A method for treating and stratifying cancer patients.

Abstract

Methods for treating and stratifying patients with cancer - Patents.com This invention relates to the stratification and treatment of cancer patients. Given that anti-PD1 therapy targets lymphocytes and the efficacy of anticancer drug treatment is measured by its effect on tumor cells, the inventors hypothesized that studying the molecular mechanisms of anti-PD1 therapy resistance should take into account the existing intercellular communication between lymphocytes and tumor cells. Because exosomes are a carrier for the intercellular transmission of miRNAs that cause chemoresistance, the inventors investigated whether exposing T cells to anti-PD1 therapy could promote the expression of exosomal miRNAs (exomiRs) that cause chemoresistance in cancer cells. They found that T cells exposed to anti-PD1 therapy promoted the enrichment of exosomal miRNA-4315. They also demonstrated that cancer cells receiving exosomal miRNA-4315 exhibited apoptosis resistance to conventional chemotherapy. At the molecular level, they found that this apoptosis resistance phenomenon was associated with miRNA-4315-mediated downregulation of the pro-apoptotic protein Bim. Furthermore, it has been confirmed that the BH3 mimetic ABT263 can circumvent this resistance in cell and mouse models.Therefore, the present invention relates to a stratification method using exosomal miRNA-4315 and a method for treating patients suffering from cancer using a BH3 mimetic.

Description

Technical Field

[0001] Field of the Invention The present invention relates to stratification and treatment of patients suffering from cancer.

[0002] Background of the Invention Immune checkpoint inhibitors are highly anticipated as anti-cancer agent therapies in combination with first-line or conventional chemotherapy (1). Anti-PD1 therapy is currently one of the most effective anti-cancer agent immunotherapies. Despite this success, a significant number of patients acquire resistance to this treatment or will acquire it in the future (2-3-4-5). Innate resistance to anti-PD1 therapy is observed in 60% of melanoma patients (6), and 25% acquire resistance after the initial stage of objective response (7). In non-small cell lung cancer, Gettinger et al. identified patients characterized by the phenomenon of acquired resistance to anti-PD1 therapy (8). Resistance to anti-PD1 therapy has been clinically observed, but its molecular cause has not been fully elucidated. Therefore, extensive research is needed to complete the description of biomarkers related to resistance to anti-PD1 therapy. Furthermore, if these innovative biomarkers are revealed, it may be possible to provide therapeutic targets for resistance to anti-PD1 therapy.

[0003] Summary of the Invention Given that anti-PD1 therapy targets lymphocytes and the effectiveness of anticancer drug treatment is measured by its impact on tumor cells, the inventors hypothesized that existing intercellular communication between lymphocytes and tumor cells should be considered to study the molecular mechanisms of resistance to anti-PD1 therapy. Since exosomes are carriers for the intercellular movement of miRNAs that cause chemotherapy resistance (9-10-11-12), they investigated whether exposure of T cells to anti-PD1 therapy would promote the expression of exosomal miRNA (exomiR) that causes chemotherapy resistance in cancer cells. As a result, it was found that T cells treated with anti-PD1 therapy promoted the enrichment of exosomal miRNA-4315. They also pointed out that cancer cells that received exosomal miRNA-4315 developed a phenomenon called apoptosis resistance to conventional chemotherapy. At the molecular level, they found that this apoptosis resistance phenomenon is associated with the downregulation of Bim, an apoptosis-promoting protein, via miRNA-4315. In cell and mouse models, the BH3 mimetic ABT263 was confirmed to evade this resistance. Longitudinal studies using exosome blood showed that miRNA-4315 and cytochrome c could be used to define the period during which cancer cell death could be effectively increased by adding ABT263 therapy, thus evading anti-PD1 resistance. Thanks to this study, we demonstrated that exosome miRNA-4315 can be used as a blood biomarker for stratifying patients who are developing resistance to anti-PD1 antibody therapy, and we identified a therapeutic alternative (use of a BH3 mimetic) to limit this resistance. Therefore, the present invention relates to a stratification method using exosome miRNA-4315 and a method for treating patients suffering from cancer using a BH3 mimetic. In particular, the present invention is defined by its claims.

[0004] Detailed description of the invention Prognostic diagnostic methods In a first embodiment, the present invention relates to a method for identifying patients who have or are at risk of developing resistance to anti-PD-1 therapy, comprising the steps of: i) determining the expression level of exosome miRNA-4315 in a sample from the patient; ii) comparing the expression level with a predetermined reference value; and iii) concluding that if the expression level of exosome miRNA-4315 is higher than the predetermined reference value, the patient has or is at risk of developing resistance to anti-PD-1 therapy, and if the expression level of exosome miRNA-4315 is lower than the predetermined reference value, the patient does not have or is not at risk of developing resistance to anti-PD-1 therapy.

[0005] In other words, the present invention relates to a method for stratifying patients treated with anti-PD-1 therapy.

[0006] The present invention also relates to a method for predicting the response to anti-PD-1 therapy in patients with cancer, comprising: i) determining the expression level of exosome miRNA-4315 in a sample obtained from a patient; ii) comparing the expression level with a predetermined reference value; and iii) concluding that the patient will not respond to anti-PD-1 therapy if the expression level of exosome miRNA-4315 is higher than the predetermined reference value, and concluding that the patient will respond to anti-PD-1 therapy if the expression level of exosome miRNA-4315 is lower than the predetermined reference value.

[0007] According to the present invention, the method of the present invention is particularly an in vitro method.

[0008] In one embodiment, the cancer may be any solid or liquid cancer. Typically, the cancer may be cholangiocarcinoma (e.g., perihepatic cancer, distal cholangiocarcinoma, intrahepatic cholangiocarcinoma), bladder cancer, bone cancer (e.g., osteoblastoma, osteochondroma, hemangioma, chondrofibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g., meningioma, astrocytoma, oligodendroglioma, glioblastoma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, germ cell tumor, craniopharyngioma), breast cancer (e.g., in Situ ductal carcinoma, invasive ductal carcinoma, invasive lobular carcinoma, in situ lobular carcinoma, gynecomastia), Castleman disease (e.g., giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g., endometrial adenocarcinoma, adenocarcinoma, papillary serous adenocarcinoma, clear cell), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g., choriocarcinoma, chorioadenoma detritus), Hodgkin's disease, non-Hodgkin lymphoma, Kaposi's sarcoma, kidney cancer (renal cell carcinoma, etc.), laryngeal and hypopharyngeal cancer, liver cancer (e.g., hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelial cancer These include tumors, plasmacytoma, nasal cavity and paranasal sinus cancers (e.g., estesioneroblastoma, median granuloma), nasopharyngeal cancer, neuroblastoma, oral and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), gastric cancer, testicular cancer (e.g., seminoma, non-seminoma germ cell cancer, etc.), thymic cancer, thyroid cancer (e.g., follicular carcinoma, undifferentiated carcinoma, poorly differentiated carcinoma, thyroid medullary carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g., uterine leiomyosarcoma).

[0009] In certain embodiments, the cancer is glioblastoma (GBM), lung cancer, breast cancer, or ovarian cancer.

[0010] In certain embodiments, glioblastoma is glioblastoma multiforme (GBM), and lung cancer is lung adenocarcinoma.

[0011] Typically, the sample according to the present invention may be blood, plasma, serum sample, T cell-derived exosomes, or cancer biopsy.

[0012] According to the present invention, the terms “patient” or “subject” refer to mammals such as rodents, cats, dogs, and primates. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In particular, the subject refers to a human having cancer, especially GBM, lung cancer, breast cancer, or ovarian cancer.

[0013] As used herein, the term “miRNA-4315” refers to the miRNA gene located on chromosome 17 (17q21.31) and accessible in the miRBase database under ID number MI0015844.

[0014] In particular, miRNA-4315 is hsa-mir-4315-1, and the nucleic acid sequence of mature miRNA-4315 is (5'-3'):CCGCUUCUGAGCUGGAC (Sequence ID 1).

[0015] As used herein, the term “exosome miRNA-4315” refers to the presence of miRNA-4315 in exosomes. In certain embodiments, “exosome miRNA-4315” refers to exosomes containing miRNA-4315 derived from T cells exposed to anti-PD-1 therapy.

