Method for diagnosing the efficacy of antitumor treatment

Abstract

The present invention relates to a method for predicting whether a subject having a tumor will respond to a tumor therapy selected from (i) immunotherapy, (ii) chemotherapy, (iii) antihormonal therapy, and (iv) anti-tyrosine kinase therapy, the method comprising: (A) determining the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from the subject, wherein the at least one nucleic acid molecule is selected from the group consisting of: (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOS: 1 to 6; (b) a nucleic acid molecule consisting of the nucleotide sequence of any one of SEQ ID NOS: 7 to 12; (c) a nucleic acid molecule encoding a polypeptide that is at least 85% identical, preferably at least 90% identical, and most preferably at least 95% identical to the amino acid sequence of (a); (d) a nucleic acid molecule consisting of a nucleotide sequence that is at least 95% identical, preferably at least 96% identical, and most preferably at least 98% identical to the nucleotide sequence of (b); (e) a nucleic acid molecule consisting of a nucleotide sequence that is degenerate to the nucleic acid molecule of (d); (f) determining a nucleic acid molecule consisting of a fragment of the nucleic acid molecule of any one of (a) to (e), wherein the fragment comprises at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides, and (g) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (f), wherein T is replaced by U, and wherein the at least one protein or peptide is selected from the protein or peptide encoded by the nucleic acid molecule of any one of (a) to (g);and (B) comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who responded to one or more of the therapies (i) to (iii), or with a corresponding predetermined standard, wherein an increase in the level of (A) compared to the level of (B) or the predetermined standard indicates that the subject is not responsive to the tumor therapy, and a decrease in the level of (A) compared to the level of (B) indicates that the subject is responsive to the tumor therapy; or (B') comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who did not respond to one or more of the therapies (i) to (iii), or with a corresponding predetermined standard, wherein a decrease in the level of (A) compared to the level of (B') or the predetermined standard indicates that the subject is responsive to the tumor therapy, and a decrease in the level of (A) compared to the level of (B') indicates that the subject is not responsive to the tumor therapy;

Description

[Technical Field]

[0001] The present invention relates to a method for predicting whether a subject having a tumor will respond to a tumor therapy selected from (i) immunotherapy, (ii) chemotherapy, (iii) antihormonal therapy, and (iv) anti-tyrosine kinase therapy, said method comprising: (A) determining the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from the subject, wherein the at least one nucleic acid molecule comprises: (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 6; (b) a nucleic acid molecule consisting of any one of the nucleotide sequences of SEQ ID NOs: 7 to 12; (c) a nucleic acid molecule encoding a polypeptide that is at least 85% identical, preferably at least 90% identical, and most preferably at least 95% identical to the amino acid sequence of (a); (d) a nucleic acid molecule consisting of a nucleotide sequence that is at least 95% identical, preferably at least 96% identical, and most preferably at least 98% identical to the nucleotide sequence of (b); (e) a nucleic acid molecule consisting of a nucleotide sequence degenerate to the nucleic acid molecule of (d); (f) a nucleic acid molecule consisting of a fragment of the nucleic acid molecule of any one of (a) to (e), wherein the fragment comprises at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides, and (g) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (f), wherein T is replaced by U, and wherein the at least one protein or peptide is selected from the protein or peptide encoded by the nucleic acid molecule of any one of (a) to (g); and (B) comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who responded to one or more of the therapies (i) to (iii), or with a corresponding predetermined standard, wherein an increase in the level of (A) compared to the level of (B) or the predetermined standard indicates that the subject is not responsive to the tumor therapy, and a decrease in the level of (A) compared to the level of (B) indicates that the subject is responsive to the tumor therapy; or (B') comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who did not respond to one or more of the therapies (i) to (iii), or with a corresponding predetermined standard, wherein a decrease in the level of (A) compared to the level of (B') or the predetermined standard indicates that the subject is responsive to the tumor therapy, and a decrease in the level of (A) compared to the level of (B') indicates that the subject is not responsive to the tumor therapy.

[0002] Many documents, including patent applications and manufacturer's manuals, have been cited herein. The disclosures of these documents, while not believed to be relevant to the patentability of this invention, are hereby incorporated by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] The human leukocyte antigen (HLA) system or complex is a complex of genes that encodes the human major histocompatibility complex (MHC) proteins. These cell surface proteins are responsible for regulating the human immune system. The HLA gene complex resides in a 3 Mbp region within chromosome 6p21. The genes in this complex are classified into three basic groups: class I, class II, and class III.

[0004] Humans have three major MHC class I genes, known as HLA-A, HLA-B, and HLA-C. The proteins produced by these genes are present on the surface of almost all cells. On the cell surface, these proteins bind to protein fragments (peptides) exported from inside the cell. MHC class I proteins present these peptides to the immune system. When the immune system recognizes foreign peptides (such as viral or bacterial peptides), it responds by triggering the self-destruction of infected cells.

[0005] There are six major MHC class II genes in humans: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. MHC class II genes provide instructions for making proteins that are found almost exclusively on the surface of certain immune system cells. Like MHC class I proteins, these proteins present peptides to the immune system.

[0006] The proteins produced by MHC class III genes have somewhat different functions, being involved in inflammation and other immune system activities. The functions of some MHC genes are unknown.

[0007] HLA genes have many possible variations, allowing each person's immune system to respond to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is given a specific number (e.g., HLA-B27). Closely related alleles are grouped together; for example, at least 40 very similar alleles are subtypes of HLA-B27. These subtypes are designated HLA-B*2701 through HLA-B*2743.

[0008] More than 100 diseases are associated with different alleles of HLA genes. For example, the HLA-B27 allele increases the risk of developing an inflammatory joint disease called ankylosing spondylitis. Many other diseases, including immune dysfunction and certain cancers, are also associated with specific HLA alleles. However, the role of HLA genes in the risk of developing these diseases is largely unknown.

[0009] Following the three major MHC class I genes, the non-classical MHC class I molecules HLA-E, HLA-F, and HLA-G are encoded by the HLA class I region. Overexpression of HLA-G, -E, and -F is a common finding across various malignancies (Kochan et al., Oncoimmunology. 2013 Nov 1; 2(11): e26491.). HLA-G and HLA-E are cancer biomarkers and have been reported to be positively correlated with poor clinical outcomes in cancer.

[0010] The HLA class I region has also been reported to contain class I pseudogenes and gene fragments (Hughes, Mol Biol Evol. 1995 Mar; 12(2):247-58). For example, HLA-H, J, and L are classified as class I pseudogenes, and HLA-N, S, and X are classified as gene fragments. In particular, Messer et al., J Immunol. 1992 Jun 15; 148(12):4043-53 reported that HLA-J is a pseudogene due to a deleterious mutation that causes translation termination in either exon 2 or exon 4. Therefore, human leukocyte antigen (HLA) genes have a long history of research as important targets in biomedical science, diagnosis, and therapy.

[0011] Furthermore, cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. Globally, approximately one in six deaths is due to cancer. Cancer incidence is currently increasing, particularly due to the increasing elderly population. Cancer mortality can be reduced if cases are detected and treated early. Without early diagnosis, patients are diagnosed at a later stage when curative treatment is no longer an option. However, even if cancer is diagnosed early, tumor heterogeneity often still complicates the identification of efficient treatments for specific patients. This is because bulk tumors can be a collection of diverse cells possessing different molecular signatures with varying levels of sensitivity to treatment. This heterogeneity can result in the uneven distribution of genetically distinct tumor cell subpopulations across and within disease sites (spatial heterogeneity) or temporal variation in the molecular composition of cancer cells (temporal heterogeneity). Heterogeneity provides the driving force for tumor resistance to specific treatment options. Therefore, there is an urgent need to predict in advance whether a tumor-bearing subject will respond to a particular tumor treatment, and there is also an urgent need for new tumor treatment methods. These needs are addressed by the present invention.

[0012] Thus, the present invention provides a method for treating a tumor-bearing subject, comprising: (i) immunotherapy; (ii) chemotherapy; (iii) antihormonal therapy, and (iv) Anti-tyrosine kinase therapy The present invention relates to a method for predicting whether a patient will respond to a tumor therapy selected from the group consisting of: (A) determining the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from the subject, wherein the at least one nucleic acid molecule comprises: (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 6; (b) a nucleic acid molecule consisting of any one of the nucleotide sequences of SEQ ID NOs: 7 to 12; (c) a nucleic acid molecule encoding a polypeptide that is at least 85% identical, preferably at least 90% identical, and most preferably at least 95% identical to the amino acid sequence of (a); (d) a nucleic acid molecule consisting of a nucleotide sequence that is at least 95% identical, preferably at least 96% identical, and most preferably at least 98% identical to the nucleotide sequence of (b); (e) a nucleic acid molecule consisting of a nucleotide sequence degenerate to the nucleic acid molecule of (d); (f) a nucleic acid molecule consisting of a fragment of the nucleic acid molecule according to any one of (a) to (e), wherein the fragment comprises at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides; and (g) A nucleic acid molecule corresponding to any one of (a) to (f), in which T is replaced with U. Selected from, and determining that at least one protein or peptide is selected from a protein or peptide encoded by the nucleic acid molecule of any one of (a) to (g); and (B) comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who responded to one or more of the therapies (i) through (iii), or with a corresponding predetermined standard, wherein an increase in the level of (A) compared to the level of (B) or the predetermined standard indicates that the subject is not responsive to the oncology therapy, and a decrease in the level of (A) compared to the level of (B) indicates that the subject is responsive to the oncology therapy; or (B') comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who have failed to respond to one or more of the therapies (i) through (iii), or with a corresponding predetermined standard, wherein a decrease in the level of (A) compared to the level of (B') or the predetermined standard indicates that the subject is responsive to the tumor therapy, and a decrease in the level of (A) compared to the level of (B') or the predetermined standard indicates that the subject is not responsive to the tumor therapy, and a decrease in the level of (A) compared to the level of (B') indicates that the subject is not responsive to the tumor therapy. Includes.

[0013] The term "subject" in relation to the present invention refers to a mammal, preferably a livestock or pet animal such as a horse, cow, pig, sheep, goat, dog or cat, most preferably a human.

[0014] A tumor is an abnormal benign or malignant new growth of tissue that has no physiological function and results from uncontrolled, usually rapid, cell proliferation. The tumor is preferably a cancer. Cancer is an abnormal malignant new growth of tissue that has no physiological function and results from uncontrolled, usually rapid, cell proliferation. The cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, cancer of the upper gastrointestinal tract, colon cancer, colorectal cancer, prostate cancer, squamous cell carcinoma of the head and neck, cervical cancer, glioblastoma, malignant ascites, lymphoma, and leukemia. Preferred cancers are defined herein below.

[0015] The tumor or cancer is preferably a solid tumor or cancer, which, in contrast to non-solid tumors (e.g., leukemia), is an abnormal mass of tissue that does not usually contain cysts or liquid areas.

[0016] Tumor treatments may also generally be, for example, surgery, but the tumor treatments herein are selected from (i) immunotherapy, (ii) chemotherapy, (iii) antihormonal therapy, and (iv) anti-tyrosine kinase therapy, among which immunotherapy is preferred.

[0017] Immunotherapy is the treatment of disease by activating or suppressing the immune system. Immunotherapy treats tumors, therefore immunotherapy is tumor immunotherapy, preferably cancer immunotherapy. Tumor immunotherapy is generally a term for artificial stimulation of the immune system to treat tumors, improving the system's natural tumor-fighting ability. Immunotherapy can be classified as active, passive, or hybrid (active and passive). Active immunotherapy instructs the immune system to target tumor antigens and attack tumor cells. Passive immunotherapy enhances existing anti-tumor responses and includes, for example, the use of monoclonal antibodies, lymphocytes, and cytokines.

[0018] The immunotherapy preferably involves the application of immune checkpoint inhibitors, and thus the immunotherapy is preferably immune checkpoint inhibitor therapy. Immune checkpoint inhibitors (also known simply as checkpoint inhibitors) are drugs that help the immune system respond more strongly to tumors. These drugs work, for example, by releasing the "brakes" that prevent T cells (a type of white blood cell and part of the immune system) from killing tumor cells. Such drugs do not directly target tumors; instead, they inhibit the ability of tumor cells to avoid the immune system's attack on them.

[0019] Thus, immune checkpoints affect immune system function. Immune checkpoints can be stimulatory or inhibitory. Tumors can use these checkpoints to protect themselves from immune system attack. Stimulatory checkpoint molecules include members of the tumor necrosis factor (TNF) receptor superfamily (CD27, CD40, OX40, GITR, and CD137) and molecules belonging to the B7-CD28 superfamily (CD28 itself and ICOS). Inhibitory checkpoint molecules include CD20, CD28, CD80, CD86, CD137, IDO1, LAG3, TIM3, TIM-4, TIGIT, BTLA, OX40, VISTA, B7-H7, CD27, GITR, CTLA4, and PD-1 and PD-L1. Most currently approved checkpoint therapies block inhibitory checkpoint receptors. Blocking negative feedback signaling to immune cells in this way enhances the immune response against tumors. Non-limiting, but preferred, examples of immune checkpoints and their inhibitors are provided and discussed herein below. Inhibition and / or activation of checkpoints can be achieved by affecting a single target or a combination of targets. By way of example and not limitation, this can be a combination of anti-CTLA4 and / or PD-1 and / or PD-L1. Furthermore, the effectiveness of checkpoint inhibitors can be improved by additional treatment with chemotherapy, and / or hormone and / or receptor tyrosine kinase inhibitors and / or DNA damage repair inhibitors.

[0020] Chemotherapy is a cancer treatment that uses drugs called cytostatics to stop tumor cells from dividing uncontrollably. Cytostatics are usually administered intravenously, but some are also available as pills. Chemotherapy can be curative (most often involving a combination of drugs), life-prolonging, or symptomatic (palliative chemotherapy). Cytostatics can act, for example, by inhibiting nucleic acid synthesis, damaging nucleic acids, altering microtubule proteins (spindle poisons), or damaging cell membranes. Chemotherapy is often combined with radiation therapy, a process called radiochemotherapy. The efficacy of chemotherapeutic agents lies in the release of tumor antigens through cell destruction, which are then presented to the immune system, ultimately increasing their recognition and potentially enhancing the effectiveness of immunotherapeutic agents such as immune checkpoint inhibitors or activators. The chemotherapy referred to herein can be adjuvant chemotherapy or neoadjuvant chemotherapy, and is preferably neoadjuvant chemotherapy. Chemotherapy uses drugs to destroy cancer cells, stop their growth, or ameliorate symptoms. Neoadjuvant chemotherapy (also called preoperative or primary chemotherapy) involves drug treatment before surgical removal of the tumor, as opposed to adjuvant chemotherapy, which involves drug treatment after surgery.

[0021] Antihormonal therapy is a treatment that inhibits the production or action of hormones. Antihormonal therapy is effective in tumor treatment because certain hormones can stimulate the growth of certain tumors. For example, endocrine therapy for breast and prostate cancer has long been established. Available treatments for blocking sex hormone receptor-mediated tumor growth are based on two principles: (i) ligand depletion, which can be achieved surgically, using luteinizing hormone-releasing hormone analogs or inhibitors of enzymes involved in steroid biosynthesis, or disrupting the feedback mechanism of sex hormone synthesis at the pituitary / hypothalamus level, and (ii) blocking sex hormone receptor function through the use of antihormones. For example, tamoxifen is used to treat breast cancer by inhibiting estrogen receptors on breast cancer cells. Furthermore, antihormonal and / or hormonal therapy also affect the immune system and antigen presentation, which may be important for immunomodulatory therapeutic strategies. The interaction of hormone activity / dependence and HLA factors is being investigated as part of this study.

