, or procaine. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by coating, such as the use of lecithin, by maintaining the required particle size in the case of suspensions, and by the use of surfactants. The prevention of microbial action can be achieved by various antibacterial and antimicrobial agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions are prepared by encapsulating the required amount of active polypeptide in a suitable solvent, along with some of the other components listed above as needed, followed by filtration sterilization. Generally, dispersions are prepared by encapsulating various sterile active ingredients in a sterile medium containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient by adding any additional desired components to the pre-sterilized filtered solution. In formulation, the solution will be administered in a manner suitable for the dosage formulation and in an amount that is therapeutically effective. The formulation can be easily administered in various forms, such as the injectable solution type described above, but drug-releasing capsules and the like can also be used.For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered as needed, and the liquid diluent should first be isotonic with sufficient physiological saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, a certain dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution or injected into the proposed injection site. Depending on the condition of the subject being treated, some dose variation will inevitably occur. In any case, the person administering the drug will determine the appropriate dose for each individual subject.

[0132] Kit or Apparatus of the Present Invention In another aspect, the present invention relates to a kit or apparatus for carrying out the method of the present invention, which includes means for determining the level of soluble CD27 in a biological sample.

[0133] In some embodiments, the kit or device includes at least one binding partner (e.g., an antibody or aptamer) specific to soluble CD27 (immobilized or unimmobilized on a solid support as described above). In some embodiments, the kit or device may include a second binding partner (e.g., an antibody or aptamer) of the present invention that generates a detectable signal. Examples of kits include, but are not limited to, ELISA assay kits and kits comprising test strips and dipsticks.

[0134] In some embodiments, the kit or apparatus of the present invention further includes a microprocessor for executing an algorithm on data including 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 apparatus of the present invention further includes a visual display and / or an audible signal indicating the probability determined by the microprocessor.

[0135] In some embodiments, the kit or apparatus of the present invention - Mass spectrometer and, - A container that can be connected to a mass spectrometer so that a biological sample can be placed inside and the mass spectrometer can quantify the level of soluble CD27 in the sample, - A microprocessor for running an algorithm on data including the level of soluble CD27 in a sample to determine the probability of responding to an immune checkpoint inhibitor, - Visual and / or audible signals indicating probabilities determined by a microprocessor. Includes.

[0136] Screening method A further object of the present invention is a method for screening agents suitable for the treatment of cancer and / or metastatic cancer, comprising: i) providing a test compound; and ii) determining the ability of the test compound to inhibit CD27 / CD70 interaction.

[0137] Any biological assay known in the art may be suitable for determining the ability of a test compound to inhibit CD27 / CD70 interaction. In some embodiments, the assay first involves determining the ability of the test compound to bind to CD27 or CD70. In some embodiments, a population of cells is then brought into contact and activated to determine the ability of the test compound to inhibit CD27 / CD70 interaction. In particular, the effect induced by the test compound is determined by comparison with the effect of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agonist (either of which is similar to a negative control condition). As used herein, the terms “control substance,” “control agonist,” or “control compound” refer to molecules that are inactive or do not possess activity related to the ability to modulate bioactivity or expression. It should be understood that test compounds capable of inhibiting CD27 / CD70 interaction are likely to exhibit similar modularity in in vivo applications, as determined using the in vitro methods described herein. Typically, the test compound is selected from the group consisting of peptides, peptide mimes, small organic molecules, antibodies, decoys, aptamers, or nucleic acids. For example, the test compound of the present invention can be selected from a library of previously synthesized compounds, a library of compounds whose structures have been determined in a database, or a library of newly synthesized compounds.

[0138] In some embodiments, the test compound can be selected from organic small molecules, decoys, or antibodies.

