Method for determining the response of a malignant disease to immunotherapy

Description

Reference to previous applications

[0001] This patent application claims priority from German patent application DE 10 2017 125 780.2, the disclosure of which is hereby incorporated by reference. Sequence protocol

[0002] The application includes an electronic sequence listing in txt format according to WIPO ST.25 standard with 78 sequences as part of the description. Field of the invention

[0003] The invention relates, among other things, to predictive molecular diagnostic methods and predictive biomarkers, as well as their uses, which are used in oncology to predict the response of a malignant disease to immunotherapy. Furthermore, the invention relates to a kit for carrying out the specified methods. Background of the invention

[0004] Personalized medicine is based on the development of therapies tailored to patients with specific diseases. Immunotherapeutic treatments with so-called immune checkpoint inhibitors represent a promising approach in oncology, as they can demonstrate outstanding results even in advanced tumors. However, the challenge is that even patients with identical clinical features can respond differently to the same therapy or drug.

[0005] Predictive molecular diagnostic methods ("companion diagnostics" and "complementary diagnostics") and predictive biomarkers ("companion biomarkers" and "complementary biomarkers") can help predict how well a patient will respond to a specific treatment or drug. For example, it is known from DE 10 2016 005 947 B3 that a DNA methylation analysis of the PD-1Gene expression of cells of a malignant disease or of T lymphocytes interacting with the cells of the malignant disease allows a prediction of whether the patient will respond to immunotherapy with drugs that inhibit the PD-1 receptor or its ligands. Micevic et al. (Clinical Epigenetics 2017, 9:34) deals with aberrant DNA methylation in melanoma. Rieke et al. (Oncotarget 2016, 7:75379-75393) investigates a correlation between the methylation of various DNA repair genes and the expression of CD274 and CTLA4. Goltz et al. (Journal of the German Society of Dermatology 2017, 15, Suppl. 3:1-88) describes a relationship between promoter methylation of CD274 and PDCD1LG2 and response to immunotherapy targeting the PD-1 / PD-L1 axis.

[0006] To further improve the benefit-risk ratio of such immunotherapies, clinical decision-making needs to be supported by even more precise subtyping of patient groups. Additional or complementary predictive methods and biomarkers are needed to guide patient selection even more reliably in light of the diverse emerging treatment options. Summary of the invention

[0007] Against this background, the present invention provides, in a first aspect, a method for predicting a response of a malignant disease to an immunotherapy designed to inhibit a PD-1 immune checkpoint signaling pathway. The method is characterized in that at least a part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOSDNA methylation analysis of cells of the malignant disease and / or of immune cells interacting with the cells of the malignant disease is then carried out. The response of the malignant disease to immunotherapy is then predicted based on the result of the DNA methylation analysis, i.e., the presence, absence, and / or extent of DNA methylation of the immunoregulatory gene. The immunotherapy comprises an active ingredient that inhibits the PD-1 immune checkpoint signaling pathway by binding the active ingredient to PD-1, PD-L1, and / or PD-L2, wherein the active ingredient comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-PD-L2 antibody.

[0008] The second aspect of the invention relates to a method for selecting a patient suffering from a malignant disease for an immunotherapy designed to inhibit a PD-1 immune checkpoint signaling pathway. The method comprises the steps of A) providing cells of the malignant disease and / or immune cells of the patient that interact with the cells of the malignant disease, B) performing a DNA methylation analysis of at least a portion of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOSfrom the cells of the malignant disease and / or from immune cells interacting with the cells of the malignant disease from A), and C) selecting the patient for immunotherapy based on the presence, absence, and / or extent of the DNA methylation of the immunoregulatory gene examined or determined in B). The immunotherapy comprises an active ingredient that inhibits the PD-1 immune checkpoint signaling pathway by binding the active ingredient to PD-1, PD-L1, and / or PD-L2, wherein the active ingredient comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-PD-L2 antibody.

[0009] The third aspect of the invention relates to the use of a DNA methylation analysis of at least a part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOSof cells of a malignant disease and / or of immune cells interacting with the cells of the malignant disease to predict a response of the malignant disease to immunotherapy, for the individualized selection of an immunotherapy for the malignant disease and / or for the selection of a patient suffering from the malignant disease for immunotherapy, wherein the immunotherapy is in turn designed to inhibit a PD-1 immune checkpoint signaling pathway. The immunotherapy comprises an active ingredient that inhibits the PD-1 immune checkpoint signaling pathway by binding the active ingredient to PD-1, PD-L1 and / or PD-L2, wherein the active ingredient comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-PD-L2 antibody.

[0010] The fourth aspect of the invention relates to the use of the presence, absence or extent of DNA methylation of at least a part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS of cells of a malignant disease and / or of immune cells interacting with the cells of the malignant disease as a biomarker for predicting a response of the malignant disease to immunotherapy, for individualized selection of an immunotherapy for the malignant disease and / or for selecting a patient suffering from the malignant disease for immunotherapy, wherein the immunotherapy is designed to inhibit a PD-1 immune checkpoint signaling pathway. The immunotherapy comprises an active ingredient that inhibits the PD-1 immune checkpoint signaling pathway by binding of the active ingredient to PD-1, PD-L1 and / or PD-L2, wherein the active ingredient comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-PD-L2 antibody.

[0011] According to a fifth aspect, the invention relates to the use of a kit for carrying out one of the aforementioned methods. The kit comprises reagents for DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS of cells of a malignant disease and / or of immune cells interacting with the cells of the malignant disease to determine the presence, absence and / or extent of DNA methylation of the immunoregulatory gene. Definitions and general explanations

[0012] Various documents are cited in this description to provide a general technical background related to the present invention. The disclosure and teachings of these documents are incorporated by reference in their entirety in the following description to avoid repetition.

[0013] The following definitions and general explanations are intended to guide and assist the skilled reader in understanding, interpreting, and practicing the present invention. Unless otherwise specified, all technical and scientific terms shall have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0014] The various aspects and variants of the invention involve techniques and methods from routine molecular biology practice. In particular, DNA methylation analysis to determine the methylation of a CpG dinucleotide is within the expertise of a molecular biologist or geneticist. Convenient laboratory manuals for these techniques and methods are readily available to the skilled person, for example, "Molecular Cloning, A Laboratory Manual" by M.R. Green and J. Sambrook, 4th Ed., 2012, Cold Spring Harbor Laboratory Press.

[0015] As used here, indefinite articles such as "a" or "an" include the possibility that two or more of these features may be present.

[0016] As used here, "malignant diseases" or "malignant disorders" encompasses those diseases characterized by a progressively destructive disease course, potentially leading to the patient's death. Malignant disorders include malignant tissue formations, such as neoplasms or tumors, where malignancy may be characterized by uncontrolled, space-occupying, displacing, infiltrative, and / or invasive growth. Malignant tumors are generally capable of forming metastases. Examples of malignant tumors include carcinomas, sarcomas, melanomas, glioblastomas, blastomas, seminomas, and teratomas. Malignant disorders also include hematological malignancies, i.e., malignant diseases that affect the blood system or the blood-forming system, such as leukemias, lymphomas, myeloproliferative disorders, and myelodysplastic syndromes.Leukemias comprise a group of malignant diseases in which immature hematopoietic cells have undergone malignant transformation, proliferate excessively, and lead to an accumulation of cells in the peripheral blood. Lymphomas comprise diseases in which cells of the lymphatic system have degenerated. Myeloproliferative diseases comprise a group of diseases in which one or more blood-forming cell lineages are greatly increased. Myelodysplastic syndromes involve a clonal expansion of precursor cells of all blood-forming cell lineages, with a chronic disorder of differentiation of the blood-forming stem cells underlying them.

[0017] "Immunotherapy" or "immunotherapeutic treatment" is used here as a collective term for all treatment approaches intended to influence the activity of the immune system. Immunotherapy can be aimed at strengthening or weakening the effect of the immune system. In certain variants of the invention, immunotherapy comprises treatment with active substances to enhance the organism's own immune response against the malignant disease. Such active substances include immune checkpoint inhibitors, such as monoclonal antibodies, which specifically bind to immune checkpoints and thus prevent their (particularly anti-inflammatory) signaling. Another possibility are active substances that already inhibit the expression of the immune checkpoints, for example, through RNA interference.