[0016] As used herein, the term “exosome miRNA-4315 expression level” or “exosome miRNA-4315 level” refers to the level of exosome miRNA-4315 compared to the total amount of miRNA-4315.

[0017] As used herein, the term “anti-PD-1 therapy” refers to the use of at least one antibody anti-PD-1 to treat a patient’s cancer. For example, the antibody anti-PD-1 may be nivolumab, pembrolizumab, or cemiprimab.

[0018] In certain embodiments, patients may receive standard chemotherapy concurrently with, separately from, or sequentially with anti-PD-1 therapy.

[0019] As used herein, the term “standard chemotherapy” includes cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosphamide, nitrosourea, platinum complexes such as cisplatin, carboplatin, oxaliplatin, mitomycin, dacarbazine, procarbidine, etoposide, teniposide, campatecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epymbim, This refers to classic anticancer agents selected from a group consisting of, but not limited to, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisol, irinotecan, estramustine, etoposide, nitrogen mustard, BCNU, nitrosourea agents such as carmstrom and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatine mesylate, hexamethylamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tilphostine, protease inhibitors, herbimicuiphosphamide, erbustatin and raventostine A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of, the following classes of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, antifolates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormone therapy, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimicrobial agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, MDR inhibitors, and Ca2+ATPase inhibitors.

[0020] In particular, standard chemotherapy may include oxaliplatin, cisplatin, temozolomide, cyclophosphamide, doxorubicin, or paclitaxel.

[0021] In another embodiment, patients with cancer, particularly glioblastoma, may also be treated with standard treatment consisting of maximum surgical resection, radiotherapy, and adjuvant standard chemotherapy such as temozolomide.

[0022] In one embodiment, and according to the method of the present invention, the expression level of exosomal miRNA-4315 of the present invention can be determined before or after the initiation of treatment with anti-PD-1 therapy in the patient.

[0023] The term “determination of expression level” as used above includes qualitative and / or quantitative detection (measuring the level), with or without reference to a control. Typically, the expression level of the miR of the present invention can be measured, for example, by RNA immunoprecipitation, cross-linked immunoprecipitation, qRT-PCR, and all RNA sequencing methods for the sample.

[0024] The "reference value" may be a healthy subject, i.e., a subject that does not have any cancer, particularly glioblastoma. In particular, the control is a subject that is not healthy. In another embodiment, the "reference value" may be a subject with cancer that is not resistant to anti-PD-1 therapy.

[0025] The expression level of miRs can be measured by various techniques well known in the art. In the present invention, before determining the level of the miRs of the present invention, T cell-derived exosomes are isolated and quantified by any technique that makes this possible (see, for example, the Materials and Methods section of this application). In particular, "ExoQuick" (see the Materials and Methods section) can be used for the isolation of exosomes.

[0026] Methods for determining the amount of miR are well known in the art. For example, nucleic acids contained in a sample (e.g., cells or tissues prepared from a patient) are first extracted according to standard methods, such as using lytic enzymes or chemical solutions, or extracted by nucleic acid binding resins according to the manufacturer's instructions. Next, the extracted miR is detected by hybridization (e.g., Northern blot analysis, in situ hybridization) and / or amplification (e.g., RT-PCR).

[0027] Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), and the like.

[0028] Nucleic acids having at least 10 nucleotides and showing sequence complementarity or homology to the miR of interest herein are useful as hybridization probes or amplification primers. Such nucleic acids need not be identical, but typically are understood to be at least about 80% identical, more particularly 85% identical, and even more particularly 90 - 95% identical with a homologous region of equivalent size. In certain embodiments, it may be advantageous to use the nucleic acid in combination with suitable means, such as a detectable label, to detect hybridization.

[0029] Typically, a nucleic acid probe includes one or more labels, for example, to enable detection of a target nucleic acid molecule using the disclosed probes. In various applications such as in situ hybridization procedures, nucleic acid probes include labels (e.g., detectable labels). A "detectable label" is a molecule or material that can be used to generate a detectable signal indicating the presence or concentration of the probe in a sample (particularly, the bound or hybridized probe). Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in a sample (to which the labeled unique nucleic acid molecule has bound or hybridized). Labels associated with one or more nucleic acid molecules (such as probes generated by the disclosed methods) can be detected directly or indirectly. Labels can be detected by any known or yet-to-be-discovered mechanism including absorption, emission, and / or scattering of photons (including photons of radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency). Detectable labels include colored, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance to another to provide a detectable difference (e.g., converting a colorless substance to a colored substance or vice versa, or by generating a precipitate or increasing sample turbidity), haptens that can be detected by antibody-binding interactions, and paramagnetic and magnetic molecules or materials.

[0030] Specific examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those skilled in the art and can be selected from, for example, Life Technologies (formerly Invitrogen), see, for example, The Handbook—A Guide to Fluorescent Probes and Labeling Technologies. Examples of specific fluorophores that can be attached (e.g., chemically bound) to nucleic acid molecules (such as uniquely specific binding regions) include, for example, Nazarenko et al., U.S. Patent No. 5,866,366, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and its derivatives such as acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 Disulfonates (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, Antre-1-ranylamide, Brilliant Yellow, coumarin and coumarin derivatives, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanosine; 4',6-diarnidino-2-phenylindole (DAPI); 5',5” dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaminepentaacetic acid; 4,4'-diisothiocyanatodihydrotilbene-2,2'-disulfonic acid; 4,4”-diisothiocyanatotilbene-2,2'-bisulfo-1 acid.5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); derivatives such as eosin and eosin isothiocyanate; derivatives such as erythrosine and erythrosine B, erythrosine isothiocyanate, etc.; ethidium; derivatives such as fluorescein and 5-carboxyfluorescein (FAM), 5-(4,6-diclotriadine-2-y-dalminofluorescein) (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorocein, fluorosentyan (FITC), and QFITC ​​Q (RITC); derivatives such as 2',7'-difluorofluorescein (OREGON GREENR); Fluoramine; IR144; IR1446; Malachite green isothiocyanate; 4-Methylumbelliferone; Orthocresolphthalein; Nitrotyrosine; Pararosanilin; Phenol Red; B-Phycoerythrin; o-Phthalaldehyde; Pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl-1 pyrene butyrate; Reactive Red 4 (Sibacron Brilliant Red 3B-A); Rhodamine and its derivatives (6-Carboxy-X-Rhodamine (ROX), 6-Carboxyrhodamine (R6G), Lisamin Rhodamine B sulfonyl chloride, Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X isothiocyanate, Rhodamine Green, Sulforhodamine B, Sulforhodamine 101 and sulfonyl chloride derivatives of Sulforhodamine 101 (Texas Red)); Examples include N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin; benzoic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates that luminescent at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem.).These include GFP, Lissamine®, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodum, and xanthene (as described in U.S. Patent No. 5,800,996 to Lee et al.), and their derivatives. Other fluorophores known to those skilled in the art may also be used, for example, those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)), including the ALEXA FLUORR series dyes (e.g., described in U.S. Patents 5,696,157, 6,130,101 and 6,716,979), the BODIPY series dyes (dipyrometeneborone difluoride dyes, e.g., U.S. Patents 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (amine-reactive derivatives of sulfonated pyrene described in U.S. Patent 1), Cascade Blue (US No. 5,132,432), Marina Contains dyes such as those listed in Blue (U.S. Patent No. 5,830,912).