[0139] The present invention will be further illustrated by the following drawings and embodiments. However, these embodiments and drawings should not be construed as limiting the scope of the present invention. [Brief explanation of the drawing]

[0140] [Figure 1A-B]Figure 1: Overexpression of CD27 and CD70 genes in ccRCC. mRNA expression of CD27(A) and CD70(B) was compared in normal kidney tissue from non-cancer individuals (n=28), tumor tissue from ccRCC patients (n=530), and normal tissue adjacent to tumors (NAT) (n=72). Data are presented as mean with SD. Unpaired t-tests were used to determine significance. (C) Correlation analysis of mRNA expression between CD27 and CD70 in ccRCC (n=530). Pearson correlation coefficient (r) and significance level (P-value) are shown. (D-F) Kaplan-Meier plots of overall survival in ccRCC patients (n=376). Patients were divided into two groups (high and low) based on median mRNA expression of CD27(8.12) and CD70(10.265). The "high CD27 and high CD70" group (n=124) refers to patients with high expression of both CD27 and CD70, while the "low CD27 and low CD70" group (n=124) refers to patients with low expression of both CD27 and CD70. Significance was determined using the log-rank Mantel-Cox test. For clarity, mRNA data values ​​are shown on a log2 scale. Values ​​of P<0.05 were considered statistically significant. *P<0.05, ****P<0.0001. [Figure 1C]Figure 1: Overexpression of CD27 and CD70 genes in ccRCC. mRNA expression of CD27(A) and CD70(B) was compared in normal kidney tissue from non-cancer individuals (n=28), tumor tissue from ccRCC patients (n=530), and normal tissue adjacent to tumors (NAT) (n=72). Data are presented as mean with SD. Unpaired t-tests were used to determine significance. (C) Correlation analysis of mRNA expression between CD27 and CD70 in ccRCC (n=530). Pearson correlation coefficient (r) and significance level (P-value) are shown. (D-F) Kaplan-Meier plots of overall survival in ccRCC patients (n=376). Patients were divided into two groups (high and low) based on median mRNA expression of CD27(8.12) and CD70(10.265). The "high CD27 and high CD70" group (n=124) refers to patients with high expression of both CD27 and CD70, while the "low CD27 and low CD70" group (n=124) refers to patients with low expression of both CD27 and CD70. Significance was determined using the log-rank Mantel-Cox test. For clarity, mRNA data values ​​are shown on a log2 scale. Values ​​of P<0.05 were considered statistically significant. *P<0.05, ****P<0.0001. [Figure 1D-F]Figure 1: Overexpression of CD27 and CD70 genes in ccRCC. mRNA expression of CD27(A) and CD70(B) was compared in normal kidney tissue from non-cancer individuals (n=28), tumor tissue from ccRCC patients (n=530), and normal tissue adjacent to tumors (NAT) (n=72). Data are presented as mean with SD. Unpaired t-tests were used to determine significance. (C) Correlation analysis of mRNA expression between CD27 and CD70 in ccRCC (n=530). Pearson correlation coefficient (r) and significance level (P-value) are shown. (D-F) Kaplan-Meier plots of overall survival in ccRCC patients (n=376). Patients were divided into two groups (high and low) based on median mRNA expression of CD27(8.12) and CD70(10.265). The "high CD27 and high CD70" group (n=124) refers to patients with high expression of both CD27 and CD70, while the "low CD27 and low CD70" group (n=124) refers to patients with low expression of both CD27 and CD70. Significance was determined using the log-rank Mantel-Cox test. For clarity, mRNA data values ​​are shown on a log2 scale. Values ​​of P<0.05 were considered statistically significant. *P<0.05, ****P<0.0001. [Figure 2] Figure 2: CD27-CD70 interaction in ccRCCs is illustrated by multiple IHC. Quantification of interacted CD27 and CD70 in ccRCCs (n=25). Data are presented as dots and mean values ​​with standard deviation. [Figure 3] Figure 3: CD27+ T cells in tumors express cleavage caspase 3. % cleavage caspase 3 in CD27+ / - T cells (n=2). [Figure 4]Figure 4: Plasma sCD27 levels correlate with tumor CD27 expression and CD27-CD70 interaction levels. (A) Plasma sCD27 concentrations were determined by ELISA from ccRCC patients (n=44) and healthy donors (n=15). Significance was determined by an unpaired t-test. Data are presented as dots and mean values ​​with SD. sCD27 was plotted against CD27+ cell counts per field (B) and interacting CD27+ cell counts per field (CD27 within 30 μm of CD70) (C) in tumors (n=25). Pearson correlation coefficients (r) and significance levels (P-values) are presented. A value of P<0.05 was considered statistically significant. **P<0.01, ***P<0.001.