[0018] Response to immunotherapy can be assessed according to the Immune-Related Response Evaluation Criteria In Solid Tumors (irRECIST) (Nishino et al., J Immunother Cancer, 2014, 2:17; Wolchok et al., Clin Cancer Res. 2009, 15:7412-20). Specifically, response to immunotherapy can be characterized by complete or partial remission, an unchanged (stable) condition, or a delay in one or more of the following events: death, recurrence, lymph node metastases, distant metastases, or progression of the malignant disease. Response to immunotherapy can also be characterized by a delayed increase or decrease in another parameter specific to the malignant disease.For example, a decrease or a delayed increase in prostate-specific antigen (PSA) in the blood can indicate the response of prostate cancer to immunotherapy. The lack of response can be characterized by an increasing or accelerated progression of the malignant disease. The extent of the malignant disease before therapy can serve as a comparison. The extent of the disease can be characterized by the number of malignant cells, the number of metastases, or the size of the malignant tumor.

[0019] As used herein, "inhibiting a PD-1 immune checkpoint pathway" or "inhibiting a CTLA4 immune checkpoint pathway" means slowing, inhibiting, or preventing one or more reactions of a chemical, biological, and / or physical nature that are mediated by an interaction of the PD-1 immune checkpoint with its ligands PD-L1 and / or PD-L2, or the CTLA4 immune checkpoint with its ligands CD86 and / or CD80, respectively.

[0020] "DNA methylation" refers to the biochemical or chemical coupling of methyl groups to specific DNA nucleotides. For the purposes of this invention, "DNA methylation" refers to the presence of a methyl group on the fifth carbon atom of a cytosine (5-methylcytosine) located in a CpG dinucleotide context. A "CpG dinucleotide" is a DNA motif that, in the generally accepted reading direction from 5' to 3', has the nucleoside sequence cytidine-phosphate-guanosine. Guanosine consists of the nucleic base guanine and the sugar β-D-ribose. Cytidine consists of the nucleic base cytosine and the sugar β-D-ribose.

[0021] A "DNA methylation analysis" within the meaning of the present invention therefore comprises the determination of such DNA methylation of one or more CpG dinucleotides from a specific sequence context. In various variants of the invention, "DNA methylation analysis" is understood to mean the determination of whether the cytosine in the CpG dinucleotide(s) is methylated. If a large number of genome copies are examined, the DNA methylation analysis can also be used to determine the extent of DNA methylation. For this purpose, for example, the average frequency of methylation of the cytosine in the CpG dinucleotide(s) in the large number of genome copies is determined, hereinafter also referred to as the "methylation degree."

[0022] "Co-methylation" refers to a correlation in DNA methylation between two or more CpG dinucleotides. Such correlated methylations regularly occur at CpG dinucleotides that are adjacent in the genome and / or that are located within neighboring, structurally and / or functionally related genes that can be expressed together. Therefore, the DNA methylation of one gene can be used to infer the DNA methylation of the other, co-methylated gene.

[0023] A "gene" within the meaning of the invention refers to a DNA segment that comprises regulatory, transcribed, and / or functional sequence regions and thus contains the basic information for the production of biologically active RNA. In particular, a gene also includes those elements, such as promoters, transcription factor binding sites, CpG islands, open chromatin, enhancers and silencers, and CTCF binding sites, which fulfill a regulatory function in the transcription of the gene.

[0024] The nomenclature for the designation of genes and their nucleotides is based on the recommendation of the Human Genome Organisation Gene Nomenclature Committee (HGNC) as of October 31, 2017. A gene strain, for example, is designated with italic Latin capital letters (e.g. CTLA4, CD86).The genes described here are publicly available via the "GenBank" of the National Institute of Health, USA, as of October 31, 2017 (Benson DA et al., Nucleic Acids Research, 2013, 41, D36-42).

[0025] When reference is made to specific DNA sequences (SEQ ID NOs) in the following description, this always includes sequence variants with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the specified DNA sequence. The sequence identity of two nucleic acid sequences can be determined, for example, using the ClustalW algorithm (Thompson et al., Nucleic Acids Research, 1994, 22, 4673-4680).

[0026] For the purposes of the present invention, "immune cells interacting with the cells of the malignant disease" encompasses those immune cells that are or can specifically contact cells of the malignant disease, for example, via a ligand-receptor bond. The ligand can be located on the surface of cells of the malignant disease, for example, an MHC:peptide complex or an MHC I:antigen complex, or an epitope. The receptor can be located on the surface of the immune cells, for example, a T-cell receptor or B-cell receptor. Alternatively, the ligand can be located on the surface of the immune cells and the receptor can be located on the surface of cells of the malignant disease."Immune cells interacting with the cells of the malignant disease" therefore also includes, for example, T lymphocytes and / or B lymphocytes that have been enabled (activated) through contact with an antigen or antigen-presenting cells to specifically interact with cells of the malignant disease via one of the aforementioned ligand-receptor bonds, without having previously come into contact with the corresponding cells of the malignant disease. An antigen-presenting cell can, for example, be a dendritic cell, a macrophage, or a B lymphocyte. Activation occurs, for example, via a ligand-receptor bond between T lymphocytes and an antigen-presenting cell, which presents an antigen originating from a cell of the malignant disease.The ligand, for example, an MHC II:antigen complex, is located on the surface of the antigen-presenting cells, and the T cell receptor is located on the surface of the T lymphocyte. Another form of interaction between T lymphocytes and cells of the malignant disease within the meaning of the invention involves the binding of adenosine produced by the cells of the malignant disease by a receptor on the surface of the T lymphocytes. It is also possible that the adenosine is produced by T lymphocytes and the receptor is located on the surface of cells of the malignant disease. Within the meaning of the present invention, "immune cells interacting with the cells of the malignant disease" further includes those immune cells, in particular T lymphocytes and / or B lymphocytes, that interact with the cells of the malignant disease via growth factors and / or cytokines.

[0027] "Biomarkers" are characteristic indicators and / or biological characteristics that can be objectively measured and allow conclusions to be drawn about the status of a normal biological or pathological process in an organism, or the response of a normal or pathological process to an intervention, such as surgery, radiation, or drug treatment. Biomarkers are often (bio)chemical substances, such as proteins, hormones, metabolites, sugars, and nucleic acids, as well as modifications thereof.

[0028] Both the foregoing general description and the following detailed description are to be considered as exemplary and explanatory of the invention as claimed. Other advantages and features of the invention will be apparent from the following description, drawings, and claims. While the invention has been described in terms of its preferred embodiments, many other variations may be made without departing from the scope of the present invention. Therefore, it is intended that the appended claims cover variations and combinations of features that are included within the true scope of the invention even if not expressly recited in the claims. Short Description of the characters

[0029] Figure 1 shows a boxplot of the distribution of DNA methylation of CTLA4in malignant melanoma patients with progressive disease, stable disease, partial remission, and complete remission during immunotherapy designed to inhibit the PD-1 immune checkpoint pathway. DNA methylation was determined prior to initiation of immunotherapy. Patients were retrospectively grouped according to irRECIST criteria. Figure 2 shows a Kaplan-Meier analysis of overall survival in 50 patients with malignant melanoma during immunotherapy designed to inhibit the PD-1 immune checkpoint pathway. Patients were evaluated in a three-stage assessment based on CTLA4 DNA methylation. The lower tertile includes the 17 patients with the lowest measured DNA methylation, the upper tertile the 17 patients with the highest measured DNA methylation, and the middle tertile the remaining 16 patients. Figure 3 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOSin 419 urothelial carcinomas of the bladder. Figure 4 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOS in 797 breast cancer tumors. Figure 5 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOS in 530 squamous cell carcinomas of the head and neck area. Figure 6 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOS in 325 clear cell renal cell carcinomas. Figure 7 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOS in 475 lung adenocarcinomas. Figure 8 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOS in 370 squamous cell carcinomas of the lung. Figure 9 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOS in 265 sarcomas. Figure 10 shows a co-methylation matrix of different gene loci of CTLA4, CD28, CD80, CD86 and ICOSin 473 cutaneous melanomas. Short description of the sequences

[0030] SEQ ID NO:1 to SEQ ID NO:78 as described under the numerical code <213> or <223> of the sequence listing as part of the description. Description of the invention

[0031] Immune checkpoints are key targets for immunotherapies. Immune checkpoint blockade (ICB) has proven particularly effective in the treatment of various malignant diseases. Immune checkpoint inhibitors disrupt the signaling pathways of immunosuppressive immune checkpoints, allowing the body's immune system to better recognize and combat the malignant cells. However, because immunotherapeutic drugs generally only work in certain patients and any effect is often only observed after several months, it is of utmost importance for clinical practice to have indications in advance of therapy as to whether a patient will respond to a particular treatment.