[0031] In addition to the fluorescent dyes mentioned above, fluorescent labels can also be fluorescent nanoparticles, such as semiconductor nanocrystals, such as QUANTUM DOT™ (e.g., Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg); see also U.S. Patents 6,815,064; 6,682,596; and 6,649,133). Semiconductor nanocrystals are tiny particles with size-dependent optical and / or electrical properties. When a semiconductor nanocrystal is irradiated with a primary energy source, secondary emission occurs of energy at a frequency corresponding to the hand gap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light or fluorescence of a specific wavelength. Semiconductor nanocrystals with different spectral properties are described, for example, U.S. Patents 6,602,660, 6,602,671, etc. See, for example, Bruchez et al., Science 281:2013-2016, 1998; Chan et al., Science Examples of semiconductor nanocrystals capable of binding to various biomolecules (including dNTPs and / or nucleic acids) or substrates are described in the techniques described in U.S. Patent Nos. 281:2016-2018, 1998; and U.S. Patent No. 6,274,323. The formation of semiconductor nanocrystals of various compositions is described, for example, in U.S. Patents Nos. 6,927,069, 6,914,256, 6,855,202, 6,709,929, 6,689,338, 6,500,622, 6,306,736, 6,225,198, 6,207,392, 6,114,038, and 6,048,616. This is also described in U.S. Patent Publication No. 5,990,479, No. 5,690,807, No. 5,571,018, No. 5,505,928, No. 5,262,357 and U.S. Patent Publication No. 2003 / 0165951, PCT Publication No. 99 / 26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can be manufactured to be distinguishable based on their different spectral properties. For example, semiconductor nanocrystals can be manufactured to emit light of different colors depending on their composition, size, or size and composition.For example, quantum dots that emit at different wavelengths based on size (565mn, 655mn, 705mn, or 800mn emission wavelengths), suitable as fluorescent labels for the probes disclosed herein, are available from Life Technologies (Carl Shad, California).

[0032] Other labels include, for example, radioactive isotopes (such as 3H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions such as Gd3+, and liposomes.

[0033] Detectable labels that can be used with nucleic acid molecules also include enzymes, such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

[0034] Alternatively, enzymes may be used in metallographic detection schemes. For example, the silver in situ hybridization (SISH) procedure includes a metallographic detection scheme for the identification and localization of hybridized genomic target nucleic acid sequences. Metallographic detection schemes involve using enzymes such as alkaline phosphatases in combination with water-soluble metal ions and the enzyme's redox-active substrate. The substrate is converted into a redox activator by the enzyme, which reduces the metal ion, causing the formation of a detectable precipitate. (See, for example, U.S. Patent Application Publication 2005 / 0100976, PCT Publication 2005 / 003777, and U.S. Patent Application Publication 2004 / 0265922). Metallographic detection methods also involve using an oxide-reductase (such as wasabi peroxidase) together with water-soluble metal ions, an oxidizer, and a reducing agent to again form a detectable precipitate. (See, for example, U.S. Patent No. 6,670,113).

[0035] Probes produced using the disclosed method can be used for nucleic acid detection in ISH procedures (e.g., fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

[0036] In situ hybridization (ISH) involves contacting a sample containing a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) with a probe that is specifically hybridizable to or specifically labeled with the target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in the context of metaphase or interphase chromosome preparation (e.g., cell or tissue samples mounted on slides). Slides are optionally pre-treated, for example, to remove paraffin or other materials that may interfere with uniform hybridization. The sample and probe are heat-treated, for example, to denature the double-stranded nucleic acids. The probe (compounded in an appropriate hybridization buffer) and sample are coupled under conditions and for a sufficient time for hybridization to occur (usually until equilibrium is reached). Chromosomes are washed to remove excess probe, and chromosome labeling is detected using standard methods.

[0037] For example, biotinylated probes can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, fluorochrome can be detected directly, or the sample can be incubated with, for example, fluorescein isothiocyanate (FITC)-labeled avidin. Amplification of the FITC signal can be performed, if necessary, by incubation with biotin-conjugated goat anti-avidin antibody, washing, and a second incubation with FITC-conjugated avidin. For detection by enzymatic activity, the sample can be pre-equilibriumized (for example, in alkaline phosphatase (AP) buffer) by incubation with, for example, streptavidin, washing, incubation with biotin-conjugated alkaline phosphatase, washing again. For a general description of in situ hybridization procedures, see, for example, U.S. Patent No. 4,888,278.

[0038] Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Patent Nos. 5,447,841, 5,472,842, and 5,427,932; and, for example, Pirkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described, for example, in Tanner et al., Am.1. Pathol. 157:1467-1472, 2000 and U.S. Patent No. 6,942,970. Additional detection methods are described in U.S. Patent No. 6,280,929.

[0039] Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As described above, probes labeled with fluorescent dyes (including fluorescent dyes and QUANTUM DOTSR) can be directly detected optically when performing FISH. Alternatively, probes can be labeled with non-fluorescent molecules such as haptens (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrazoles, thiazoles, nitroallyls, benzofurazans, triterpenes, urea, thiourea, rotenone, coumarin, coumarin compounds, podophyllotoxins, podophyllotoxin compounds and combinations thereof), ligands, or other indirectly detectable parts). Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting a sample (e.g., a cell or tissue sample to which the probe is bound) with a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific to the selected hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOTR) or another indirectly detectable moiety, or can be contacted with one or more additional specific conjugates (e.g., a secondary antibody or specific antibody) that can be labeled with a fluorophore.

[0040] In other examples, a probe, or specific binder (such as an antibody, e.g., a primary antibody, a receptor, or other binder), is labeled with an enzyme that can convert a fluorescent or chromogenic composition into a detectable fluorescence, color, or other detectable signal (e.g., in the deposition of detectable metal particles in SISH). As described above, the enzyme can be attached directly or indirectly to the relevant probe or detection reagent via a linker. Examples of suitable reagents (e.g., binding reagents) and chemicals (e.g., linkers and attachment chemicals) are described in U.S. Patent Applications Publication Nos. 2006 / 0246524; 2006 / 0246523, and 2007 / 0117153.

[0041] Those skilled in the art will understand that by appropriately selecting labeled probe-specific binder pairs, it is possible to construct a multiple detection scheme that facilitates the detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample, or on multiple cell or tissue samples). For example, a first probe corresponding to a first target sequence can be labeled with a first hapten such as biotin, and a second probe corresponding to a second target sequence can be labeled with a second hapten such as DNP. After exposing a sample to the probe, to detect the bound probe, the sample is bound with a first specific binder (in this case, a first fluorescent, e.g., avidin labeled with a first spectrally different QUANTUM DOTR, e.g., emitting at 585 mn) and a second specific binder (in this case, an anti-DNP antibody or antibody fragment labeled with a second fluorescent (e.g., a second fluorescent that emits at 705 mn)). Probe-binding pairs can be added to the multiple detection scheme using other fluorescent dyes. Many variations of direct and indirect methods (one-step, two-step, or more) are conceivable, all of which are appropriate to the context of the disclosed probes and assays.

[0042] Probes typically consist of single-stranded nucleic acids with a length of 10 to 1000 nucleotides, e.g., 10 to 800, more specifically 15 to 700, and typically 20 to 500. Primers are typically short single-stranded nucleic acids with a length of 10 to 25 nucleotides, designed to perfectly or nearly perfectly match the target nucleic acid to be amplified. Probes and primers are "specific" to the nucleic acid they hybridize, i.e., they are particularly suitable for high-stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x SCC (SCC is 0.15M NaCl, 0.015M Na-citric acid)).

[0043] The nucleic acid primers or probes used in the amplification and detection methods described above can be assembled as kits. Such kits include consensus primers and molecular probes. Certain kits also include components necessary to determine whether amplification has occurred. Kits may also include, for example, PCR buffer and enzyme; a positive control sequence, reaction control primers; and instructions for amplifying and detecting specific sequences.

[0044] In certain embodiments, the method of the present invention includes the steps of providing total miRs extracted from cumulus cells and subjecting the miRs to amplification and hybridization to a specific probe, more specifically by quantitative or semi-quantitative RT-PCR.

[0045] In another specific embodiment, the expression level is determined by DNA chip analysis. Such a DNA chip or nucleic acid microarray consists of different nucleic acid probes chemically attached to a substrate which may be a microchip, a glass slide, or microsphere-sized beads. The microchip can be made of polymer, plastic, resin, polysaccharide, silica or silica-based material, carbon, metal, inorganic glass, or nitrocellulose. The probes consist of nucleic acids such as cDNA or oligonucleotides of about 10 to about 60 base pairs. To determine the expression level, a sample from a subject is optionally first subjected to reverse transcription, labeled, and contacted with a microarray under hybridization conditions, leading to the formation of a complex between target nucleic acids complementary to 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, such as the use of radiolabeling or fluorescent labeling. Many variations of microarray hybridization techniques are available to those skilled in the art (see, for example, Hoheisel's review, Nature Reviews, Genetics, 2006, 7:200-210).