[0141] Examples Materials and methods Patients and samples

[0142] Two cohorts from a prospective study were enrolled at the Hopital Europeen Georges Pompidou (HEGP) (Paris, France). Patients from the Colcheckpoint cohort (CPP Ile-de-France 2015-08-04 MS2) were diagnosed with metastatic ccRCC and treated with anti-PD-1 / PD-L1. Patients from the ExhauCRF cohort (CPP Ile-de-France 2016-07-08) were diagnosed with focal ccRCC. For ELISA, 26 plasma samples were collected from Colcheckpoint before anti-PD-1 / PD-L1 treatment and 18 plasma samples were collected from ExhauCRF at the time of diagnosis. Plasma from 15 healthy donors from Etablissement Francais du Sang was selected as a control. For multiplex immunofluorescence (mIF), seven formalin-fixed paraffin-embedded (FFPE) tumor tissues from Colcheckpoint and 18 from the ExhauCRF cohort were selected, collected prior to immunotherapy. For flow cytometry, two fresh tumors were collected from patients confirmed to have ccRCC on the day of surgery. Clinical features, such as histopathology, Performance Status Scale (ECOG), TNM stage, and survival data were prospectively collected for each patient.

[0143] Multiple immunofluorescence (mIF) staining The inventors developed two mIF panels: a CD4 panel (CD4-CD27-CD70-PAX8) and a CD8 panel (CD8-CD27-CD70-PAX8). Pax8 is a transcription factor that exhibits potent nuclear expression in most neoplastic cells in all tissue types of human primary or metastatic RCC. These mIF panels, composed of various markers, were manually developed and then automatically applied to all FFPE tumor tissues on LEICA Bond RX using the same protocol. Slides from the FFPE tissues were heated at 57°C for 2 hours before staining. Residual paraffin was removed in three consecutive Bioclear New dewaxing solutions (Biognost, Zagreb, Croatia) for 3 minutes each. The tissue was rehydrated for 2 minutes each using three sequential dilutions of ethanol (100%, 75%, and 50%) and distilled water. Then, the tissue was fixed in formaldehyde-fixed neutral buffer (NB) (Biognost) for 15 minutes and washed with distilled water. Antigen recovery was then performed using pH9 Target Retrieval (Agilent, California, United States) with microwave treatment at 1000 watts for 45 seconds, followed by 100 watts for 30 minutes. Blocking was performed for 15 minutes with animal-derived protein-free blocking buffer (Cell Signaling Technology (CST), Massachusetts, United States), and then the slides were incubated for 30 minutes with primary antibody diluted in SignalStain® Antibody Diluent (CST). After washing with Tris-buffered saline and 0.1% Tween® 20 washing agent (TBST) (Agilent), the slides were incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) (ImmunoReagents, North Carolina, United States) for 15 minutes. The slides were washed with TBST and treated with CF® Dye Tyramide (Biotium, California, United States) for 10 minutes.Next, the slides were microwaved to strip the primary and secondary antibodies, washed, and blocked again using a blocking solution. This process was repeated until the fourth marker was labeled (Figure 1). In the final step, staining with DAPI (PerkinElmer, Massachusetts, United States) was applied, followed by washing with distilled water. The slides were mounted using EverBrite Mounting Medium (Biotium). Finally, the slides were read using a Vectra Polaris fluorescence microscope (Akoya Biosciences, California, United States). The antibodies used in the mIF panel are listed in Table 1.