[0032] The inventor of the present invention has already been able to show in DE 10 2016 005 947 B3 that in cells of malignant diseases or in T lymphocytes interacting with the cells of the malignant disease, the DNA methylation of central immunoregulatory genes such as the gene coding for PD-1 PDCD1 directly correlates with the expression of the immune checkpoints encoded by these genes. The inventor has further recognized that, due to this connection, DNA methylation analysis of these immunoregulatory genes can be used to predict a patient's response to immunotherapy. Accordingly, for example, a response to a drug that inhibits the PD-1 receptor is more likely if the DNA methylation analysis of the corresponding PDCD-1gene of cells of the malignant disease or of T lymphocytes interacting with the cells of the malignant disease indicates that the PD-1 receptor is initially expressed by the cells.

[0033] In the context of intensive further research and complex clinical studies, the inventor has now been able to show that, surprisingly, DNA methylation of the immunoregulatory genes CTLA4, CD86, CD28, CD80 and ICOSindicates with particularly high reliability whether a malignant disease responds to immunotherapy that inhibits a PD-1 immune checkpoint signaling pathway. Reference is also made to the following exemplary embodiments in this regard. This new finding was not to be expected from a technical point of view because these genes encode immune checkpoints that are not directly related to the PD-1 immune checkpoint signaling pathway. Therefore, the inventor's new findings differ fundamentally from the disclosure and

[0034] Teaching of DE 10 2016 005 947 B3, which is limited to the fact that a DNA methylation analysis of an immunoregulatory gene is suitable for predicting the response to an immunotherapy directed against the immune checkpoint encoded by the same immunoregulatory gene.

[0035] Against this background, the first aspect of the present invention relates to a method for predicting a response of a malignant disease to an immunotherapy designed to inhibit a PD-1 immune checkpoint signaling pathway. At least a portion of an immunoregulatory gene of cells of the malignant disease and / or of immune cells interacting with the cells of the malignant disease is subjected to DNA methylation analysis in order to examine the immunoregulatory gene for DNA methylation. The immunoregulatory gene is selected from the group consisting of CTLA4, CD86, CD28, CD80, ICOS or any combination thereof. Based on this DNA methylation analysis, the response of the malignant disease to immunotherapy is then predicted based on the presence, absence, and / or extent of DNA methylation of the immunoregulatory gene.

[0036] A particular feature of the present invention is that DNA methylation analysis of the immunoregulatory genes according to the invention is universally suitable for predicting the response behavior of a wide variety of malignant diseases or tumor entities. This discovery by the inventor is consistent with the latest findings of the US Food and Drug Administration, which recently approved, for the first time, an immunotherapy for cancers based on a common genetic characteristic of the diseases, instead of defining approval based on the diseased organ, as was previously customary (Chang et al., Appl Immunohistochem Mol Morphol. 2017, Epub ahead of print). The malignant disease can include, in particular, melanoma, carcinoma, sarcoma, glioblastoma, lymphoma, and / or leukemia.The carcinoma may include, for example, adenocarcinoma, squamous cell carcinoma, small cell carcinoma, neuroendocrine carcinoma, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, anal carcinoma, bronchial carcinoma, endometrial carcinoma, cholangiocarcinoma, hepatocellular carcinoma, testicular carcinoma, colorectal carcinoma, head and neck carcinoma, esophageal carcinoma, gastric carcinoma, breast carcinoma, renal carcinoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, thyroid carcinoma and / or cervical carcinoma. A sarcoma can be, for example, an angiosarcoma, a chondrosarcoma, an Ewing's sarcoma, a fibrosarcoma, a Kaposi's sarcoma, a liposarcoma, a leiomyosarcoma, a malignant fibrous histiocytoma, a neurogenic sarcoma, an osteosarcoma or a rhabdomyosarcoma.A leukemia can be, for example, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML). A lymphoma can be Hodgkin's lymphoma or non-Hodgkin's lymphoma. A non-Hodgkin's lymphoma can be a B-cell lymphoma or a T-cell lymphoma. In particular, the malignant disease is a malignant melanoma, possibly metastatic.

[0037] The immunotherapy comprises an active agent that binds to PD-1, PD-L1, and / or PD-L2 and, through this binding, inhibits the PD-1 immune checkpoint pathway by disrupting the natural interaction between the PD-1 receptor and its ligands. The active agent comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-PD-L2 antibody. Preferably, this antibody is a monoclonal antibody. The active ingredient can, for example, consist of Nivolumab (BMS-936558, trade name: Opdivo, manufacturer: Bristol-Myers Squibb), Pembrolizumab (MK-3475, SCH900475, trade name: Keytruda ®< ; manufacturer: Merck / MSD Sharp & Dohme), Pidilizumab (CT-011, MDV9300; manufacturer: CureTech Ltd., licensed by Medivation), MGD013 (Macrogenics), AMP-224 (manufacturer: GlaxoSmithKline), MEDI0680 (AMP-514, manufacturer: MedImmune LLC), AUNP-12 (manufacturer: Aurigene Discovery Technologies Ltd.), BMS935559 (MDX-1105, manufacturer: Bristol-Myers Squibb), CA-170 and CA-237 (manufacturer: Curis Inc.), MPDL3280A (manufacturer: Roche), MEDI4736 (manufacturer: AstraZeneca), Avelumab (MSB0010718C, manufacturer: Pfizer) and / or rHIgM12B7 (B7-DC cross-linking antibody rHIgM12B7, Mayo Clinic), TSR-042 (manufacturer: Tesaro), SHR-1210 (manufacturer: Jiangsu Hengrui Medicine Co., Ltd.), Sym021 (manufacturer: Symphogen A / S), REGN2810 (manufacturer: Regeneron), JNJ-63723283 (manufacturer: JoJanssen Research & Development, LLC), BGB-A317 (manufacturer: BeiGene), PDR001 (manufacturer: Novartis), JTX-4014 (manufacturer: Jounce Therapeutics), Atezolizumab (MPDL3280A, manufacturer: Genentech / Roche), Durvalumab (MEDI4736, MEDI-4736, manufacturer: Medimmune / AstraZeneca), LY3300054 (manufacturer: Lilly), KN035 (manufacturer: Suzhou Alphamab Co. Ltd.), CX-072 (manufacturer: CytomX Therapeutics) and any combination thereof.

[0038] DNA methylation analysis can essentially be performed using all common methods known to the skilled person from the relevant literature. A suitable method comprises, for example, the following steps: A) Providing DNA from the cells of the malignant disease or from the immune cells interacting with the cells of the malignant disease; B) Converting at least a portion of the cytosines contained in the DNA from A) into uracil or another base with a base pairing behavior and / or molecular weight distinguishable from cytosine; C) Examining the DNA obtained from step B) for DNA methylation of the immunoregulatory gene.

[0039] The DNA to be analyzed in step A) can originate from various sources and include, for example, cells of the malignant disease or infiltrating immune cells, in particular tumor-infiltrating T lymphocytes, from tissue removed surgically or by biopsy. The (immune) cells can also originate from smears and aspirates such as lavage fluids, fine needle aspirates, or sputum. The DNA can also originate from blood, blood serum, and blood plasma, for example in the form of freely circulating DNA, exosomal DNA, or in the form of freely circulating cells of the malignant disease and / or peripheral immune cells from which the DNA is obtained. The DNA can also originate from other body fluids such as lymph fluid, urine, pleural effusions, or ascites, for example in the form of free DNA or in the form of cells of the malignant disease or immune cells from which the DNA is obtained.DNA can also be obtained from non-preserved (fresh) cells, tissues, and body fluids, as well as from fixed cells, tissues, and body fluids. The fixation of (immune) cells, tissues, and body fluids can be achieved using precipitating fixatives such as ethanol and other alcohols or cross-linking fixatives such as formaldehyde. This can be, for example, formalin-fixed and paraffin-embedded tissue (FFPET). The DNA can also come from any combination of these sources. It can also be extracted DNA from the aforementioned sources. It is also possible to enrich the DNA, for example, by precipitation or extraction. This can be useful, for example, for freely circulating DNA from the aforementioned body fluids.It is also possible to enrich the (immune) cells, for example, by size filtration or using magnetic particles bearing antibodies on their surface whose antigens are located on the surface of the (immune) cells to be enriched. This can be useful, for example, for freely circulating cells of the malignant disease or immune cells from the aforementioned body fluids. Other suitable sources for the DNA to be analyzed are homogenates of fresh tissue and lysates of fixed tissue. Compared to conventional prediction methods based on immunohistochemical methods or mRNA expression analysis, a particular advantage of the present invention is that DNA methylation delivers particularly robust and accurate prediction results even with preserved sample materials, minimal cell quantities, or completely cell-free DNA samples.