[0022] Anti-tyrosine kinase therapy uses tyrosine kinase inhibitors (TKIs), pharmaceutical drugs that inhibit tyrosine kinases. Tyrosine kinases are enzymes responsible for the activation of many proteins through signal transduction cascades. Proteins are activated by adding a phosphate group to the protein (phosphorylation), a process that TKIs inhibit. TKIs are used as anti-cancer drugs. TKIs act by four different mechanisms: they can compete with adenosine triphosphate (ATP), a phosphorylated entity, a substrate, or both, or they can act allosterically; that is, they bind to a site outside the active site and affect its activity by changing its configuration. The interaction of receptor tyrosine kinases with HLA factors is explored as part of the present invention.

[0023] The nucleic acid sequences of SEQ ID NOs: 7 to 12 are the human HLA genes for membrane-bound HLA-G, HLA-L, soluble HLA-G, HLA-H, HLA-J, and HLA-L, respectively. Furthermore, the membrane-bound isoforms can be released by proteolytic activity, thereby increasing the soluble fractions of HLA-G and HLA-L. The nucleic acid molecules of the present invention are preferably genomic DNA or mRNA. In the case of mRNA, the nucleic acid molecule may further include a poly(A) tail.

[0024] Surprisingly, as discovered in accordance with the present invention and as shown in the following examples, HLA-G is expressed as a full-length transcript and a splice form containing only exons 1-5 of HLA-G. While full-length HLA-G contains a transmembrane domain and is therefore membrane-bound, soluble HLA-G lacks this transmembrane domain. Furthermore, the examples show that high levels of expression of mRNA encoding full-length HLA-G (i.e., as indicated, for example, by high levels of expression measured for exons 5 and 8 or only for exon 8) and high expression of mRNA encoding the soluble form (i.e., as indicated, for example, by high and low levels of exon 8 measured for exon 5 or only for exon 5) are associated with tumor patients who do not respond to tumor therapy as defined herein. As noted above, membrane-bound HLA isoforms can also be released by post-translational proteolytic cleavage, resulting in the release of soluble HLA fragments.

[0025] In addition, the gene encoding HLA-L contains a sequence encoding a transmembrane domain.Therefore, it is believed that HLA-L can also be found in tumors in its full-length membrane-bound form (SEQ ID NO: 2) as well as in its soluble form (SEQ ID NO: 8).Full-length HLA-L can also be released by post-translational proteolytic cleavage, resulting in the release of soluble HLA fragments.

[0026] On the other hand, the genes encoding HLA-H and HLA-J (SEQ ID NOs: 11 and 12) do not contain open reading frames encoding transmembrane domains. In the following examples, it is shown that HLA-H and HLA-J are soluble. The following examples also show that high expression of mRNA encoding such soluble HLA is associated with tumor patients who do not respond to tumor treatment as defined hereinabove.

[0027] SEQ ID NOs: 1 to 6 are the amino acid sequences of the human HLA genes HLA-G, HLA-L, soluble HLA-G, HLA-H, HLA-J, and HLA-L proteins, respectively.

[0028] According to the present invention, the term "nucleic acid sequence" or "nucleic acid molecule" includes DNA, such as cDNA, or double- or single-stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) refers to any chain or sequence of chemical building blocks, called nucleotide bases, adenine (A), guanine (G), cytosine (C), and thymine (T), linked together on a deoxyribose sugar backbone. DNA can have a single strand of nucleotide bases or two complementary strands that can form a double-helical structure. "RNA" (ribonucleic acid) refers to any chain or sequence of chemical building blocks, called nucleotide bases, adenine (A), guanine (G), cytosine (C), and uracil (U), linked together on a ribose sugar backbone. RNA typically has a single strand of nucleotide bases, such as mRNA. Also included are single- and double-stranded hybrid molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA. Nucleic acid molecules can also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps," substitution of one or more naturally occurring nucleotide analogs, and internucleotide modifications, such as those with uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules (hereinafter also referred to as polynucleotides) may contain one or more additional covalently linked moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. Polynucleotides can be derivatized by forming methyl or ethyl phosphotriester or alkyl phosphoramidate linkages. Additionally included are nucleic acid mimetic molecules known in the art, such as synthetic or semisynthetic derivatives and mixed polymers of DNA or RNA.Such nucleic acid mimic molecules or nucleic acid derivatives include phosphorothioate nucleic acids, phosphoramidate nucleic acids, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acids, hexitol nucleic acids (HNA), peptide nucleic acids (PNA), and locked nucleic acids (LNA) (see Braasch and Corey, Chem Biol 2001, 8:1). LNA is an RNA derivative in which the ribose ring is constrained by a methylene bond between the 2'-oxygen and the 4'-carbon. Also included are nucleic acids containing modified bases, such as thiouracil, thioguanine, and fluorouracil. Nucleic acid molecules typically carry genetic information, including information used by cellular machinery to make proteins and / or polypeptides. Nucleic acid molecules may further include promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5' and 3' non-coding regions, and the like.

[0029] The term "protein," when used interchangeably with the term "polypeptide" herein, refers to a linear molecular chain of amino acids, including a single-chain protein or fragment thereof, containing at least 50 amino acids. The term "peptide" as used herein describes a group of molecules consisting of up to 49 amino acids, while the term "polypeptide" (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids. The term "peptide" as used herein preferably refers to a group of molecules of at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, and at least 40 amino acids. The group of peptides and polypeptides is referred to together using the term "(poly)peptide." (Poly)peptides can also form oligomers consisting of at least two identical or different molecules. The corresponding higher-order structures of such multimers are correspondingly called homo- or heterodimers, homo- or heterotrimers, etc. For example, HLA proteins contain cysteines and therefore contain potential dimerization sites. Furthermore, peptidomimetics of such proteins / (poly)peptides, in which amino acids and / or peptide bonds are replaced by functional analogs, are also encompassed by the present invention.Such functional analogs include all known amino acids other than the 20 genetically encoded amino acids, such as selenocysteine.The terms "(poly)peptide" and "protein" also refer to naturally modified (poly)peptides and proteins, where modification is achieved, for example, by glycosylation, acetylation, phosphorylation, and similar modifications well known in the art.

[0030] According to the present invention, the term "percent (%) sequence identity" refers to the number of identical nucleotide / amino acid matches ("hits") in two or more aligned nucleic acid or amino acid sequences compared to the number of nucleotides or amino acid residues that make up the entire length of a template nucleic acid or amino acid sequence. In other terms, alignment can be used to determine the percentage of amino acid residues or nucleotides that are the same (e.g., 80%, 85%, 90%, or 95% identical) for two or more sequences or subsequences over a comparison window, or over a designated region as measured using sequence comparison algorithms known in the art, or when manually aligned and visually inspected, where (sub)sequences are compared and aligned for maximum correspondence. This definition also applies to the complement of any sequence being aligned.

[0031] Analysis and alignment of nucleotide and amino acid sequences relevant to the present invention is preferably performed using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402). BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST). Those skilled in the art will be aware of additional suitable programs for aligning nucleic acid sequences.

[0032] As defined herein, at least 85% identity, preferably at least 90% identity, and most preferably at least 95% identity are contemplated by the present invention, however the present invention also preferably contemplates sequence identities of at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identity.

[0033] The sample may be a bodily fluid of the subject or a tissue sample from a subject's organ. Non-limiting examples of bodily fluids include whole blood, plasma, serum, urine, peritoneal and pleural fluid, cerebrospinal fluid, tears, or cells in solution therefrom. Non-limiting examples of tissues include colon, liver, breast, ovary, and testis. Tissue samples can be collected by aspiration or puncture, excision, or any other surgical method that leads to biopsy or excised cellular material. The sample may also be a processed sample, such as a frozen, fixed, embedded, etc. sample. A preferred type of sample is a formalin-fixed, paraffin-embedded (FFPE) sample. Preparation of FFPE samples is standard medical practice, and these samples can be stored for long periods of time.

[0034] Means for obtaining levels of nucleic acid molecules or proteins or peptides are established in the art.

[0035] For example, the level of a nucleic acid molecule can be obtained by real-time quantitative PCR (RT-qPCR), electrophoresis techniques, or DNA microarray (Roth (2002), Curr. Issues Mol. Biol., 4: 93-100), with RT-qPCR being preferred. In these methods, the expression level can be normalized to the (average) expression level of one or more reference genes in the sample. The term "reference gene" as used herein refers to a gene that has a relatively invariant level of expression at the RNA transcript / mRNA level in the system being examined, i.e., tumor. Such genes are sometimes called housekeeping genes. Non-limiting examples of reference genes are CALM2, B2M, RPL37A, GUSB, HPRT1, and GAPDH, preferably CALM2 and / or B2M. Other suitable reference genes are known to those skilled in the art.

[0036] RT-qPCR is demonstrated by example. RT-qPCR is performed in a thermal cycler, irradiating each sample with light of at least one specific wavelength and detecting the fluorescence emitted by the excited fluorophore. Thermal cyclers can also rapidly heat and cool samples, thereby taking advantage of the physicochemical properties of nucleic acids and DNA polymerase. Two common methods for detecting PCR products in real-time qPCR are (1) nonspecific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides labeled with a fluorescent reporter that allow detection only after hybridization with its complementary sequence (e.g., TaqMan probes). The probes are generally fluorescently labeled probes. Preferably, fluorescently labeled probes consist of oligonucleotides labeled with both a fluorescent reporter dye and a quencher dye (= dual-labeled probe). Suitable fluorescent reporter and quencher dyes / moieties are known to those of skill in the art and include, but are not limited to, reporter dye / moieties 6-FAM™, JOET™, Cy5®, Cy3®, and quencher dye / moieties Dabcyl, TAMRA™, BHQ™-1, -2, or -3. Preferably, primers for use in accordance with the present invention have a length of 15-30 nucleotides, particularly deoxyribonucleotides. In one embodiment, primers (1) are specific for or derived from a target mRNA sequence of an HLA gene, (2) provide an amplicon size of less than 120 bp (preferably less than 100 bp), (3) are mRNA-specific (exon / intron consideration; preferably no amplification of genomic DNA), (4) are not prone to dimerization, and / or (5) have a temperature range of 58°C to 62°C (preferably T m The melting point T m As mentioned above, a probe is required for RT-qPCR using (2), but in the case of RT-qPCR using (1), it can be replaced with an intercalator dye such as SYBR green.

[0037] As an alternative to qPCR, electrophoretic techniques, or as a further alternative, DNA microarrays, can be used to obtain levels of nucleic acid molecules according to the first aspect of the present invention. The traditional approach for identifying and quantifying mRNA relies on a combination of gel electrophoresis, which provides information about size and sequence-specific probing. Northern blots are the most commonly applied technique in this latter class. Ribonuclease protection assays (RPA) have been developed as a more sensitive and less labor-intensive alternative to Northern blots. Hybridization is performed using a labeled ribonucleotide probe in solution, after which the unhybridized sample and probe are digested with a mixture of ribonucleases (e.g., RNase A and RNase T1) that selectively degrade single-stranded RNA. Subsequent denaturing polyacrylamide gel electrophoresis provides a means for quantification and also yields the size of the region hybridized by the probe. For both Northern blots and RPA, the accuracy and precision of quantification are a function of the detection method and the label or standard used. Most commonly, probes are radiolabeled with 32P or 33P. In this case, the final gel is exposed to X-ray film or a phosphor screen, and the intensity of each band is quantified with a densitometer or a phosphor imager, respectively. In both cases, exposure times can be adjusted to suit the required sensitivity, but fluorescent-based techniques generally offer greater sensitivity and a greater dynamic range. Instead of using radioactivity, probes can be labeled with antigens or haptens, followed by binding with horseradish peroxidase- or alkaline phosphatase-conjugated antibodies, and quantified by chemiluminescence on film or a phosphor imager after the addition of substrate. In all of these imaging applications, background subtraction from adjacent areas of the gel without probe should be performed. A major advantage of the gel format is that any reference standard can be imaged simultaneously with the samples. Similarly, detection of housekeeping genes is performed under the same conditions for all samples.

[0038] Additionally, next-generation sequencing (NGS) can be used (Behjati and Tarpey, Arch Dis Child Educ Pract Ed. 2013 Dec; 98(6):236). NGS is an RNA or DNA sequencing technology that has revolutionized genomic research. With NGS, the entire human genome can be sequenced within a day. In contrast, the previous Sanger sequencing technology used to decipher the human genome took more than 10 years to deliver a final draft. In light of the present invention, NGS can be used to quantify in an open configuration (genome-wide exome sequencing) or as a focused panel with each of the HLA genes and isoforms disclosed in this application.

[0039] Two techniques have emerged for constructing DNA microarrays. Generally, the starting point in each case for designing an array is a set of sequences corresponding to the genes or putative genes to be probed. In the first approach, oligonucleotide probes are chemically synthesized on a glass substrate. Due to the variable efficiency of oligonucleotide hybridization to cDNA probes, multiple oligonucleotide probes are synthesized complementary to each gene of interest. In addition, for each perfectly complementary oligonucleotide on the array, an oligonucleotide with a mismatch at a single nucleotide position is constructed and used for normalization. Oligonucleotide arrays are typically constructed with approximately 10 4 -10 6 probes / cm 2 DNA microarrays are routinely produced at densities of 1000 kJ / s. The second major technology for constructing DNA microarrays is the direct robotic printing of cDNA probes onto glass slides or other suitable substrates. DNA clones are obtained for each gene of interest, purified, and amplified from a common vector by PCR using universal primers. The probes are robotically deposited in spots on the order of 50-200 μm in size. At this spacing, for example, approximately 10 3 probes / cm 2 A density of 0.1% can be achieved.

[0040] The level of a protein or peptide can be determined, for example, by using a "molecule that binds to a protein or peptide," and preferably a "molecule that specifically binds to a protein or peptide." A molecule that binds to a protein or peptide refers to a molecule that primarily binds to a protein or peptide under known conditions. A "molecule that binds to a protein or peptide" is one of the binding molecules described herein below, preferably an inhibitor of the protein or peptide, such as an antibody, an aptamer, etc. The level of a protein or peptide can also be obtained by using Western blot analysis, mass spectrometry, FACS analysis, ELISA, and immunohistochemistry. These techniques are non-limiting examples of means that can be used to qualitatively, semi-quantitatively, and / or quantitatively detect a protein or peptide.

[0041] Western blot analysis is a common and well-known analytical technique used to detect specific proteins or peptides in a given sample, such as a tissue homogenate or body extract. It uses gel electrophoresis to separate native or denatured proteins or peptides by (poly)peptide length (denaturing conditions) or by the protein's 3-D structure (native / non-denaturing conditions). The proteins or peptides are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) with antibodies specific to the target protein.

[0042] Mass spectrometry (MS) analysis is a widespread and well-known analytical technique in which the mass-to-charge ratio of charged particles is measured. Mass spectrometry is used to determine the mass of particles, determine the elemental composition of samples or molecules, and elucidate the chemical structure of molecules such as proteins, peptides, and other compounds. The MS principle involves ionizing chemical compounds to produce charged molecules or molecular fragments and measuring their mass-to-charge ratios.

[0043] Fluorescence-activated cell sorting (FACS) analysis is a popular and well-known analytical technique in which biological cells are sorted based on the specific light scattering of each cell's fluorescent properties. Cells can be fixed in 4% formaldehyde, permeabilized with 0.2% Triton-X-100, and incubated with fluorophore-labeled antibodies (e.g., monoclonal or polyclonal anti-HLA antibodies).

[0044] Enzyme-linked immunosorbent assay (ELISA) is a popular and well-known sensitive analytical technique in which an enzyme is conjugated to an antibody or antigen as a marker for the detection of a specific protein or peptide.