[0144] [Table 1]

[0145] Multispectral imaging, phenotypic analysis, and spatial analysis Multiplex stained slides were acquired using the Vectra® Polaris® Automated Quantitative Pathology Imaging system version 2 (Akoya). A target region was selected, and 10 representative images per patient were used for analysis. For each marker, a spectral library containing fluorophores emitting spectral peaks was created using multispectral images obtained from single-stained slides with inForm (version 2.4.6) image analysis software (PerkinElmer). This spectral library was then used to separate each multispectral image into its individual components. This allows for color-based identification of all five markers in a single image using the inForm software.

[0146] The selected images were exported as inForm component data for phenotypic and spatial analysis in HALO software (Indica labs, New Mexico, United States). The Highplex FL module in HALO was used for cell phenotyping. Cells were segmented according to DAPI (nuclear) staining. Phenotyping was achieved by setting appropriate thresholds for each marker according to fluorescence intensity, and the same algorithm was applied to all images for uniformity. To determine cell interactions, spatial analysis was performed based on the cell phenotype obtained from HALO. The distance between cells was calculated from the center of each cell. CD27-positive cells within 30 μm of CD70-positive cells were named "interacted CD70," and the central CD70-positive cell with interacted CD27 was named "interacted CD70." Interacted CD27 and interacted CD70 were calculated.

[0147] Flow cytometry of fresh tumors for characterizing TILs in ccRCC Two fresh tumors were collected from a ccRCC patient. For digestion, the fresh tumors were first cut into small pieces and then incubated at 37°C for 1 hour with 25 mL of Hanks' balanced salt solution (HBSS) containing calcium and magnesium (Lonza, Basel, Switzerland), 1 mg / ml final concentration collagenase (Roche, Basel, Switzerland), and 15 mg / ml DNAase (Roche). The tumor tissue was filtered and washed with 200 μl of EDTA (Sigma-Aldrich, Missouri, United States) and HBSS at 1000r for 10 minutes. After removing the supernatant, the remaining cells were resuspended in HBSS. Cell counts were recorded using trypan blue.

[0148] One million cells per tube were used for staining. First, the cells were stained with Zombie Nir (Biolegend, California, United States) for 30 minutes to distinguish live cells from dead cells. After washing with cell staining buffer (Biolegend), the cells were then stained in the dark for 30 minutes with the following monoclonal antibodies (Table 2) directly labeled with a fluorescent agent. After staining, the cells were washed again and resuspended in 200 μl of cell staining buffer. Samples were acquired using a cytometer (Navios 10 colors, Beckman Coulter), and the data were analyzed using Kaluza software (version 1.2).

[0149] [Table 2]

[0150] Measurement of sCD27 in plasma 10 μL of plasma from ccRCC patients and healthy donors was used to analyze sCD27 concentrations using a CD27 (soluble) human instant ELISA kit (ThermoFisher Scientific, Massachusetts, United States) according to the manufacturer's instructions. Data were acquired using an MRX Revelation microplate reader (DYNEX Technologies, Virginia, United States).

[0151] Gene expression analysis from the TCGA database Gene expression analysis was performed using the UC Santa Cruz Cancer Genomics Browser (http: / / xena.ucsc.edu / ). To compare the gene expression of CD27 and CD70 across 31 types of human solid tumors, the TCGA PanCan study was created using normalized gene-level RNA sequence data (n=9575) downloaded from The Cancer Genome Atlas (TCGA) and Pan-Cancer Atlas databases. To compare the gene expression of CD27 and CD70 in tumors and normal tissues, normalized mRNA data from ccRCC tumor tissue (n=530), normal kidney tissue (n=28), and normal tissue adjacent to tumors (NAT) (n=72) were downloaded from the TCGA-PanCan and GTEX studies. Tumor and NAT tissues were derived from ccRCC patients, while normal kidney tissue was derived from individuals without cancer. Corresponding clinical data (n=376), such as overall survival rates, were also downloaded for Kaplan-Meier survival analysis.