[0040] In a preferred variant, the DNA therefore comprises freely circulating DNA, DNA from exosomes, and / or DNA from freely circulating (immune) cells from a body fluid, so-called liquid biopsies. Liquid biopsies currently represent a central area of ​​oncology research. Instead of analyzing the suspicious tissue itself, such as tumor tissue, a sample of body fluid is analyzed, but rather a sample of body fluid, such as a blood sample or a lymph fluid sample. Various substances originating from the tumor can be examined in this sample, since freely circulating genomic DNA, exosomal DNA, or freely circulating cells or immune cells are released from the tumor into the bloodstream. These can also be immune cells originating from the thymus or a lymph node and capable of specifically interacting with cells of the malignant disease, but not originating from the tumor itself.The method according to the invention is advantageously used for analyzing liquid biopsies when the tumor or metastasis cannot be biopsied or when a biopsy would pose too great a risk to the patient in the late tumor stage. The present invention is characterized by the fact that DNA methylation of immunoregulatory genes can be easily measured in body fluids, whereas conventional detection of expression of immunoregulatory genes using mRNA or immunohistochemistry is difficult or even impossible.

[0041] The conversion of the DNA in step B) can, in principle, be carried out using any method known and suitable for this purpose in the prior art. Typically, this involves a chemical or enzymatic conversion, for example, by contacting the DNA with bisulfite, for example, sodium bisulfite or ammonium bisulfite.

[0042] If necessary, the DNA can be purified after the conversion in step B) and before the DNA methylation analysis in step C). Suitable purification methods and protocols are known to the person skilled in the art and may include, for example, DNA extraction, precipitation, or polymer-mediated enrichment. Reference is also made to the above explanations in this regard.

[0043] DNA methylation analysis determines the presence, absence, or extent of DNA methylation in the analyzed portion of the immunoregulatory gene. The analyzed portion therefore contains at least one CpG dinucleotide that is analyzed for DNA methylation, preferably several CpG dinucleotides that are analyzed for DNA methylation. The presence of DNA methylation therefore means that at least one methylated CpG dinucleotide is detected in the analyzed portion of the immunoregulatory gene. The absence of DNA methylation means that no methylation is detectable in any of the CpG dinucleotides contained in the portion. Determining the extent of DNA methylation of the immunoregulatory gene can involve analyzing several CpG dinucleotides contained in the analyzed portion for DNA methylation.Determining the extent of DNA methylation of the immunoregulatory gene may also involve examining the same CpG dinucleotide for DNA methylation in multiple copies of the immunoregulatory gene. Combinations of these variants are also possible.

[0044] The investigation of DNA methylation of the immunoregulatory gene in step C) is not subject to any particular restrictions. Suitable methods can easily be determined by a person skilled in the art on the basis of this disclosure. Reference is also made to the above-mentioned laboratory manuals in this regard. In a preferred variant, a polymerase chain reaction (PCR) is first performed using oligonucleotides, so-called primers, which are designed to amplify a section of the DNA converted in step B) that comprises the part of the immunoregulatory gene to be analyzed or the at least one CpG dinucleotide to be analyzed.Subsequently, at least a portion of the amplicon is preferably sequenced, for example, by Sanger sequencing, pyrosequencing, mass spectrometric sequencing, or second- or third-generation sequencing, which are also referred to as "massive parallel sequencing," "next-generation sequencing" (NGS), or nanopore sequencing. It is also possible to perform hybridization with methylation-specific oligonucleotides (probes) following the PCR, for example, in the form of a DNA microarray. DNA methylation can also be determined by quantitative real-time PCR (qPCR), optionally followed by melting curve analysis. In particular, quantitative real-time PCR can be performed with methylation-specific primers as in WO 1997 / 046705 A1 and / or methylation-specific blocker oligonucleotides as in WO 2002 / 072880 A2.In a preferred variant, methylation-specific detection probes are used.

[0045] In other preferred variants, PCR can be omitted, for example, with whole genome shotgun bisulfite sequencing (WGSBS) or direct nanopore sequencing. In WGSBS, the DNA is fragmented, and adapters are then ligated to the DNA fragments. The adapters then allow for amplification and sequencing. It is also possible to omit the fragmentation step in WGSBS, as the DNA may already be fragmented, for example, due to conversion through bisulfite treatment. Protocols for performing WGSBS are readily available to those skilled in the art (Johnson, MD et al., Curr. Protoc. Mol. Biol., 2012, 99, 21.23.1-21.23.28; Lister, R. et al., Nature, 2009, 462, 315-322; Berman, BP et al., Nat. Genet., 2011, 44, 40-46).

[0046] In another preferred variant, hybridization with specific oligonucleotides (probes) can be performed prior to PCR amplification. Upon binding, these probes are ligated and subsequently amplified by PCR. Suitable methods and protocols, such as multiplex ligation-dependent probe amplification (MLPA), are readily available to those skilled in the art, for example, in "PCR Mutation Detection Protocols" by BDM Theophilus and R. Rapley, 2nd Edition, 2011, Springer.

[0047] In another preferred variant, DNA methylation analysis is performed using the Infinium HumanMethylation450 BeadChip. Suitable protocols can be found, for example, in the chapter "Determination of DNA Methylation Levels Using Illumina HumanMethylation450 BeadChips" by MA Carless, which can be found in the book "Chromatin Protocols" by SP Chellappan, Volume 1288, 2015, of the book series "Methods in Molecular Biology," Springer Science+Business Media, New York. Further suitable protocols are evident from the following examples.

[0048] The inventor has discovered that the lower the degree of DNA methylation of the immunoregulatory gene, the more likely the malignant disease will respond to immunotherapy. Consequently, the response of the malignant disease to immunotherapy is particularly likely when DNA methylation of the immunoregulatory gene approaches zero, i.e., particularly when DNA methylation is actually absent or, due to technical detection limits, at least de facto absent. It is therefore advantageous if DNA methylation analysis is performed under conditions that allow a quantitative determination of DNA methylation of the immunoregulatory gene.For example, if the DNA methylation analysis includes copies of the immunoregulatory gene from multiple cells of the malignant disease and / or from multiple immune cells interacting with the cells of the malignant disease, a proportion of those copies of the immunoregulatory gene that exhibit DNA methylation can be determined. For this purpose, the number or quantity of methylated copies of the immunoregulatory gene can be compared to the total number or quantity of analyzed copies of the immunoregulatory gene. In this way, a response of the malignant disease to immunotherapy can be predicted even more accurately and, for example, is likely if less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, or less than or equal to 5% of the copies of the immunoregulatory gene exhibit DNA methylation.In still other variants, it is possible that the malignant disease is unlikely to respond to immunotherapy if more than 30%, more than 35%, more than 40% or more than 45% of the gene copies of the immunoregulatory gene have DNA methylation or in principle in the presence of DNA methylation.

[0049] The immune cells interacting with the cells of the malignant disease can be T lymphocytes, B lymphocytes, antigen-presenting cells, or natural killer cells (NKs). Combinations of these immune cells are also possible. In certain variants, the immune cells include tumor-infiltrating T lymphocytes and / or B lymphocytes, particularly tumor-infiltrating CD8+ lymphocytes and / or regulatory T lymphocytes (Tregs). However, they can also be peripheral and / or lymphatic T lymphocytes, B lymphocytes, antigen-presenting cells, and / or NKs.

[0050] The DNA methylation analysis can comprise one or more parts or one or more CpG dinucleotides within and in the vicinity of the immunoregulatory gene. Preferably, the DNA methylation analysis comprises at least part of a regulatory gene region, in particular a transcription factor binding site, a promoter, a CpG island, a silencer, an enhancer, or a CTCF binding site. The DNA methylation analysis can also comprise at least part of a sequence coding for a transcript of the immunoregulatory gene. Combinations of the aforementioned parts are also possible. Enhancers can, for example, be located away from the gene as distal enhancers. Enhancers can also be located close to the gene and are then called proximal enhancers. Regulatory gene regions are well known to the person skilled in the art and are described, for example, in "Gene Control" by DS Latchman, 2.Edition, 2015, Garyland Science, Taylor & Francis Group, LLC. For the method according to the invention, those parts or CpG dinucleotides whose methylation state correlates with the transcriptional activity or expression of the immunoregulatory gene are also suitable. Transcriptional activity can be detected, for example, by an altered chromatin structure. So-called "open chromatin" can be associated with high transcriptional activity of a gene, as described, for example, in "Genetik" by W. Janning and E. Kunst, 2004, Georg Thieme Verlag, Stuttgart and New York. Regions of "open chromatin" are therefore suitable for the DNA methylation analysis according to the invention.