[0046] Gene expression levels can be expressed as absolute or normalized expression levels. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing it to the expression of genes not relevant to determining a patient's cancer stage, such as constitutively expressed housekeeping genes. Suitable genes for normalization include the actin gene ACTB, the ribosomal 18S gene, GUSB, PGK1, and TFRC, which are housekeeping genes. In this invention, the housekeeping genes used were GAPDH, GUSB, TBP, and ABL1. This normalization allows expression levels in one sample, such as a patient sample, to be compared with another sample or between samples from different sources.

[0047] Typically, a “threshold,” “threshold level,” “reference value,” or “cutoff value” can be determined experimentally, empirically, or theoretically. The threshold can also be arbitrarily selected based on existing experimental and / or clinical conditions that would be recognized by those skilled in the art. In particular, those skilled in the art can compare the expression level of the miR obtained according to the method of the present invention to a defined threshold.

[0048] In particular, the threshold is the mean expression level of the miR of the present invention in a population of healthy individuals. As used herein, the term “healthy individual” means a person known to be healthy, i.e., a person who does not have cancer, particularly glioblastoma, and does not require medical treatment.

[0049] Typically, a person skilled in the art can determine the expression level of the miR of the present invention in 100 biological samples, for example, in biopsies of glioblastoma cancer, from individuals whose health status is known. The mean value of the obtained expression levels is then determined according to a well-known statistical analysis to obtain the mean expression level of the miR of the present invention. This value is considered normal and therefore serves as a threshold. By comparing the expression level of the miR of the present invention to this threshold, a physician can classify cancer and diagnose its prognosis.

[0050] Therefore, physicians will be able to adapt and optimize appropriate medical care for patients with life-threatening conditions due to cancer. The aforementioned prognosis determination is highly relevant to follow-up care and clinical decision-making.

[0051] The present invention also relates to a kit useful for the method of the present invention, which includes means for detecting the miR of the present invention.

[0052] Treatment method A second aspect of the present invention relates to a BH3 mimetic for use in the treatment of cancer in subjects identified as having, or likely to have, or likely to develop resistance to anti-PD-1 therapy according to the present invention.

[0053] In other words, the present invention relates to a BH3 mimetic agent for use in the treatment of subjects that do not respond to anti-PD-1 therapy according to the present invention.

[0054] Accordingly, the present invention also relates to a method for identifying patients who are resistant to or at risk of resistant to anti-PD-1 therapy, comprising the steps of: i) determining the expression level of exosome miRNA-4315 in a sample from the patient; ii) comparing the expression level with a predetermined reference value; and iii) concluding that if the expression level of exosome miRNA-4315 is superior to the predetermined reference value, the patient has or is at risk of developing resistance to anti-PD1 therapy, and concluding that the patient is not at risk of having or not developing resistance to anti-PD1 therapy. PD1 therapy is administered when the expression level of exosome miRNA-4315 is inferior to a predetermined reference value, and a BH3 mimetic is administered to patients who have or are at risk of developing resistance to anti-PD1 therapy.

[0055] In other words, the present invention relates to a method for predicting the effectiveness of anti-PD-1 therapy in patients with cancer who require it, comprising: i) determining the expression level of exosome miRNA-4315 in a sample obtained from a patient; ii) comparing the expression level with a predetermined reference value; and iii) concluding that if the expression level of exosome miRNA-4315 is high, the patient will not respond to anti-PD-1 therapy; and concluding that if 4315 is above the predetermined reference value and the expression level of exosome miRNA-4315 is below the predetermined reference value, the patient will respond to anti-PD-1 therapy, and administering a BH3 mimoid to patients who do not respond to anti-PD-1 therapy.

[0056] As used herein, the term “BH3 mimetics” refers to all compounds that have an effect (inhibition) on the anti-apoptotic protein Bcl-2 and can mimic only the BH3 protein, and therefore include all small molecules that induce apoptosis (see, for example, Haiming Dai et al. Mitochondrial apoptosis and BH3 mimetics, 2016). BH3 mimetics may be selected from the group consisting of ABT-737, venetoclax (ABT-199), and navitoclax (ABT-263).

[0057] In another embodiment, the present invention also relates to anti-miRNA-4315 (antago-miR) for use in the treatment of cancer in subjects identified as having, or likely to have, or likely to develop resistance to anti-PD-1 therapy according to the present invention.

[0058] In other words, the present invention also relates to an anti-miRNA-4315 (antago-miR) for use in the treatment of subjects that do not respond to anti-PD-1 therapy according to the present invention.

[0059] As used herein, the terms “treatment” or “to treat” include both prophylactic or preventive treatment and curative or disease-modifying treatment, and include treatment for subjects at risk of or suspected of having the disease, as well as subjects diagnosed with suffering from the disease or condition, and include suppression of clinical relapse. Treatments may be administered to subjects with a medical disability, or may be applied to subjects who may ultimately acquire such disability, in order to prevent, cure, delay the onset, reduce the severity of, or improve one or more symptoms of the disease or recurrent disorder, or to extend the subject’s survival time beyond what would be expected in the absence of such treatment. “Treatment regimen” means a pattern of treatment for a disease, e.g., a pattern of medication used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The terms “induction regimen” or “induction period” mean a treatment regimen (or part of a treatment regimen) used for the initial treatment of the disease. The general goal of an induction regimen is to provide the subject with a high level of medication during the initial period of the treatment regimen. An induction regimen may employ a “loading regimen” (partially or entirely), which may include administering a larger dose of the drug than the physician employs during the maintenance regimen, administering the drug more frequently than the physician employs during the maintenance regimen, or both. The terms “maintenance regimen” or “maintenance period” refer to a treatment regimen (or part of a treatment regimen) used to maintain a subject during treatment for a disease, for example, to maintain the subject in remission for an extended period (several months or several years). A maintenance regimen may employ continuous therapy (e.g., administering the drug at regular intervals, e.g., weekly, monthly, or yearly) or intermittent therapy (e.g., interruption therapy, intermittent therapy, therapy on relapse, or therapy when certain predetermined criteria [e.g., disease onset] are met).

[0060] The present invention also relates to combination preparations, either simultaneous, separate, or sequential, for use in the treatment of cancer in subjects that have, will have, or will develop resistance to, the anti-PD-1 therapy according to the present invention, i) anti-PD-1 therapy and ii) BH3 mimetic agents.

[0061] The present invention also relates to a combination formulation of i) an anti-PD1 therapy and ii) a BH3 mimetic agent for use in the treatment of cancer in subjects that do not respond to the anti-PD1 therapy according to the present invention, either simultaneously, separately, or sequentially.

[0062] The present invention also relates to simultaneous, separated, or sequential combination preparations for use in the treatment of cancer in subjects identified as having, becoming resistant to, or developing resistance to the anti-PD-1 therapy according to the present invention, ii) a BH3 mimetic, and iii) standard chemotherapy. The present invention also relates to simultaneous, separated, or sequential combination preparations for use in the treatment of cancer in subjects that do not respond to the anti-PD-1 therapy according to the present invention, ii) a BH3 mimetic, and iii) standard chemotherapy.

[0063] In certain embodiments, the subject may also be treated with radiotherapy or radiotherapy agents.

[0064] As used herein, “radiotherapy” may consist of gamma-ray irradiation, X-ray irradiation, electron or photon irradiation, external beam radiation therapy, or curitherapy.

[0065] As used herein, the term “radiotherapy agent” is intended to mean any radiotherapy agent known to those skilled in the art to be effective in treating or improving cancer, without limitation. For example, a radiotherapy agent could be an agent such as one administered in brachytherapy or radionuclide therapy. Such a method may optionally further include the administration of chemotherapy and / or other radiotherapy, but is not limited to, one or more additional cancer therapeutic agents.

[0066] In one embodiment, the cancer according to the present invention is GBM, lung cancer, breast cancer, or ovarian cancer.