[0152] statistical analysis One-way Anova was performed using the UCSC Xena tool to compare the gene expression of CD27 and CD70 across various types of solid tumors. Other statistical analyses were performed using GraphPad Prism software version 8. Where appropriate, two-sided unpaired t-tests were used to assess quantitative differences between the two groups. For correlation analysis, Pearson correlation coefficients (r) and p-values ​​were calculated. Kaplan-Meier plots were presented for survival analysis. Statistical differences were assessed using the log-rank Mantel-Cox test. A p-value < 0.05 was considered statistically significant.

[0153] Tissue processing RNA extraction, single-cell RNA sequencing Fresh tumor cells were collected as described above. After tumor dissociation, cells were stained with a viability-staining FVS 520 (eBioscience), anti-CD8a (Biolegend) labeled with APC fluorophores, and anti-NKP46 (Biolegend) labeled with BV421 to remove natural killer cells from analysis during cell sorting. FACS-enriched T cells were loaded onto 10×Chromium (10×Genomics), and libraries were prepared using the Single Cell 5' Reagent Kit (V1 chemistry, 10×Genomics) according to the manufacturer's protocol, targeting 3000 recovered cells per sample. Single cells were divided into droplets with gel beads coated with oligos having unique barcodes, molecular identifiers (UMIs), and template switch oligo (TSO) sequences, barcoded, and subsequently reverse transcribed in the droplets to generate barcoded full-length cDNA. Next, the cDNA was recovered from the droplet, purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific), and then amplified using the following protocol: 98°C - 45 seconds; 12 × (98°C - 20 seconds, 67°C - 30 seconds, 72°C - 1 minute), 72°C - 1 minute; held at 4°C. The amplified cDNA product was purified using SPRI select Reagent Kit (Beckman Coulter). Gene expression libraries were constructed according to these steps: (1) fragmentation, end repair, and A-tailing; (2) size selection using SPRI select Beads; (3) adapter ligation; (4) post-ligation purification using SPRI select Beads; (5) sample index PCR according to the protocol below: 98°C-45 sec; 12 × (98°C-20 sec, 54°C-30 sec, 72°C-20 sec), 72°C-1 min; hold at 4°C and final purification using SPRI select Beads. Library quality was evaluated using the dsDNA High Sensitivity Assay Kit and Bioanalyzer Agilent 2100 System. Libraries were quantified using the dsDNA HS Kit and Qubit 2.0.The indexed library was pooled and sequenced on Illumina HiSeqX using paired-end 150bp sequencing mode, targeting at least 50,000 reads per cell.

[0154] scRNA-seq data analysis All scRNA-seq data were processed using the Cellranger pipeline (version 2.1.1). This process included demultiplexing of raw base call (BCL) files into FASTQ files, read alignment on the human genome assembly GRCh38-3.0.0 using STAR, and counting of unique molecular identifiers (UMIs). The Seurat (v3.1.1) workflow was used to read the raw data into R (3.6.1). Raw counts for each donor were normalized using the Seurat SCTransform function with default parameters and integrated into a single object using the Seurat CCA integration algorithm. As a quality control step, low-quality cells were first filtered: cells with fewer than 200 detected genes and cells with more than 5,000 detected genes. Cells with more than 5,000 mitochondrial reads and cells with a read-to-mitochondrial read ratio greater than 6.5 were removed. Reads aligned to mitochondrial genes or ribosomal proteins were removed from the analysis. Following these quality control criteria, 13,389 T CD8 cells (Patient P1 = 2,006 cells; Patient P2 = 7,002 cells; Patient P3 = 1,654 cells; Patient P4 = 2,727 cells) were ultimately preserved. For each patient, single cells were selected as either "CD27 positive" (nUMI corresponding to CD27 gene > 0) or "CD27 negative". Genes differentially expressed between CD27-positive and CD27-negative cells were identified using Student's t-test and Benjamini-Hochberg p-values. Genes were considered differentially expressed if the corrected p-value was ≤0.05 and log FC ≥ 0.4. 412 genes were upregulated in CD27-negative cells, and 330 genes were upregulated in CD27-positive cells. The Morpheus tool (https: / / software.broadinstitute.org / morpheus / ) was used for heatmap visualization.scRNA-seq data selected from renal cell carcinoma samples is available on the NCBI's Gene Expression Omnibus (GEO) archiving platform (https: / / www.ncbi.nlm.nih.gov / geo / ) under accession number GSE160243.