[0051] The identification of regulatory gene elements is readily possible for the skilled person using suitable databases. For example, the "Ensembl" database annotates such regulatory elements, as described, for example, in "The Ensembl Regulatory Build" by DR Zerbino, SP Wilder, N. Johnson, T. Juettemann, and PR Flicek, 2015, Genome Biology, Issue 16, doi:10.1186 / s13059-015-0621-5.

[0052] A suitable primary sequence of the human genome, by means of which suitable and preferred regions and sequences of the immunoregulatory genes for the DNA methylation analysis according to the invention can be determined, is, for example, the human genome version of the Genome Reference Consortium Human Build 38 (GRCh38) or Reference Consortium Human Build 38 patch release 10 (GRCh38.p10) as of October 15, 2017. Regions of the genome are referred to below in the notation "chromosome number: position of the first base of the region - position of the last base of the region", for example "2:203583059-203583108" for the region from base 203583059 to base 203583108 of chromosome 2.

[0053] The DNA methylation analysis of the CTLA4 The gene preferably comprises at least part of one or more of the following regions: a region encoding the gene CTLA4 and the neighboring comethylated genes CD28and ICOS comprises (2:203551590-204126647, SEQ ID NO:1), a region encoding the transcripts (2:203867786-203873960), a promoter (2:203866174-203868926 and 2:203869477-203874095), an enhancer (2:203874152-203875266, 2:203876672-203878051 and 2:203879313-203881585), a region between CTLA4 and the neighboring immunoregulatory gene ICOS (2:203872383-203939876), an area between CTLA4 and the neighboring immunoregulatory gene CD28 (2:203738066-203867984), one or more regions selected from SEQ ID NO:51, SEQ ID NO:27, SEQ ID NO:48, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:50, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:30, SEQ ID NO:49, SEQ ID NO:31 and SEQ ID NO:52.

[0054] The DNA methylation analysis of the CD28 The gene preferably comprises at least a part of one or more of the following regions: a region which contains a coding sequence of CD28and comprises a promoter (2:203551590-203754454), a region containing the coding sequence (2:203706475-203738912) and a region containing a promoter (2:203677265-203707326).

[0055] The DNA methylation analysis of the ICOS The gene preferably comprises at least part of one or more of the following regions: a region encoding a transcript (2:203936748-203961577), a promoter (2:203934590-203941036 and 2:203948548-203953636), an enhancer (2:203931099-203937863 and 2:203940518-203949061), one or more regions selected from SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:34, SEQ ID NO:54, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:35 and SEQ ID NO:55.

[0056] The DNA methylation analysis of the CD86 The gene preferably comprises at least a part of one or more of the following regions: a region which contains a coding sequence of CD86and comprises a promoter (3:122039741-122154807, SEQ ID NO:3), a region encoding transcripts (3:122054701-122121475), a promoter (3:122054261-122061082, 3:122073513-122079893 and 3:122087703-122093314), an enhancer (3:122098924-122104974), one or more regions selected from SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:71, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:77.

[0057] The DNA methylation analysis of the CD80 The gene preferably comprises at least a part of one or more of the following regions: a region which contains a coding sequence of CD80and comprises a promoter (3:119523584-119573836, SEQ ID NO:2), a region encoding transcripts (3:119524293-119559602), a promoter (3:119554042-119563668 and 3:119568227-119573274), an enhancer (3:119563379-119568778, 3:119538188-119543511 and 3:119545840-119554325), one or more regions selected from SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:60, SEQ ID NO:59, SEQ ID NO:64, SEQ ID NO:58 and SEQ ID NO:63.

[0058] In a preferred variant, the DNA methylation analysis comprises at least a part of CTLA4. In a further preferred variant, the DNA methylation analysis comprises at least a part of CD86. In yet another preferred variant, the DNA methylation analysis comprises at least a part of CD28. In yet another preferred variant, the DNA methylation analysis comprises at least a part of CD80.In yet another preferred variant, the DNA methylation analysis comprises at least a part of ICOS. In particularly preferred variants, the DNA methylation analysis comprises at least a portion of at least two of the immunoregulatory genes. In this way, a reliable prediction of the response to the malignant disease is achieved, especially when only small amounts of DNA are available for the DNA methylation analysis. In this regard, reference is also made to the following embodiment 2. Preferably, the DNA methylation analysis comprises at least a portion of CTLA4 and at least part of at least one other of the immunoregulatory genes.

[0059] In addition to the aforementioned genes, DNA methylation analysis can also include other immunoregulatory genes in order to achieve even more precise patient subtyping by combining the results. In particular, DNA methylation analysis can also include at least some of PDCD1, CD274 and / or PDCD1LG2 which encode the immune checkpoints PD-1, PD-L1 and PD-L2. For example, DNA methylation analysis can detect at least a portion of CTLA4 and at least part of PDCD1, CD274 and / or PDCD1LG2 DNA methylation analysis may also include at least part of CTLA4 and at least part of PDCD1, CD274, PDCD1LG2, CD86, CD28, CD80 and / or ICOS include.

[0060] The DNA methylation analysis of the PDCD1The gene preferably comprises at least part of one or more of the following regions: a region encoding a transcript (2:241849881-241858908), a region with open chromatin (2:241849051-241853001 and 2:241861820-241862593), an enhancer (2:241852997-241855201), a CTCF binding site (2:241859081-241860074), a promoter (2:241856912-241861429 and 2:241862929-241865230).

[0061] The DNA methylation analysis of the CD274 The gene preferably comprises at least part of one or more of the following regions: a region encoding a transcript (9:5450503-5470566), a promoter (9:5445402-5456799 and 9:5458041-5461360), an enhancer (9:5457122-5457702, 9:5463574-5468340, 9:5440647-5441785 and 9:5472191-5473149), a CTCF binding site (9:5440970-5441435 and 9:5446325-5446870).

[0062] The DNA methylation analysis of the PDCD1LG2The gene preferably comprises at least part of one or more of the following regions: a region coding for a transcript (9:5510570-5571254), a promoter (9:5507688-5523442, 9:5491444-5503289, 9:5528150-5534251 and 9:5547972-5571492), an enhancer (9:5479110-5491616, 9:5522642-5528253, 9:5534822-5547690 and 9:5572730-5580962), a region upstream of the coding sequence (9:5496357-5510570).

[0063] The aforementioned features and preferred embodiments of the first aspect are also fully contained in the subsequent aspects of the invention and are merely not mentioned again in order to avoid repetition.

[0064] In personalized oncology, therapies are tailored to patients with specific cancer types. A high patient response rate to targeted immunotherapy with a PD-1 immune checkpoint inhibitor can only be achieved if patients with correspondingly sensitive diseases are reliably identified and specifically selected for treatment. In this sense, according to the second aspect of the invention, a method is provided by which a patient suffering from a malignant disease is selected for immunotherapy designed to inhibit a PD-1 immune checkpoint signaling pathway. The method comprises the following steps: A) Providing cells of the malignant disease and / or immune cells of the patient that interact with the cells of the malignant disease, B) Conducting a DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOSfrom the cells of the malignant disease and / or from immune cells interacting with the cells of the malignant disease from A), C) selecting the patient for immunotherapy based on the presence, absence and / or extent of DNA methylation of the immunoregulatory gene determined in B), wherein the immunotherapy comprises an active ingredient which inhibits the PD-1 immune checkpoint signaling pathway by binding of the active ingredient to PD-1, PD-L1 and / or PD-L2, wherein the active ingredient comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-PD-L2 antibody.

[0065] In particular, the patient will be selected if DNA methylation of the immunoregulatory gene indicates that the malignant disease is likely to respond to immunotherapy. In this regard, reference is made to the relevant comments on the first aspect.

[0066] According to a third aspect of the invention, a DNA methylation analysis of at least a part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOSof cells of a malignant disease and / or of immune cells interacting with the cells of the malignant disease can be used in various ways in personalized medicine. For example, a use is possible for predicting a response of the malignant disease to immunotherapy, a use for the individualized selection of an immunotherapy for the malignant disease, and / or a use for selecting a patient suffering from the malignant disease for immunotherapy if the immunotherapy is designed to inhibit a PD-1 immune checkpoint signaling pathway and comprises an active ingredient that inhibits the PD-1 immune checkpoint signaling pathway through binding of the active ingredient to PD-1, PD-L1 and / or PD-L2, wherein the active ingredient comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-PD-L2 antibody.