[0045] Immunohistochemistry (IHC) is a common application of immunostaining. It involves selectively identifying antigens (proteins) in cells of tissue sections by utilizing the principle of antibodies specifically binding to antigens in biological tissues. In combination with specific devices, IHC can be used for quantitative in situ evaluation of protein expression (for a review, see Kreger et al. (2006) Arch Pathol Lab Med, 130:1026-1030). Quantitative IHC utilizes the correlation of staining intensity with absolute protein levels.

[0046] Methods for determining whether a subject has responded to one or more tumor treatments, and methods for determining whether a subject has responded to one or more tumor treatments, are known in the art. Generally, a tumor patient responds to treatment if the tumor shrinks (in the case of solid tumors), the number of tumor cells in non-solid tumors decreases (such as blood cancers), or the symptoms caused by neoplastic disease decrease or remain the same ("stabilization"). Generally, a tumor patient does not respond if the tumor worsens (increases in size, increases in cell number, or the tumor-related disease worsens, etc.) during treatment. In relation to response, tumor shrinkage is preferred.

[0047] Definitive evidence of the efficacy of a treatment is improvement in clinical symptoms and survival rate, whereas definitive evidence of the ineffectiveness of a treatment is worsening of clinical symptoms and ultimately death of the subject. As part of this invention, disease-specific survival is often used, which is defined by the onset of the treatment option under investigation until cancer-specific death. Imaging, especially in the case of tumor lesions, is commonly used to assess treatment efficacy earlier. At present, efficacy assessment is primarily based on changes in tumor size measured by CT (computed tomography) or other anatomical imaging methods, where a reduction in tumor size indicates a response. Also, PET (positron emission tomography) and glucose analogs are used to assess the efficacy of treatment. 18 Imaging of tumor metabolism with F-FDG is an attractive technique for objectively and quantitatively assessing the efficacy of treatment.

[0048] For the evaluation of solid tumors, the Response Evaluation Criteria in Solid Tumors (RECIST) is preferred. RECIST is a set of rules that defines when a patient's tumor improves, remains stable, or worsens during treatment. The criteria were published in February 2000 by an international collaboration including the European Organization for Research and Treatment of Cancer (EORTC), the National Cancer Institute of the United States, and the National Cancer Institute of Canada Clinical Trials Group. Currently, the majority of clinical trials evaluating cancer treatments aimed at objective response in solid tumors use RECIST. These criteria were updated in 2009. For the evaluation of solid tumors, the PET Response Criteria in Solid Tumors (PERCIST) is also preferred. PERCIST is an alternative set of rules that uses positron emission tomography (PET) to define when a patient's tumor improves, remains stable, or worsens during treatment. These criteria were established in 2009.

[0049] More preferably, one or more subjects each responded or did not respond, such as at least 2, at least 5, at least 10, at least 25, and at least 50. Examining multiple subjects has the advantage of biasing the level of difference between each patient in responding or non-responding.

[0050] The predetermined criteria specify values ​​previously obtained from one or more subjects who have responded to one or more oncology treatments or from one or more subjects who have not responded to one or more oncology treatments.

[0051] The increased levels of (B) and (B') are more preferably at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, or 4-fold increased compared to the level of (A). The decreased levels of (B) and (B') are more preferably at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, or 4-fold decreased compared to the level of (A). The substantially same levels of (B) and (B') are preferably less than 10%, more preferably less than 5%, different (i.e., higher or lower) from the control or predetermined standard. For example, if the level of (A) is set to 100%, the substantially same levels may be between less than 110% and more than 90% of the 100% control level.

[0052] The following examples of this specification surprisingly revealed that in patients with bladder cancer receiving immune checkpoint therapy (anti-PD-1 or anti-PDL-1), high expression levels of membrane-bound HLA-G (exon 8 probe), soluble or membrane-bound HLA-G (exon 3 probe), membrane-bound HLA-L (exon 7 probe), soluble HLA-H (exon 2 / 3 probe), and soluble HLA-J (exon 4 / 5 probe) were inversely associated with patient survival. The higher the expression levels of these HLA genes, the higher the patient's chance of dying from cancer within two years. It is important to take into account that post-transcriptional events may affect membrane-bound HLA isoforms. Therefore, the determination of membrane-bound HLA-G mRNA isoforms measured by quantification of exon 8 may ultimately result in biologically active soluble fragments after proteolytic cleavage events following translation into the protein structure. However, HLA mRNA expression levels were measured in tumor tissue samples obtained from bladder cancer patients before they began immune checkpoint therapy. Thus, the data of the Examples demonstrate that expression levels of HLA-G, L, H, and J genes or proteins can be used to predict whether a subject is likely to benefit from treatment before immune checkpoint therapy is initiated. Low expression levels are associated with superior disease-specific survival, while high expression levels are associated with inferior disease-specific survival.

[0053] The predictive value of HLA-G, L, H and J expression levels shown in the example of the survival of bladder cancer patients undergoing immune checkpoint therapy is also believed to be applicable to other tumors and anti-tumor treatments, such as general immunotherapy, chemotherapy, antihormonal therapy and anti-tyrosine therapy.This is because it is assumed that when any effective anti-cancer treatment leads to tumor cell destruction and antigen exposure to the immune system, thereby unmasking the tumor, high expression levels of HLA-G, L, H and J can help tumor cells or a subpopulation of tumor cells escape anti-tumor therapy.Therefore, the cellular strategy for reducing immune recognition conferred by HLA-G, L, H and J expression is generally important not only for immunotherapy, but also for chemotherapy and / or antihormonal and / or tyrosine kinase inhibitor therapy, or any combination of these therapies.

[0054] It is further noted that, with regard to the sequences of membrane-bound human HLA-L and soluble HLA-H, HLA-J, and HLA-L, it has been surprisingly discovered herein that HLA-L, HLA-H, and HLA-J have been erroneously annotated as pseudogenes in the art. In fact, these genes encode proteins, and the expression of HLA-L, HLA-H, and HLA-J can be detected in various cancers, as illustrated in the appendix examples. Because HLA-L, HLA-H, and HLA-J have all been erroneously annotated in the art, HLA-L, HLA-H, and HLA-J can be collectively described as a new HLA-group. Furthermore, the following examples show that high expression levels of HLA-L, HLA-H, and HLA-J in patients with bladder cancer are adversely associated with the survival of these patients. A growing body of evidence showing that the higher the expression levels of these HLA genes, the more likely a patient is to die from cancer within two years suggests that expression of HLA patterns L, H, and J is likely used by tumors as a mechanism to evade the immune system of tumor patients. These genes and the proteins they encode are functional and are not pseudogenes that do not encode any functional proteins.

[0055] In a preferred embodiment of the first aspect of the present invention, any one of SEQ ID NOs: 1 to 6 is any one of SEQ ID NOs: 6, preferably SEQ ID NO: 4 or 5, and any one of SEQ ID NOs: 7 to 12 is any one of SEQ ID NOs: 9 to 12, preferably SEQ ID NO: 11 or 12.

[0056] SEQ ID NOs: 9 and 10 are nucleic acid sequences encoding soluble HLA forms of membrane-bound HLA-G and HLA-L, and SEQ ID NOs: 11 and 12 are soluble HLA-H and HLA-J. SEQ ID NOs: 3 to 6 are the corresponding amino acid sequences.

[0057] The data of the Examples show that, based on the HLA classes G, H, L and J, HLA genes and proteins can predict the response of tumor patients to tumor treatments as defined herein.

[0058] In a preferred embodiment of the first aspect of the invention, the method further comprises determining the mRNA expression level or protein level of one or more selected from ErbB2, EGFR, CD20, CTLA4, IDO1, LAG3, TIM3, TIM-4, CXCL9, CXCL13, TIGIT, BTLA, CD137, OX40, VISTA, B7-H7, CD27, GITR, TGF-β signaling pathway, IL-15, PD-1 and PD-L1, preferably PD-1 or PD-L1.

[0059] In connection with this preferred embodiment, it should be understood that mRNA expression levels or protein levels are determined in a subject and then compared with respective controls or predetermined standards from known responders or non-responders and / or known survivors or non-survivors, as explained above in connection with HLA genes.

[0060] The mRNA expression level or protein level of one or more selected from ERBB2, EGFR, CD20, CTLA4, IDO1, LAG3, TIM3, TIM-4, CXCL9, CXCL13, TIGIT, BTLA, CD137, OX40, VISTA, B7-H7, CD27, GITR, TGF-β signaling pathway, IL-15, PD-1, and PD-L1, preferably PD-1 or PD-L1 alone, is not sufficiently predictive for determining whether a subject is likely to respond to tumor therapy, particularly immunotherapy, and more particularly checkpoint therapy, as defined herein, which is useful in combination with the present invention. Therefore, additional analysis of one or more of these levels is expected to further improve the predictive value of the present invention.

[0061] PD-1 (programmed cell death protein 1, also known as CD279) is a protein on the surface of cells that regulates the immune system's response to the body's own cells by downregulating the immune system and promoting self-tolerance through suppressing T cell inflammatory activity.

[0062] PD-L1 (Programmed death-ligand 1, also known as CD274 or B7-H1) is a 40 kDa type 1 transmembrane protein that is speculated to play a major role in suppressing the immune system during certain events, such as pregnancy, tissue allografts, autoimmune diseases, and other disease states, such as hepatitis. Upregulation of PD-L1 may enable cancer to evade the host immune system. Importantly, PD-L1 can be expressed by tumor or non-tumor cells, such as macrophages.

[0063] ErbB2 (receptor tyrosine-protein kinase erbB-2, also known as CD340 or proto-oncogene Neu) is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer.

[0064] EGFR (epidermal growth factor receptor, also known as HER1) is a transmembrane protein that is the receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands.

[0065] CD20 is an activation glycosylated phosphorylated protein expressed on the surface of all B cells, beginning at the pro-B stage (CD45R+, CD117+) and increasing in concentration until they mature. CD20 is the target of the monoclonal antibodies rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, and ublituximab, all of which are effective drugs for the treatment of B-cell lymphomas, leukemias, and B-cell autoimmune diseases.

[0066] CTLA4 (cytotoxic T-lymphocyte-associated protein 4, also known as CD152) is a protein receptor that functions as an immune checkpoint (or checkpoint inhibitor) to downregulate immune responses. CTLA4 is constitutively expressed on regulatory T cells, but is only upregulated on conventional T cells after activation. This phenomenon is particularly noteworthy in cancer.

[0067] IDO1 (indoleaminepyrrole 2,3-dioxygenase) is a heme-containing enzyme. IDO1 is involved in immune regulation through its ability to limit T cell function and participate in mechanisms of immune tolerance. IDO1 is activated during tumor development, helping malignant cells escape elimination by the immune system.

[0068] LAG3 (lymphocyte activation gene 3, also known as CD223) is a cell surface molecule that exerts diverse biological effects on T cell function. It is an immune checkpoint receptor and, as such, is the target of various drug development programs by pharmaceutical companies seeking to develop new treatments for cancer and autoimmune disorders.

[0069] TIM-3 (T-cell immunoglobulin and mucin-domain containing-3, also known as hepatitis A virus cellular receptor 2 (HAVCR2)) mediates CD8+ T-cell exhaustion. TIM-3 has also been shown to regulate macrophage activation and is a CD4+ Th1-specific cell surface protein that enhances the severity of experimental autoimmune encephalomyelitis in mice.

[0070] TIM-4 (T-cell immunoglobulin and mucin-domain-containing-4) is a phosphatidylserine receptor that enhances the engulfment of apoptotic cells. TIM-4 is involved in the regulation of T-cell proliferation and lymphotoxin signaling.

[0071] CXCL9 (chemokine (C-X-C motif) ligand 9) is a small cytokine belonging to the C-X-C chemokine family, also known as monokines, that are induced by gamma interferon (MIG). CXCL9 is a T cell chemoattractant and is induced by IFN-γ.

[0072] CXCL13 (chemokine (C-X-C motif) ligand 1, also known as B lymphocyte chemoattractant (BLC) or B cell-attractant chemokine 1 (BCA-1)) is a small chemokine that belongs to the C-X-C chemokine family. As its name suggests, this chemokine is selectively chemotactic for B cells belonging to both the B-1 and B-2 subsets, and it exerts its effects by interacting with the chemokine receptor CXCR5.

[0073] TIGIT, also known as T cell immunoreceptor with Ig and ITIM domains, is an immunoreceptor present on a subset of T cells and natural killer (NK) cells. It is also identified as WUCAM and Vstm3. TIGIT and PD-1 have been shown to be overexpressed on tumor antigen-specific (TA-specific) CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) from melanoma patients.

[0074] BTLA (B and T lymphocyte attenuator, also known as CD272) expression is induced during T cell activation, and BTLA remains expressed on Th1 cells but not on Th2 cells. BTLA activation inhibits the function of human CD8+ cancer-specific T cells.

[0075] CD137, also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB, is induced by lymphocyte activation (ILA). The best-characterized activity of CD137 is its costimulatory activity on activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. Furthermore, it can enhance immune activity and eliminate tumors.

[0076] Ox40 (also known as tumor necrosis element receptor superfamily, member 4 (TNFRSF4) and CD134) is a secondary costimulatory immune checkpoint molecule that is expressed 24–72 hours after activation; its ligand, Ox40L, is also not expressed on resting antigen-presenting cells but is expressed following their activation. OX40 expression is dependent on full activation of T cells; in the absence of CD28, OX40 expression is delayed and levels are fourfold lower.

[0077] VISTA (V-domain Ig inhibitor of T-cell activation) is a type I transmembrane protein that functions as an immune checkpoint. VISTA acts as both a ligand and a receptor on T cells, inhibiting T-cell effector functions and maintaining peripheral immune tolerance.

[0078] B7-H7 (also known as human endogenous retrovirus-H-terminal repeat-associated 2 (HHLA2)) is a B7 family member that regulates human T cell function. B7-H7 was previously known to have unidentified functions. B7-H7 has been identified as a specific ligand for human CD28H. The B7-H7-CD28H pathway strongly promotes CD4+ T cell proliferation and cytokine production through an AKT-dependent signaling cascade in the presence of TCR signaling, suggesting that B7-H7 comprises a novel costimulatory pathway. The first IgV domain of B7-H7, which likely binds to the putative receptor, shows the highest homology with other B7 family members.

[0079] CD27 is required for the generation and long-term maintenance of T cell immunity. It binds to its ligand CD70 and plays an important role in regulating B cell activation and immunoglobulin synthesis.

[0080] GITR (glucocorticoid-inducible TNFR-related protein, also known as tumor necrosis factor receptor superfamily member 18 (TNFRSF18) and activation-induced TNFR family receptor (AITR)) has been shown to be upregulated during T cell activation and is thought to play an important role in the dominant immunological self-tolerance maintained by CD25+ / CD4+ regulatory T cells. Knockout studies in mice also suggest that this receptor is involved in the regulation of CD3-mediated T cell activation and programmed cell death.

[0081] The transforming growth factor beta (TGFβ) signaling pathway is involved in many cellular processes in both adult organisms and developing embryos, including cell proliferation, cell differentiation, apoptosis, cell homeostasis, and other cellular functions. Despite the wide variety of cellular processes regulated by the TGFβ signaling pathway, the process is relatively simple. TGFβ superfamily ligands bind to type II receptors, which recruit and phosphorylate type I receptors. The type I receptors then phosphorylate receptor-regulated SMADs (R-SMADs), which can then bind to the coSMAD SMAD4. The R-SMAD / coSMAD complexes accumulate in the nucleus, where they act as transcription factors and are involved in regulating target gene expression.

[0082] IL-15 (interleukin-15) is a cytokine structurally similar to interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex consisting of the IL-2 / IL-15 receptor beta chain (CD122) and common gamma chain-C (CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) after viral infection. This cytokine induces cell proliferation of natural killer cells, cells of the innate immune system whose primary role is to kill virus-infected cells.