[0155] result Different expression patterns of CD70 and CD27 in ccRCC correlate with patient survival. To investigate the high frequency of CD70 expression in ccRCC (Adam et al., 2006; Jilaveanu et al., 2012), we compared the expression of CD70 and its ligand CD27 at the mRNA level across 31 human solid tumors from the TCGA cohort and Pan-Can Atlas (n=9575). The results showed that CD70 was most frequently expressed in renal clear cell carcinoma (KIRC) (data not shown). Similarly, CD27 expression in KIRC was also higher compared to other solid tumors (data not shown). Focusing on ccRCC, CD27 and CD70 expression were found to be significantly higher in tumors than in normal kidney tissue and normal tissue adjacent to tumors (NAT) (Figure 1A, Figure 1B). Furthermore, CD27 was significantly correlated with CD70 in tumors (Figure 1C). To study the impact of these different expression patterns of CD27 and CD70 on clinical outcomes, overall survival analyses were performed using data from the TCGA-PanCan study (n=530). Patients were divided into two groups (high and low) based on the median mRNA data on a log2 scale. The median threshold was 8.12 for CD27 and 10.265 for CD70. Neither CD27 nor CD70 alone affected the patients' overall survival (Figure 1D, Figure 1E). Considering the correlation between CD27 and CD70 in tumors, the two markers were combined, and patients were divided into two groups: "high CD27, high CD70" (n=124) and "low CD27, low CD70" (n=124). Interestingly, significantly poorer OS was observed in the "high CD27, high CD70" group (Figure 1F). This suggests that the synergistic role of CD27 and CD70 has a negative impact on the prognosis of ccRCC patients.

[0156] CD70 at ccRCC + Tumor cells and CD27 + Interaction with T cells To better understand the synergistic role of CD27 and CD70 in the clear cell renal cell carcinoma (ccRCC) tumor microenvironment (TME), five-color multiplex immunofluorescence (mIF) was performed on formalin-fixed paraffin-embedded (FFPE) tumor tissues from 25 patients. Two staining panels were designed: a CD4 panel (CD4, red; CD27, yellow; CD70, green; PAX8, orange; DAPI (nuclear stain), blue) and a CD8 panel (CD8, red; CD27, yellow; CD70, green; PAX8, orange; DAPI, white). PAX8 was used to identify tumor cells. Composite images with five markers after multispectral imaging and corresponding single-stained images were presented (data not shown). After nuclear, cytoplasmic, and membrane cell segmentation and measurement of fluorescence intensity in each compartment, cells were phenotyped and counted using HALO software (data not shown). An algorithm composed of appropriate intensity thresholds for each marker was applied to all patients for homogeneity. Flow cytometry enables accurate cell phenotyping but cannot capture spatial relationships that better reflect cell interactions. To investigate this, after phenotyping, coordinates were assigned to each cell that could be used to determine intracellular distances. The number of cells within a certain distance of another cell population could also be calculated.