[0067] A fourth aspect of the invention relates to the use of the presence, absence or extent of DNA methylation of at least a part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS of cells of a malignant disease and / or of immune cells interacting with the cells of the malignant disease as a biomarker for predicting a response of the malignant disease to immunotherapy, for individualized selection of an immunotherapy for the malignant disease, and / or for selecting a patient suffering from the malignant disease for immunotherapy, wherein the immunotherapy is in each case designed to inhibit a PD-1 immune checkpoint signaling pathway and comprises an active ingredient which inhibits the PD-1 immune checkpoint signaling pathway by binding of the active ingredient to PD-1, PD-L1 and / or PD-L2, wherein the active ingredient comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-PD-L2 antibody.

[0068] In the aforementioned uses, the immunotherapy can also be additionally designed to inhibit a CTLA4 immune checkpoint signaling pathway. In his clinical studies, the inventor has recognized not only that DNA methylation analysis of the genes according to the invention also reliably indicates a likely response of the malignant disease to such CTLA4 immune checkpoint pathway-inhibiting immunotherapies, but also that the combination of PD-1 and CTLA4 immune checkpoint inhibition in the context of immunotherapy can be associated with particular treatment success.The invention is therefore unique in that it can provide the clinician with at least three treatment options using a single diagnostic test based on the presence, absence, or extent of DNA methylation of the immunoregulatory gene: selective inhibition of the PD-1 immune checkpoint pathway (e.g., as monotherapy), combined inhibition of the PD-1 and CTLA4 immune checkpoint pathways, and sequential inhibition, first of the PD-1 immune checkpoint pathway and then of the CTLA4 immune checkpoint pathway. The latter option may be appropriate, for example, if inhibition of the PD-1 immune checkpoint pathway causes severe side effects in a patient and therefore must be discontinued, or if PD-1 inhibition is no longer effective and the patient's disease progresses.

[0069] The immunotherapy designed to inhibit the CTLA4 immune checkpoint signaling pathway preferably comprises an active agent that inhibits the CTLA4 immune checkpoint signaling pathway by binding the active agent to CTLA4, CD80, CD86, or CD28. The immunotherapy or the active agent can, for example, comprise an anti-CTLA4 antibody, an anti-CD80 antibody, an anti-CD86 antibody, and / or an anti-CD28 antibody. In particular, these are monoclonal antibodies.

[0070] A fifth aspect of the invention relates to the use of a kit for carrying out the methods according to the first or second aspect. The kit comprises reagents for DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOSof cells of a malignant disease and / or immune cells interacting with the cells of the malignant disease to determine the presence, absence, and / or extent of DNA methylation of the immunoregulatory gene. The kit may also include instructions for predicting the response of the malignant disease to immunotherapy based on the presence, absence, and / or extent of DNA methylation of the immunoregulatory gene.

[0071] In particular, the kit contains at least a first oligonucleotide pair for DNA methylation analysis, which is designed to hybridize to a sequence of the immunoregulatory gene in DNA from the cells of the malignant disease and / or from the immune cells after cytosines contained in the DNA have been converted to uracil or another base with a base pairing behavior and / or molecular weight distinguishable from cytosine, in order to amplify and / or detect the sequence. At least one of the oligonucleotides can be designed to distinguish between converted methylated and converted unmethylated DNA, so that the sequence is amplified in a methylation-dependent manner. For this purpose, the oligonucleotide can be reverse-complementary to a binding sequence containing at least one CpG dinucleotide to be analyzed.For example, the oligonucleotide can be reverse complementary to the binding sequence if the cytosine in the CpG dinucleotide has been converted, meaning it was originally unmethylated. Alternatively, the oligonucleotide can be reverse complementary to the binding sequence if the cytosine in the CpG has not been converted, meaning it was originally methylated. This ensures that amplification only occurs if the sequence is methylated or unmethylated.

[0072] It is also possible for the oligonucleotides to be designed to amplify the sequence independently of DNA methylation. Preferably, the oligonucleotides are then reverse complementary to binding sequences that do not contain any CpG dinucleotides to be analyzed. Preferably, the CpG dinucleotides to be analyzed are located between the binding sequences of the oligonucleotides. The kit can additionally contain hybridization probes that distinguish between converted methylated sequence and converted unmethylated sequence, so that the amplified sequence is detected in a methylation-dependent manner. The extent of DNA methylation can then be determined from the signal ratio of the probes.

[0073] Furthermore, the kit can comprise at least a second oligonucleotide pair designed to hybridize to a sequence of the converted DNA that does not contain CpG dinucleotides in order to amplify and / or detect the sequence independently of methylation. Using an oligonucleotide pair designed in this way, it is possible, for example, to determine the total number or total amount of genome or gene copies, in particular the gene copies of the immunoregulatory gene, present in the converted DNA. In this way, according to the above statements regarding the first aspect, the relative proportion of those gene copies of the immunoregulatory gene that exhibit DNA methylation can be determined. In this regard, reference is also made to the first embodiment.

[0074] In preferred variants, the kit contains two or more first oligonucleotide pairs designed to bind to sequences of at least two different immunoregulatory genes selected from CTLA4, CD86, CD28, CD80 and / or ICOS to hybridize in the converted DNA in order to amplify and / or detect the sequences for DNA methylation analysis. The combined DNA methylation analysis of at least two of the immunoregulatory genes according to the invention allows a particularly robust measurement value and thus a particularly reliable prediction of the response to immunotherapy to be achieved. Reference is also made to the second embodiment in this regard.

[0075] Preferred regions and sequences of the immunoregulatory gene to be amplified and / or detected using the oligonucleotide pairs correspond to those of the first aspect.

[0076] The kit preferably comprises instructions for use for carrying out the method according to the first and / or second aspect and / or for use according to the third or fourth aspect.

[0077] All aforementioned aspects of the invention relate to deductive steps in connection with a preceding in vitro Process such that no technical step essential to the invention takes place on the human or animal body.

[0078] However, it is also possible in principle to incorporate the method according to the invention into a tailored treatment approach for a patient with a malignant disease in order to improve the probability of a therapeutic response. Consequently, the sixth aspect of the invention relates to a method for the immunotherapeutic treatment of a patient suffering from a malignant disease with an active ingredient designed to inhibit a PD-1 immune checkpoint signaling pathway. In the method, in step I) before and / or during the immunotherapeutic treatment, a DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOSof cells of the malignant disease and / or of immune cells interacting with the cells of the malignant disease, and the response of the malignant disease to the active ingredient is predicted based on the presence, absence, and / or extent of DNA methylation of the immunoregulatory gene. Subsequently, in step II), the active ingredient is administered if step I) indicates that the malignant disease is likely to respond to immunotherapeutic treatment with the active ingredient. However, if I) indicates that the malignant disease is unlikely to respond or no longer responds to immunotherapeutic treatment with the active ingredient, as an alternative to step II), the active ingredient is not administered, or administration of the active ingredient is stopped or reduced (step III). With regard to the likely response, reference is made to the explanations regarding the first aspect.In this way, the invention contributes to better typing of the malignant disease through diagnostic tests, thus enabling tailored treatment with an improved benefit-risk ratio.

[0079] During treatment, in step II) in addition to the active ingredient and / or in step III) instead of the active ingredient, another or different active ingredient designed to inhibit a CTLA4 immune checkpoint signaling pathway can be administered. As already explained in the fourth aspect, a particular advantage of the invention is that both complementary and alternative immunotherapeutic treatment options can be presented to the clinician using a single diagnostic test: selective inhibition of the PD-1 immune checkpoint signaling pathway or alternatively of the CTLA4 immune checkpoint signaling pathway, combined inhibition of the PD-1 and CTLA4 immune checkpoint signaling pathways, and sequential inhibition first of the PD-1 immune checkpoint signaling pathway and subsequently of the CTLA4 immune checkpoint signaling pathway. In this way, the clinician can exploit the treatment options for the malignant disease even more specifically and efficiently.

[0080] The additional or other active ingredient may, for example, comprise an anti-CTLA4 antibody, an anti-CD80 antibody, an anti-CD86 antibody, and / or an anti-CD28 antibody. In particular, it may be a monoclonal antibody. Detailed description of implementation examples

[0081] The invention is described in more detail below using exemplary embodiments and experimental results. These exemplary embodiments serve to illustrate the invention and are not intended to limit it to specific details. Example 1: Clinical study to predict the response of a malignant disease to inhibition of the PD-1 immune checkpoint pathway based on DNA methylation of CTLA4

[0082] The patient cohort studied included a total of 50 patients diagnosed with metastatic malignant melanoma. Before initiating immunotherapy, tumor tissue samples were collected, formalin-fixed, and paraffin-embedded. The patients were treated with anti-PD-1 immune checkpoint blockade using pembrolizumab or nivolumab between October 2014 and April 2017.

