Hla-h in medicine and diagnostics

Abstract

To provide a method for detecting, in a sample obtained from a subject, a nucleic acid molecule and / or a protein having a specific sequence, wherein the presence thereof indicates that the subject has a tumor.SOLUTION: The present invention provides a method for detecting the presence of a nucleic acid molecule having a specific amino acid sequence, a nucleic acid molecule encoding a polypeptide having at least 95% identity to the specific amino acid sequence, a nucleic acid molecule having a specific nucleotide sequence, a nucleotide sequence having at least 95% identity to the specific nucleotide sequence, a nucleic acid molecule comprising a degenerate nucleotide sequence with respect to a nucleic acid molecule consisting of the specific nucleotide sequence, a fragment of any one of the nucleic acid molecules as described above, the fragment comprising at least 250 nucleotides, a nucleic acid molecule as described in any of the foregoing in which T is replaced with U, or a protein or peptide encoded by the nucleic acid molecule.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to nucleic acid molecules, vectors, host cells, or proteins or peptides, or combinations thereof, for use as immunosuppressants, as tumor vaccines, or as pregnancy promoters, wherein (I) the nucleic acid molecule is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or (b) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 2; or (c) a nucleic acid molecule encoding a polypeptide that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, and most preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 1; or (d) a nucleic acid molecule consisting of a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, and most preferably at least 95% identical to the nucleotide sequence of SEQ ID NO: 2; or (e) a nucleic acid molecule consisting of a degenerate nucleotide sequence relative to the nucleic acid molecule of (d); or (f) a fragment of any one of the nucleic acid molecules 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; or (g) a nucleic acid molecule corresponding to any one of the nucleic acid molecules of (a) to (f) in which T is replaced by U; (II) the vector contains the nucleic acid molecule of (I), (III) the host cell is transformed, transduced or transfected with the vector of (II), and (IV) the protein or peptide is encoded by the nucleic acid molecule of (I).

Background Art

[0002] Numerous documents are cited in this specification, including patent applications and manufacturer manuals. The disclosures of these documents are not considered relevant to the patentability of the present invention, but are hereby incorporated by reference in their entirety. More specifically, all reference documents are incorporated by reference to the same extent as if each individual document had been specifically and individually indicated to be incorporated by reference.

[0003] The human leukocyte antigen (HLA) system or complex is a gene complex that encodes the major histocompatibility complex (MHC) proteins in humans. These cell surface proteins are responsible for regulating the human immune system. The HLA gene complex is located 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] There are three major MHC class I genes in humans known as HLA-A, HLA-B, and HLA-C. The proteins produced from these genes are present on the surface of almost all cells. At the cell surface, these proteins bind to protein fragments (peptides) exported from within the cell. MHC class I proteins present these peptides to the immune system. When the immune system recognizes foreign peptides (such as those from viruses or bacteria), it responds by triggering the infected cell to self-destruct.

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

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

[0007] The HLA gene has many possible variations, enabling each person's immune system to respond to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each given a specific number (such as HLA-B27). Closely related alleles are grouped together. For example, at least 40 very similar alleles are subtypes of HLA-B27. These subtypes are named HLA-B*2701 to HLA-B*2743.

[0008] Over 100 diseases are associated with different alleles of the HLA gene. 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, it is often unclear what role the HLA gene plays in the development risk of these diseases.

[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 have a positive correlation with poor clinical outcomes in cancer.

[0010] The HLA class I region has also been reported to contain class I pseudogenes as well as gene fragments (Hughes, Mol Biol Evol.1995 Mar; 12(2):247-58). For example, HLA-H, J, K, and L are classified as class I pseudogenes, and HLA-N, S, and X are classified as gene fragments.

[0011] Therefore, the human leukocyte antigen (HLA) gene has a long research history as an important target in biomedical science and therapy. However, considering the clinical importance of the HLA system, it is necessary to focus on the HLA gene and, in particular, to focus research on identifying further targets for biomedical and therapeutic applications based on the HLA system. This need is addressed by the present invention. In connection with the present invention, surprisingly, HLA-H has been found to be a target for the treatment and detection of diseases, particularly by activating or inhibiting the activity of HLA-H.

Summary of the Invention

[0012] Accordingly, the present invention relates to a nucleic acid molecule, a vector, a host cell, or a protein or peptide, or a combination thereof, for use as an immunosuppressive agent, as a tumor vaccine, or as a pregnancy promoter, wherein (I) the nucleic acid molecule is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or (b) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 2; or (c) a nucleic acid molecule encoding a polypeptide that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, and most preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 1; or (d) a nucleic acid molecule consisting of a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, and most preferably at least 95% identical to the nucleotide sequence of SEQ ID NO: 2; or (e) a nucleic acid molecule consisting of a degenerate nucleotide sequence relative to the nucleic acid molecule of (d); or (f) a fragment of any one of the nucleic acid molecules 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; or (g) a nucleic acid molecule corresponding to any one of the nucleic acid molecules of (a) to (f) in which T is replaced by U; (II) the vector contains the nucleic acid molecule of (I), (III) the host cell is transformed, transduced or transfected with the vector of (II), and (IV) the protein or peptide is encoded by the nucleic acid molecule of (I).

[0013] A first aspect of the present invention likewise relates to nucleic acid molecules, vectors, host cells, or proteins or peptides, or combinations thereof, for use as immunosuppressive agents, as tumor vaccines, or as pregnancy promoters, wherein (I) the nucleic acid molecule is: (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 54; or (b) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 2; or (c) a nucleic acid molecule encoding a polypeptide that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 1 or 54; or (d) a nucleic acid molecule consisting of a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the nucleotide sequence of SEQ ID NO: 2; or (e) a nucleic acid molecule consisting of a degenerate nucleotide sequence relative to the nucleic acid molecule of (d); or (f) a fragment of any one of the nucleic acid molecules of (a) to (e), wherein the fragment comprises at least 250 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, most preferably at least 600 nucleotides; or (g) a nucleic acid molecule corresponding to any one of the nucleic acid molecules of (a) to (f) in which T is replaced by U; (II) the vector contains the nucleic acid molecule of (I); (III) the host cell is transformed, transduced or transfected with the vector of (II); and (IV) the protein or peptide is encoded by the nucleic acid molecule of (I).

[0014] As used herein, the term "nucleic acid molecule" includes DNA such as cDNA, or double-stranded or single-stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) means any strand or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C), and thymine (T), which are linked together on a deoxyribose sugar backbone and are called nucleotide bases. DNA can have a single strand of nucleotide bases or two complementary strands that form a double helix structure. "RNA" (ribonucleic acid) means any strand or sequence of the chemical building blocks that make up adenine (A), guanine (G), cytosine (C), and uracil (U), which are called nucleotide bases and are linked together on a ribose sugar backbone. RNA typically has a single strand of nucleotide bases, such as mRNA. Also included are single-stranded 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 with analogs of one or more naturally occurring nucleotides, and internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules (hereinafter also referred to as polynucleotides) may include one or more additional covalently attached moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridines, psoralens, etc.), chelators (e.g., metals, radioactive metals, iron, oxidized metals, etc.), and alkylators. Polynucleotides can be derivatized by the formation of methyl or ethyl phosphotriester or alkyl phosphoramidate linkages. Further included are nucleic acid mimetic molecules known in the art, such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers.Examples of such nucleic acid mimetic molecules or nucleic acid derivatives include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA), and locked nucleic acid (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 that includes the information used by cellular machinery to make proteins and / or polypeptides. The nucleic acid molecules according to the present invention may further include a promoter, an enhancer, a response element, a signal sequence, a polyadenylation sequence, an intron, 5' and 3' untranslated regions, and the like.

[0015] The nucleic acid molecule according to the present invention encodes a polypeptide or a fragment thereof derived from the HLA-H protein of SEQ ID NO: 1 or 54, and the protein is encoded by SEQ ID NO: 2. Therefore, the nucleic acid molecule of the present invention is preferably genomic DNA or mRNA. In the case of mRNA, the nucleic acid molecule may further include a poly A tail.

[0016] The term "protein", when used interchangeably with the term "polypeptide" in this specification, refers to a linear molecular chain of amino acids that includes single-chain proteins or fragments thereof, containing at least 50 amino acids. The term "peptide" as used in this specification describes a group of molecules consisting of up to 49 amino acids, while the term "polypeptide" (also referred to as "protein") as used in this specification 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 groups of peptides and polypeptides are referred to together using the term "(poly)peptide". (Poly)peptides can further form oligomers consisting of at least two identical or different molecules. The corresponding higher-order structures of such multimers are correspondingly referred to as homo- or hetero-dimers, homo- or hetero-trimers, etc. The HLA-H protein of SEQ ID NO: 1 contains cysteines at positions 93, 127, 229, and 285, and thus contains potential dimerization sites. Similarly, the HLA-H protein of SEQ ID NO: 54 contains cysteines at positions 89, 124, 225, and 281, and thus contains 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 the modification is achieved, for example, by glycosylation, acetylation, phosphorylation, and similar modifications well known in the art.

[0017] According to the present invention, the term "percent sequence identity (%)" refers to the number of identical nucleotides / amino acids ( "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 full length of the template nucleic acid or amino acid sequence. In other words, using alignment, for two or more sequences or subsequences, the percentage of amino acid residues or nucleotides that are the same (e.g., 70%, 75%, 80%, 85%, 90% or 95% identical) is measured over a window of comparison, or over a specified region as measured using sequence comparison algorithms known in the art, or when aligned manually and visually inspected, the (sub)sequences are compared and determined when aligned for maximum correspondence. This definition also applies to the complement of any sequence being aligned.

[0018] Analysis and alignment of nucleotide and amino acid sequences related to the present invention are 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 know additional suitable programs for aligning nucleic acid sequences.

[0019] As defined herein, at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, and most preferably at least 95% sequence identity are contemplated by the present invention. However, the present invention also preferably contemplates at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, and at least 99.8% sequence identity.