[0083] In a second aspect the invention relates to an inhibitor of a binding molecule, preferably at least one nucleic acid molecule as defined in relation to the first aspect of the invention, or at least one protein or peptide as defined in relation to the first aspect of the invention, for use in the treatment of a tumour in a subject, wherein the inhibitor is used in combination with (i) immunotherapy; (ii) chemotherapy; (iii) antihormonal therapy; and / or (iv) anti-tyrosine kinase therapy.

[0084] Definitions are provided herein above in which the first aspect of the invention applies mutatis mutandis to the second aspect of the invention.

[0085] The inhibitor of the binding molecule, preferably the nucleic acid molecule defined in relation to the first aspect of the present invention, is preferably selected from small molecules, aptamers, siRNAs, shRNAs, miRNAs, ribozymes, antisense nucleic acid molecules, CRISPR-Cas9-based constructs, CRISPR-Cpf1-based constructs, meganucleases, zinc finger nucleases, and transcription activator-like (TAL) effector (TALE) nucleases. Further details about these classes are provided herein below.

[0086] The binding molecules, preferably inhibitors of HLA proteins according to the present invention, are preferably selected from small molecules, antibodies or antibody mimetics, and aptamers, with the antibody mimetics preferably being selected from affibodies, adnectins, anticalins, DARPins, avimers, nanophytins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies.

[0087] As used herein, the term "antibody mimetic" refers to a compound that can specifically bind to an antigen like an antibody, e.g., the HLA proteins of SEQ ID NOS: 1-6 herein, but is not structurally related to an antibody. Antibody mimics are typically artificial peptides or proteins with a molar mass of approximately 3-20 kDa. For example, the antibody mimetic can be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanophytins, affilins, Kunitz domain peptides, and Fynomers®. These polypeptides are well known in the art and are described in further detail below.

[0088] The term "affibody" as used herein refers to a family of antibody mimics derived from the Z domain of Staphylococcus aureus protein A. Structurally, affibody molecules are based on a three-helix bundle region that can also be incorporated into fusion proteins. Affibodies themselves have a molecular weight of approximately 6 kDa and are stable at high temperatures and under acidic or alkaline conditions. Target specificity is achieved by randomizing 13 amino acids located in two α-helices involved in the binding activity of the parent protein region (Feldwisch J, Tolmachev V.; (2012) Methods: Mol Biol. 899:103-26).

[0089] The term "Adnectin" (also referred to as "monobody"), as used herein, refers to molecules based on the tenth extracellular domain of human fibronectin III (10Fn3), which adopt a 94-residue Ig-like β-sandwich fold with two to three exposed loops but lacking a central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectins with desired target specificity, i.e., against HLA proteins, can be engineered by introducing modifications into specific loops of the protein.

[0090] The term "anticalin" as used herein refers to a modified protein derived from lipocalin (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci US A. 96(5):1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Anticalins have an eight-stranded β-barrel, forming a highly conserved core unit among lipocalins, and naturally form a binding site for ligands via four structurally variable loops at the open end. Although anticalins are not homologous to the IgG superfamily, they exhibit characteristics previously considered typical of antibody binding sites: (i) high structural plasticity as a result of sequence variation, and (ii) increased conformational flexibility, allowing for induced adaptation to targets with different shapes.

[0091] The term "DARPin" as used herein refers to a designed ankyrin repeat domain (166 residues) that provides a rigid interface, typically resulting from three repeated β-turns. DARPins usually have three repeat sequences corresponding to an artificial consensus sequence, with six positions randomized per repeat. Therefore, DARPins lack structural flexibility (Gebauer and Skerra, 2009).

[0092] As used herein, the term "avimer" refers to a class of antibody mimics consisting of two or more peptide sequences, each 30-35 amino acids long, derived from the A-domains of various membrane receptors and linked by a linker peptide. Target molecule binding occurs via the A-domains, and the desired binding specificity, i.e., domains specific for HLA proteins, can be selected, for example, by phage display technology. The binding specificities of the different A-domains contained in an avimer can be identical, but do not have to be (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0093] "Nanophytins" (also known as affitins) are antibody-mimetic proteins derived from the DNA-binding protein Sac7d of Sulfolobus acidocaldarius. Nanophytins typically have a molecular weight of approximately 7 kDa and are engineered to specifically bind to target molecules (e.g., HLA proteins) by randomizing the amino acids on the binding surface (Mouratou B, Béhar G, Paillard-Laurance L, Colinet S, Pecorari F., (2012) Methods: Mol Biol.; 805:315-31).

[0094] The term "affilin" as used herein refers to an antibody mimic that is developed by using either gamma-B crystalline or ubiquitin as a scaffold and modifying the amino acids on the surface of these proteins by random mutagenesis. The selection of affilin for the desired target specificity, i.e., HLA protein, is carried out, for example, by phage display or ribosome display technology. Depending on the scaffold, the molecular weight of affilin is approximately 10 or 20 kDa. As used herein, the term affilin also refers to the dimeric or multimeric form of affilin (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

[0095] "Kunitz domain peptides" are derived from the Kunitz domains of Kunitz-type protease inhibitors, such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP), or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of approximately 6 kDA, and domains with the required target specificity, i.e., HLA protein, can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

[0096] As used herein, the term "Fynomer®" refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well known in the art and are described, for example, in Grabulovski et al. (2007) JBC, 282, pp. 3196-3204, WO 2008 / 022759, Bertschinger et al. (2007) Protein Eng Des Sel 20(2):57-68, Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255, or Schlatter et al. (2012), MAbs 4:4, 1-12.

[0097] As used herein, the term "trispecific binding molecule" refers to a polypeptide molecule having three binding domains and thus capable of binding, preferably specifically binding, to three different epitopes. At least one of these three epitopes is an epitope of an HLA protein of the present invention. The two other epitopes may also be epitopes of an HLA protein of the present invention, or may be epitopes of one or two different antigens. The trispecific binding molecule is preferably TriTac. TriTac is a T-cell engager for solid tumors that is composed of three binding domains, has an extended serum half-life, and is approximately one-third the size of a monoclonal antibody.

[0098] The term "probody" as used herein refers to a protease-activatable antibody prodrug. Probodies consist of an authentic IgG heavy chain and a modified light chain. A masking peptide is fused to the light chain via a peptide linker cleavable by a tumor-specific protease. The masking peptide prevents probe-like binding to healthy tissue, thereby minimizing toxic side effects. For example, in probodies, small molecules, antibodies, or protein drugs or aptamers can be conjugated to a masking peptide that limits or prevents binding to HLA proteins according to the present invention, which can be cleaved by a protease. Proteases are enzymes that digest proteins into smaller fragments by cleaving specific amino acid sequences, known as substrates. In normal, healthy tissues, protease activity is tightly regulated. In cancer cells, protease activity is upregulated. In healthy tissues or cells where protease activity is regulated and minimal, the target binding site of the probody remains masked and therefore cannot bind. On the other hand, in diseased tissues or cells where protease activity is upregulated, the target binding site of the probody is unmasked and therefore able to bind and / or inhibit.

[0099] The binding molecule of the second embodiment is a compound capable of binding to a nucleic acid molecule, protein, or peptide defined herein. The binding molecule preferably specifically binds to the nucleic acid molecule, protein, or peptide. Specific binding indicates that the binding molecule essentially does not bind to, or essentially does not bind to, other nucleic acid molecules, proteins, or peptides other than the nucleic acid molecules, proteins, or peptides defined herein. In particular, the binding molecule preferably cannot bind to HLA proteins other than the respective selected HLA proteins. The binding molecules of the present invention are suitable, for example, for research or diagnostic purposes. For example, antibodies that bind to the proteins of the present invention can be used in immunoassays such as ELISA or Western blot. Immunoassays are biochemical tests that can measure the presence or enrichment of a protein of the second embodiment in a sample (e.g., a solution). Furthermore, the antibodies can be used for tissue or cell staining, including, but not limited to, IHC, FACS, immunofluorescence, etc. The binding molecule of the protein of the second embodiment preferably inhibits the nucleic acid molecule, protein, or peptide defined herein. In this case, the binding molecule is called an inhibitor.

[0100] According to the present invention, a compound that inhibits the expression of a nucleic acid molecule and / or protein of the present invention is (i) a compound that reduces or prevents the transcription of a gene encoding the nucleic acid molecule and / or protein of the present invention, or (ii) a compound that reduces or prevents the translation of an mRNA encoding the protein of the present invention. Compounds (i) include compounds that interfere with the transcription machinery and / or its interaction with expression control elements distant from the promoter, such as the promoter and / or enhancer of the gene. Compounds (ii) include compounds that interfere with the translation machinery. A compound that inhibits the expression of a nucleic acid molecule and / or protein of the present invention specifically inhibits the expression of a nucleic acid molecule and / or protein of the present invention, for example, by specifically interfering with the promoter region that controls expression. Preferably, transcription of a nucleic acid molecule and / or protein, or translation of a protein of the present invention, is reduced by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, preferably at least 90% or 95%, even more preferably at least 98%, and most preferably about 100% (e.g., compared to the same experimental setting in the absence of the compound).

[0101] Compounds that inhibit the activity of nucleic acid molecules, proteins, and / or proteins according to the present invention cause the nucleic acid molecules, peptides, and / or proteins to perform their / their functions less efficiently. Compounds that inhibit the activity of nucleic acid molecules, peptides, and / or proteins specifically inhibit the activity of the nucleic acid molecules, peptides, and / or proteins. As described in further detail below, compounds that inhibit the activity of nucleic acid molecules, peptides, and / or proteins according to the present invention can specifically inhibit the activity of the nucleic acid molecules, peptides, and / or proteins by interacting with the nucleic acid molecules, peptides, and / or proteins themselves, or by specifically inhibiting (preferably killing) cells that produce the nucleic acid molecules, peptides, and / or proteins, and / or by binding to the peptides or proteins. Preferably, the activity of the nucleic acid molecules, peptides, and / or proteins according to the present invention is reduced by at least 50%, more preferably at least 75%, for example at least 90% or 95%, even more preferably at least 98%, and most preferably about 100% (e.g., compared to the same experimental setting in the absence of the compound).

[0102] Alternatively, the nucleic acid molecules, proteins, and / or compounds inhibiting the activity of proteins according to the present invention also include nucleic acids or their analogs used to vaccinate patients against specific HLA isoforms. Vaccination methods can be based on RNA, protein, or peptide levels, which require further modification for stabilization in vivo within the human body. Such methods can be adapted from personalized mutagenesis vaccination approaches (Sahin U. Personalized RNA vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 2017).

[0103] As a further option, the nucleic acid molecules, proteins and / or compounds that inhibit the activity of proteins according to the present invention also comprise the isolation of naturally occurring autoantibodies or cells that produce naturally occurring autoantibodies against the respective HLA genes, isoforms and fragments, which can be modified or expanded before reintroduction into the respective patient.

[0104] The activity of the nucleic acid molecules, peptides and / or proteins according to the present invention is preferably its / their ability to induce resistance to tumor treatment as defined herein above in cancer patients. Means and methods for determining this activity are established in the art and are described in the examples below. Therefore, according to the medical aspect of the present invention, this activity of the nucleic acid molecules and / or proteins according to the present invention should be inhibited.

[0105] The effectiveness of an inhibitor's suppression can be quantified by comparing the level of activity in the presence of the inhibitor with that in the absence of the inhibitor. For example, changes in the amount of the nucleic acid molecules and / or proteins of the present invention formed can be measured. The effectiveness of several inhibitors can be determined simultaneously in a high-throughput format. High-throughput assays, independent of biochemical, cellular, or other assays, are typically performed in the wells of microtiter plates, where each plate may contain 96, 384, or 1536 wells. Plate handling, including incubation at temperatures other than ambient temperature and contacting the test compounds with the assay mixture, is preferably performed by one or more computer-controlled robotic systems, including pipetting devices. For screening large libraries of test compounds and / or rapid screening, a mixture of, for example, 10, 20, 30, 40, 50, or 100 test compounds can be added to each well. In cases where a well exhibits the expected activity, the mixture of test compounds can be deconvoluted to identify one or more test compounds in the mixture that produce the activity.

[0106] Compounds that inhibit the expression and / or activity of nucleic acid molecules and / or proteins of the present invention can be formulated as vesicles (e.g., liposomes or exosomes). Liposomes have attracted great interest from the perspective of drug delivery due to the specificity and duration of action they offer. Liposomal cell-based delivery systems have been used to effectively deliver nucleic acids, such as siRNA, to cells in vivo (Zimmermann et al. (2006) Nature, 441:111-114). Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse efficiently with the cell wall, but are phagocytosed by macrophages and other cells in vivo. Exosomes are lipid packages that can carry a variety of different molecules, including RNA (Alexander et al. (2015), Nat Commun; 6:7321). Exosomes containing the molecules contained therein can be taken up by recipient cells. Therefore, exosomes are important mediators of intercellular communication and regulators of cellular niches. Exosomes can be used as delivery vehicles, for example, as imaging agents or drugs, making them useful for diagnostic and therapeutic purposes.

[0107] Compounds that inhibit the expression and / or activity of nucleic acid molecules, peptides, and / or proteins can be administered to a subject at an appropriate dosage and / or therapeutically effective amount. The therapeutically effective amount for a given situation is readily determined by routine experimentation and is within the skill and judgment of an ordinary clinician or physician. Generally, a regular administration regimen of the pharmaceutical composition should be in the range of 1 μg to 5 g units per day. However, a more preferred dosage may be 0.01 mg to 100 mg, even more preferably 0.01 mg to 50 mg, and most preferably 0.01 mg to 10 mg per day. Further, for example, when the compound is an iRNA agent, such as an siRNA, the total pharmaceutically effective amount of the pharmaceutical composition administered is typically less than about 75 mg per kg of body weight, e.g., less than about 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of body weight. More preferably, the amount is less than 2000 nmoles of iRNA agent (e.g., about 4.4 x 10) per kg of body weight, such as less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, or 0.00015 nmoles of iRNA agent per kg of body weight. 16 The length of treatment required to observe changes and the interval after treatment for response to occur will vary depending on the desired effect. The specific amount can be determined by conventional testing well known to those skilled in the art. The length of treatment required to observe changes and the interval after treatment for response to occur will vary depending on the desired effect. The specific amount can be determined by conventional testing well known to those skilled in the art. Suitable tests are described, for example, in Tamhane and Logan (2002), "Multiple Test Procedures for Identifying the Minimum Effective and Maximum Safe Doses of a Drug", Journal of the American Statistical Association, 97(457):1-9.

[0108] The compound that inhibits the expression and / or activity of a nucleic acid molecule, peptide and / or protein is preferably mixed with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition. According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention comprises the compound described above. It may optionally contain additional molecules that can alter the properties of the compound of the present invention, thereby, for example, stabilizing, regulating, and / or activating their function. The composition may be in solid, liquid, or gaseous form, and may, inter alia, be in the form of powder(s), tablet(s), solution(s), or aerosol(s). The pharmaceutical composition of the present invention may optionally and additionally comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solution, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, organic solvents including DMSO, and the like. Compositions containing such carriers can be formulated by well-known conventional methods. Means and methods for preparing pharmaceutical compositions are described, for example, in Pharmaceutical Development (2005), ISBN-10: 1907568999 or Handbook of Pharmaceutical Manufacturing Formulations, ISBN-10: 9781420081169.

[0109] The pharmaceutical composition can be administered by any suitable route. The actual route to be selected depends, for example, on the physical and chemical properties of the drug, the desired site of action, the rate of absorption of the drug from various routes, the metabolism of the drug, and the condition of the patient. Examples of administration routes are enteral / gastrointestinal, topical, and parenteral. Furthermore, the pharmaceutical composition can be applied as an infusion therapy to the bladder in cases of bladder cancer or neoplastic lesions thereof. Administration as an infusion therapy is particularly contemplated for the combination of immunological, chemotherapeutic, antihormonal, or anti-tyrosine kinase compounds with anti-HLA agents, as described as part of this application.