[0157] As a result, CD27 was observed to be expressed on CD4 + T cells and CD8 + T cells, while CD70 was expressed on tumor cells (PAX8). More importantly, CD27 + T cells and CD70 + tumor cell interactions could be seen in the TME (data not shown). After phenotyping, CD27 + cells were 65% of CD4 + CD27 + [[ID=I8]]T cells, 19% of CD8 + CD27 +Composed of T cells and 16% other cells (data based on 7 samples) (data not shown). This suggests that CD27 is mostly expressed on T cells. To better measure the intracellular interaction of CD27-CD70, spatial analysis was performed using HALO software (data not shown). CD70 + CD27 within 30 μm of a cell + We define cells as "interacting CD27" and CD27 around them within a radius of 30 μm. + CD70 with cells + Cells were defined as "interacted CD70" (data not shown). Quantitative results for interacted CD27 and interacted CD70 are shown in Figure 2. These results suggest a significant interaction between CD27-positive cells and CD70-positive cells. CD27 + Cells CD70 + More CD70 interacts with cells than other cells. + The cells are CD27 + It is thought to interact with cells. CD70 + Since CD27 is expressed by tumor cells, and CD27 is primarily expressed by T cells, this result can be explained by the larger surface area occupied by tumor cells compared to T cells.

[0158] CD27 in ccRCC + T cell apoptosis Since the role of CD27-CD70 interaction in ccRCC is unknown, next, CD27 in tumors + We investigated the phenotypic characteristics of T cells. Previous reports have shown that CD27-induced apoptosis is mediated by exposure to CD70 in ccRCCs in vitro (Diegmann et al., 2006). Subsequently, we investigated how CD27 is mediated in humans by ccRCCs that highly express CD70. + We investigated whether T cell apoptosis was induced. To confirm this hypothesis, we performed flow cytometry analysis on fresh tumors from two ccRCC patients. CD27 was compared with CD4. + on T cells and CD8 +It is expressed on T cells (data not shown). Caspase 3 is associated with cell apoptosis. Cleavage caspase 3 remains intact during apoptosis and can be detected using a substrate. In our research, cleavage caspase 3 is observed in TILs (data not shown). More importantly, CD27 - Compared to T cells, CD27 + A higher proportion of cleavage-type caspase 3 was found in T cells: CD4 + CD27 + T cells (28%) vs. CD4 + CD27 - T cells (13%); CD8 + CD27 + T cells (15.5%) vs. CD8 + CD27 - T cells (9%) (Figure 3). Although this is a limited sample, this suggests that CD27 + T cells CD27 - This still suggests that the T cells are more apoptotic, which may be due to the CD27-CD70 interaction in tumors. However, this result needs to be confirmed in larger patient series.

[0159] In situ, the CD27-CD70 interaction correlates with elevated sCD27 concentrations in the peripheral blood of patients with ccRCC. It is known that CD27 is cleaved into its soluble form, sCD27, on activated T cells during CD27-CD70 interaction by proteases (Hintzen et al., 1991; Loenen et al., 1992a). Previous studies have shown that CD70-expressing glioma and RCC cell lines may induce sCD27 release from PBMCs (Ruf et al., 2015; Wischhusen et al., 2002). To investigate whether CD27-CD70 interaction induces sCD27 release in situ, ELISA was first performed to measure plasma sCD27 from ccRCC patients. Elevated sCD27 levels were observed in ccRCC patients (n=44) compared to healthy donors (n=15) (Figure 4A). Next, a correlation analysis was performed between sCD27 and CD27 cell count in situ. sCD27 in peripheral blood is CD27 in situ + It was significantly correlated with the cells (Figure 4B). More importantly, a significant correlation was observed with the CD27 that interacted in situ. + This was observed in cells and in sCD27 in peripheral blood (Figure 4C). This suggests that the elevated sCD27 levels in peripheral blood may originate from the CD27-CD70 interaction in ccRCC.