[0020] MHC class I molecules generally consist of two chains, an MHC alpha chain (heavy chain) and a beta2-microglobulin chain (light chain). Only the alpha chain spans the membrane. The alpha chain has three extracellular domains (named alpha1, 2, 3, with alpha1 at the N-terminus). The alpha1 and alpha3 domains of the HLA-H alpha chain mainly determine the immunosuppressive ability of HLA-H, and the alpha3 domain is considered the most important. It is noted that HLA-H is composed of a truncated alpha3 domain of only 13 amino acids, while the alpha3 domains of other HLA classes are approximately 93 amino acids. The nucleotide sequences of SEQ ID NOs: 3 and 4 encode the alpha1 and alpha3 domains of HLA-H, respectively. The amino acid sequences of SEQ ID NOs: 5 and 6 are the amino acid sequences of the alpha1 and alpha3 domains of HLA-H, respectively.

[0021] Therefore, a nucleotide sequence having any one of the preferred higher identities, including a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO: 2 or at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 4, is preferred. A nucleotide sequence having any one of the preferred higher identities, including a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO: 2 or at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 6, is preferred. A nucleotide sequence having any one of the preferred higher identities, including a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO: 2 or at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 4, and / or including a nucleotide sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 3, is preferred. Also, a nucleotide sequence having any one of the preferred higher identities, including a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO: 2 or at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 6, and / or including a nucleotide sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 5, is preferred.

[0022] A nucleotide sequence having at least 70% identity with the nucleotide sequence of SEQ ID NO: 2 or having any one of preferably higher identities is (i) a nucleotide sequence having at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identity with SEQ ID NO: 4, and (ii) at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identity with SEQ ID NO: 3. Most preferably, it comprises a nucleotide sequence having increased preference.

[0023] A particularly preferred example of an amino acid sequence sharing at least 95% identity with SEQ ID NO: 1 is the amino acid sequence of SEQ ID NO: 54. SEQ ID NO: 54 lacks the first 4 amino acids of SEQ ID NO: 1 but is otherwise identical to SEQ ID NO: 1. The first 4 amino acids of SEQ ID NO: 1 have been found not to be important for the function of the HLA-H protein. Thus, SEQ ID NO: 54 can also replace or supplement SEQ ID NO: 1 as an alternative sequence of the HLA-H polypeptide in any of the embodiments as described herein.

[0024] The term "degeneracy" as used in the present invention refers to the degeneracy of the genetic code. Since the triplet code specifies 20 amino acids and a stop codon and there are 4 bases available to encode genetic information, the triplet code is required to make at least 21 different codes. The possible 4 bases in the triplet 3The possibility gives 64 possible codons. That is, it means that some degeneracy must exist. As a result, some amino acids are encoded by two or more triplets, i.e., up to six. The degeneracy mainly results from the change in the third position in the triplet. This means that nucleic acid molecules having a nucleotide sequence different from that specified above but still encoding the same polypeptide are within the scope of the present invention. Thus, with respect to the first aspect of the present invention, those skilled in the art will understand that "consisting of a nucleotide sequence degenerate to the nucleic acid molecule of (d)" listed in item (I)(e) indicates a nucleic acid molecule encoding the same amino acid sequence as the nucleic acid molecule of item (I)(d). This amino acid sequence is either the amino acid sequence of SEQ ID NO: 1 or 54 or an amino acid sequence derived therefrom, and the latter amino acid sequence is identical to SEQ ID NO: 1 or 54 to the extent required and implied by the sequence identity values listed in item (I)(d) of the main embodiment.

[0025] The fragments of the nucleic acid molecules of (I)(a) to (f) of the first aspect of the present invention contain at least 150 nucleotides. In this regard, the fragments according to the present invention are preferably polynucleotides of at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, or at least 650 nucleotides, and most preferably fragments that lack only the 5´ ATP start codon and / or the 3´-TAG stop codon. Furthermore, it is preferred that the fragment contains a nucleotide sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 4 with increasing preference, or encodes an amino acid sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 6 with increasing preference. It is more preferred that the fragment contains a nucleotide sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 4 with increasing preference, and / or a nucleotide sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 3 with increasing preference. Similarly, it is more preferred that the present fragment encodes an amino acid sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 6 with increasing preference, and / or encodes an amino acid sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 5 with increasing preference.The fragment is a nucleotide sequence that is (i) at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 4, and (ii) most preferably a nucleotide sequence that is at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% identical to SEQ ID NO: 3.

[0026] According to a preferred embodiment of the first aspect of the present invention, the nucleic acid molecule is fused to a heterologous nucleotide sequence and is preferably operably linked to a heterologous promoter.

[0027] The heterologous nucleotide sequence can be fused directly or indirectly to the nucleic acid molecule of the present invention. In the case of indirect fusion, preferably, a nucleotide sequence encoding a peptide linker is used, such as (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 7), where n is 1 to 3.

[0028] As used herein, a heterologous nucleotide sequence is a sequence that is not naturally found fused to the nucleotide sequence of SEQ ID NO: 2. Noting that SEQ ID NO: 2 is of human origin, it is preferred that the heterologous nucleotide sequence is also of human origin.

[0029] Accordingly, a heterologous promoter is a promoter that is not naturally found operably linked to the nucleotide sequence of SEQ ID NO: 2. The heterologous promoter is preferably of human origin.

[0030] A promoter is a nucleic acid sequence that initiates the transcription of a specific gene, where said gene is derived from the HLA-H gene of SEQ ID NO: 2 or is according to the present invention of SEQ ID NO: 2. In this context, "operably linked" means that a heterologous promoter is fused to the nucleic acid molecule of the present invention, such that as a result, transcription of the nucleic acid molecule of the present invention can be initiated via the promoter, for example, in prokaryotic or eukaryotic cells. The heterologous promoter can be a constitutively active promoter, a tissue-specific or developmental stage-specific promoter, an inducible promoter, or a synthetic promoter. Constitutive promoters direct expression in virtually all tissues and are largely, if not completely, independent of environmental and developmental factors. Since their expression is not usually conditioned by endogenous factors, constitutive promoters are usually activated across species and even across kingdoms. Tissue-specific or developmental stage-specific promoters direct the expression of a gene in a particular tissue or at a particular stage of development. The activity of inducible promoters is induced by the presence or absence of biotic or abiotic factors. Inducible promoters are very powerful tools in genetic engineering because the expression of genes functionally linked to them can be turned on or off as needed. Synthetic promoters are constructed by combining primary elements of promoter regions from diverse origins.

[0031] Non-limiting examples of heterologous promoters used in the art for heterologous gene expression are (for mammalian systems) SV40, CMV, HSV, UBC, EF1A, PGK, Vlambda1, RSV, and CAGG; (for Drosophila systems) COPIA and ACT5C; and (for yeast systems) GAL1, GAL10, GAL7, GAL2, and can also be used in connection with the present invention.

[0032] Alternatively or additionally, the heterologous nucleic acid sequence may be a coding sequence such that the nucleic acid sequence of the present invention gives rise to a fusion protein. Such fusion proteins are discussed in more detail hereinbelow.

[0033] When the nucleic acid molecule is not fused to a heterologous promoter, for the purpose of expression, the nucleic acid molecule is fused to its own promoter.

[0034] As used herein, the term "vector" preferably means a plasmid, cosmid, virus, bacteriophage, or another vector customarily used in genetic engineering, for example, having the nucleic acid molecule of the present invention. The nucleic acid molecules of the present invention can be inserted, for example, into several commercially available vectors. Non-limiting examples include expression vectors of the pUC series, pBluescript (Stratagene), pET series (Novagen) or pCRTOPO (Invitrogen), as well as vectors suitable 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 plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen).

[0035] The nucleic acid molecule inserted into the vector can be synthesized, for example, by standard methods or isolated from natural resources. Ligation of the coding sequence to transcriptional regulatory elements and / or other amino acid coding sequences can also be carried out using established methods. Transcriptional regulatory elements (part of the expression cassette) that ensure expression in prokaryotic or eukaryotic cells are well known to those skilled in the art. These elements include regulatory sequences that ensure the initiation of transcription (e.g., promoters such as translation initiation codons, native-related or heterologous promoters and / or insulators; see above), internal ribosome entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476), and optionally a polyA signal that ensures the termination of transcription and the stabilization of the transcript. Further regulatory elements may include transcriptional and translational enhancers. Preferably, the polynucleotide encoding the polypeptide / protein or fusion protein of the present invention is operably linked to such expression control sequences that enable expression in prokaryotic or eukaryotic cells. The vector may further contain a nucleic acid sequence encoding a secretion signal as a further regulatory element. Such sequences are well known to those skilled in the art. Furthermore, depending on the expression system used, a leader sequence that can direct the expressed polypeptide to an intracellular compartment can be added to the coding sequence of the polynucleotide of the present invention. Such leader sequences are well known in the art.

[0036] Furthermore, the vector preferably contains a selectable marker. Examples of selectable markers include genes encoding resistance to neomycin, ampicillin, hygromycin, and kanamycin. Specifically designed vectors allow for DNA shuffling between different hosts such as bacterial-fungal cells or bacterial-animal cells (e.g., the Gateway system available from Invitrogen). The expression vector according to the present invention can direct the replication and expression of the polynucleotide of the present invention and the encoded peptide or fusion protein. Apart from introduction via vectors such as phage vectors or viral vectors (e.g., adenovirus, retrovirus), the above nucleic acid molecules can be designed for direct introduction or for introduction into cells via liposomes. Furthermore, baculovirus systems or systems based on vaccinia virus or Semliki Forest virus can be used as eukaryotic expression systems for the nucleic acid molecules of the present invention.

[0037] The term "host cell" means any cell of any organism that is selected, modified, transformed, propagated, or used, or manipulated in any way for the production of the protein or peptide or fusion protein of the present invention by the cell.