[0083] For DNA methylation analysis, 10 µm thin sections of the tumor tissue samples were first prepared and mounted on glass slides. Based on a HE section, tumor areas were identified by pathological examination and scraped from the glass slides with a scalpel for further processing. Bisulfite-converted DNA was prepared from the tumor areas using the innuCONVERT Bisulfite All-In-One Kit (Analytik Jena, Jena, Germany) according to the manufacturer's instructions. The total amount of converted DNA was then quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

[0084] In the next step, a DNA methylation analysis was carried out, for example, by removing part of the CTLA4Gene locus was amplified methylation-specifically using a quantitative real-time PCR and simultaneously quantified. For example, a duplex PCR was used, in which, in addition to DNA methylation of CTLA4 The total DNA, i.e. the total amount and / or total number of genome copies of the converted DNA, was also determined. The methylation-specific amplification of the CTLA4 Locus was performed using primers of the sequences SEQ ID NO:9 and SEQ ID NO:10. These primers amplify the sequence resulting from bisulfite conversion of the sequence SEQ ID NO:13. In the case of complete methylation, this converted region in the genome has the sequence SEQ ID NO:12. Methylation-specific detection was performed using a probe of the sequence SEQ ID NO:11, which carried the fluorescent dye 6-FAM at 5' and the quencher BHQ-1 at 3'. For the determination of total DNA, a locus in the ACTBThe gene was amplified independently of methylation. This locus has the sequence SEQ ID NO:8 in the genome and, after bisulfite conversion, the sequence SEQ ID NO:7. This sequence was amplified using primers with the sequences SEQ ID NO:4 and SEQ ID NO:5. Sequence-specific detection of the amplificate was performed using the probe with the sequence SEQ ID NO:6, which carried the fluorescent dye Atto 647N at the 5' and the quencher BHQ-2 at the 3'.

[0085] The real-time PCR was carried out in 20 µl PCR reactions in three independent measurements each, for example the following reaction composition was suitable: 35 mM Tris-HCl, pH 8.4, 6 mM MgCl 2 , 50 mM KCl, 4% glycerol, 0.25 mM each dNTP (dTTP, dATP, dGTP, dCTP), 2 U FastStart TaqDNA polymerase (Roche Applied Science, Penzberg, Germany), 0.4 µM of each primer, and 0.2 µM of each detection probe. The qPCR was performed using an AB 7500 Fast Real-Time PCR System (Life Technologies Corporation, Carlsbad, CA, USA). A suitable temperature profile included, for example, the following steps: 20 min at 95 °C, followed by 45 cycles of 45 s at 56 °C and 15 s at 95 °C.

[0086] The share of CTLA4 Methylation in the converted DNA was calculated using the DeltaDelta-CT method and expressed as a percentage compared to a standard DNA with 100% methylation. Artificially methylated DNA (CpGenome™ Universal Methylated DNA; Merck Millipore, Darmstadt, Germany) was used as the standard DNA, which had previously been converted using the innuCONVERT Bisulfite All-In-One Kit according to the manufacturer's instructions.

[0087] The response to immunotherapy was retrospectively assessed according to the criteria for the evaluation of immune-related response in solid tumors ( immune-related response evaluation criteria in solid tumors -irRECIST). For the survival analysis, death was considered the endpoint. Survival was defined as the period from the first administration of the immune checkpoint inhibitor until the time of death. A Kaplan-Meyer analysis with log-rank test was performed on the survival data. Hazard ratios were calculated using the univariate Cox proportional hazard model, and DNA methylation values ​​of CTLA4 logarithmized to the base 2. Comparisons were performed using one-way ANOVA and Bonferroni post-hoc tests. Categorical variables were tested using the chi-square test (χ 2< test). SPSS version 23.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

[0088] Figure 1 shows a boxplot evaluation of the relationship between the relative CTLA4-methylation (in %, y-axis) and the response of patients grouped according to the irRECIST criteria (x-axis). The mean CTLA4 Methylation was 46.1% (95% confidence interval CI: 31.2-61.0) in the patient group with progressive disease, 21.6% (95% CI: 11.2-31.9) in the group with stable disease, 7.6% (95% CI: 1.1-14.2) in the group with partial remission, and 4.9% in the group with complete remission. These results clearly show, with high statistical significance (p = 0.018), that low DNA methylation of the CTLA4 gene is associated with a response of the malignant disease to immunotherapy for inhibiting the PD-1 immune checkpoint pathway. Furthermore, the results show that the strength of the response also depends on the extent of DNA methylation or the methylation level of the CTLA4 gene. Consequently, the lower the level of the gene, the better the response. CTLA4gene is methylated.

[0089] Figure 2 In addition, a Kaplan-Meier analysis of the overall survival of 50 patients with malignant melanoma during immunotherapy is shown. The patients were classified according to a three-stage evaluation commonly used in pathological classification using CTLA4 -methylation tertiles. The analysis shows a highly significant (p = 0.002) prolonged survival of patients in the lowest CTLA4- Methylation tertile compared to the middle and highest CTLA4 -tertile. Of the 17 patients with the lowest CTLA4- Methylation in the tumor, which affects the lower CTLA4 -methylation tertile, more than 80% of the patients were still alive or censored 30 months after the start of immunotherapy. Of the 17 patients with the highest CTLA4 -Methylation in the tumor, which affects the upper CTLA4 -methylation tertile, as well as the 16 patients of the middle CTLA4-methylation tertile, less than 40% survived longer than 12 months after initiation of immunotherapy.

[0090] Thus, the inventor was able to show for the first time that a DNA methylation analysis of the immunoregulatory gene CTLA4 of cells of a malignant disease and / or of immune cells interacting with the cells of the malignant disease, allows a high degree of reliability in predicting the response of the malignant disease to immunotherapy designed to inhibit a PD-1 immune checkpoint pathway. Accordingly, it has also been shown that the presence, absence, or extent of DNA methylation of CTLA4 represents a reliable biomarker for predicting the response of the malignant disease to such immunotherapy. Example 2: Determination of co-methylation of CTLA4, CD86, CD28, CD80 and ICOS in various malignant diseases

[0091] Within the scope of the present invention, it was further recognized that the response of a patient to immunotherapy can be determined not only by DNA methylation analysis of CTLA4 but also by DNA methylation analysis of an immunoregulatory gene whose DNA methylation is correlated with the DNA methylation of CTLA4 The immunoregulatory genes proved to be particularly suitable for this purpose CD28, CD80, CD86 and / or ICOS. These genes are functionally related to CTLA4. In addition, CD28 and ICOS in the genome also in close proximity to CTLA4 localized.

[0092] The comethylation of CTLA4, CD28, CD80, CD86 and ICOSDNA methylation analysis was performed using a genome-wide DNA methylation analysis, for which the Infinium HumanMethylation450 BeadChip (Illumina, Inc., San Diego, CA, USA) was used according to the manufacturer's instructions. The HumanMethylation450 BeadChip raw data were generated as described by the TCGA Research Network. (http: / / cancergenome.nih.gov / ) are described. In total, raw data from 419 urothelial carcinomas of the bladder, 797 breast cancer tumors, 530 squamous cell carcinomas of the head and neck, 325 clear cell renal cell carcinomas, 475 adenocarcinomas of the lung, 370 squamous cell carcinomas of the lung, 265 sarcomas, and 473 cutaneous melanomas were used and retrospectively analyzed.

[0093] The DNA methylation analysis included different regions of the genes covered by the beads of the HumanMethylation450 BeadChip: SEQ ID NO:16 to SEQ ID NO:21 and SEQ ID NO:37 to SEQ ID NO:42 detect DNA methylation immediately upstream of the CD28Gen. SEQ ID NO:22 to SEQ ID NO:26 and SEQ ID NO:43 to SEQ ID NO:47 detect DNA methylation in the coding region of CD28. SEQ ID NO:27 to SEQ ID NO:29 and SEQ ID NO:48 detect DNA methylation in a region between CD28 and CTLA4. SEQ ID NO:30 to SEQ ID NO:33, SEQ ID NO:49 and SEQ ID NO:50 detect DNA methylation in the coding region of the CTLA4 Gens. SEQ ID NO:52 allows DNA methylation analysis of a region between CTLA4 and ICOS. SEQ ID NO:34 to SEQ ID NO:36 and SEQ ID NO:53 to SEQ ID NO:56 detect DNA methylation of a coding region of the ICOS Gens. SEQ ID NO:57 to SEQ ID NO:64 detect DNA methylation in the coding region and the promoter of the CD80 Gens. SEQ ID NO:65 to SEQ ID NO:77 detect DNA methylation in the coding region and the promoter of the CD86The sequence listing shows which beads of the HumanMethylation450 BeadChip were used for DNA methylation analysis of the respective sequences.