[0067] In one embodiment and according to the method of treatment, cancer may be any solid or liquid cancer. Typically, cancer is cancer, cancer is cholangiocarcinoma (e.g., perihepatic cancer, distal cholangiocarcinoma, intrahepatic cholangiocarcinoma), bladder cancer, bone cancer (e.g., osteoblastoma, osteochondroma, hemangioma, chondrofibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g., meningioma, astrocytoma, oligodendroglioma, glioblastoma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, germ cell tumor, craniopharyngioma), breast cancer (e.g., in Situ ductal carcinoma, invasive ductal carcinoma, invasive lobular carcinoma, in situ lobular carcinoma, gynecomastia), Castleman disease (e.g., giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g., endometrial adenocarcinoma, adenocarcinoma, papillary serous adenocarcinoma, clear cell), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g., choriocarcinoma, chorioadenoma detritus), Hodgkin's disease, non-Hodgkin lymphoma, Kaposi's sarcoma, kidney cancer (renal cell carcinoma, etc.), laryngeal and hypopharyngeal cancer, liver cancer (e.g., hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelial cancer These include tumors, plasmacytoma, nasal cavity and paranasal sinus cancers (e.g., estesioneroblastoma, median granuloma), nasopharyngeal cancer, neuroblastoma, oral and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), gastric cancer, testicular cancer (e.g., seminoma, non-seminoma germ cell cancer, etc.), thymic cancer, thyroid cancer (e.g., follicular carcinoma, undifferentiated carcinoma, poorly differentiated carcinoma, thyroid medullary carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g., uterine leiomyosarcoma).

[0068] In certain embodiments, glioblastoma is GBM, lung cancer, breast cancer, or ovarian cancer.

[0069] Another object of the present invention relates to a method for treating cancer, comprising administering a therapeutically effective amount of a BH3 mimetic to a subject identified as having, or likely to have, or likely to develop resistance to anti-PD-1 therapy according to the present invention.

[0070] therapeutic composition Another object of the present invention relates to a therapeutic composition comprising a BH3 mimetic agent according to the present invention for use in the treatment of cancer in subjects identified as having, or likely to have, or likely to develop resistance to anti-PD-1 therapy according to the present invention.

[0071] A further object of the present invention relates to a therapeutic composition comprising a BH3 mimetic agent according to the present invention for use in the treatment of cancer in subjects that do not respond to anti-PD-1 therapy according to the present invention.

[0072] Any therapeutic agent of the present invention can be combined with pharmaceutically acceptable excipients and optionally a sustained-release matrix such as a biodegradable polymer to form a therapeutic composition.

[0073] "Pharmacologically" or "pharmaceutically acceptable" means, as appropriate, molecular entities and compositions that, when administered to mammals, particularly humans, do not produce adverse reactions, allergic reactions, or other unpleasant reactions. A pharmacopoeia is a pharmacopoeia of any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0074] The form of the pharmaceutical composition, the route of administration, the dosage, and the regimen naturally depend on the condition to be treated, the severity of the disease, the patient's age, weight, and sex, etc.

[0075] The pharmaceutical composition of the present invention can be formulated for external use, oral administration, intranasal administration, parenteral administration, intraocular administration, intravenous administration, intramuscular administration, or subcutaneous administration.

[0076] In particular, pharmaceutical compositions include vehicles that are pharmaceutically acceptable as injectable formulations. These are particularly isotonic, sterile, physiological saline (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of these salts), or dry, particularly lyophilized compositions, which can be made into injectable solutions by optionally adding sterile water or physiological saline.

[0077] The dosage used for administration can be adapted as a function of various parameters, particularly as a function of the mode of administration used, the associated pathological condition, or alternatively, as a function of the desired duration of treatment.

[0078] Furthermore, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; time-release capsules; and any other form currently available.

[0079] The pharmaceutical composition of the present invention may contain further therapeutic activators. The present invention also relates to a kit comprising the compound according to the present invention and further therapeutic activators.

[0080] In one embodiment, the therapeutic activator may be an anticancer agent.

[0081] Possible anticancer drugs include melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), and bendamustine (Treanda).

[0082] Other anticancer drugs include, for example, cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosphamide, nitrosourea, platinum complexes such as cisplatin, carboplatin, and oxaliplatin, mitomycin, dacarbazine, procarbidine, etoposide, teniposide, campatecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mytoxantrone, and L-asparagus. Laginase, doxorubicin, epymbicil, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisol, irinotecan, estramustine, etoposide, nitrosourea such as BCNU, carmstome and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatine mesylate, hexamethylamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPa ifosphamide phostine, protease inhibitors, inhibitors such as Herbimic A, genistin, erbustatin, and raventostine A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of, the following classes of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, antifolates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormone therapy, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimicrobial agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, MDR inhibitors, and Ca2+ATPase inhibitors.

[0083] Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, monospecific, bispecific or polyspecific antibodies, monobodies, or polybodies.

[0084] Additional anticancer agents may be selected from, but are not limited to, growth factors or hematopoietic factors such as erythropoietin and thrombopoietin, and their growth factor mimics.

[0085] In the method of the present invention for treating cancer, a further therapeutic activator may be an antiemetic. Suitable antiemetics include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine monoethanolamine, arizaprid, azasetron, and benzkinamide. Other suitable antiemetics include, but are not limited to, vietanautin, bromopride, buclidine, clevopride, cyclidine, dunenhydrinate, diphenidol, drasetron, mecrisum, metalatale, metopimazine, nabilone, oxypemdil, pivamazine, scopolamine salpiride, tetrahydrocanbinol, cefilperazine, thioproperazine, and tropisetron. In preferred embodiments, the antiemetic is granisetron or ondansetron.

[0086] In another embodiment, further therapeutic activators may be hematopoietic colony-stimulating factors. Preferred hematopoietic colony-stimulating factors include, but are not limited to, filgrastim, salglamostim, morglamostim, and epoetin-α.

[0087] In yet another embodiment, other therapeutic agents may be opioid or non-opioid analgesics. Suitable opioid analgesics include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, methopone, apomorphine, nomioipuhine, etoipubin, buprenorphine, mepezine, lopamide, anilezin, etheptadine, pimidine, betaprozin, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodone, pemazosin, cyclazosin, methadone, isomethadone, and propoxyfene. Suitable non-opioid analgesics include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, difludinal, etodolac, fenoprofen, fluviprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam, and sulindac.

[0088] In yet another embodiment, the further therapeutic activator may be an anxiolytic. Suitable anxiolytics include, but are not limited to, buspirone and benzodiazepines such as diazepam, lorazepam, oxazapam, clorazepate, clonazepam, chlordiazepoxide, and alprazolam.

[0089] In yet another embodiment, further therapeutic activators may be checkpoint-blocking cancer immunotherapy agents.

[0090] Typically, checkpoint inhibitor cancer immunotherapies are drugs that inhibit immunosuppressive receptors expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1 (PDCD1), (best known as PD-1), or drugs that block the major ligands of these receptors, such as NK cells, as well as various members of the killer cell immunoglobulin-like receptor (KIR) family, or PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

[0091] Generally, checkpoint inhibitor cancer immunotherapy agents are antibodies.

[0092] In some embodiments, the checkpoint-blocking cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-IDO1 antibody, anti-TIGIT antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-BTLA antibody, and anti-B7H6.