[0038] The host cells of the present invention are typically produced by introducing the nucleic acid molecule or vector(s) of the present invention into the host cell, and by virtue of the host cell / its presence, the expression of the nucleic acid molecule of the present invention encoding the protein or peptide or fusion protein of the present invention is mediated. The host from which the host cell is derived or isolated can be any prokaryotic or eukaryotic cell or organism, preferably excluding human embryonic stem cells directly derived by the destruction of a human embryo.

[0039] Suitable prokaryotes (bacteria) useful as hosts for the present invention are, for example, Escherichia coli (e.g., E. coli strains BL21, HB101, DH5a, XL1 Blue, Y1090 and JM101), Salmonella typhimurium, Serratia marcescens, Burkholderia glumae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Streptomyces lividans, Lactococcus lactis, Mycobacterium smegmatis, Streptomyces coelicolor or Bacillus subtilis, which are commonly used for cloning and / or expression. Suitable culture media and conditions for the above host cells are well known in the art.

[0040] Suitable eukaryotic host cells can be vertebrate cells, insect cells, fungal / yeast cells, nematode cells or plant cells. Fungal / yeast cells can be Saccharomyces cerevisiae cells, Pichia pastoris cells or Aspergillus cells. Preferred examples of host cells genetically engineered using the nucleic acid molecules or vectors of the present invention are cells of yeast, Escherichia coli and / or Bacillus species (e.g., Bacillus subtilis). In one preferred embodiment, the host cell is a yeast cell (e.g., S. cerevisiae).

[0041] In different preferred embodiments, the host cells are mammalian host cells such as Chinese hamster ovary (CHO) cells, mouse myeloma lymphoblastoid cells, human embryonic kidney cells (HEK-293), human embryonic retina cells (Crucell's Per.C6), or human amniotic fluid cells (Glycotope and CEVEC). The cells are frequently used in the art for producing recombinant proteins. CHO cells are the most commonly used mammalian host cells for the industrial production of recombinant protein therapeutics for humans.

[0042] The terms "protein" and "peptide" and their preferred embodiments are defined above in connection with the first aspect of the present specification. These definitions and preferred embodiments are applied mutatis mutandis to the second aspect. The peptides of the present invention are preferably at least 80%, preferably at least 90%, most preferably at least 95% identical to a subsequence of SEQ ID NO: 1 or 54.

[0043] The proteins or peptides of the present invention can be produced by molecular cloning techniques well known in the art. Recombinant expression can be achieved, for example, by using vectors and host cells as described above herein.

[0044] According to a preferred embodiment, the protein or peptide is a fusion protein.

[0045] A "fusion protein" according to the present invention comprises at least one additional heterologous amino acid sequence. Often, but not necessarily, these additional sequences are located at the N-terminus or C-terminus of the (poly)peptide. For example, it may be advantageous to first express the polypeptide as a fusion protein with additional amino acid residues that can be removed by a protease that specifically trims the fusion protein to release the (poly)peptide of the present invention. The amino acid sequence compound can be directly or indirectly fused to the nucleic acid molecule of the present invention. In the case of indirect fusion, generally, a GS-linker (e.g., (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 7), where n is 1 to 3) can be used.

[0046] At least one additional heterologous amino acid sequence of the fusion protein comprises an amino acid sequence that confers desired properties such as modified / enhanced stability, modified / enhanced solubility and / or the ability to target one or more specific cell types. For example, a fusion protein with an antibody. The term "antibody" is further defined below and includes, inter alia, antibody fragments and derivatives. The antibody may be specific for a cell surface marker, for example, or may be an antigen recognition fragment of said antibody. The protein or peptide of the invention may be fused to the N-terminus and / or C-terminus of the light chain and / or heavy chain of an antibody. The protein or peptide of the invention is preferably fused to the N-terminus of the light chain and / or heavy chain of an antibody such that the Fc portion of the antibody is free to bind to the Fc receptor.

[0047] The fusion protein may also contain protein domains that are known to function in signal transduction and / or are known to be involved in protein-protein interactions. Examples of such domains are ankyrin repeats; arm, Bcl-homology, Bromo, CARD, CH, Chr, C1, C2, DD, DED, DH, EFh, ENTH, F-box, FHA, FYVE, GEL, GYF, hect, LIM, MH2, PDZ, PB1, PH, PTB, PX, RGS, RING, SAM, SC, SH2, SH3, SOCS, START, TIR, TPR, TRAF, tsnare, Tubby, UBA, VHS, W, WW and 14-3-3 domains. Further information on these and other protein domains is available from the databases InterPro (http: / / www.ebi.ac.uk / interpro / , Mulder et al., 2003, Nucl. Acids. Res. 31: 315-318), Pfam (http: / / www.sanger.ac.uk / Software / Pfam / , Bateman et al., 2002, Nucleic Acids Research 30(1): 276-280) and SMART (http: / / smart.embl-heidelberg.de / , Letunic et al., 2002, Nucleic Acids Res. 30(1), 242-244).

[0048] At least one further heterologous amino acid sequence of the fusion protein according to the invention may comprise or consist of (a) a cytokine, (b) a chemokine, (c) a coagulation promoting factor, (d) a proteinaceous toxic compound, and / or (e) an enzyme for prodrug activation.

[0049] Cytokines are preferably selected from the group consisting of IL-2, IL-12, TNF-alpha, IFN-alpha, IFN-beta, IFN-gamma, IL-10, IL-15, IL-24, GM-CSF, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, LIF, CD80, B70, TNF-beta, LT-beta, CD-40 ligand, Fas-ligand, TGF-beta, IL-1 alpha and IL-1 beta. As is well known in the art, cytokines can be advantageous for the pro-inflammatory or anti-inflammatory responses of the immune system. Thus, depending on the disease to be treated, either a pro-inflammatory or an anti-inflammatory cytokine fusion protein may be advantageous. For example, generally for the treatment of inflammatory diseases, a fusion construct containing an anti-inflammatory cytokine is preferred, and for the treatment of cancer, a fusion construct containing a pro-inflammatory cytokine is generally preferred.

[0050] Chemokines are preferably selected from the group consisting of IL-8, GROα, GROβ, GROγ, ENA-78, LDGF-PBP, GCP-2, PF4, Mig, IP-10, SDF-1α / β, BUNZO / STRC33, I-TAC, BLC / BCA-1, MIP-1α, MIP-1β, MDC, TECK, TARC, RANTES, HCC-1, HCC-4, DC-CK1, MIP-3α, MIP-3β, MCP-1-5, eotaxin, Eotaxin-2, I-309, MPIF-1, 6Ckine, CTACK, MEC, lymphotactin and fractalkine. The main role of chemokines is to act as chemoattractants that induce cell migration. The cells attracted by chemokines follow a signal in which the chemokine concentration increases towards the chemokine source. As a result, within the fusion protein, chemokines can be used to induce the movement of the proteins or peptides of the present invention to specific cell types or body sites, for example.

[0051] The coagulation promoting factor is preferably tissue factor. The coagulation promoting factor promotes the process by which blood changes from a liquid to a gel to form a blood clot. The procoagulant factor can assist, for example, in wound healing.

[0052] The proteinaceous toxic compounds are preferably Ricin-A chain, modeccin, truncated Pseudomonas exotoxin A, diphtheria toxin and recombinant gelonin. The toxic compounds can have toxic effects on the whole organism as well as on the substructures of the organism such as specific cell types. Toxic compounds are often used in the treatment of tumors. Tumor cells generally proliferate faster than normal somatic cells, and as a result, they preferentially accumulate, and accumulate in higher amounts, the toxic compounds.

[0053] The enzymes for prodrug activation are preferably enzymes selected from the group consisting of carboxypeptidase, glucuronidase and glucosidase. Among the wide range of genes that have been evaluated for tumor therapy, the genes encoding prodrug activation enzymes are particularly attractive because they directly complement ongoing clinical chemotherapy regimens. These enzymes can activate prodrugs with low inherent toxicity using both bacterial and yeast enzymes, or enhance prodrug activation by mammalian enzymes.

[0054] According to a preferred embodiment, the protein or peptide is fused to a heterologous non-proteinaceous compound.

[0055] As used herein, a heterologous compound is a compound that cannot be found naturally fused to the amino acid sequence of SEQ ID NO: 1 or 54.

[0056] The heterologous non-proteinaceous compound can be fused directly or indirectly to the nucleic acid molecule of the invention. For example, chemical linkers can be used. Chemical linkers can contain a variety of functional groups such as primary amines, sulfhydryls, acids, alcohols and bromides. Many of our cross-linking agents are functionalized with maleimides (sulfhydryl reactive) and succinimidyl esters (NHS) or isothiocyanates (ITC) groups that react with amines.

[0057] Heterologous non-proteinaceous compounds are preferably pharmaceutically active compounds or diagnostically active compounds. The pharmaceutically active compounds or diagnostically active compounds are preferably selected from the group consisting of (a) fluorescent dyes, (b) photosensitizers, (c) radionuclides, (d) medical contrast agents, (e) toxic compounds, or (f) ACE inhibitors, renin inhibitors, ADH inhibitors, aldosterone inhibitors, angiotensin receptor blockers, TSH receptors, LH- / HCG receptors, estrogen receptors, progesterone receptors, androgen receptors, GnRH receptors, GH (growth hormone) receptors, or receptors for IGF-I or IGF-II.

[0058] The fluorescent dye is preferably a component selected from Alexa Fluor or Cy dyes.

[0059] The photosensitizer is preferably the phototoxic red fluorescent protein KillerRed or hematoporphyrin.

[0060] The radionuclide is preferably a group of gamma-ray emitting isotopes, more preferably 99m Tc, 123 I, 111 In, and / or a group of positron emitters, more preferably 18 F, 64 Cu, 68 Ga, 86 Y, 124 I, and / or a group of beta emitters, more preferably 131 I, 90 Y, 177 Lu, 67 Cu, 90 Sr, or a group of alpha emitters, preferably 213 Bi, 211 selected from any of At.