[0094] The co-methylation of these gene loci was compared with the DNA methylation of the sequence SEQ ID NO:51 (Bead cg08460026) of the CTLA4 This gene contains the same four CpG dinucleotides as SEQ ID NO:13, which were investigated in Example 1 using quantitative real-time PCR. This allows a direct correlation to the results presented in Example 1 for predicting the response of the malignant disease to immunotherapy.

[0095] First, a methylation value was calculated for each of the bead pairs considered and for each patient sample using the HumanMethylation450 BeadChip raw data. To do this, the signal of the bead in a pair that binds to the methylated variant (S_M) was compared to the signal of the bead in the pair that binds to the unmethylated DNA (S_U). A bead contains a bound oligonucleotide and is referred to here as a probe. DNA methylation was calculated based on the ratio using the equation: Methylation = (Probe Intensity S_M) / ((Probe Intensity S_M) + (Probe Intensity S_U)).

[0096] The Figures 3 to 10show the extent of co-methylation of the analyzed gene loci in the various types of malignant diseases. The gene loci are labeled vertically and horizontally consecutively from SEQ ID NO:16 (vertical: top, horizontal: left) to SEQ ID NO:77 (vertical: bottom, horizontal: right). SEQ ID NO:51 (black outline) includes SEQ ID NO:13, which was analyzed in Example 1 using quantitative real-time PCR to detect DNA methylation of the CTLA4 gene was examined.

[0097] The matrices show whether the DNA methylation of a particular sequence under investigation is correlated with the DNA methylation of the other sequences. A statistically significant correlation between the DNA methylation of two sequences, i.e., a p-value of the Spearman rank correlation of less than 0.05, is represented in the matrix as a gray box. White boxes indicate that there is no significant correlation (p ≥ 0.05) between the DNA methylation of the two corresponding sequences.

[0098] The results clearly show that all CD28, CD80, CD86 and ICOS analyzed gene regions exhibit DNA methylation that correlates significantly positively with the DNA methylation of CTLA4 Due to this co-methylation, the inventor was able to prove that analogous to CTLA4 also an association between the DNA methylation of the genes CD28, CD80, CD86 and ICOSand the response of the malignant disease to immunotherapy according to Example 1. Consequently, alternatively or in addition to DNA methylation analysis of CTLA4 - a DNA methylation analysis of one or more of the genes CD28, CD80, CD86 and ICOS predicting the response of a malignant disease to immunotherapy designed to inhibit a PD-1 immune checkpoint pathway.

[0099] The co-methylation described above results in the further particular advantage of the invention that the DNA methylation analyses of the genes CTLA4, CD28, CD80, CD86 and ICOScan also functionally influence each other. Since, for example, as shown above, low DNA methylation of one gene according to the invention correlates with low DNA methylation of another gene according to the invention, a combined DNA methylation analysis of both genes has the effect that both individual results complement each other to form a particularly robust overall result with regard to predicting the response of the malignant disease to immunotherapy. This mutual "consolidation effect" of the DNA methylation analysis of the genes according to the invention in predicting response is particularly advantageous for small sample quantities in which the available amount of DNA of an individual immunoregulatory gene is close to the lower detection limit of a DNA methylation analysis.The present invention thus solves the problem that in everyday clinical practice, often only small tissue or liquid biopsies with small amounts of DNA from cells of the malignant disease and / or immune cells are available, on the basis of which the response behavior of the malignant disease must be reliably predicted.

Claims

1. A method for predicting a response of a malignant disease to an immunotherapy configured to inhibit a PD-1 immune checkpoint signalling pathway, wherein a DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS is carried out on cells of the malignant disease and / or on immune cells interacting with cells of the malignant disease, and the response of the malignant disease to the immunotherapy is predicted on the basis of the presence, absence and / or level of DNA methylation of the immunoregulatory gene, wherein the immunotherapy comprises a pharmaceutical compound that inhibits the PD-1 immune checkpoint signalling pathway by binding of the pharmaceutical compound to PD-1, PD-L1 and / or PD-L2, wherein the pharmaceutical compound comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-PD-L2 antibody.

2. The method according to claim 1, wherein the DNA methylation analysis comprises at least part of at least two of the immunoregulatory genes.

3. The method according to any one of claims 1 or 2, wherein the immune cells interacting with cells of the malignant disease are selected from T-lymphocytes, B-lymphocytes, antigen-presenting cells and / or natural killer cells.

4. The method according to any one of claims 1 to 3, wherein the malignant disease comprises a melanoma, a carcinoma, in particular a squamous cell carcinoma or an adenocarcinoma, a leukaemia, a glioblastoma, a sarcoma and / or a lymphoma.

5. The method according to any one of claims 1 to 4, wherein the lower the level of DNA methylation of the immunoregulatory gene, the more likely the malignant disease is to respond to the immunotherapy, in particular wherein the absence of DNA methylation of the immunoregulatory gene indicates that the malignant disease is likely to respond to the immunotherapy.

6. The method according to any one of claims 1 to 5, wherein the DNA methylation analysis comprises gene copies of the immunoregulatory gene of several cells of the malignant disease and / or of several of the immune cells interacting with cells of the malignant disease, and wherein the malignant disease is likely to respond to the immunotherapy if less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10% or less than or equal to 5% of the gene copies of the immunoregulatory gene comprise the DNA methylation.

7. A method for selecting a subject suffering from a malignant disease for an immunotherapy configured to inhibit a PD-1 immune checkpoint signalling pathway, comprising A) providing cells of the malignant disease and / or immune cells interacting with cells of the malignant disease of the subject, B) performing a DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS of the cells of the malignant disease and / or of the immune cells interacting with cells of the malignant disease from A), C) selecting the subject for the immunotherapy on the basis of the presence, absence and / or level of a DNA methylation of the immunoregulatory gene determined in B), wherein the immunotherapy comprises a pharmaceutical compound that inhibits the PD-1 immune checkpoint signalling pathway by binding of the pharmaceutical compound to PD-1, PD-L1 and / or PD-L2, wherein the agent comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or anti-PD-L2 antibody.

8. Use of a DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS of cells of a malignant disease and / or of immune cells interacting with cells of the malignant disease a) for predicting a response of the malignant disease to an immunotherapy, b) for the individualised selection of an immunotherapy for the malignant disease, and / or c) for selecting a subject suffering from the malignant disease for an immunotherapy, wherein the immunotherapy is designed to inhibit a PD-1 immune checkpoint signalling pathway and comprises a pharmaceutical compound which inhibits the PD-1 immune checkpoint signalling pathway by binding of the pharmaceutical compound to PD-1, PD-L1 and / or PD-L2, wherein the pharmaceutical compound comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or anti-PD-L2 antibody.

9. Use of the presence, absence or level of DNA methylation of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS of cells of a malignant disease and / or of immune cells interacting with cells of the malignant disease as a biomarker i) for predicting a response of the malignant disease to an immunotherapy, ii) for the individualised selection of an immunotherapy for the malignant disease, and / or iii) for selecting a subject suffering from the malignant disease for an immunotherapy, wherein the immunotherapy is configured to inhibit a PD-1 immune checkpoint signalling pathway and comprises a pharmaceutical compound which inhibits the PD-1 immune checkpoint signalling pathway by binding of the pharmaceutical compound to PD-1, PD-L1 and / or PD-L2, wherein the pharmaceutical compound comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or anti-PD-L2 antibody.

10. The use according to any one of claims 8 or 9, wherein the immunotherapy is further configured to inhibit a CTLA4 immune checkpoint signalling pathway, in particular wherein the immunotherapy further comprises an anti-CTLA4 antibody, an anti-CD80 antibody, an anti-CD86 antibody and / or an anti-CD28 antibody.

11. Use of a kit for carrying out a method according to any one of claims 1 to 7, comprising reagents for DNA methylation analysis of at least part of an immunoregulatory gene selected from CTLA4, CD86, CD28, CD80 and / or ICOS of cells of a malignant disease and / or of immune cells interacting with cells of the malignant disease, to determine presence, absence and / or level of DNA methylation of the immunoregulatory gene.

12. The use according to claim 11, wherein the DNA methylation analysis comprises at least part of at least two of the immunoregulatory genes.