[0093] The present invention is further illustrated by the following figures and examples. However, these examples and figures should not be construed in any way as limiting the scope of the present invention. [Brief explanation of the drawing]

[0094] [Figure 1A] Figure 1: Exosomes of T cells exposed to anti-PD-1 therapy reduce temozolomide-induced cell death via miR-4315. A. On day 14, RT-qPCR was performed to show that anti-PD-1 exposure increases exosome levels of miR-4315. B. RT-qPCR was performed to verify the miR-4315 levels of A1 exosomes after exposure to indicated exosomes. [Figure 1B]Figure 1: Exosomes of T cells exposed to anti-PD-1 therapy reduce temozolomide-induced cell death via miR-4315. A. On day 14, RT-qPCR was performed to show that anti-PD-1 exposure increases the exosome level of miR-4315. B. RT-qPCR was performed to verify the miR-4315 level in A1 exosomes after exposure to indicated exosomes. [Figure 2] Figure 2: Exosomes derived from T cells exposed to αPD1 (Exo / αPD1) promote the cisplatin-induced apoptosis phenotype in A549 cells via miR-4315. Using cisplatin-induced cell death assays, PARP, and Caspase-3 cleavage, we demonstrated that exosomes derived from T cells exposed to αPD1 (Exo / αPD1) promote the cisplatin-induced apoptosis phenotype. RT-qPCR and intracellular ELISA revealed that this phenomenon is associated with Bim downregulation via miR-4315. [Figure 3A] Figure 3: Effects of exosome pretreatment on oxaliplatin or paclitaxel-induced cell death in ovarian (OV90) (3A) and breast (MCF7) cancer cells (3B). [Figure 3B] Figure 3: Effects of exosome pretreatment on oxaliplatin or paclitaxel-induced cell death in ovarian (OV90) (3A) and breast (MCF7) cancer cells (3B). [Figure 4A] Figure 4: ABT263 neutralizes anti-PD1 / exomiR-4315-induced chemotherapy resistance in an in vivo model of lung cancer. A. Cisplatin-induced cell death measurements and PARP and caspase-3 cleavage tests are applied to show that the cisplatin-resistant phenotype induced by exosomes derived from αPD1 (Exo / αPD1)-exposed T cells is inhibited by the use of ABT263.A cells. B. Graph showing the effect of treatment on tumor burden. Each treatment includes 4 mice. [Figure 4B]Figure 4: ABT263 neutralizes anti-PD1 / exomiR-4315-induced chemotherapy resistance in an in vivo model of lung cancer. A. Cisplatin-induced cell death measurements and PARP and caspase-3 cleavage tests are applied to show that the cisplatin-resistant phenotype induced by exosomes derived from αPD1 (Exo / αPD1)-exposed T cells is inhibited by the use of ABT263.A cells. B. Graph showing the effect of treatment on tumor burden. Each treatment includes 4 mice. [Examples]

[0095] Materials and methods and Cell culture. T cells were obtained from Stem Cell Technology (France) and cultured in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. A172 and MCF7 cells were cultured in DMEM (4.5 g / L glucose) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. SKOV3 and A549 cells were cultured in RMPI medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. MCF7 cells were cultured in EMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were cultured in a 5% CO2 incubator at 37°C.

[0096] FOXO1 activity. For the estimation of FOXO1 activity, we used the TransAM® FKHR / FOXO1 kit (Active Motif, France). In short, cells were collected at the indicated time and conditions and used for nuclear extraction of the protein using the Nuclear Extraction Kit (Active Motif, France). At each point (technical duplication, independent biological triplication), 15 μg of nuclear extract was used according to the Active Motif instructions. The OD of the ELISA plates was read using a Victor® x3 spectrophotometer (Perkin-Elmer, France).

[0097] Cellular cytotoxicity assay Cell viability was estimated using the Colorimetric Cell Cytotoxicity Assay Kit (Abcam, France). In short, cells were seeded in 96-well plates. Following the manufacturer's instructions, absorbance was read at 570 nm and 605 nm using a Victor® x3 spectrophotometer (Perkin-Elmer, France).

[0098] Measurement of caspase-3 and poly-ADP-ribose polymerase (PARP) cleavage. Levels of cleaved caspase-3 and cleaved PARP are both considered biomarkers of apoptosis. Measurements of these two parameters were performed using Human Cleaved PARP1 and Human Cleaved Caspase-3 ELISA Kits (Abcam, France) according to the manufacturer's protocol.

[0099] In-cell ELISA. Intracellular ELISA was performed using the Bim Colorimetric Cell-Based ELISA Kit (Aviva Systems Biology, France) according to the manufacturer's instructions. Briefly, 5,000 cells were seeded into a 96-well plate and it was determined whether or not they would be exposed to the indicated exosomes or miRNAs. Subsequently, the cells were treated with fixative (4% paraformaldehyde solution) at room temperature for 10 minutes, and the exosomes were incubated overnight at 4°C. A suitable HRP-labeled secondary antibody was incubated at room temperature for 1 hour. Detection was performed at 450 nm.

[0100] After washing, the cells in each well were incubated in crystal violet solution at room temperature for 5 minutes, according to the manufacturer's instructions. Absorbance was read at 595 nm and used to normalize the "Bim signal".

[0101] Luciferase promoter and 3'UTR reporter assay. Cells were seeded in 24-well plates and transfected with the indicated firefly luciferase construct along with the SV40-renilla control vector. After 40 hours, lysates were prepared, and luciferase activity was measured using a Dual Luciferase Reporter Assay system (Promega, France) and a luminometer (MicroLumat Plus, EG&G Berthold, France).

[0102] Isolation of cell-derived exosomes Following anti-PD1 exposure, T cell-derived exosomes were isolated using the ExoQuick kit (Ozyme, France) according to the manufacturer's instructions. In short, cell culture supernatant was collected and centrifuged at 3000 g / 15 min. ExoQuick solution was incubated with the supernatant at 4°C overnight. After the initial centrifugation (1500 g / 30 min), the supernatant was aspirated, and the residual solution was centrifuged (1500 g / 5 min). The exosome pellet was resuspended in PBS. The total protein concentration of the purified exosomes was measured using the Bradford assay (Bio-Rad Laboratories, France), and the purified exosomes were stored at -80°C until use.

[0103] Nanosite experiments have shown that the Exoquick formulation consists mainly of extracellular vescules, i.e., exosomes, within a size range of 80–120 nm (Addendum 1). Therefore, the term "exosome" is used in this paper.

[0104] Cell treatment with exosomes and / or miRs. In T0, 7,105 cells were seeded in a 12-well plate. After 1 day, the cells were co-incubated with T cell-derived exosomes (150 μg) and α-amanitin (50 ug / ml) (Sigma, France). Residual exosomes were removed by three cell washes with PBS solution. α-amanitin was used to block the transcription of putative miRNAs caused by the experimental conditions because it inhibits DNA-dependent RNA polymerase II activity.39 The amount of miRNA delivered by intracellular exosomes was estimated by the difference in Ct values ​​between α-amanitin-treated cells with and without exosomes using qRT-PCR.

[0105] Exosome loading using anti-miRNA. Exosomes were transfected with anti-miRNA using Exo-Fect Exosome Transfection reagent (Ozyme, France). Briefly, anti-miRNA was incubated with exosomes (300 μg of exosomal protein) in a shaker at 37°C for 15 minutes. After stopping the reaction by adding ExoQuick-TC solution, the mixture was incubated on ice for 30 minutes. After centrifugation, the transfected exosome pellet was resuspended in 300 μL of PBS before use. The total protein concentration of purified exosomes was measured using the Bradford assay (Bio-Rad Laboratories, France).

[0106] For cell transfection using mimic-miR, mimic-mutant-miR, and miR inhibitors (also called anti-miR), HiPerFect Transfection Reagents (Qiagen, France) were used. These reagents include mimic-miR-4315:5'CCGCUUCUGAGCUGGAC (Syn-hsa-miR-4315 miScript miRNA mimic), mimic-mutant-miR-4315:5'CCGAAAUCUGAGCUGGAC, and anti-miR-4315 (miScript miRNA Inhibitor and miScript Inhibitor Neg. Control) (Qiagen, France).

[0107] Plasma sample. Plasma was collected from GBM patients treated at the "Institut de Cancerologie de l'Ouest" (ICO, http: / / www.ico-cancer.fr). All patients from whom samples were collected provided signed informed consent. All collected samples and associated clinical information were registered in a database (N°DC-2018-3321) validated by the French Ministry of Research. The biological resources were stored at the "Centre de Ressources Biologiques-Tumorotheque" (Institut de Cancerologie de l'Ouest, Saint-Herblain, F44800, France).