[0061] The contrast agent used herein is a substance used to enhance the contrast of structures or fluids in the body in medical imaging. General contrast agents act based on X-ray attenuation and magnetic resonance signal enhancement.

[0062] The toxic compound is preferably a small organic compound, more preferably a toxic compound selected from the group consisting of calicheamicin, maytansinoid, neocarzinostatin, esperamicin, dynemicin, kedarcidin, maduropeptin, doxorubicin, daunorubicin, and auristatin. In contrast to the proteinaceous toxic compounds described above herein, these toxic compounds are non-proteinaceous.

[0063] The nucleic acid molecule, vector, host cell, or protein or peptide, or combination thereof according to the present invention can be formulated as a pharmaceutical composition. According to the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention contains the above-mentioned compounds. Optionally, it may further contain additional molecules that can change the properties of the compounds of the present invention, thereby, for example, stabilizing, regulating and / or activating their functions. The composition may be in solid, liquid or gaseous form, and in particular may be in the form of (one or more) powders, (one or more) tablets, (one or more) solutions or (one or more) aerosols. The pharmaceutical composition of the present invention can optionally and additionally contain a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include aqueous phosphate buffered saline solution, water, emulsions (for example, oil / water emulsions), various types of wetting agents, sterile solutions, organic solvents including DMSO, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject at an appropriate dosage. The method of dosage administration is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors including the patient's size, body surface area, age, the specific compound being administered, gender, time and route of administration, general health, as well as other drugs being administered simultaneously. A therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of an ordinary clinician or physician. Generally, the regimen as regular administration of the pharmaceutical composition should be in the range of 1 μg to 5 g per day. However, more preferred dosages can 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 / day.Furthermore, for example, when the compound is an iRNA agent such as siRNA, the total pharmaceutically effective amount of the pharmaceutical composition to be administered is typically less than about 75 mg per kg of body weight, for example 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 nmol of the iRNA agent 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 nmol of the iRNA agent per kg of body weight (for example, about 4.4×10. 16 copy). The length of treatment required to observe a change and the interval after treatment at which an effect occurs will vary depending on the desired effect. A specific amount can be determined by conventional tests well known to those skilled in the art.

[0064] Immunosuppressants are drugs that can suppress the immune response. These can be used, for example, (i) to prevent rejection of transplanted organs and tissues (e.g., bone marrow, heart, kidney, liver), (ii) to treat diseases or disorders that are most likely of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, psoriasis, vitiligo, systemic lupus erythematosus, sarcoidosis, focal segmental glomerulosclerosis, Crohn's disease, Behcet's disease, pemphigus, scleroderma, and ulcerative colitis), and / or (iii) to treat non-autoimmune inflammatory diseases (e.g., long-term allergic asthma control and ankylosing spondylitis) in immunosuppressive therapy.

[0065] Tumor vaccines can be used to treat existing tumors or to prevent the occurrence of tumors. Vaccines for treating existing cancers are also known as therapeutic cancer vaccines. The vaccine may be "autologous", i.e., prepared from a sample taken from the patient and specific to that patient. The approach of cancer vaccination generally involves separating proteins from cancer cells and immunizing the patient with that protein as an antigen, with the aim of stimulating the immune system to kill cancer cells. The antigen is according to the present invention derived from HLA-H protein / peptide.

[0066] Accordingly, the present invention also relates to a method for preparing a tumor vaccine, which comprises mixing a nucleic acid molecule, a vector, a host cell, a protein or a peptide, a binding molecule, preferably an inhibitor of the present invention or a combination thereof, with at least one pharmaceutically acceptable excipient, carrier and / or diluent.

[0067] A pregnancy promoter is a compound that increases the likelihood of pregnancy, particularly the likelihood of embryo implantation. Implantation is the stage of pregnancy in which a fertilized egg attaches to the uterine wall. This attachment allows the embryo to receive oxygen and nutrients from the mother for growth. In humans, the implantation of a fertilized egg is most likely to occur around 5 to 6 days after ovulation. Implantation failure is thought to be caused by insufficient uterine receptivity in two-thirds of cases and problems with the embryo itself in the other one-third. This also depends on the mother's age. Insufficient uterine receptivity is more common in younger mothers, and problems with the embryo itself (e.g., chromosomal abnormalities) are more common in older mothers (especially those over 35 years old). Insufficient uterine receptivity may be caused by abnormal cytokine and hormonal signaling as well as epigenetic changes. Recurrent implantation failure is a cause of female infertility. Therefore, the pregnancy rate can be improved by optimizing endometrial receptivity for transplantation.

[0068] A nucleic acid molecule, vector, host cell, protein or peptide, binding molecule, preferably an inhibitor of the present invention or a combination thereof, can be used, for example, in in vitro fertilization, where the oocyte is cultured in the presence of the nucleic acid molecule, vector, host cell, protein or peptide, preferably an inhibitor of the present invention or a combination thereof, and then fertilized and transplanted into the mother.

[0069] The detection of HLA-H expression in tissue samples from cancer patients is shown in the attached examples. More specifically, Examples 1 and 2 show HLA-H expression in bladder cancer patients, Example 3 shows HLA-H expression in bladder cancer patients before and after chemotherapy, and Example 4 shows HLA-H expression in ovarian cancer patients before and after chemotherapy. Examples 2 to 4 show that high levels of HLA-H expression are associated with adverse outcomes, such as low survival rates in checkpoint therapy or chemotherapy resistance. Furthermore, Example 4 shows that an increase in HLA-H expression is positively associated with a higher tumor stage. Thus, it can be reasonably assumed that HLA-H expression helps tumors escape from the immune system. This in turn indicates that HLA-H acts as an immunosuppressive agent.

[0070] This series of evidence indicates that HLA-H is not a pseudogene but is actually a functional gene encoding a protein. In this regard, reference is made to the pseudogene HLA-H gene entry in the database GeneCards (GC06P032554). The database entry refers to the amino acid sequence UniPortKB: P01893, warns that the protein may be a product of a pseudogene, and characterizes the protein as "putative". The experimental data herein have revealed that HLA-H is not a pseudogene but actually encodes a functional protein. Even more unexpectedly, this functional protein has the amino acid sequence of SEQ ID NO: 1 rather than the amino acid sequence of UniPortKB: P01893.

[0071] The amino acid sequence UniPortKB: P01893 is based on a wrongly assumed open reading frame. For this reason, SEQ ID NOs: 1 and 54 provided herein share only about 90% sequence identity with subparts of UniPortKB: P01893. Further, UniPortKB: P01893 contains an HLA transmembrane domain and does not contain the correct HLA-H disclosed herein. Putative HLA-H UniPortKB: P01893- is membrane-bound like HLA-G, but unexpectedly it has been found that HLA-H is actually a soluble HLA. It was not obvious from the prior art that the available sequences of HLA-H pseudogenes and putative HLA-H proteins contained in public gene and protein databases were wrong, let alone that SEQ ID NOs: 1 and 2 were the correct sequences.

[0072] The above data of the examples also make it at least plausible that vaccination of cancer patients with HLA-H serves to suppress or prevent tumor escape from the immune system via HLA-H expression. Thus, the nucleic acid molecules, vectors, host cells, proteins or peptides or combinations of the present invention can be used as immunosuppressive agents or as tumor vaccines. The nucleic acid molecule is preferably the nucleic acid molecule of item (g) of the first aspect. WO 2018 / 140525 pamphlet contemplates the use of HLA-H antibodies for the treatment of cancer, but WO 2018 / 140525 pamphlet does not disclose any HLA-H, let alone the correct HLA-H sequences of SEQ ID NOs: 1 and 2 provided herein. Similarly, WO 2018 / 183921 pamphlet mentions a long list of potential new immunotherapy targets, where HLA-H is among this list. Again, the HLA-H sequence is not disclosed.

[0073] Furthermore, it is contemplated that nucleic acids, vectors, host cells, proteins or peptides, or combinations thereof, can be used to optimize endometrial receptivity for implantation, thereby promoting pregnancy. The nucleic acid is preferably the nucleic acid of item (g) of the first aspect. This is because HLA-G is thought to play an important role in implantation by controlling the invasion of trophoblast cells and regulating cytokine secretion to maintain local immune tolerance (see Roussev and Coulam and J Assist Reprod Genet 2007 Jul; 24(7): 288-295). Furthermore, preimplantation embryos are known to express soluble HLA-G and soluble HLA-F. The higher the expression concentration of soluble HLA-G and soluble HLA-F, the higher the embryo implantation rate. Therefore, it is also expected that high levels of HLA-H expression coincide with successful implantation, while low levels of HLA-H expression coincide with implantation failure.

[0074] In a second aspect, the invention relates to an inhibitor of a binding molecule of a nucleic acid molecule defined in relation to the first aspect of the invention and / or a protein defined in relation to the first aspect of the invention, preferably for use as an immune activator, preferably an inhibitor of a protein defined in relation to the first aspect of the invention for use in the treatment of tumors.

[0075] The binding molecule of the protein according to the present invention is a compound capable of binding to the protein according to the present invention. The binding molecule preferably binds specifically to the protein according to the present invention. Specific binding indicates that the binding molecule essentially does not bind or essentially binds to other proteins or peptides other than this protein. In particular, it is preferable that the binding molecule cannot bind to other HLA proteins other than HLA-H. The binding molecule of the protein according to the present invention is suitable for research purposes, for example. In immunoassays such as ELISA or Western blot, an antibody that binds to the protein of the present invention can be used. The binding molecule of the protein according to the present invention preferably can inhibit the protein according to the present invention. In this case, the binding molecule is called an inhibitor.