[0110] These pharmaceutical compositions can be administered to subjects at suitable dosage.The method of administering dosage is determined by attending physician and clinical factors.As is well known in the medical field, the dosage of any one patient depends on many factors, including patient's size, body surface area, age, the specific compound that is administered, sex, administration time and route, general health, and other drugs that are administered at the same time.

[0111] As mentioned above, the data in the Examples show that high expression levels of these HLAs are associated with poor disease-specific survival. Furthermore, high HLA-G, L, H, and J expression levels may help tumor cells or subpopulations of tumor cells escape antitumor therapy.

[0112] Therefore, it is also envisioned that combination therapy, in which classical antitumor treatments (e.g., (i) immunotherapy; (ii) chemotherapy; (iii) antihormonal therapy; and / or (iv) antityrosine kinase therapy) are combined with inhibitors of HLA-G, L, H, or J, may further improve antitumor treatment. Such combination antitumor treatments can be performed as a preventative measure, particularly in patients diagnosed with high levels of HLA-G, L, H, and / or J at the start of treatment. In such patients, treatment failure can be converted into treatment success.

[0113] In a preferred embodiment of the second aspect of the invention, the subject is predicted by the method of the first aspect of the invention to not respond to (i) immunotherapy; (ii) chemotherapy; (iii) anti-hormonal therapy; and / or (iv) anti-tyrosine kinase therapy.

[0114] Previous diagnosis of a subject to be treated as not responding to (i) immunotherapy; (ii) chemotherapy; (iii) anti-hormonal therapy; and / or (iv) anti-tyrosine kinase therapy indicates the need to further treat the subject with a binding molecule, preferably an inhibitor, of the present invention.

[0115] This is because expression of HLA genes as discussed herein above is believed to protect malignant cells in a subject from (i) immunotherapy; (ii) chemotherapy; (iii) antihormonal therapy; and / or (iv) anti-tyrosine kinase therapy, and as a result, combination of a binding molecule, preferably an inhibitor of the present invention, with (i) immunotherapy; (iii) chemotherapy; (iii) antihormonal therapy; and / or (iv) anti-tyrosine kinase therapy can convert a (predicted) treatment failure into a treatment success.

[0116] In a preferred embodiment of the second aspect, the inhibitor is a small molecule inhibitor, a nucleotide-based inhibitor, or an amino acid-based inhibitor.

[0117] As used herein, "small molecules" are preferably organic molecules. Organic molecules relate to or belong to a class of compounds with a carbon base, where the carbon atoms are linked together by carbon-carbon bonds. The original definition of the term "organic" relates to the source of the chemical compound: organic compounds are carbon-containing compounds obtained from plant, animal, or microbial sources, while inorganic compounds are obtained from mineral sources. Organic compounds may be natural or synthetic. Organic molecules are preferably aromatic molecules, more preferably heteroaromatic molecules. In organic chemistry, the term aromaticity is used to describe cyclic (ring-shaped), planar (flat) molecules with resonance-bonded rings that are more stable than other geometric or bonding arrangements with the same set of atoms. Aromatic molecules are highly stable and do not easily decompose to react with other substances. In heteroaromatic molecules, at least one of the atoms in the aromatic ring is an atom other than carbon, such as N, S, or O. For all of the above organic molecules, the molecular weight is preferably in the range of 200 Da to 1500 Da, more preferably 300 Da to 1000 Da.

[0118] Alternatively, a "small molecule" according to the present invention may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (excluding carbon dioxide, carbon monoxide, and carbonates). Preferably, small molecules have a molecular weight of less than about 1000 Da, such as less than about 2000 Da, or less than about 500 Da, and even more preferably less than about 250 Da. The size of a small molecule can be determined by methods well known in the art, such as mass spectrometry. Small molecules can be designed, for example, based on the crystal structure of a target molecule, where sites likely responsible for biological activity can be identified and validated in in vivo assays, such as in vivo high-throughput screening (HTS) assays.

[0119] Nucleotide-based inhibitors comprise or consist of nucleic acid sequences. Nucleotide-based inhibitors may comprise or consist of RNA, DNA, or both. The nucleotide-based or nucleotide analog-based inhibitors of the present invention are molecules that specifically bind to the HLA genes of SEQ ID NOS: 7-12 and inhibit the activity of the HLA encoded by the genes. As used herein, specific binding means that the inhibitor specifically targets HLA and does not substantially exert any off-target inhibitory effects, particularly on other cellular nucleic acid molecules.

[0120] Amino acid-based inhibitors comprise or consist of an amino acid sequence, and preferably an amino acid sequence of at least 25, more preferably at least 50 amino acids. Amino acid-based inhibitors are molecules that specifically bind to HLAs of SEQ ID NOS: 1-6 and inhibit the activity of said HLAs. Amino acid-based inhibitors preferably comprise natural amino acids, but may also comprise unnatural amino acids. Amino acid-based inhibitors are preferably selected or designed to specifically bind to an amino acid sequence selected from SEQ ID NOS: 1-6.

[0121] In relation to the second embodiment, the binding molecule, preferably the inhibitor, may be a cell such as a T cell, wherein the T cell is preferably a CAR-T cell.

[0122] The cells generally carry on their surface a binding molecule, preferably at least one nucleic acid molecule according to the present invention or at least one protein or peptide inhibitor according to the present invention. In the case of T cells, the binding molecule is preferably a naturally occurring or chimeric T cell receptor, the inhibitor of which specifically targets at least one protein or peptide. Chimeric antigen receptor T cells (also known as CAR T cells) are T cells genetically engineered to produce an artificial T cell receptor for use in immunotherapy.

[0123] Thus, a chimeric antigen receptor (CAR, also known as a chimeric immune receptor), chimeric T cell receptor, or artificial T cell receptor is a receptor protein that has been engineered to confer a new function on T cells: to specifically target at least one protein or peptide. The receptor is chimeric because it combines both antigen-binding and T cell activation functions into one receptor.

[0124] In a more preferred embodiment of the second aspect of the present invention, the nucleotide-based inhibitor or amino acid-based inhibitor is an aptamer, ribozyme, siRNA, shRNA or antisense oligonucleotide, CRISPR endonuclease-based construct, meganuclease, zinc finger nuclease, or transcription activator-like (TAL) effector (TALE) nuclease, and the amino acid-based inhibitor is an antibody or a protein drug.

[0125] Aptamers are nucleic acid or peptide molecules that bind to specific target molecules. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used as macromolecular drugs for both basic research and clinical purposes. Aptamers can be combined with ribozymes, which self-cleave in the presence of their target molecules. These compound molecules have further research, industrial, and clinical applications.

[0126] Nucleic acid aptamers are nucleic acid species, usually consisting of short strands of oligonucleotides, that have typically been engineered through repeated rounds of in vitro selection or equivalently SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and organisms.

[0127] Peptide aptamers are typically peptides or proteins designed to disrupt other protein interactions within cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of peptide aptamers (nanomolar range), to levels comparable to those of antibodies. The variable peptide loop typically contains 10–20 amino acids, and the scaffold can be any protein with good solubility properties. Currently, the bacterial protein thioredoxin-A is the most commonly used scaffold protein. The variable peptide loop is inserted into the redox-active site, which in the wild-type protein is a -Cys-Gly-Pro-Cys-loop (SEQ ID NO: 13), and the two cysteine ​​side chains can form disulfide bridges. Peptide aptamer selection can be performed using different systems, but the yeast two-hybrid system is currently the most widely used.

[0128] Aptamers offer molecular recognition properties comparable to those of commonly used biomolecules, particularly antibodies, making them useful for biotechnology and therapeutic applications. In addition to their discriminatory recognition, aptamers offer advantages over antibodies because they can be fully modified in vitro, are easily produced by chemical synthesis, have desirable storage properties, and induce little or no immunogenicity in therapeutic applications. Unmodified aptamers are rapidly cleared from the bloodstream, with half-lives ranging from minutes to hours. This is primarily due to their inherently low molecular weight, resulting in nuclease degradation and clearance from the body by the kidneys. Applications of unmodified aptamers currently focus on treating transient conditions such as blood clotting or organs such as the eye where localized delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) conjugation, fusion to albumin or other half-life-extending proteins, are available to scientists, allowing aptamers to extend their half-lives over days or even weeks.

[0129] Ribozymes (derived from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been shown to catalyze the aminotransferase activity of ribosomes. Non-limiting examples of well-characterized small self-cleaving RNAs include hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes, while group I introns are examples of larger ribozymes. The principle of catalytic self-cleavage has become well established in recent years. Hammerhead ribozymes are the best characterized RNA molecules with ribozyme activity. Since it has been shown that hammerhead structures can be incorporated into heterologous RNA sequences and ribozyme activity can thereby be transferred to these molecules, it appears possible to create catalytic antisense sequences for almost any target sequence, provided the target sequence contains a potentially matching cleavage site. The basic principle for constructing a hammerhead ribozyme is as follows: a region of interest in an RNA containing a GUC (or CUC) triplet is selected. Two oligonucleotide strands, usually 6-8 nucleotides long, are taken and a catalytic hammerhead sequence is inserted between them.

[0130] Best results are usually obtained with short ribozymes and target sequences. A recent development is the combination of hammerhead ribozymes with aptamers that recognize small compounds. Conformational changes induced in the aptamer upon binding to the target molecule can modulate the catalytic function of the ribozyme.

[0131] According to the present invention, the term "small interfering RNA (siRNA)," also known as short interfering RNA or silencing RNA, refers to double-stranded RNA molecules 18-30, preferably 19-25, most preferably 21-23, or even more preferably 21 nucleotides in length that play a variety of roles in biology. Most notably, siRNAs participate in the RNA interference (RNAi) pathway, disrupting the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also function in RNAi-related pathways, for example, as an antiviral mechanism or in shaping the chromatin structure of the genome.

[0132] Naturally occurring siRNAs have a well-defined structure: short double-stranded RNA (dsRNA) with 2-nt 3' overhangs on both ends. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by Dicer, an enzyme that converts either long dsRNA or small hairpin RNAs into siRNA. siRNAs can also be exogenously (artificially) introduced into cells to specifically knockdown a gene of interest. In this way, essentially any gene whose sequence is known can be targeted based on sequence complementarity with an appropriately tailored siRNA. Double-stranded RNA molecules or their metabolic processing products can mediate target-specific nucleic acid modifications, particularly RNA interference and / or DNA methylation. Exogenously introduced siRNAs may lack overhangs at their 3' and 5' ends; however, it is preferred that at least one RNA strand possesses a 5' and / or 3' overhang. Preferably, one end of the duplex has a 3'-overhang of 1 to 5 nucleotides, more preferably 1 to 3 nucleotides, and most preferably 2 nucleotides. The other end may be blunt or may have a 3'-overhang of up to 6 nucleotides. In general, any RNA molecule suitable for acting as an siRNA against a target according to the present invention is contemplated by the present invention. To date, the most efficient silencing has been obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands paired to have a 2-nt 3'-overhang. The sequence of the 2-nt 3'-overhang contributes little to the specificity of target recognition, which is limited to the unpaired nucleotide adjacent to the first base pair. 2'-deoxynucleotides in the 3'-overhang are as efficient as ribonucleotides but are cheaper to synthesize and likely more nuclease-resistant.Delivery of siRNA can be achieved using any method known in the art, for example, by combining the siRNA with saline and administering the combination intravenously or intranasally, or by formulating the siRNA in glucose (e.g., 5% glucose) or cationic lipids, and polymers can be used for siRNA delivery in vivo via either intravenous (IV) or intraperitoneal (IP) systemic routes (Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol. 437: Drug Delivery Systems - Chapter 3: Delivering Small Interfering RNA for Novel Therapeutics).

[0133] Short hairpin RNAs (shRNAs) are RNA sequences that form tight hairpin turns and can be used to silence gene expression via RNA interference. shRNAs are introduced into cells using a vector that utilizes a U6 promoter to ensure constant expression of the shRNA. This vector is usually inherited by daughter cells, allowing gene silencing to be inherited. The shRNA hairpin structure is cleaved into siRNA by the cellular machinery and then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves the mRNA that matches the bound siRNA. The si / shRNAs used in the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA) and Cruachem (Glasgow, UK). Most conveniently, siRNA or shRNA can be obtained from a commercial RNA oligo synthesis supplier, which sells RNA synthesis products of different quality and cost. Generally, the RNA applicable to the present invention can be synthesized by conventional techniques and easily provided with quality suitable for RNAi.

[0134] Other molecules that affect RNAi include, for example, microRNA (miRNA).The RNA species is a single-stranded RNA molecule.Endogenously existing miRNA molecules bind to complementary mRNA transcripts and trigger the degradation of the mRNA transcripts through a process similar to RNA interference, thereby regulating gene expression.Therefore, exogenous miRNA can be used as an inhibitor of HLA gene after being introduced into respective cells.

[0135] The term "antisense nucleic acid molecule" as used herein refers to the nucleic acid that is complementary to target nucleic acid.Antisense molecules according to the present invention can interact with target nucleic acid, more specifically, can hybridize with target nucleic acid.The formation of hybrid reduces or blocks the transcription of target gene and / or the translation of target mRNA.Standard methods for antisense technology are described (see, for example, Melani et al., Cancer Res. (1991) 51:2897-2901).

[0136] CRISPR / Cas9, like CRISPR-Cpf1, can be applied to almost any cell or model organism and can be used for knockout mutations, chromosomal deletions, DNA sequence editing, and gene expression regulation. Regulation of gene expression can be engineered by using a catalytically dead Cas9 enzyme (dCas9) coupled to a transcriptional repressor to suppress transcription of a specific gene, in this case, the HLA gene. Similarly, catalytically inactive "dead" Cpf1 nuclease (CRISPR derived from Prevotella and Francisella-1) can be fused to a synthetic transcriptional repressor or activator to downregulate endogenous promoters (e.g., promoters controlling HLA gene expression). Alternatively, the DNA-binding domain of zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) can be designed to specifically recognize the HLA-J gene, its promoter region, or its 5'-UTR, thereby inhibiting HLA gene expression.

[0137] Also contemplated herein is an inhibitor that inhibits nucleic acid molecules that target HLA genes or regulatory molecules involved in HLA expression.Such molecules that reduce or eliminate the expression of target HLA or regulatory molecules include, but are not limited to, meganucleases, zinc finger nucleases and transcription activator-like (TAL) effector (TALE) nucleases.Such methods are described in Silva et al., Curr Gene Ther.2011;11(1):11-27; Miller et al., Nature biotechnology.2011;29(2):143-148, and Klug, Annual review of biochemistry.2010;79:213-231.

[0138] The term "antibody" as used in accordance with the present invention includes, for example, any kind of polyclonal or monoclonal antibody and humanized versions thereof. Furthermore, derivatives or fragments thereof that still retain the binding specificity for the target, for example, the HLA proteins of SEQ ID NOs: 1 to 6, are also included in the term "antibody." Antibody fragments or derivatives include, inter alia, Fab or Fab' fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VH or V-like domains such as VhH or V-NAR domains, as well as multimeric formats such as minibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically linked Fab'-multimers (see, for example, Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 198; Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc.), vol. 75(13), 1584; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 1126). Multimeric formats particularly include bispecific antibodies that can simultaneously bind to two different types of antigens. The first antigen can be found on the HLA protein of the present invention. The second antigen can be, for example, a tumor marker specifically expressed on cancer cells or a specific type of cancer cell. Non-limiting examples of bispecific antibody formats are Biclonics (bispecific, full-length human IgG antibodies), DART (Dual-affinity Re-targeting Antibody), and BiTE (composed of two single-chain variable fragments (scFvs) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).