[0160] Apoptosis and CD27+ T cell dysfunction in RCCs To confirm the phenotype of CD27+ T cells, CD27+ and CD27-CD8+ T cells were selected from four renal tumor samples and subjected to single-cell RNA sequencing (scRNA-seq) analysis. CD8+CD27+ TILs had expression profiles of apoptosis-related enriched genes from a gene ontology database consisting of BAX, FASLG, BCL2L11, CYCS, FBXO32, LGALS1, PIK3R1, TERF1, TXNIP, and CDKN2A. In addition, the TRM phenotype of CD27+CD8+ T cells was confirmed. This is because CD27+CD8+ T cells express the ITGAE gene (CD103) and other transcription factors (PRDM1, RBPJ, ZNF683) associated with the TRM phenotype at a higher frequency. Furthermore, single-cell RNA-seq analysis confirmed that CD27+CD8+ T cells also express exhaustion markers (PDCD1, CTLA4, HAVCR2, LAG3, TIGIT, TNFRSF9, SIRPG, ICOS, LAYN, CXCL13, CD38, TOX) (data not shown). Increased levels of cytotoxicity-related transcripts (GZMA, GZMB, GZMH, CTSC) were also observed in CD27+CD8+ T cells. Finally, CD27-CD8+ T cells were shown to express IL-2 and IL-7R, which are associated with the naive central memory phenotype, at a higher frequency.

[0161] In summary, using TCGA data, frequent expression patterns of CD27 and CD70 in ccRCC were found to correlate with patient survival. The inventors aim to understand the roles of CD27 and CD70 in ccRCC. In the study cohort, interactions between CD27+ T cells and CD70+ tumor cells were observed in situ. The CD27-CD70 interaction outcome may lead to apoptosis of CD27+ T cells, as suggested by flow cytometry analysis of TILs. In addition, plasma levels of sCD27 were elevated in ccRCC and correlated with in situ CD27-CD70 interaction. This study demonstrates that CD27-CD70 interaction leads to T cell dysfunction and sCD27 release.

[0162] References Throughout this application, various references describe the current state of the art to which the present invention pertains. The disclosures of these references are incorporated into this disclosure by reference.

[0163] [Table 3] TIFF0007856627000012.tif249165 TIFF0007856627000013.tif243165 TIFF0007856627000014.tif249165 TIFF0007856627000015.tif250165 TIFF0007856627000016.tif242165 TIFF0007856627000017.tif243165 TIFF0007856627000018.tif24165

Claims

1. A method for predicting tumor T lymphocyte (LT) dysfunction by determining the interaction between CD27 and CD70, i) A step of determining the level of soluble CD27 (sCD27) in a plasma sample, ii) A step of comparing the level of sCD27 quantified in step i) with its corresponding predetermined reference value, iii) A method comprising the step of concluding that an interaction exists between CD27 and CD70 if the level of sCD27 is higher than its corresponding predetermined reference value, or concluding that no interaction exists between CD27 and CD70 if the level of sCD27 is lower than its corresponding predetermined reference value.

2. The method according to claim 1, further comprising the step of providing data to conclude that there is a tumor lymphocyte (LT) dysfunction when the level of sCD27 is higher than the corresponding predetermined reference value, or providing data to conclude that there is no tumor lymphocyte (LT) dysfunction when the level of sCD27 is lower than the corresponding predetermined reference value.

3. A method for predicting whether a subject will develop or is susceptible to developing cancer and / or metastatic cancer that expresses CD70, i) A step of determining the level of soluble CD27 (sCD27) in a plasma sample, ii) A step of comparing the level of sCD27 quantified in step i) with its corresponding predetermined reference value, iii) A method comprising the step of providing data to conclude that a subject has or has been diagnosed with a CD70-expressing cancer and / or metastatic cancer if the level of sCD27 is higher than the corresponding predetermined reference value, or providing data to conclude that a subject does not have or does not have a CD70-expressing cancer and / or metastatic cancer if the level of sCD27 is lower than the corresponding predetermined reference value.

4. A kit or apparatus for predicting tumor T lymphocyte (LT) dysfunction by determining the interaction between CD27 and CD70, comprising means for determining the level of soluble CD27 in a plasma sample.

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