[0076] A compound that inhibits the expression of the nucleic acid molecule and / or protein of the present invention is (i) a compound that reduces or prevents the transcription of the nucleic acid molecule of the present invention and / or the gene encoding the protein of the present invention, or (ii) a compound that reduces or prevents the translation of the mRNA encoding the protein of the present invention. The compounds of (i) include compounds that interfere with the transcription mechanism and / or its interaction with expression control elements away from the promoter, such as the promoter and / or enhancer of the gene. The compounds of (ii) include compounds that interfere with the translation mechanism. A compound that inhibits the expression of the nucleic acid molecule and / or protein of the present invention specifically inhibits the expression of the nucleic acid molecule and / or protein of the present invention, for example, by specifically interfering with the promoter region that controls expression. Preferably, the transcription of the nucleic acid molecule and / or the protein of the present invention, or the translation of the protein of 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%, most preferably about 100% (compared to the same experimental setting in the absence of the compound).

[0077] Compounds that inhibit the activity of the nucleic acid molecules and / or the proteins of the present invention cause the nucleic acid molecules and / or the proteins to perform their functions with low efficiency. Compounds and / or proteins that inhibit the activity of nucleic acid molecules specifically inhibit the activity of said nucleic acid molecules and / or proteins. As will be described in more detail below, compounds and / or proteins of the present invention that inhibit the activity of nucleic acid molecules interact with the nucleic acid molecules and / or the proteins themselves, or specifically inhibit (preferably kill) the cells that produce the nucleic acid molecules and / or produce and / or bind to the proteins, thereby specifically inhibiting the activity of the nucleic acid molecules and / or the proteins. Preferably, the activity of the nucleic acid molecules and / or proteins of the present invention is reduced by at least 50%, more preferably by at least 75%, for example by at least 90% or 95%, even more preferably by at least 98%, most preferably by approximately 100% (compared to the same experimental setting in the absence of the compound).

[0078] The activity of the nucleic acid molecules and / or proteins of the present invention is according to the present invention, preferably its ability to induce resistance to chemotherapy in cancer patients and / or to reduce progression-free and overall survival in cancer patients (see also the attached examples). 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 improve symptoms. In neoadjuvant chemotherapy (also called preoperative or primary chemotherapy), drug treatment is carried out before surgical removal of the tumor. This is in contrast to adjuvant chemotherapy, which is drug treatment after surgery. Means and methods for determining this activity are established in the art and are described in the following examples. Thus, according to the medical aspect of the present invention, these activities of the nucleic acid molecules and / or proteins of the present invention should be inhibited.

[0079] The effectiveness of an inhibitor can be quantified by a method that compares the concentration of activity in the presence of the inhibitor to the concentration in the absence of the inhibitor. For example, changes in the amount of nucleic acid molecules formed and / or proteins can be used for measurement. The effectiveness of some inhibitors can be determined simultaneously in a high-throughput format. High-throughput assays can generally be performed in the wells of a microtiter plate, independent of biochemical assays, cell assays, or other assays, where each plate can contain 96, 384, or 1536 wells. The handling of plates, including incubation at temperatures other than ambient temperature and the contact of test compounds with the assay mixture, is preferably performed by one or more computer-controlled robotic systems including pipetting devices. When screening a large library of test compounds and / or performing the screening in a short time, for example, a mixture of 10, 20, 30, 40, 50, or 100 test compounds can be added to each well. In cases where a well exhibits a predicted activity, the mixture of test compounds may be deconvoluted to identify one or more of the test compounds in the mixture that give rise to the activity.

[0080] Compounds that inhibit the expression and / or activity of nucleic acids and / or proteins 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. Liposome cell-type 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 single- or multi-layered vesicles with a membrane formed from lipophilic materials 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 various different molecules, including RNA (Alexander et al. (2015), Nat Commun; 6:7321). Exosomes containing the molecules they enclose can be taken up by recipient cells. Thus, exosomes are important mediators of intercellular communication and regulators of the cellular niche. Exosomes can be used as delivery vehicles, for example, as contrast agents or drugs, and are effective for diagnostic and therapeutic purposes.

[0081] The compounds of the present invention that inhibit the expression and / or activity of nucleic acids and / or proteins can be administered to a subject in a suitable dosage and / or a therapeutically effective amount. This is further discussed below in the context of the pharmaceutical compositions of the present invention.

[0082] The length of treatment required to observe a change and the interval after treatment at which efficacy occurs vary depending on the desired effect. Specific amounts can be determined by conventional tests well known to those of ordinary skill in the art. Appropriate 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.

[0083] Compounds that inhibit the expression and / or activity of the nucleic acid molecules and / or proteins of the present invention are preferably mixed with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition. Suitable pharmaceutically acceptable carriers or excipients and formulations of pharmaceutical compositions are discussed above herein.

[0084] An immunostimulant is a drug that can promote an immune response. Immunostimulants can be used in immunostimulatory therapy, for example, to promote and / or initiate an immune response against diseased cells. The immune response is preferably a cytotoxic immune response and / or a T cell response against diseased cells.

[0085] As mentioned, immunostimulants are preferably used in the context of treating tumors. As is apparent from the appended examples, HLA-H is expressed in tumors. HLA-H is a secreted protein, and the data in the following examples show that HLA-H is secreted by tumor cells, thereby forming a "cloud" of HLA-H protein around the tumor cells, and this cloud prevents the tumor cells from being recognized and removed by the immune system. Binding molecules, preferably inhibitors, remove this protective cloud from the tumor cells, thereby promoting and / or initiating an immune response against the tumor cells. This immunostimulatory mechanism also applies mutatis mutandis to diseased cells other than tumor cells.

[0086] 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. A solid tumor is, in contrast to a non-solid (or liquid) tumor, usually a mass of abnormal tissue that does not contain cysts or liquid portions.

[0087] As discussed above herein, based on the data in the following examples herein, it can be reasonably assumed that HLA-H expression is used by tumors to evade the immune system and become resistant to established anti-tumor therapies (e.g., chemotherapy and immune checkpoint therapies). HLA-H is thought to help tumors by acting as an immunosuppressant. Thus, it can also be reasonably assumed that inhibitors of HLA-H are particularly suitable for use as immune activators for the treatment of tumors.

[0088] Inhibitors of HLA-H are preferably used in combination with established anti-tumor therapies, preferably chemotherapy or immune checkpoint therapies, more preferably immune checkpoint therapies, and most preferably anti-PD-L1 therapy. Example 1 shows a positive correlation between HLA-H expression and the immune checkpoint PD-L1, and Example 2 further shows that the survival rate decreases when tumor patients expressing high levels of HLA-H are treated with anti-PD-L1 antibodies. This indicates that patients expressing both PD-L1 and HLA-H must be treated with anti-PD-L1 therapy as well as an HLA-H inhibitor in order for the anti-PD-L1 therapy not to fail.

[0089] According to a preferred embodiment of the second aspect of the present invention, (I) the inhibitor of the nucleic acid molecule is selected from small molecules, aptamers, siRNA, shRNA, miRNA, 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, and / or (II) the binding molecule of the present protein, preferably the inhibitor of the present protein, is selected from small molecules, antibodies or antibody mimetics, aptamers, and the antibody mimetic is preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanobodies, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules, and probodies.

[0090] As used herein, "small molecule" preferably refers to an organic molecule. An organic molecule relates to or belongs to a class of compounds having a carbon basis, and the carbon atoms are linked together by carbon-carbon bonds. This is the original definition of the term "organic" in relation to the source of chemical compounds. 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 represent cyclic (ring-shaped), planar (flat) molecules having a resonance-bonded ring that is more stable than other geometric or bonding arrangements having the same set of atoms. Aromatic molecules are very stable and do not easily decompose to react with other substances. In a heteroaromatic molecule, 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 in the range of 300 Da to 1000 Da.

[0091] Alternatively, the "low molecule" according to the present invention may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds that do not have a carbon atom (excluding carbon dioxide, carbon monoxide, and carbonates). Preferably, the low molecule has a molecular weight of less than about 2000 Da, or less than about 1000 Da such as less than about 500 Da, and even more preferably less than about Da amu. The size of the low molecule can be determined by methods well known in the art, such as mass spectrometry. The low molecule can be designed, for example, based on the crystal structure of the target molecule, where sites considered to be the cause of biological activity are identified and can be verified in in vivo assays such as in vivo high-throughput screening (HTS) assays.

[0092] The term "antibody" includes, for example, polyclonal or monoclonal antibodies, and further includes derivatives or fragments thereof that still retain the binding specificity for a target, such as the HLA-H protein of SEQ ID NO: 1 or 54. Antibody fragments or derivatives include, inter alia, single domain VH or V-like domains such as Fab or Fab´ fragments, Fd, F(ab´)2, Fv or scFv fragments, VhH or V-NAR domains, and 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 include, in particular, bispecific antibodies that can bind simultaneously to two different types of antigens. The first antigen can be found on the protein of the present invention. The second antigen can be, for example, a tumor marker that is 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 (consisting of two single-chain variable fragments (scFvs) of various antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).

[0093] The term "antibody" also includes embodiments such as chimeric (human constant domain, non-human variable domain), single-chain, and humanized (human antibody except for non-human CDRs) antibodies.

[0094] A variety of techniques for the production of 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 an animal after immunization with an antigen in a mixture with an additive and an adjuvant, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line culture. Examples of such techniques are 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, for example, 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 the production of 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. Systems suitable for the expression of recombinant (humanized) antibodies can be selected, for example, from bacteria, yeast, insects, mammalian cell lines, or transgenic animals or plants (see, for example, U.S. Patent No. 6,080,560; Holliger P, Hudson PJ.2005, Nat Biotechnol., vol.23(9), 11265). Additionally, techniques described for the production of single-chain antibodies (see particularly U.S. Patent No. 4,946,778) can be adapted to produce single-chain antibodies specific for epitopes of HLA-H.Surface plasmon resonance, such as used in a BIAcore system, can be used to increase the efficiency of phage antibodies.

[0095] As used herein, the term "antibody mimetic" refers to a compound that can specifically bind to an antigen, such as the HLA-H protein of SEQ ID NO: 1 or 54 herein, like an antibody, but is not structurally related to an antibody. Antibody mimetics are typically artificial peptides or proteins having a molar mass of about 3 to 20 kDa. For example, antibody mimetics can be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanobodies, affilins, knotted domain peptides, Fynomer®, trispecific binding molecules, and prododies. These polypeptides are well known in the art and are described in further detail below.