[0139] The term "antibody" also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanized (human antibody minus the non-human CDRs) antibodies.

[0140] Various techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane (1988) and (1999) and Altshuler et al. 2010 (supra). Thus, polyclonal antibodies can be obtained from the blood of animals after immunization with an antigen in a mixture with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line cultures. Examples of such techniques include those described, for example, in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, and include the hybridoma technique first described by Kohler and Milstein, 1975, the trioma technique, the human B cell hybridoma technique (see, e.g., Kozbor D, 1983, Immunology Today, vol. 4, 7; Li J, et al. 2006, PNAS, vol. 103(10), 3557), and the EBV-hybridoma technique (Cole et al., 1985, Alan R. Liss, Inc., 77-96) for producing human monoclonal antibodies. Furthermore, recombinant antibodies can be obtained from monoclonal antibodies or prepared de novo using various display methods such as phage, ribosome, mRNA, or cell display. Suitable systems for the expression of recombinant (humanized) antibodies can be selected from, for example, bacterial, yeast, insect, or mammalian cell lines, or transgenic animals or plants (see, for example, U.S. Pat. No. 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Furthermore, techniques described for the production of single-chain antibodies (see, in particular, U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies specific for epitopes of the HLA genes of the present invention.Surface plasmon resonance, such as that used in the BIAcore system, can be used to increase the efficiency of phage antibodies.

[0141] As used herein, the term "protein drug" refers to a protein or peptide that exhibits therapeutic (curative or prophylactic) efficacy when administered to a subject. Examples of protein drug classes are discussed herein below.

[0142] As described above, the above-mentioned small molecules, antibodies, or protein drugs and aptamers can specifically bind to the proteins of the present invention. This binding blocks the immunosuppressive properties of the proteins of the present invention and, preferably, the ability of the proteins to induce resistance to tumor treatments as defined herein and / or reduce progression-free and overall survival in cancer patients. In this case, the small molecules, antibodies, or protein drugs and aptamers are also called blocking small molecules, antibodies, or protein drugs and aptamers. The blocking small molecules, antibodies, or protein drugs and aptamers block the interaction of the HLA proteins of the present invention with other cellular components, such as ligands and receptors, that normally interact with the HLA proteins of the present invention.

[0143] Small molecules, antibodies, or protein drugs and aptamers can also be produced in the form of drug conjugates. In this case, the small molecules, antibodies, or protein drugs and aptamers themselves may not have an inhibitory effect, but the inhibitory effect is conferred solely by the drug. The small molecules, antibodies, or protein drugs and aptamers allow the drug to bind site-specifically to cells that produce and / or bind HLA proteins. The drug preferably kills cells that produce and / or bind HLA proteins. Therefore, by combining the targeting ability of the molecule that binds to the HLA protein according to the present invention with the cell-killing ability of the drug, the drug conjugate becomes an inhibitor that can distinguish between healthy and diseased tissues and cells. Cleavable and non-cleavable linkers for designing drug conjugates are known in the art. Non-limiting examples of drugs that can kill cells include cytostatics and radioisotopes that deliver radiation directly to cancer cells.

[0144] Furthermore, the binding and / or inhibitory activity of small molecule, antibody or protein drugs and aptamers can be restricted to particular tissues or cell types, particularly diseased tissues or cell types. For example, probodies, as further described below, can be designed.

[0145] In an even more preferred embodiment of the second aspect of the present invention, the protein drug is an antibody mimetic, preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanophytins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies.

[0146] In another preferred embodiment of the second aspect of the present invention, the nucleotide-based inhibitor comprises: (a) a nucleic acid sequence comprising or consisting of a nucleic acid sequence that is complementary to or at least 80% identical to at least 12 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 7-12; (b) a nucleic acid sequence comprising or consisting of a nucleic acid sequence that is at least 80% identical to the complementary strand of one or more nucleic acid sequences selected from SEQ ID NOs: 7-12; (c) a nucleic acid sequence comprising or consisting of the nucleic acid sequence set forth in (a) or (b), wherein the nucleic acid sequence is DNA or RNA; (d) an expression vector that expresses the nucleic acid sequence defined in any one of (a)-(c), preferably under the control of a tumor-specific promoter; or (e) a host comprising the expression vector of (d).

[0147] The nucleic acid sequences defined in items (a) to (c) of this preferred embodiment comprise or consist of a sequence that is complementary to the nucleotides of the HLA genes defined by one or more of SEQ ID NOs: 7 to 12. Thus, the nucleic acid sequences defined in items (a) to (c) comprise or are antisense nucleic acid sequences.

[0148] The nucleic acid sequence described in item (a) of this further preferred embodiment of the present invention preferably comprises or consists of a sequence complementary to one or more of at least 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides selected from SEQ ID NOs: 7 to 12. These at least 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides are preferably one or more consecutive portions selected from SEQ ID NOs: 7 to 12, i.e., the nucleotides are consecutive in each SEQ ID NO. The format of the nucleic acid sequence described in item (a) is not particularly limited, as long as it comprises or consists of at least 12 consecutive nucleotides complementary to a nucleic acid sequence selected from SEQ ID NOs: 7 to 12. The nucleic acid sequence described in item (a) may comprise or consist of an antisense oligonucleotide. Therefore, the nucleic acid sequence according to item (a) reflects the above-mentioned basic principle of antisense technology, and is the use of oligonucleotide to silence selected target RNA through the exquisite specificity of complementary pairing.Therefore, it should be understood that the nucleic acid sequence according to item (a) is preferably in the format of antisense oligonucleotide, or forms part of the siRNA or shRNA defined hereinabove.Antisense oligonucleotide is preferably LNA-GapmeRs, AntagomiRs or antimiRs.

[0149] The nucleic acid sequence described in item (b), which requires at least 70% identity to the complementary strand of one or more nucleic acid sequences selected from SEQ ID NOs: 7 to 12, typically comprises an antisense oligonucleotide and is significantly longer than the nucleic acid sequence described in item (a), comprising at least 12 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 7 to 12. The nucleic acid sequence described in item (b) of the above preferred embodiment can interact with the HLA gene of the present invention, more specifically, can hybridize with the HLA gene of the present invention. Upon hybrid formation, the function of HLA is reduced or blocked.

[0150] The sequence identity of the molecule described in item (b) relative to a sequence selected from SEQ ID NOs: 7-12 is, in increasing order of preference, at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99%, and 100%. The sequence identity relative to each of SEQ ID NOs: 7-12 can be selected individually. Means and methods for determining sequence identity are known in the art. Preferably, the BLAST (Basic Local Alignment Search Tool) program is used to determine sequence identity relative to one or more of SEQ ID NOs: 7-12.

[0151] In the nucleic acid sequence described in item (c), the nucleotide sequence may be RNA or DNA. RNA or DNA includes chemically modified RNA or DNA nucleotides. As is generally known, RNA contains U nucleotides and DNA contains T nucleotides.

[0152] According to items (d) and (e) of the above preferred embodiments, the inhibitor may also be an expression vector or a host capable of producing the nucleic acid sequence defined in any one of items (a) to (c), respectively.

[0153] An expression vector can be a plasmid used to introduce a specific transcript into a target cell. Once inside the cell, the protein encoded by that gene is produced by the ribosomal complex, the cellular transcription and translation machinery. Plasmids are typically engineered to contain regulatory sequences that act as enhancer and / or promoter regions and direct efficient transcription of the transcript.

[0154] Non-limiting examples of expression vectors, including prokaryotic plasmid vectors, include the pUC series, pBluescript (Stratagene), pET series expression vectors (Novagen) or pCRTOPO (Invitrogen), as well as vectors adapted for expression in mammalian cells, such as pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Examples of suitable plasmid vectors for Pichia pastoris include, for example, the plasmids pAO815, pPIC9K, and pPIC3.5K (all from Invitrogen). For the formulation of pharmaceutical compositions, appropriate vectors are selected in accordance with good manufacturing practice. Such vectors are known in the art, for example, Ausubel et al., Hum Gene Ther. 2011 Apr; 22(4):489-97 or Allay et al., Hum Gene Ther. May 2011; 22(5):595-604.

[0155] A typical mammalian expression vector contains a promoter element that mediates the initiation of mRNA transcription, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements, such as an origin of replication, a drug resistance gene, and a regulator (as part of an inducible promoter), may also be included. The lac promoter is a typical inducible promoter useful in prokaryotic cells; it can be induced using the lactose analog isopropylthiol-bD-galactoside ("IPTG"). For recombinant expression and secretion, the polynucleotide of interest may be ligated, for example, between the PelB leader signal, which directs the recombinant protein in the periplasm, and gene III in a phagemid called pHEN4 (described in Ghahroudi et al., 1997, FEBS Letters 414:521-526). Additional elements may include enhancers, Kozak sequences, and intervening sequences flanking donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved using the early and late promoters from SV40, the long terminal repeats (LTRs) from retroviruses such as RSV, HTLV-1, and HIV-1, and the early promoter of cytomegalovirus (CMV). However, cellular elements (e.g., the human actin promoter) can also be used. Expression vectors suitable for use in the practice of the present invention include vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), and pBC12MI (ATCC 67109). Alternatively, recombinant (poly)peptides can be expressed in stable cell lines containing chromosomally integrated gene constructs. Cotransfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows for identification and isolation of transfected cells. Transfected nucleic acids can also be amplified to express large amounts of the encoded (poly)peptide. The dhfr (dihydrofolate reductase) marker is useful for developing cell lines that carry hundreds or thousands of copies of a gene of interest.Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al., 1991, Biochem J. 227:277-279; Bebbington et al., 1992, Bio / Technology 10:169-175). Using these markers, mammalian cells are grown in selective medium and the most resistant cells are selected. As noted above, expression vectors preferably contain at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin, or ampicillin resistance genes for culture in E. coli and other bacteria. For vector modification techniques, see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, Vol. 3. Generally, vectors may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in the host (e.g., antibiotic resistance), and one or more expression cassettes. Suitable origins of replication (ori) include, for example, Col E1, SV40 viral and M13 origins of replication.

[0156] Sequences to be inserted into vectors can be synthesized by standard methods or isolated from natural sources. Ligation of coding sequences to transcriptional regulatory elements and / or other amino acid coding sequences can be performed using established methods. Transcriptional regulatory elements (part of an expression cassette) ensuring expression in prokaryotic or eukaryotic cells are well known to those skilled in the art. These elements include regulatory sequences (e.g., translation initiation codon, promoter, enhancer, and / or insulator) ensuring transcription initiation, an internal ribosome entry site (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98(2001), 1471-1476), and optionally a poly(A) signal ensuring transcription termination and transcript stabilization. Additional regulatory elements may include transcriptional and translational enhancers, and / or naturally associated or heterologous promoter regions. Preferably, the nucleotide sequence defined in item (a) of the above preferred embodiment is operably linked to such expression control sequences enabling expression in prokaryotic or eukaryotic cells.

[0157] The host may be a prokaryotic or eukaryotic cell. Suitable eukaryotic hosts may be mammalian cells, amphibian cells, fish cells, insect cells, fungal cells, or plant cells. Representative bacterial cells include Escherichia coli, Streptomyces, and Salmonella typhimurium cells; representative fungal cells include yeast cells; and representative insect cells include Drosophila S2 and Spodoptera Sf9 cells. Preferably, the cell is a mammalian cell, such as a human cell. Mammalian host cells that can be used include human Hela, 293, H9, and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7, and CV1, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells. The cell may be part of a cell line, preferably a human cell line or a CHO cell line. Appropriate culture media and conditions for the above host cells are known in the art. The host is preferably a host cell, more preferably an isolated host cell. The host is also preferably a non-human host.

[0158] According to preferred embodiments of the first and second aspects of the present invention, the immunotherapy comprises the application of an immune checkpoint inhibitor, preferably an inhibitor of ErbB2, EGFR, CD20, PD-1, PDL-1, CTLA4, IDO1, LAG3, TIM3, TIM-4, CXCL9, CXCL13, TIGIT, BTLA, CD137, OX40, VISTA, B7-H7, CD27, GITR, the TGF-β signaling pathway, IL-15, PD-1 or PD-1L, preferably PD-1 and / or PD-1L.

[0159] The prior art has shown that the mRNA expression level or protein level of one or more selected from ErbB2, EGFR, CD20, CTLA4, IDO1, LAG3, TIM3, TIM-4, CXCL9, CXCL13, TIGIT, BTLA, CD137, OX40, VISTA, B7-H7, CD27, GITR, TGF-β signaling pathway, IL-15, PD-1, and PD-1L are involved in immune checkpoints. Therefore, the mRNA or protein of ErbB2, EGFR, CD20, CTLA4, IDO1, LAG3, TIM3, TIM-4, CXCL9, CXCL13, TIGIT, BTLA, CD137, OX40, VISTA, B7-H7, CD27, GITR, TGF-β signaling pathway, IL-15, PD-1, and PD-1L are targets for immune checkpoint inhibitors. Specific preferred examples of such immune checkpoint inhibitors are provided below.

[0160] According to more preferred embodiments of the first and second aspects of the present invention, the immune checkpoint inhibitor is selected from the group consisting of trastuzumab, cetuximab, rituximab, nivolumab, pembrolizumab, cemiplimab, atezolizumab, durvalumab, avelumab, ipilimumab, leratolimuab, LY3321367, MBF453, TSR-022, urelumab, PFZ-05082566, 1-7F9 (IPH2101), GSK2831781, MEDI16469, MEDI16383, MOXR0916, varilumab, TRX518, NKG2D ligand anti-tumor Fv fusion (preclinical development), galunisertib, ALT-803 (IL-15-IL-15α-Sushi-Fc fusion complex) epacadostat, IMP321, and JNJ-63723283.

[0161] Trastuzumab is a therapeutic antibody that binds to the HER2 receptor, thereby slowing down cell replication.

[0162] Cetuximab is an antibody directed against the epidermal growth factor receptor (EGFR) and is used to treat cancers such as metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer.

[0163] Rituximab is a chimeric monoclonal antibody directed against the protein CD20. It is used to treat autoimmune diseases and cancer.

[0164] Nivolumab (marketed as Opdivo) is an anti-PD-1 monoclonal antibody used to treat cancer. Pembrolizumab (formerly MK-3475 and lambrolizumab, known as Keytruda) and cemiplimab are also anti-PD-1 antibodies used to treat cancer.

[0165] Atezolizumab is an antibody against the protein programmed cell death-ligand 1 (PD-L1) and is used in cancer immunotherapy. Durvalumab and avelumab are additional antibodies against PD-L1 that are useful in cancer treatment.

[0166] Ipilimumab is a monoclonal antibody directed against CTLA-4. It is used to treat cancer, particularly melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, and metastatic hormone-refractory prostate cancer.

[0167] Leratolimab (BMS-986016) is an anti-LAG3 antibody designed for the treatment of melanoma.

[0168] LY3321367, MBF453, and TSR-022 are anti-HAVCR2 monoclonal antibodies that are effective in treating cancer.

[0169] Urelumab (BMS-663513 or anti-4-1BB antibody) and utomimurab (PF-05082566) are anti-CD137 antibodies that specifically bind to and activate CD137-expressing immune cells, thereby stimulating immune responses, particularly cytotoxic T cell responses against tumor cells.

[0170] IPH2101 is an anti-KIR(1-7F9) human monoclonal antibody developed for the treatment of patients with acute myeloid leukemia.

[0171] GSK2831781 is an anti-Lag3 antibody used to treat autoimmune diseases.

[0172] MEDI16469 is an anti-OX40 antibody used in immunotherapy.

[0173] MEDI16383 is a human OX40 fusion protein and is also used in immunotherapy.

[0174] MOXR0916 is an anti-Ox40 antibody used to treat solid tumors.

[0175] Varlilumab specifically binds to CD27 and is used to treat cancers such as advanced breast and ovarian cancer.