[0108] Isolation of exosomal miRNAs from blood. From blood samples collected in K+EDTA tubes, 4-5 ml of plasma was separated by two centrifugations of 10 ml of whole blood (10 min / 1900 g / 4°C, 10 min / 16000 g / 4°C). 1 ml of plasma was processed for miRNA isolation using the ExomiRNeasy serum / plasma kit (Qiagen, France) according to the manufacturer's instructions.

[0109] miRNA RT-qPCR. RT-qPCR was performed on Rotor-Gene Q (Qiagen, France) using miScript II RT with miScriptHiSpec buffer, miScript SYBR Green PCR kits, and miScript Primer Assays (Qiagen, France). MiRNA quantification and purity were analyzed using Qubit (Thermo, France) and Agilent 2100 (Small RNA kit, Agilent, France), respectively, according to the manufacturer's instructions.

[0110] RT-qPCR analysis. RNA extraction was performed using the RNeasy Mini QIAcube Kit and QIAcube (Qiagen, France). RT-qPCR was performed using the QuantiTect Reverse Transcription Kit, Rotor-Gene SYBR Green PCR Kit, QuantiTect Primer Assays, and Rotor-Gene Q as real-time thermocycler (Qiagen, France). The 2-ΔΔCt relative quantification method was used with the reference gene RPLP0.

[0111] In vivo experiments. The experimental procedures using animals followed the Institutional Animal Care guidelines and the French National Committee of Ethics. Furthermore, all experiments were conducted in accordance with the regulations for animal experimentation at the "Plate-forme Animalerie" within the "Institut de Recherche en Sante de l'Universite de Nantes (IRS-UN)" and were approved by the French National Committee of Ethics. Cultured A549 cells were harvested by trypsin treatment, washed, and resuspended in physiological saline. The cell suspension was subcutaneously injected (sc) into the flanks of 7-8 week old mice (Janvier, France). Tumor volume, based on caliper measurements, was calculated using the modified ellipsoid formula (tumor volume = 1 / 2 (length × width 2)).

[0112] Statistical analysis and results Unless otherwise specified, data are representative values ​​of the mean and standard deviation calculated from three independent experiments. Significance of the difference between the mean ± standard deviation was calculated using the Student's t-test. Significance of the correlation between two parameters was calculated using the Pearson's test. A statistical significance criterion of P < 0.05 was used.

[0113] result T cell exosomes treated with anti-PD1 therapy reduced temozolomide-induced cell death via miR-4315. The effects of anti-PD1 antibody (αPD1) therapy on T cells were analyzed by exposing purified human T cells to αPD1 (data not shown). It has been previously demonstrated that anti-PD1 therapy promotes the transcriptional activity of FoxO1 in T lymphocytes.1 In this model, the transcriptional activity of FoxO1 was strongly increased in T cells treated with 1 μg / mL of αPD1 (p<0.0001) (data not shown). Therefore, following a predictive study performed with the miRGen.v3 program, the expression of five miRNAs regulated by FoxO1 (miR-101-5p, miR-612, miR-3671, miR-4315, miR-let7i) was analyzed. RT-qPCR confirmed the expression of these five miRNAs in T cells (Figure 1A), and it appeared that their expression was not altered by αPD1 treatment. Surprisingly, however, miR-4315 was expressed 10 times more in exosomes derived from T cells exposed to αPD1 (Exo / αPD1) than in exosomes derived from T cells exposed to IgG control (Exo) (Figure 1A).

[0114] Next, we analyzed the effects of exosomes produced by control or αPD1-treated T cells on the glioma cell line U172. RT-qPCR revealed that intracellular levels of miR-4315 in A172 glioblastoma cells increased in a dose-dependent manner in the presence of exosomes derived from T cells exposed to Exo / αPD1, α-amanitin, and IgG control (Exo) (Figure 1B). This observation suggests the possibility of uptake and transport of miR-4315 from Exo / αPD1 to tumor cells (α-amanitin was used to block de novo production of miR-4315).

[0115] Temozolomide (TMZ) is a standard chemotherapeutic agent used to kill glioblastoma (GBM) cells. Therefore, we investigated the effect of TMZ on A172 cells in the presence or absence of Exo / αPD1 by measuring its cytotoxicity and apoptosis-inducing ability through the detection of cleavage forms of PARP1 and Caspase-3 (apoptotic biomarkers). In this experiment, cells were exposed to the indicated exosomes for 48 hours before treatment with TMZ (50 μM, 72 hours). The results confirmed that Exo / αPD1 suppresses TMZ-induced apoptosis (data not shown). To determine the contribution of miR-4315 in this process, Exo / αPD1 was transfected with anti-miR-4315. The data showed that the presence of anti-miR-4315 significantly reduced TMZ resistance associated with the addition of Exo / αPD1. Overall, our data demonstrate that exosome miR-4315 limits chemotherapeutic-induced apoptosis. Furthermore, this effect is similar to that observed with miR-4315 alone (data not shown).

[0116] Exposure to anti-PD1 promotes chemotherapy-resistant phenotypes in several cancer cell types via the exomiR-4315 / Bim axis. Previous data showing that exosome miR-4315 limits apoptosis suggested that this miR might target pro-apoptotic proteins of the BCL2 family, as these proteins are central to apoptotic execution. The Target Scan Human website suggests that the pro-apoptotic protein Bim may be a target of miR-4315 (data not shown). Therefore, we focused our research on this protein. Although not an inactive mutant, mimics of miR-4315 downregulated Bim at the protein and mRNA levels (data not shown) and reduced luciferase activity associated with the 3'UTR / Bim plasmid in A172 (data not shown). Exo / αPD1 reduced Bim expression in A172 cells, and anti-miR-4315 significantly limited the Exo / αPD1-induced reduction in Bim expression. GW182-CLIP-qPCR revealed that in A172 cells treated with T cell-derived Exo / αPD1, miR-4315 co-precipitated with 3'UTR / Bim, but not with T cell-derived Exo (data not shown). Similarly, anti-miR-4315 reduced the levels of miR-4315 co-precipitated with 3'UTR / Bim and GW182, but anti-miR-Ctrl had no effect (data not shown).

[0117] Similar studies were conducted on A549 (lung cancer cell line), OV90 (ovarian cancer cell line), and MCF7 (breast cancer cell line), with or without the respective cancer chemotherapy regimens of oxaliplatin, cisplatin, and paclitaxel. As observed in the A172 glioma cell line, Exo / αPD1 reduced drug-induced cell death, and these effects were counteracted by anti-miR-4315 (Figures 2, 3A, and 3B). As expected, the suppression of cell death was associated with downregulation of Bim expression and reduced PARP and Caspase-3 cleavage (Figures 2, 3A, and 3B).

[0118] Here, we revealed that exosomes derived from T cells exposed to αPD1 reduce Bim expression via exomiR-4315. Furthermore, we demonstrated that incorporating miR-4315 into cancer cell lines suppresses apoptosis and improves resistance to chemotherapy.

[0119] In lung cancer patients treated with anti-PD1 therapy, longitudinal expression of exomiR-4315 is associated with a serum biomarker of Apotot resistance. To determine the clinical relevance of these observations, we examined exomiR-4315 expression and serum cytochrome c concentration in four lung cancer patients treated with αPD1 (data not shown). Serum cytochrome c concentration was selected as a biomarker for cell death.13 Serum concentrations of exomiR-4315 and cytochrome c were dynamically regulated during the anti-PD1 treatment period (data not shown). By comparing exomiR-4315 expression between two αPD1 administrations in patients, we noted an increase in exomiR-4315 expression in 10 out of 15 patients. Furthermore, we confirmed that exomiR-4315 and serum cytochrome c concentration changed antiparallel or mirror-image during the αPD1 administration period (data not shown). In addition, Pearson's correlation test revealed that longitudinal exomiR-4315 expression was inversely correlated with serum cytochrome c concentration in all patients included (data not shown). Overall, these data demonstrate that longitudinal expression of exomiR-4315 was inversely correlated with serum biomarkers of apoptosis resistance in lung cancer patients treated with αPD1.