[0096] As used herein, the term "affibody" refers to a family of antibody mimetics derived from the Z domain of staphylococcal protein A. Structurally, the affibody molecule is based on a three-helix bundle region that can also be incorporated into a fusion protein. An affibody has a molecular weight of about 6 kDa by itself and is stable under high temperature and acidic or alkaline conditions. Target specificity is obtained by randomization of 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).

[0097] The term "adnectin" (also referred to as "monobody") as used herein refers to a molecule based on the 10th extracellular domain of human fibronectin type III (10Fn3), adopting a 94-residue Ig-like β-sandwich fold with 2-3 exposed loops, but lacking a central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). The desired target specificity, i.e., the adnectin against HLA-H, can be genetically engineered by introducing modifications into specific loops of the protein.

[0098] 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 U S A. 96(5):1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Anticalins have an 8-stranded β-barrel, form a highly conserved core unit within lipocalins, and naturally form a binding site for ligands via four structurally variable loops at the open ends. Anticalins are not homologous to the IgG superfamily, but exhibit features that have hitherto been considered typical of antibody binding sites: (i) high structural plasticity as a result of sequence variation, and (ii) increased conformational flexibility, enabling induced fit to targets of different shapes.

[0099] The term "DARPin" as used herein refers to a designed ankyrin repeat domain (166 residues), which typically provides a rigid interface arising from 3 repeated β-turns. DARPin usually has three repeat sequences corresponding to artificial consensus sequences, with six positions randomized for each repeat. Therefore, DARPin lacks structural flexibility (Gebauer and Skerra, 2009).

[0100] As used herein, the term "abimer" refers to a class of antibody mimetics consisting of two or more peptide sequences of 30 to 35 amino acids each, derived from the A-domains of various membrane receptors and linked by a linker peptide. Binding to the target molecule occurs via the A-domain, and the desired binding specificity, i.e., the domain for HLA-H, can be selected, for example, by phage display technology. The binding specificities of the different A-domains contained in the abimer may be the same, but do not have to be the same (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0101] "Nanobody" (also known as affibody) is an antibody mimetic protein derived from the DNA-binding protein Sac7d of Sulfolobus acidocaldarius. Nanobodies usually have a molecular weight of about 7 kDa and are designed to specifically bind to a target molecule (e.g., HLA-H) by randomizing the amino acids on the binding surface (Mouratou B, Behar G, Paillard-Laurance L, Colinet S, Pecorari F., (2012) Methods: Mol Biol.; 805:315-31).

[0102] As used herein, the term "affilin" refers to an antibody mimetic developed by using either gamma-B crystallin or ubiquitin as a scaffold and modifying the amino acids on the surface of these proteins by random mutagenesis. Selection of the desired target specificity, i.e., the affilin for HLA-H, is performed, for example, by phage display or ribosome display technology. Depending on the scaffold, the molecular weight of the affilin is approximately 10 or 20 kDa. As used herein, the term affilin also refers to the dimeric or multimeric form of the affilin (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

[0103] "Knitted domain peptide" is derived from the knitted domain of a knitted type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). The knitted domain has a molecular weight of about 6 kDa, and the required target specificity, i.e., the domain for HLA-H, can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

[0104] 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, p. 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.

[0105] As used herein, the term "trispecific binding molecule" means that a polypeptide molecule has three binding domains and can thus bind, preferably specifically bind, to three different epitopes. At least one of these three epitopes is an epitope of the protein of the fourth aspect of the present invention. The two other epitopes can also be epitopes of the proteins of the present invention or can 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.

[0106] As used herein, the term "probody" refers to a protease-activatable antibody prodrug. A probody consists of a native IgG heavy chain and a modified light chain. The masking peptide is fused to the light chain via a peptide linker that is cleavable by tumor-specific proteases. The masking peptide prevents probe-like binding to healthy tissue, thereby minimizing toxic side effects.

[0107] An aptamer is a nucleic acid molecule or a peptide molecule that binds to a specific target molecule. Aptamers are usually prepared by selecting them from a large random sequence pool, although 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 and self-cleave in the presence of their target molecules. These compound molecules have further research, industrial, and clinical applications (Osborne et. al. (1997), Current Opinion in Chemical Biology, 1:5-9; Stull & Szoka (1995), Pharmaceutical Research, 12, 4:465-483).

[0108] Nucleic acid aptamers are nucleic acid species usually consisting of (usually short) strands of oligonucleotides. Typically, they are modified via iterative rounds of in vitro selection or equivalently SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms.

[0109] Peptide aptamers are peptides or proteins usually designed to interfere with other protein interactions within cells. They consist of a variable peptide loop flanked at both ends by protein scaffolds. This dual-structural constraint greatly increases the binding affinity of peptide aptamers to a level comparable to that of antibodies (in the nanomolar range). 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, and the variable peptide loop is inserted within the redox active site, which is the -Cys-Gly-Pro-Cys- loop (SEQ ID NO: 8) in the wild protein, and the two cysteine side chains can form a disulfide bridge. The selection of peptide aptamers can be carried out using different systems, but currently the most widely used is the yeast two-hybrid system.

[0110] Aptamers provide useful properties for biotechnology and therapeutic applications as they offer molecular recognition properties comparable to those of commonly used biomolecules, especially antibodies. In addition to their discriminatory recognition, aptamers 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, making them advantageous over antibodies. Unmodified aptamers are rapidly removed from the bloodstream with a half-life of minutes to hours. This is mainly due to the fact that aptamers are inherently of low molecular weight, resulting in nuclease degradation and clearance from the body by the kidneys. The applications of unmodified aptamers currently focus on the treatment of transient conditions such as blood clotting or the treatment of organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo imaging diagnostics. Several modifications such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) conjugation, fusion to albumin or other proteins that extend the half-life are available to scientists and can increase the half-life of aptamers over several days or weeks.

[0111] As discussed above, the low molecular weight molecules, antibodies or antibody mimetics and aptamers described above can specifically bind to the protein of the present invention. This binding can block the immunosuppressive properties of the protein of the present invention and preferably its ability to induce resistance to chemotherapy in cancer patients and / or to reduce progression-free and overall survival in cancer patients. In this case, the low molecular weight molecules, antibodies or antibody mimetics and aptamers are also referred to as blocking low molecular weight molecules, antibodies or antibody mimetics and aptamers. The blocking low molecular weight molecules, antibodies or antibody mimetics and aptamers block the interaction of the protein of the present invention with other cellular components such as ligands and receptors that normally interact with the protein of the present invention.

[0112] Small molecules, antibodies or antibody mimetics and aptamers can also be generated in the format of drug-conjugates. In this case, the small molecules, antibodies or antibody mimetics and aptamers may not have an inhibitory effect by themselves, but the inhibitory effect is conferred only by the drug. Small molecules, antibodies or antibody mimetics and aptamers confer site-specific binding of the drug to cells that produce and / or bind the protein of the present invention. The drug can preferably kill the cells that produce and / or bind the protein. Thus, by combining the targeting ability of the molecule that binds to the protein of the present invention with the cell-killing ability of the drug, the drug conjugate becomes an inhibitor that enables discrimination between healthy tissue and diseased tissue 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 are cytostatic agents and radioisotopes that directly deliver radiation to cancer cells.

[0113] Furthermore, the binding and / or inhibitory activity of small molecules, antibodies or antibody mimetics and aptamers can be limited to a specific tissue or cell type, particularly the diseased tissue or cell type. For example, a prodrug can be designed. In the prodrug, the small molecule, antibody or antibody mimetic or aptamer is bound to a masking peptide that restricts or prevents binding to the protein of the present invention, and this masking peptide can be cleaved by a protease. A protease is an enzyme that digests a protein into smaller fragments by cleaving a specific amino acid sequence known as the substrate. In normal healthy tissue, protease activity is tightly regulated. In cancer cells, protease activity is upregulated. In healthy tissue or cells where protease activity is regulated and minimal, the target binding site of the prodrug remains masked and thus cannot bind. On the other hand, in diseased tissue or cells where protease activity is upregulated, the target binding site of the prodrug is not masked and thus can bind and / or inhibit.

[0114] 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 that are 18 to 30, preferably 19 to 25, most preferably 21 to 23 or even more preferably 21 nucleotides in length and play various roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway that interferes with the expression of specific genes. In addition to its role in the RNAi pathway, siRNA also acts in RNAi-related pathways, for example, as an antiviral mechanism or when forming the chromatin structure of the genome.

[0115] Naturally occurring siRNA has a defined structure. That is, it is a short double-stranded RNA (dsRNA) with 2-nt 3´ overhangs at 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 RNA into siRNA. It is also possible to exogenously (artificially) introduce siRNA into cells to effect specific knockdown of a target gene. In this way, essentially any gene of known sequence can be targeted based on sequence complementarity with appropriately engineered siRNA. Double-stranded RNA molecules or their metabolic processing products can mediate target-specific nucleic acid modification, particularly RNA interference and / or DNA methylation. Exogenously introduced siRNA may lack overhangs at its 3´ and 5´ ends, however, it is preferred that at least one RNA strand has 5´ and / or 3´-overhangs. Preferably, one end of the duplex has a 3´-overhang of 1 to 5 nucleotides, more preferably 1 to 3 nucleotides, 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 siRNA is contemplated in the present invention. The most efficient silencing to date has been obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands paired to have 2-nt 3´ overhangs. The sequence of the 2-nt 3´ overhang contributes slightly to the specificity of target recognition limited to nucleotides not paired adjacent to the first base pair (Elbashir et al. 2001). 2´-Deoxynucleotides of the 3´ overhang are as efficient as ribonucleotides, but are less expensive to synthesize and are presumably more nuclease resistant.Delivery of siRNA can be carried out using any method known in the art, for example, by combining siRNA with physiological saline and administering the combination intravenously or intranasally, or by formulating siRNA in glucose (e.g., 5% glucose, etc.), or using cationic lipids, which can be used either intravenously (IV) or intraperitoneally (IP) for in vivo siRNA delivery (De Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol. 437: Drug Delivery Systems ー Chapter 3: Delivering Small Interfering RNA for Novel Therapeutics).