[0176] TRX518 is an antibody that blocks the interaction of glucocorticoid-induced TNF superfamily receptors (GITRs), and is effective in treating tumors.

[0177] Galunisertib is a small molecule inhibitor of TGF-β and is used as a cancer drug.

[0178] ALT-803 (IL-15-IL-15α-Sushi-Fc fusion complex) is an IL-15 superagonist complex containing an IL-15 mutant (IL-15N72D) fused to an IL-15 receptor α / IgG1 Fc fusion protein. ALT-803 can induce antigen-specific antitumor responses.

[0179] Epacadostat is a small molecule inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) and is used to treat cancer.

[0180] IMP321 (efthirazimod alfa) is a soluble version of LAG3 that is used to boost the immune response against tumors.

[0181] JNJ-63723283 is a monoclonal antibody directed against the negative immunoregulatory human cell surface receptor programmed cell death 1 protein (PD-1, PCDC-1) and has potential immune checkpoint inhibitory and antitumor activities. Upon administration, the anti-PD-1 monoclonal antibody JNJ-63723283 binds to PD-1 and blocks its interaction with its ligands, programmed cell death 1 ligand 1 (PD-L1, PD-1L1) and PD-1 ligand 2 (PD-L2, PD-1L2). Inhibition of ligand binding prevents PD-1-mediated signaling, leading to both T cell activation and the induction of T cell-mediated immune responses against tumor cells.

[0182] According to another more preferred embodiment of the first and second aspects of the present invention, the antihormonal therapy comprises an antiestrogen therapy and / or an antiprogesterone and / or an antiandrogen therapy.

[0183] Estrogen (or estrogens) is the primary female hormone. It is normally responsible for the development and regulation of the female reproductive system and secondary sexual characteristics. Progesterone (P4) is an endogenous steroid involved in the menstrual cycle, pregnancy, and embryogenesis in humans and other species. Androgens are the primary male hormones. It is normally responsible for the development and regulation of the male reproductive system and secondary sexual characteristics. Estrogen, progesterone, and androgens are all hormones involved in tumorigenesis. In particular, estrogen-, androgen-, or progesterone-receptor-positive cancers are treated with drugs that suppress the production of or block the action of these hormones in the body.

[0184] According to a further preferred embodiment of the first and second aspects of the present invention, the tumor is a cancer, preferably a carcinoma, most preferably a bladder cancer.

[0185] In the following examples, expression levels of HLA-G, L, H and J genes or proteins were determined in samples from bladder cancer patients.

[0186] In the case of bladder cancer or neoplastic lesions thereof, the use preferably comprises infusion therapy into the bladder. Administration as infusion therapy is particularly contemplated for the combination of immunological, chemotherapeutic, antihormonal, or anti-tyrosine kinase compounds together with anti-HLA agents as described as part of this application.

[0187] In a third aspect, the present invention relates to a method for preparing a kit for predicting whether a subject with a tumor will respond to a tumor treatment selected from (i) immunotherapy, (ii) chemotherapy, (iii) anti-hormonal therapy, and (iv) anti-tyrosine kinase therapy, and instructions for using the kit, which method comprises combining the kit with means for detecting the level of at least one nucleic acid molecule as defined herein above and / or at least one protein or peptide as defined herein above.

[0188] The kits prepared embody the necessary means for practicing the invention of the present invention in kit format, and for this reason the definitions and preferred embodiments provided herein above in relation to the first aspect of the invention are equally applicable to the kits of the present invention.

[0189] The means for detecting and / or quantifying nucleic acid molecules as exemplified as part of the present invention may be one or more of the primers and probes as shown in Table 1 below. However, any detection module capable of quantifying nucleic acids, such as arrays, NGS, or other molecular systems, is suitable as part of the present invention. The means for detecting proteins or peptides are preferably antibodies and / or protein binders and / or peptide binders (?) as described hereinabove. For detection and / or quantification, the antibodies and / or protein binders and / or peptide binders (?) may be labeled, for example, with fluorescent dyes or radioactive labels. Examples of fluorescent dyes and radioactive labels are also described hereinabove.

[0190] The various components of the kit may be packaged in one or more containers, such as one or more vials.The vial may contain preservatives or buffers for storage in addition to the above-mentioned components.The kit may also include instructions on how to use the kit, preferably informing how to use the components of the kit to predict whether a subject with tumor will respond to the tumor treatment defined herein.

[0191] In a preferred embodiment of the third aspect of the present invention, the means comprises a primer pair and, optionally, a hydrolysis probe or other labeled primer or probe detection approach for target sequence quantification known to those skilled in the art, such as a scorpion primer, a FRET probe, or a molecular beacon used for sequence-specific detection of at least one nucleic acid molecule as defined herein above.

[0192] Primer pairs and optionally hydrolysis probes are generally used in real-time quantitative PCR a described hereinabove for the specific detection of at least one nucleic acid molecule as described hereinabove. Preferred primer pairs and hydrolysis probes are shown in Table 1 below.

[0193] A hydrolysis probe refers to a sequence-specific DNA probe consisting of an oligonucleotide labeled with a fluorescent reporter that can be detected only after hybridization between the probe and its complementary sequence (e.g., a TaqMan probe). More specifically, a hydrolysis probe is a dual-labeled oligonucleotide. The 5' end of the oligonucleotide is labeled with a fluorescent reporter molecule, while the 3' end is labeled with a quencher molecule. The probe sequence is specific to a region of interest in the amplified target molecule. The length of the hydrolysis probe sequence is designed to position the 5' fluorophore and 3' quencher in sufficient proximity to suppress fluorescence. During the extension phase of the PCR cycle, DNA polymerase synthesizes a complementary strand downstream of the PCR primer. When extension reaches the bound hydrolysis probe, the 5'-3' exonuclease activity of the DNA polymerase degrades the hydrolysis probe. Cleavage of the hydrolysis probe separates the fluorescent reporter molecule from the remainder of the probe, allowing the reporter molecule to fluoresce.

[0194] With respect to the embodiments characterized herein, and in particular the claims, it is intended that each embodiment referred to in a dependent claim may be combined with each embodiment (independent or dependent) of each claim. With respect to the embodiments characterized herein, and in particular the claims, it is intended that each embodiment referred to in a dependent claim may be combined with each embodiment (independent or dependent) of each claim. For example, if independent claim 1 recites three alternatives A, B, and C, dependent claim 2 recites three alternatives D, E, and F, and claim 3 depends from claims 1 and 2 and recites three alternatives G, H, and I, it will be understood that the specification, unless otherwise stated, expressly discloses embodiments corresponding to the combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I;

[0195] Similarly, it is understood that even if an independent and / or dependent claim does not recite alternatives, if a dependent claim refers back to multiple preceding claims, the combination of subject matter covered thereby will be considered explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 which refers back to claim 1, and dependent claim 3 which refers back to both claims 2 and 1, the combination of subject matter of claims 3 and 1 will be clearly and unambiguously disclosed, as will the combination of subject matter of claims 3, 2, and 1. If there is a further dependent claim 4 which refers back to any one of claims 1-3, the combinations of subject matter of claims 4 and 1, claims 4, 2, and 1, claims 4, 3, and 1, and claims 4, 3, 2, and 1 will be clearly and unambiguously disclosed. [Brief explanation of the drawings]

[0196] [Figure 1] Consort diagram of the advanced or metastatic urothelial carcinoma cohort. After excluding formalin-fixed, paraffin-embedded (FFPE) blocks with insufficient and / or lymph node tissue, tissue from 55 patients was available for analysis.

[0197] [Figure 2] Distribution of luminal and basal subtype markers, checkpoint target genes, and FGFR1-4 gene expression data measured by RT-qPCR from tissues of patients with muscle-invasive bladder cancer.

[0198] [Figure 3] Quantification of HLA-G, -H, -J, -L, and -V mRNA expression by RT-qPCR of different exon regions. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene. A higher 40-DCT value indicated higher gene expression.

[0199] [Figure 4] Intergenic Spearman correlations of HLA-G luminal and basal subtype markers, checkpoint target genes, FGFR1-4 genes, and exon 8 mRNA expression analysis were measured by RT-qPCR from tissues of patients with muscle-invasive bladder cancer.

[0200] [Figure 5] Spearman correlations between genes in luminal and basal subtype markers, checkpoint target genes, FGFR1-4 genes, and HLA-G exons 3-6 mRNA expression analysis were measured using RT-qPCR in tissues from patients with muscle-invasive bladder cancer (n=61).

[0201] [Figure 6] Correlation of HLA-H mRNA expression with FGFR receptors, PD-1, PD-L1, and basal and luminal cell type markers in patients with urothelial carcinoma.

[0202] [Figure 7] Cluster analysis of HLA genes with immunohistologically and molecularly assessed urothelial markers. Red highlights high gene expression, while blue indicates low gene expression. Genes are depicted to the left of the cluster analysis. Each column represents a patient-derived cystectomy UBC sample.

[0203] [Figure 8] Cluster analysis of PD-1, PD-L1, and FGF receptor genes with basal and luminal markers. Red highlights high gene expression, while blue indicates low gene expression. Genes are depicted to the left of the cluster analysis. Each column represents a patient-derived cystectomy UBC sample.

[0204] [Figure 9] Kaplan-Meier plot showing disease-specific survival (DSS) probability from patients with muscle-invasive bladder cancer stratified by HLA-G exon 8 expression quantified by RT-qPCR. Relative mRNA expression is measured by the 40-DCT method using CALM2 as the reference gene.

[0205] [Figure 10] Kaplan-Meier plot showing disease-specific survival (DSS) probability from patients (n=57) with locally advanced or metastatic muscle-invasive bladder cancer (UBC) stratified by HLA-G exon 8 expression quantified by RT-qPCR. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0206] [Figure 11] Kaplan-Meier plot showing disease-specific survival (DSS) probability from patients (n=57) with locally advanced or metastatic muscle-invasive bladder cancer (UBC) stratified by HLA-G exon 3 expression quantified by RT-qPCR. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0207] [Figure 12]Kaplan-Meier plot showing disease-specific survival (DSS) probability from patients (n=57) with locally advanced or metastatic muscle-invasive bladder cancer (UBC) stratified by HLA-J exon 4 / 5 expression quantified by RT-qPCR. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0208] [Figure 13] Kaplan-Meier plot showing disease-specific survival (DSS) probability from patients (n=20) with muscle-invasive bladder cancer and locally advanced or node-positive UBC, stratified by HLA-G exon 8 expression quantified by RT-qPCR. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0209] [Figure 14] Kaplan-Meier plot showing disease-specific survival (DSS) probability from patients (n=20) with muscle-invasive bladder cancer and locally advanced or node-positive UBC, stratified by HLA-G exon 3 expression quantified by RT-qPCR. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0210] [Figure 15] Kaplan-Meier plot showing disease-specific survival (DSS) probability from patients (n=19) with muscle-invasive bladder cancer and locally advanced or node-positive UBC, stratified by HLA-L exon 7 expression quantified by RT-qPCR. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0211] [Figure 16]Kaplan-Meier plot showing the disease-specific survival (DSS) probability of lung, bone, or liver metastasis in patients with muscle-invasive bladder cancer (n=17) stratified by HLA-L exon 7 expression quantified by RT-qPCR. Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0212] [Figure 17] Kaplan-Meier plot showing disease-specific survival (DSS) probability for patients with muscle-invasive bladder cancer who developed lung and liver metastases, stratified by HLA-H exon 2 / 3 expression quantified by RT-qPCR assay (n=17). Relative mRNA expression was measured by the 40-DCT method using CALM2 as the reference gene.

[0213] The present invention will now be illustrated by examples. [Example]

[0214] Example 1: HLA profiling in chemotherapy-resistant advanced urothelial cancer Transurethral resection (TUR) biopsies and cystectomy specimens from primary tumors refractory to chemotherapy and subsequently receiving first- or second-line immuno-oncology ("IO") treatment with PD-1 and PD-L1 checkpoint inhibitor drugs (i.e., atezolizumab, nivolumab, and pembrolizumab) were analyzed for HLA expression and correlated with histopathological and molecular parameters as well as response to IO treatment and post-IO disease-specific survival.

[0215] Between 2016 and 2018, 72 patients newly diagnosed with histologically confirmed urothelial carcinoma, including bladder cancer and upper tract urothelial carcinoma, were enrolled in this study. Nivolumab, pembrolizumab, and atezumab were administered as first-, second-, and third-line monotherapy according to approved indications. All hematoxylin-eosin (HE)-stained tumor tissue sections from the sample population were evaluated and classified according to the UICC TNM classification (2017) by two uropathologists. Rare histologic variants were classified according to the World Health Organization (WHO) classification of genitourinary tumors (2016). After central histopathological review, 18 tissues were excluded due to insufficient tumor material or non-urothelial carcinoma. Five patients only had lymph node tissue available and were therefore excluded from the primary analysis of the prognostic and / or predictive efficacy of HLA gene expression (Figure 1; see consort diagram). In addition, on October 16, 2018, in conjunction with the parallel FDA submission, the clinical trial data database was closed.

[0216] For mRNA detection, RNA was extracted from FFPE tissues from TUR biopsies, cystectomies, and corresponding mapping bladder tissues using a commercially available kit (Xtract, Stratifyer). For each reaction, 2.5 μl of total RNA extracted from FFPE sections was mixed with 2.5 μl of assay mix, 2.5 μl of enzyme mix, and 2.5 μl of water in one well of a 96-well optical reaction plate. PCR reactions were measured using a Versant kPCR Cycler (Siemens) or Light Cycler 480 (Roche) according to the manufacturer's instructions under the appropriate conditions (5 min at 50°C, 1 cycle; 20 s at 95°C, 1 cycle; 15 s at 95°C, 1 min at 60°C, 40 cycles). Relative mRNA expression was correlated with response to IO treatment, as determined based on RECIST (Response Evaluation Criteria in Solid Tumors) criteria assessed at each site, and with disease-specific survival, as determined from the start of IO treatment until cancer-specific death. Partition testing using biostatistical JMP SAS 9.0.0 (SAS, Cary, North Carolina, USA) was performed to assess possible differences in response to IO treatment.

[0217] For detailed analysis of gene expression by RT-qPCR, primers flanking the site of interest and a fluorescently labeled probe hybridizing in the middle were utilized. Target-specific primers and probes were selected using the NCBI primer design tool (www.ncbi.nlm.nih.go). RNA-specific primer / probe sequences were used to enable RNA-specific measurement by positioning the primer / probe sequences across exon / exon boundaries. Furthermore, primers / probes were selected to avoid binding to sequence regions with known single nucleotide polymorphisms (SNPs). When multiple isoforms of the same gene exist, primers were selected to adequately amplify all relevant or selected splice variants. All primer pairs were checked for specificity by conventional PCR reactions. After further primer / probe optimization, the primers and probes listed in Table 1 provided the best results. These primers / probes are superior to primers / probes known from the prior art, for example, in terms of specificity and amplification efficiency. To normalize the amount of sample RNA, CALM2 was selected as a reference gene because it was not differentially regulated in the analyzed samples. Paired specimens with low RNA content (i.e., raw CALM2 C values ​​< 22) due to pre-treatment raw biopsies or post-treatment resections were excluded. TaqMan® validation experiments were performed and showed that the amplification efficiencies of target and control were approximately equal, which is a prerequisite for relative quantification of gene expression by the comparative ΔC method. Table 1: Primers and probes used for HLA mRNA quantification [Table 1]

[0218] Determination of luminal and basal subtypes in the UC cohort by RT-qPCR revealed a similarly wide dynamic range of KRT5 and KRT20 mRNA, with 40-DCT values ​​ranging from 19 to 48. PD-1 and PD-L1 mRNA expression ranged from 19 to 41. The dynamic range of FGFR genes differed significantly within the FGFR family: relative FGFR1 mRNA ranged from 29 to 37, FGFR2 mRNA from 19 to 39, FGFR3 mRNA from 19 to 43, and FGFR4 mRNA from 19 to 36 (Figure 2).