[0120] ABT263 neutralizes anti-PD1 / exomiR-4315-induced chemotherapy resistance in an in vivo model of lung cancer. The dynamic and antiparallel expression of serum levels of exomiR-4315 and cytochrome c suggests that the effectiveness of cancer cell death-inducing therapies fluctuates through different stages of effectiveness and ineffectiveness, promoting cell death throughout the treatment. In light of the above data linking the ineffectiveness of cisplatin + αPD1 therapy with exomiR-4315-induced Bim down expression, we then investigated the effect of "Bim / BH3 mimetic drugs" such as ABT263 on cisplatin resistance in A549 cells exposed to Exo / αPD1. Next, A549 lung cancer cells were exposed to Exo / αPD1 and Exo before the addition of cisplatin (CIS, 5 μM) and / or ABT263 (15 μM). ABT263 abolished the resistance to CIS induced by Exo / αPD1 (Figure 4A).

[0121] Next, the efficacy of ABT263 was evaluated in xenograft mice inoculated with A549 (data not shown). As expected, CIS administration reduced the volume of A549-induced tumors (Figure 4B). Exo administration did not alter the effect of CIS on A549 tumors, in contrast to Exo / αPD1 inoculation, which neutralized the antitumor activity of CIS. Exo / αPD1 inoculation also confirmed a decrease in Bim expression (protein and mRNA levels) in tumors. ABT263 induced similar activity and suppressed the harmful effects of Exo / αPD1 on cancer cell resistance to CIS (Figure 4B). These results suggest that ABT263 can be used to neutralize resistance to cisplatin treatment induced by Exo / αPD1. Furthermore, a significant inverse correlation was observed between the therapeutic effect on tumors and serum cytochrome c levels (r=-0.9566; p=0.0028) (data not shown).

[0122] conclusion Recent advances in understanding the molecular mechanisms governing anti-PD1 resistance have made it possible to identify several major causes of this phenomenon (14-15): the evolution of the neoantigen landscape (16), the presence of JAK1 / 2 mutations (17), the presence of β2-microglobulin mutations (18), and the limited acquisition of memory capacity in CD8+ T cells (19-20). Recent papers have identified or explained signatures related to the molecular mechanisms governing anti-PD1 resistance, with Bertrand et al. (2017) demonstrating that TNFα blockade overcomes resistance to anti-PD1 in a mouse experimental melanoma model, suggesting that the use of anti-PD1 antibodies and anti-TNF antibodies could be therapeutic solutions to suppress the process of anti-PD1 resistance (21). By identifying the T cell-derived ExomiR-4315 expression / Bimdown-regulation axis as a "pan-cancer cascade" of events related to resistance to anti-PD1 therapy, this study completed a list of molecular mechanisms governing this phenomenon. Thus, our research is the first to reveal that the process of horizontal RNA transfer is important in the transmission of resistance to anti-PD1 therapy. In this horizontal RNA transfer (22), the donor cells are T cells exposed to anti-PD1 therapy, the recipient T cells are tumor cells, the exosomes are the means of transfer, and miR-4315 is the transferred biological information.

[0123] Over the past decade, miR research in the field of cancer has identified miRs as biomarkers for diagnosis, prognosis, and drug efficacy prediction, as well as therapeutic agents such as onco or tumor suppressor miRs (23-24). Amidst this diverse literature, the literature on miR-4315 appears to be scarce. However, it has been confirmed that miR-4315 expression is increased in cancerous tissue compared to non-cancerous tissue in breast cancer (25), decreased in colorectal cancer (26), and increased in primary lung adenocarcinoma tissue compared to non-cancerous tissue (27). Thus, our study is the first to correlate exomiR-4315 expression with a predictive biomarker value for predicting the anticancer drug efficacy of miR-4315.

[0124] It has already been reported in the literature that Bim expression levels can be used to predict the response to anti-PD-1 therapy in patients with metastatic melanoma (28-29). These papers state that measuring Bim levels in CD8+ T cells is a promising minimally invasive strategy for predicting the response to anti-PD-1 therapy. This study differs significantly from these findings in that 1) it includes T cells as well as CD8+ T cells, 2) Bim regulation occurs in tumor cells rather than immune cells, and 3) it is involved not only in T cells but also in intercellular communication between T cells and cancer cells. Furthermore, while Dronca et al. (2016) (29) did not point to a molecular cause, this study identified exosomal miRNA-4315 as the molecular cause of Bim expression regulation. Thus, our data focuses on Bim in tumor cells and not on Bim in T cells, and therefore does not overlap with data existing in the literature.

[0125] This paper opens up the possibility of detecting certain patients who do not respond to anti-PD-1 drugs or anti-PD-1 drugs in combination with chemotherapy (by monitoring exomiR-4315 levels).

[0126] References Throughout this application, various references describe the state of the art to which the present invention relates. The disclosures of these documents are incorporated into this disclosure by reference.

[0127] [Table 1]

[0128] [Table 2]

[0129] [Table 3]

[0130] Table 4

[0131] Table 5

Claims

1. An in vitro method for stratifying patients with cancer who are resistant to or at risk of developing resistance to anti-PD-1 antibody therapy, or who develop resistance to anti-PD-1 antibody therapy, comprising: i) determining the expression level of exosome miRNA-4315 in a sample obtained from the patient; ii) comparing the expression level with a predetermined reference value; and iii) stratifying the patient as one who is resistant to or at risk of developing resistance to anti-PD-1 antibody therapy, or who develops resistance to anti-PD-1 antibody therapy, and / or stratifying the patient as one who is not resistant to or at risk of developing resistance to anti-PD-1 antibody therapy, or who does not develop resistance to anti-PD-1 antibody therapy, if the expression level of exosome miRNA-4315 is lower than a predetermined reference value.

2. An in vitro method for stratifying patients with cancer who respond to or do not respond to anti-PD-1 antibody therapy, comprising: i) determining the expression level of exosome miRNA-4315 in a sample obtained from the patient; ii) comparing the expression level with a predetermined reference value; and iii) stratifying the patient as a patient who does not respond to anti-PD-1 antibody therapy if the expression level of exosome miRNA-4315 is better than the predetermined reference value, and / or stratifying the patient as a patient who responds to anti-PD-1 antibody therapy if the expression level of exosome miRNA-4315 is worse than the predetermined reference value.

3. The method according to claim 1 or 2, wherein the anti-PD-1 antibody therapy is nivolumab, pembrolizumab, or semiprimab.

4. The method according to claim 1 or 2, wherein the sample is blood, plasma, serum sample, T cell-derived exosomes, or cancer biopsy.

5. The method according to any one of claims 1 to 4, wherein a patient can receive standard chemotherapy simultaneously with, separately from, or sequentially with anti-PD-1 antibody therapy.

6. The method according to claim 5, wherein the standard chemotherapy is oxaliplatin, cisplatin, temozolomide, cyclophosphamide, doxorubicin, or paclitaxel.

7. A pharmaceutical composition for use in the treatment of cancer in patients stratified as having resistance to anti-PD-1 antibody therapy, or being at risk of developing resistance to anti-PD-1 antibody therapy, by the method of claim 1, comprising a BH3 mimetic agent.

8. A pharmaceutical composition for use in the treatment of cancer in patients stratified as unresponsive to anti-PD-1 antibody therapy by the method of claim 2, comprising a BH3 mimetic agent.

9. The pharmaceutical composition according to claim 7 or 8, wherein the BH3 mimetic agent is selected from the group consisting of ABT-737, venetoclax (ABT-199), and navitocrax (ABT-263).

10. The method according to claim 1 or 2, wherein the cancer is glioblastoma (GBM), lung cancer, breast cancer, or ovarian cancer.

11. The pharmaceutical composition according to claim 7 or 8, wherein the cancer is glioblastoma (GBM), lung cancer, breast cancer, or ovarian cancer.

12. A pharmaceutical composition according to claim 7 or 8, to be used in combination with standard chemotherapy, wherein the standard chemotherapy is oxaliplatin, cisplatin, temozolomide, cyclophosphamide, doxorubicin or paclitaxel, the BH3 mimetic is selected from the group consisting of ABT-737, venetoclax (ABT-199) and navitoclax (ABT-263), and the cancer is glioblastoma (GBM), lung cancer, breast cancer or ovarian cancer.

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