[0116] Short hairpin RNA (shRNA) is a sequence of RNA that forms a tight hairpin turn and can be used to silence gene expression via RNA interference. shRNA is introduced into cells using a vector that utilizes the U6 promoter to ensure constant expression of the shRNA. This vector is typically passed on to daughter cells and can inherit gene silencing. The shRNA hairpin structure is cleaved by cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that matches the bound siRNA. The si / shRNA used in the present invention is preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. 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 is obtained from commercial RNA oligo synthesis suppliers that sell RNA synthesis products of different qualities and costs. Generally, the RNA applicable in the present invention is synthesized by the prior art and is readily available in a quality suitable for RNAi.

[0117] Additional molecules that affect RNAi include, for example, microRNA (miRNA). The RNA species are single-stranded RNA molecules. Endogenously present miRNA molecules bind to complementary mRNA transcripts and regulate gene expression by triggering the degradation of the mRNA transcripts through a process similar to RNA interference. Thus, exogenous miRNA can be used as an inhibitor of HLA-H after being introduced into respective cells.

[0118] A ribozyme (also called ribonucleic acid enzyme-derived, RNA enzyme, or catalytic RNA) is an RNA molecule that catalyzes chemical reactions. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, although they have also been shown to catalyze the aminotransferase activity of ribosomes. Non-limiting examples of well-characterized small self-cleaving RNAs are the 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. The hammerhead-type ribozyme is the best characterized among RNA molecules with ribozyme activity. Since it has been shown that the hammerhead-type structure can be incorporated into heterologous RNA sequences and ribozyme activity can thereby be transferred to these molecules, it seems possible to create catalytic antisense sequences for almost any target sequence if the target sequence contains a cleavage site that potentially matches. The basic principle for constructing a hammerhead-type ribozyme is as follows: A region of interest of the RNA containing the GUC (or CUC) triplet is selected. Two oligonucleotide strands, usually 6 to 8 nucleotides in length, are taken, and the catalytic hammerhead sequence is inserted between them. The best results are usually obtained with short ribozymes and target sequences.

[0119] Also, recent developments are combinations of aptamers that recognize small compounds having a hammerhead-type ribozyme. The conformational changes induced in the aptamer when it binds to the target molecule can regulate the catalytic function of the ribozyme.

[0120] As used herein, the term "antisense nucleic acid molecule" refers to a nucleic acid that is complementary to a target nucleic acid. The antisense molecules according to the present invention can interact with the target nucleic acid, and more specifically, can hybridize with the target nucleic acid. By forming a hybrid, the transcription of the target gene and / or the translation of the target mRNA are reduced or blocked. Standard methods for antisense technology are described (see, for example, Melani et al., Cancer Res. (1991) 51:2897-2901).

[0121] Similar to CRISPR-Cpf1, CRISPR / Cas9 is applicable to almost all cells / model organisms and can be used for knockout mutations, chromosomal deletions, DNA sequence editing, and regulation of gene expression. Regulation of gene expression can be manipulated by using a catalytically dead Cas9 enzyme (dCas9) bound to a transcriptional repressor to suppress the transcription of a specific gene, here the HLA-H gene. Similarly, a catalytically inactive "dead" Cpf1 nuclease (CRISPR from Prevotella and Francisella-1) can be fused to a synthetic transcriptional repressor or activator to down-regulate an endogenous promoter (e.g., the promoter controlling HLA-H expression). Alternatively, the DNA-binding domain of a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN) can be designed to specifically recognize the HLA-H gene or its promoter region or its 5'-UTR, thereby inhibiting the expression of the HLA-H gene.

[0122] Inhibitors provided as inhibiting nucleic acid molecules targeting the HLA-H gene or regulatory molecules involved in HLA-H expression are also contemplated herein. Such molecules that reduce or ablate the expression of HLA-H or regulatory molecules include, but are not limited to, meganucleases, zinc finger nucleases, and transcription activator-like (TAL) 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.

[0123] In connection with the second aspect, a binding molecule of the protein as defined in connection with the first aspect, preferably an inhibitor of the protein as defined in connection with the first aspect, may also be a cell such as a T cell, where the T cell is preferably a CAR-T cell.

[0124] Cells generally carry on their surface a binding molecule, preferably an inhibitor of the protein as defined in connection with the first aspect. In the case of T cells, the binding molecule, preferably the inhibitor, is a naturally occurring T cell receptor or a chimeric T cell receptor that specifically targets the protein as defined in connection with the first aspect. Chimeric antigen receptor T cells (also known as CAR T cells) are T cells that have been genetically engineered to produce an artificial T cell receptor for use in immunotherapy.

[0125] A chimeric antigen receptor (CAR, also known as a chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) is thus a receptor protein engineered to give T cells a new function of specifically targeting a protein as defined in connection with the first aspect. The receptor is chimeric because it combines both an antigen-binding function and a T cell activation function into one receptor.

[0126] In a third aspect, the present invention relates to the use of a nucleic acid molecule defined in item (I)(g) of the first aspect of the present invention or a protein or peptide defined in relation to the first aspect of the present invention for diagnosing a tumor, and / or grading a tumor, and / or diagnosing a tumor prognosis, and / or classifying a tumor as an HLA-H low-expression tumor or an HLA-H high-expression tumor, and / or diagnosing transplant rejection, in a sample obtained from a subject.

[0127] The sample may be a body fluid of the subject or a tissue sample derived from an organ of the subject. Non-limiting examples of body fluids are whole blood, plasma, serum, urine, peritoneal fluid, and pleural fluid, cerebrospinal fluid, tear fluid, or cells from these in solution. Non-limiting examples of tissues are colon, liver, breast, ovary, and testis. The tissue sample can be collected by aspiration or puncture, excision, or any other surgical method leading to excised cell material. The sample may be a processed sample, for example, a frozen, fixed, embedded, etc. sample. A preferred type of sample is a formalin-fixed paraffin-embedded (FFPE) sample. The preparation of FFPE samples is a standard medical practice and these samples can be stored for a long time.

[0128] As used herein, the term "diagnose" is directed to the identification of a disease in a subject suffering from symptoms of the disease. According to the present invention, the disease is a tumor or a transplant failure. As used herein, the term "determine malignancy" means the identification of the degree of cellular dedifferentiation of tumor cells in a subject diagnosed with a tumor. The system most commonly used for cancer malignancy classification is the system according to the guidelines of the American Joint Commission on Cancer. According to these guidelines, the following malignancy classification categories are distinguished: GX (malignancy cannot be evaluated), G1 (well-differentiated; low malignancy), G2 (moderately differentiated; medium malignancy), G3 (poorly differentiated, high malignancy); G4 (undifferentiated, high malignancy). As used herein, the term "prognosis" is directed to the outlook or chance of recovery from a disease such as a tumor and / or the outlook or chance of survival of a disease such as a tumor. In the case of a tumor, the prognosis may include one or more of tumor size change of the target lesion, disease-specific survival (DSS), recurrence-free survival (RFS), progression-free survival (PFS), and distant recurrence-free survival, where DSS is preferred.

[0129] As used in connection with the present invention, the term "subject" refers to a mammal, preferably a farm animal or a pet animal, such as a horse, cow, pig, sheep, goat, dog or cat, most preferably a human.

[0130] As described above, the increase in HLA-H expression level in tumor patients is associated with a significant shortening of the progression-free survival period and the overall survival period of tumor patients. Therefore, the level of HLA-H expression also correlates with a higher malignancy. Furthermore, since HLA-H expression was observed in all tumor samples, it has been demonstrated that HLA-H expression serves not only as a prognostic marker but also as a diagnostic marker for tumors.

[0131] In the above use, positive and / or negative samples and predetermined criteria may be incorporated. The control can be obtained from a sample of one or more subjects, such as at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, or at least 2000 subjects. The predetermined criteria indicate values previously obtained from positive and / or negative samples.

[0132] For the diagnosis of tumors, since healthy subjects are expected not to express HLA-H, it is considered that no criteria are needed at all. Also, HLA-H expression was detected in all tumor patient cases examined. Nevertheless, positive and / or negative samples and predetermined criteria can be incorporated into the diagnostic tumor use of the present invention. For diagnosis, positive samples are derived from one or more subjects known to have a tumor, preferably a tumor of the same body site as the one to be diagnosed. Similarly, negative samples are derived from one or more subjects known not to have a tumor. When the expression concentration of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample is preferably increased by at least 1.5-fold, 2-fold, 3-fold, or 4-fold compared to the negative control or the predetermined criteria derived therefrom, the subject is diagnosed as having a tumor. When the expression concentration of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample is preferably different by less than 50%, less than 25%, and less than 10% from the positive control or the predetermined criteria derived therefrom, the subject is diagnosed as having a tumor. For example, when the positive control is set at 100%, patients showing values of 150% to 50%, preferably 125% or 75% are diagnosed as having a tumor. Also, for the diagnosis of poor transplantation, positive and / or negative samples and predetermined criteria may be used.

[0133] For diagnosis, positive samples are from one or more female subjects having at least one implantation failure, preferably at least two implantation failures, and most preferably at least three implantation failures. Two or more implantation failures are also referred to as recurrent or repeated implantation failures. Similarly, negative samples are from one or more female subjects having at least one successful pregnancy, preferably at least two successful pregnancies, and most preferably at least three successful pregnancies. When the expression concentration of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample is preferably reduced by at least 1.5-fold, 2-fold, 3-fold, or 4-fold compared to a negative control or a predetermined criterion derived therefrom, the female subject is diagnosed as having an implantation failure. When the expression concentration of the nucleic acid molecule of the first aspect or the protein or peptide of the fourth aspect in the sample differs from a positive control or a predetermined criterion derived therefrom by preferably less than 50%, less than 25%, and less than 10% (i.e., higher or lower), the subject is diagnosed as having an implantation failure. For example, if the positive control is set at 100%, a patient displaying a value of 125% or 75% is diagnosed as having an implantation failure.