[0219] In addition to mRNA expression analysis of luminal and basal markers, PD-1, PD-L1, and FGFR family, expression profiles of classical HLA as well as exon expression of HLA genes and pseudogenes have been performed (Figure 3).

[0220] Nonparametric Spearman correlations of FGFR genes 1-4, PD-1, PD-L1, basal and luminal markers, and HLA primer sets for exon 8 reveal a strong and significant correlation of PD-1 (Spearman rho 0.2904, p=0.0232) in urothelial tumors expressing HLA-G exon 8. In addition to PD-1, high FGFR1 (Spearman rho 0.2724, p=0.0337) expression is also associated with HLA-G exon 8 expression. However, no significant correlation was observed with any HLA for luminal urothelial carcinoma (Figure 4).

[0221] Surprisingly, Spearman correlations of luminal and basal subtype markers, checkpoint target genes, and FGFR1-4 genes with the remaining HLA-G exons reveal a strong significant association with all HLA-G exons, as indicated by similarly high co-expression of HLA-G with the checkpoint marker PD-1 (PD-1 at the 3' end of exon 3: Spearman rho 0.2768, p=0.0308; PD-1 at exon 4: Spearman rho 0.2768, p=0.0308; PD-1 at exon 5: Spearman rho 0.3220, p=0.0114; PD-1 at exon 6: Spearman rho 0.3805, p=0.0025) (Figure 5). These interesting findings were confirmed only for HLA-G exon 5 relative to PD-L1 (Spearman rho 0.2695, p=0.0357). For the 3' end of exon 3, highly significant correlations were also observed for FGF receptors 3 (Spearman rho 0.2990, p=0.0193) and 4 (Spearman rho 0.2703, p=0.0352). This association could not be determined for exon 4, although exon 4 expression was associated with high mRNA expression of the basal cell marker KRT5 (Spearman rho 0.2931, p=0.0219). The basal marker KRT5 (Spearman rho 0.3526, p=0.0053) also showed a significant correlation with HLA-G exon 6. Furthermore, FGF receptors 3 (spearman rho 0.2972, p=0.0200) and 4 (spearman rho 0.3552, p=0.0050) also showed a significant correlation with HLA-G exon 6 in mRNA expression.

[0222] Furthermore, nonparametric Spearman correlation analysis of HLA-H with luminal and basal subtype markers, PD1, PD-L1, and FGFR1-4 genes was also performed (Figure 6). However, no correlation was found between HLA-H expression and luminal or basal markers or checkpoint inhibitors.

[0223] Furthermore, we performed a cluster analysis of FGF receptor genes with PD-1, PD-L1, and basal and luminal markers. The results revealed that PD-1 and PD-L1 expression was more prevalent in basal urothelial carcinoma subtypes. Furthermore, FGFR1 mRNA was also more highly expressed in cytokeratin 5-positive tumors, whereas FGF receptors 2-4 were more highly expressed in luminal urothelial carcinoma subtypes.

[0224] Cluster analysis of HLA genes using immunohistochemical evaluation of urothelial cancer markers revealed that HLA-G expression occurs primarily in the basal urothelial carcinoma subtype (IHC_ST_CK5), which can be further divided by HLA mRNA expression. Some basal tumor subtypes exhibit high levels of HLA-G expression (Figure 7A). Finally, cluster analysis of HLA exon 8 expression with immunohistochemical cell and subclassification markers (CK5, CD44, CK20, FOXA1, GATA3), PD-1, and PD-L1, as well as HLA-H expression, was performed. The analysis revealed that HLA-G exon 5 and exon 8 expression, HLA-H expression, and PD-1 and PD-L1 expression could be assigned to the basal subtype. However, HLA-G, HLA-H, PD-1, and PD-L1 expression were also observed in luminal urothelial tumors (Figure 7B). Additionally, in silico promoter analysis revealed several estrogen response elements (EREs) as well as progesterone response elements (PREs) in the HLA-G gene. This suggests that HLA-G expression may be important not only in basal cancer but also in luminal cancer subtypes. Because mRNA exon and exon / exon junction expression varies within luminal and basal cancer subtypes, single-exon expression and exon / exon junction analysis as stratification tools should be applied to both basal and luminal urothelial cancer subtypes. Surprisingly, in silico analysis of the HLA-H promoter region also revealed several estrogen response elements. Together with cluster analysis, this highlights the important role of the pseudogene HLA-H as a further stratification tool in urothelial cancer. As shown in Figure 8, cluster analysis of FGF receptor genes has been further performed using PD-1, PD-L1, and basal and luminal markers. PD-1 and PD-L1 expression was found to be predominantly in the basal urothelial tumor subtype. Furthermore, FGFR1 mRNA is also more highly expressed in cytokeratin 5-positive tumors, whereas FGF receptors 2-4 are more highly expressed in luminal urothelial carcinoma subtypes.This indicates the representativeness of the cohort analyzed for HLA gene interactions. Example 2: Exon expression of various HLA genes in urothelial carcinoma (DSS) as a marker of disease-specific survival

[0225] The predictive value of HLA gene expression in bladder cancer tissue from patients with advanced or metastatic urothelial carcinoma receiving immuno-oncology checkpoint therapy (IO therapy) (i.e., atezolizumab, nivolumab, or pembrolizumab) was evaluated based on detailed clinical follow-up data (i.e., WHO grading, primary metastatic site, initiation of IO therapy, time of cancer-specific death or last contact date). Immuno-oncology disease-specific survival was calculated from the initiation of IO therapy to cancer-specific death or last contact date, and to censoring, respectively.

[0226] The association of altered HLA-G mRNA expression with disease-specific survival (DSS) in patients with urothelial carcinoma was analyzed, as shown in Figure 9. When all available tissues, including metastatic lymph nodes, were taken into account (n = 60), Kaplan-Meier analysis revealed that increased HLA-G exon 8 mRNA expression above a 40-DCT value of 28.43 indicated worse disease-specific survival (p = 0.0102).

[0227] However, to exclude non-cancer-related effects of HLA expression by non-tumor-associated lymphocytes in lymph nodes, metastatic lymph node tissues were excluded from subsequent analysis, leaving 57 samples for survival analysis as shown in Figure 1. As shown in Figure 10, high HLA-G exon 8 mRNA expression (>=28.43) was significantly associated with poor disease-specific survival, with HLA-G exon 8-positive patients having a 35% survival probability after 2 years, whereas HLA-G exon 8-negative patients had a 65% survival probability after 2 years (p=0.298).

[0228] Because the HLA-G-specific exon 8 region examined is not translated into protein, further confirmation analysis is being performed by determining the HLA-G exon 3 region, which is a part of the translation region close to the HLA-G signal peptide. As shown in Figure 11, high HLA-G exon 3 mRNA expression (>=28.23) was significantly associated with poor disease-specific survival in HLA-G exon 3-positive patients, who had a 30% chance of survival after 2 years, while HLA-G exon 8-negative patients had a 70% chance of survival after 2 years (p=0.0156).

[0229] Next, we analyzed the prognostic value of other HLA genes in the overall population, with a particular focus on currently classified "pseudogenes," as exemplified by HLA-J, H, V, or L. As shown in Figure 12, high HLA-J exon 4 / 5 mRNA expression (>=25.08) was associated with poor disease-specific survival in 36 HLA-J exon 4 / 5-positive patients, who had a 35% chance of survival after two years, while 19 HLA-J exon 4 / 5-negative patients had a 70% chance of survival after two years.

[0230] To further elucidate the relationship of HLA-G expression to survival after IO treatment, we analyzed only primary tumor tissues and further specified the analysis by taking into account the site of primary metastasis. This is based on the initial finding that the efficacy of IO therapy varies depending on the site of metastasis, for example, visceral metastasis to the liver is less effective. This is likely due to the exclusion of PD1-positive T cells from the liver in patients with metastatic urothelial carcinoma, independent of classical checkpoint mechanisms (Eckstein M, Sikic D, Strissel PL, Erlmeier F. Evolution of PD-1 and PD-L1 Gene and Protein Expression in Primary Tumors and Corresponding Liver Metastases of Metastatic Bladder Cancer. Eur Urology 2018.). Therefore, patients were classified by the first presentation of metastases with local progression, locoregional lymph nodes, or extraregional retroperitoneal lymph nodes were classified as 0 or 0.5, respectively, while bone, liver, lung, lung, and bone or lung and liver dissemination were classified with increasing indices (1, 2, 3, 4, 5, respectively). For this analysis, 54 datasets from primary tumor tissues with sufficient clinical data and primary tumor tissue material were available; 19 patients had local progression or lymph node metastases, 17 patients had initial bone or liver metastases, and 18 patients had metastases with lung involvement as the sole site or in combination with bone or liver lesions; all of them (74%) had been treated with IO drugs and primarily with first-line therapy.

[0231] In patients with urothelial bladder cancer who had advanced or node-positive disease, high HLA-G mRNA expression was associated with poorer disease-specific survival (DSS) from the start of IO treatment to cancer-specific death. As illustrated in Figure 13, high HLA-G exon 8 mRNA expression (>=28.545) was associated with significantly poorer outcomes in 11 HLA-G exon 8-positive patients, who had only a 25% chance of survival after two years, while nine HLA-G exon 8-negative patients had a 100% chance of survival after two years (p=0.0068).

[0232] Because the HLA-G specific exon 8 region examined is not translated into protein, further confirmatory analysis is being performed by determining the exon 3 region of HLA-G, which is part of the translated region close to the signal peptide of HLA-G.

[0233] As shown in Figure 14, high HLA-G exon 3 mRNA expression (>=26.535) was associated with significantly poorer disease-specific survival (p=0.0013), with the 10 HLA-G exon 3-positive patients having only a 15% chance of survival after two years, compared with a 100% chance of survival after two years for the 10 HLA-G exon 3-negative patients. This is similar to the predictive value of HLA-G exon 8 mRNA expression, further demonstrating that HLA-G expression is associated with worse outcomes in advanced, node-positive patients despite checkpoint therapy inhibiting IO drugs.

[0234] We next examined whether other HLA genes, classical or non-classical, known genes, or still pseudogenes, predict IO outcome in urothelial bladder cancer.

[0235] As an example, an assay was developed to quantify the mRNA of the "pseudogene" HLA-L in a region similar to the exon 8 region of HLA-G at the 3' end of the "pseudogene." As shown in Figure 15, high HLA-L exon 7 mRNA expression (>=29.89) was associated with poor disease-specific survival, with 10 HLA-L exon 7-positive patients having a 2-year survival rate of only 30%, compared with 10 HLA-L exon 7-negative patients having an 80% 2-year survival rate. However, this association did not reach statistical significance using the log-rank test due to the crossing of survival curves. It can be argued that the log-rank test may not be effective in this case because, on the one hand, the sample size was still small, and, on the other hand, very early cases after 1 month may have an excessive influence on the p-value and therefore may not be optimal for assessing risk.

[0236] This indicates that not only HLA-G but also other HLA genes and / or pseudogenes are associated with worse outcomes despite checkpoint-based therapy inhibiting IO drugs, suggesting that from a therapeutic perspective, not only HLA-G but also other HLA-genes and / or pseudogenes should be targeted simultaneously to avoid or destroy resistance to IO drugs.

[0237] Next, we investigated whether HLA genes could predict tumor biology even in the most aggressive cases, especially when multiple organs, including the lungs, had already metastasized on CT scans at diagnosis before IO treatment. As shown in Figure 16, high HLA-L exon 7 mRNA expression (>=30.195) was associated with poor disease-specific survival in 16 HLA-L exon 7-positive patients, with a 1-year survival rate of only 0%, while the 11 HLA-L exon 7-negative patients had a 1-year survival rate of 70% (p=0.0418).

[0238] In this highly metastatic setting, other "pseudogenes," as exemplified by HLA-H, were also significant. As shown in Figure 17, high HLA-H exon 2 / 3 mRNA expression (>= 29.95) was associated with poor disease-specific survival in HLA-H exon 2 / 3 mRNA-positive patients, who had only a 30% chance of survival after one year, whereas HLA-H exon 2 / 3 mRNA-negative patients had an 80% chance of survival after one year.

Claims

1. A method for predicting whether a subject with bladder cancer will respond to immune checkpoint inhibitor therapy, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody; (A) determining the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from the subject, wherein the at least one nucleic acid molecule comprises: (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 2 and 4-6; (b) a nucleic acid molecule consisting of the nucleotide sequence of any one of SEQ ID NOs: 8 and 10-12; (c) a nucleic acid molecule encoding a polypeptide that is at least 90% identical, or at least 95% identical, to the amino acid sequence of (a); (d) a nucleic acid molecule consisting of a nucleotide sequence that is at least 95% identical, at least 96% identical, or at least 98% identical to the nucleotide sequence of (b); (e) a nucleic acid molecule consisting of a nucleotide sequence degenerate to the nucleic acid molecule of (d); (f) a nucleic acid molecule consisting of a fragment of the nucleic acid molecule of any one of (a) to (e), wherein the fragment comprises at least 250 nucleotides, at least 300 nucleotides, at least 450 nucleotides, or at least 600 nucleotides; and (g) a nucleic acid molecule corresponding to any one of (a) to (f), wherein T is replaced by U. Selected from, and determining that the at least one protein or peptide consists of an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2 and 4-6 and is selected from proteins or peptides or fragments thereof that exhibit immunosuppressive function; and (B) comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who responded to immune checkpoint inhibitor therapy, or with a corresponding predetermined standard, wherein an increased level of (A) compared to the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who responded to immune checkpoint inhibitor therapy, or with a corresponding predetermined standard, indicates that the subject does not respond to the tumor therapy, and a substantially similar or decreased level of (A) compared to the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who responded to immune checkpoint inhibitor therapy, or with a corresponding predetermined standard, indicates that the subject responds to the tumor therapy; or (B') comparing the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who did not respond to immune checkpoint inhibitor therapy, or with a corresponding predetermined standard, wherein a decrease in the level of (A) compared to the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who did not respond to immune checkpoint inhibitor therapy, or with a corresponding predetermined standard, indicates that the subject is responsive to the tumor therapy, and a decrease in the level of (A) compared to the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who did not respond to immune checkpoint inhibitor therapy, or with a corresponding predetermined standard, indicates that the subject is not responsive to the tumor therapy. A method comprising:

2. The method of claim 1, wherein any one of SEQ ID NOs: 2 and 4 to 6 is SEQ ID NO: 5 or 6, and any one of SEQ ID NOs: 8 and 10 to 12 is SEQ ID NO: 11 or 12.

3. 3. The method of claim 1 or 2, further comprising determining the mRNA expression level or protein level of one or more selected from ErbB2, EGFR, CD20, CTLA4, IDO1, LAG3, TIM3, TIM-4, CXCL9, CXCL13, TIGIT, BTLA, CD137, OX40, VISTA, B7-H7, CD27, GITR, TGF-β signaling pathway, IL-15, PD-1, and PD-1L.

4. 3. The method of claim 1 or 2, further comprising determining the mRNA expression level or protein level of one or more selected from PD-1 and PD-1L.

5. A kit for a method for predicting whether a subject with bladder cancer according to claim 1 or 2 will respond to immune checkpoint inhibitor therapy, comprising: The method comprises combining a means for detecting the level of at least one nucleic acid molecule described in claim 1 or 2 and / or at least one protein or peptide described in claim 1 or 2 with instructions for use of the kit.

6. 6. The kit of claim 5, wherein said means comprises a primer pair and optionally a hydrolysis probe used for the specific detection of at least one nucleic acid molecule of claim 1.

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