[0134] Regarding classifying tumors as HLA-H low-expression tumors or HLA-H high-expression tumors, it is noted that not every tumor and all tumors are expected to express or express a substantial amount of HLA-H. Thus, in a subject having a tumor-binding molecule, preferably to determine whether the inhibitor of the present invention can be a treatment option, the tumor can be classified as an HLA-H low-expression tumor or HLA-H high-expression only in the latter case as an option for the binding molecule or inhibitor. For classification, the control can be one or more subjects known to have tumors that express HLA-H and preferably tumors that were known to be treatable by a binding molecule, preferably an inhibitor of the present invention. In cases where the HLA-H expression of the tumor to be classified is reduced, preferably at least 2-fold, at least 3-fold, at least 3-fold, at least 4-fold, and at least 5-fold compared to the control, the tumor is an HLA-H low-expression tumor. On the other hand, in cases where the HLA-H expression of the tumor to be classified is increased, preferably at least 2-fold, at least 3-fold, at least 3-fold, at least 4-fold, and at least 5-fold compared to the control, the tumor is an HLA-H high-expression tumor.

[0135] For prognostic diagnosis, the positive control can be from one or more subjects who died of a tumor (preferably a tumor in the same body part as the prognostic diagnosis), and the negative sample can be from one or more subjects who survived the tumor (preferably a tumor in the same body part as the prognostic diagnosis) for a substantial period of time without tumor progression. The substantial period preferably indicates at least 1 year, at least 2 years, at least 3 years, at least 4 years, and at least 5 years. When the expression concentration of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample is preferably reduced by at least 1.5-fold, 2-fold, 3-fold, 4-fold compared to the positive control or a predetermined criterion derived therefrom, the subject has a favorable prognostic diagnosis. Also, when the expression concentration of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample is different from the negative control or a predetermined criterion derived therefrom and is less than 50%, less than 25%, and less than 10%, the subject has a favorable prognostic diagnosis. When the expression concentration of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample is preferably increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold compared to the negative control or a predetermined criterion derived therefrom, the subject has an unfavorable prognostic diagnosis. Also, when the expression concentration of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample is preferably different from the positive control or a predetermined criterion derived therefrom by less than 50%, less than 25%, and less than 10%, the subject has an unfavorable prognostic diagnosis. Therefore, the prognostic diagnosis is preferably a prognostic diagnosis of the expected success of tumor treatment, where the anti-tumor treatment is preferably chemotherapy, and / or the patient to be diagnosed preferably has breast cancer.

[0136] For malignancy determination, the positive sample may be from one or more subjects that are malignancy-determined into one of the categories G1 - G4. Two or more positive samples can be used for malignancy determination, where the positive samples are all of the two, preferably three, most preferably four categories G1 - G4. When the difference between the expression level of the nucleic acid molecule of the present invention or the protein or peptide of the present invention in the sample and a predetermined criterion derived from the positive G1 control is preferably less than 50%, less than 25%, and less than 10%, the subject is malignancy-determined to have a G1 tumor. This is applied mutatis mutandis to stages G2 - G4.

[0137] Means for obtaining the level of the nucleic acid molecule of the present invention or the protein or peptide of the present invention are established in the art.

[0138] For example, the level of the nucleic acid molecule of the present invention 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. As used herein, the term "reference gene" means a gene having a relatively invariant level of expression on the RNA transcript / mRNA level in the system being examined, i.e., cancer. Such genes are sometimes referred to as 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.

[0139] RT-qPCR is shown by the examples. RT-qPCR is performed in a thermal cycler with the ability to irradiate each sample with light of at least one specific wavelength and detect the fluorescence emitted by the excited phosphor. The thermal cycler can also rapidly heat and cool the sample, thereby utilizing the physicochemical properties of nucleic acids and DNA polymerase. Two common methods for the detection of PCR products in real-time qPCR are (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides labeled with a fluorescent reporter that allows detection only after hybridization with a probe and its complementary sequence (e.g., TaqMan probe). The latter detection method is used in the following examples. The probe is generally a fluorescently labeled probe. Preferably, the fluorescently labeled probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye (= dual-labeled probe). Suitable fluorescent reporters and quencher dyes / parts are known to those skilled in the art and include, but are not limited to, reporter dye / parts 6-FAMTM, JOETM, Cy5®, Cy3® and quencher dye / parts Dabcyl, TAMRATM, BHQTM-1, -2 or -3. Preferably, the primers for use according to the present invention have a length of 15 to 30 nucleotides and are particularly deoxyribonucleotides. In one embodiment, the primer is (1) specific for or derived from the target mRNA sequence of HLA-H, (2) provides an amplicon size of less than 120 bp (preferably less than 100 bp), (3) is mRNA-specific (consideration of exons / introns; preferably no amplification of genomic DNA), (4) has no tendency to dimerize, and / or (5) has a melting point T m of about 60°C) T m and is designed to have.

[0140] As an alternative to qPCR, the levels of the nucleic acid molecules of the present invention can also be obtained using electrophoresis techniques or DNA microarrays. Conventional methods for the identification and quantification of mRNA are by a combination of gel electrophoresis that provides information on size and sequence-specific probing. Northern blotting is the most commonly applied technique in this class. The ribonuclease protection assay (RPA) was developed as a more sensitive and less labor-intensive alternative to Northern blotting. Hybridization is performed using a labeled ribonucleotide probe in solution, and subsequently, 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 gives the size of the region hybridized by the probe. For both Northern blotting and RPA, the accuracy and precision of quantification are functions of the detection method and the description or standard utilized. Most commonly, the probe is radiolabeled with 32P or 33P, in which case the final gel is exposed to X-ray film or a fluorescent screen, and the intensity of each band is quantified with a densitometer or a fluorescence imager, respectively. In both cases, the exposure time can be adjusted to match the required sensitivity, but phosphor-based technologies are generally more sensitive and have a larger dynamic range. Instead of using radioactivity, the probe can be labeled with an antigen or hapten, subsequently bound with a horseradish peroxidase or alkaline phosphatase-conjugated antibody, and quantified by chemiluminescence on film or a fluorescence imager after addition of a substrate. In all of these imaging applications, subtraction of the background from an adjacent region 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 sample. Similarly, detection of housekeeping genes is performed under the same conditions for all samples. Two techniques have emerged for the construction of DNA microarrays. Generally, the starting point in each case for the design of the array is a set of sequences corresponding to the genes or putative genes to be probed.

[0141] In the first approach, oligonucleotide probes are chemically synthesized from 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. Further, for each fully complementary oligonucleotide on the array, an oligonucleotide having a mismatch at a single nucleotide position is constructed and used for normalization. Oligonucleotide arrays are routinely created at a density of about 10 4 -10 6 probes / cm 2 The second major technique for DNA microarray construction is the direct robotic system printing method of cDNA probes onto a glass slide or other suitable substrate. DNA clones are obtained, purified for each gene of interest, 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, a density of about 10 3 probes / cm 2 can be achieved.

[0142] The level of the protein or peptide of the present invention can be determined, for example, by using a "molecule that binds to the protein or peptide", and preferably a "molecule that specifically binds to the protein or peptide". A molecule that binds to a protein or peptide refers to a molecule that mainly binds to the protein or peptide under known conditions. One of the binding molecules described above in the present specification, such as an antibody, an aptamer, etc., preferably an "inhibitor of the protein or peptide of the present invention" of a molecule that binds to the protein or peptide can be used. The level of the protein or peptide of the present invention 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 detect proteins or peptides qualitatively, semi-quantitatively, and / or quantitatively.

[0143] Western blot analysis is a well-known and widespread analytical technique used to detect specific proteins or peptides in a given sample, such as a tissue homogenate or a body extract. It uses gel electrophoresis to separate native or denatured proteins or peptides either by the length of the (poly)peptide (denaturing conditions) or by the 3-D structure of the protein (native / non-denaturing conditions). The protein or peptide is then transferred to a membrane (typically nitrocellulose or PVDF), where it is probed (detected) using an antibody specific to the target protein.

[0144] Mass spectrometry (MS) analysis is a well-known and widespread analytical technique where the mass-to-charge ratio of charged particles is measured. Mass spectrometry is used to determine the mass of particles, to determine the elemental composition of a sample or molecule, and to elucidate the chemical structure of molecules such as proteins, peptides, and other compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecular fragments and measuring their mass-to-charge ratios.

[0145] Fluorescence-activated cell sorting (FACS) analysis is a well-known and widespread analytical technique where biological cells are sorted based on specific light scatter of the fluorescence characteristics of each cell. The cells can be fixed in 4% formaldehyde, made permeable with 0.2% Triton-X-100, and incubated with a fluorophore-labeled antibody (e.g., monoclonal or polyclonal anti-HLA-H antibody).

[0146] Enzyme-linked immunosorbent assay (ELISA) is a well-known and widespread highly sensitive analytical technique where an enzyme is conjugated to an antibody or antigen as a marker for the detection of specific proteins or peptides.

[0147] Immunohistochemistry (IHC) is a common application of immunostaining. It involves methods for selectively identifying antigens (proteins) in cells of tissue sections by utilizing the principle of antibodies that specifically bind to antigens in biological tissues. In combination with specific devices, IHC can be used for quantitative in situ assessment of protein expression (as a review, see Creager et al. (2006) Arch Pathol Lab Med, 130:1026 - 1030). Quantitative IHC takes advantage of the fact that staining intensity correlates with absolute protein levels.

[0148] Next, the nucleic acid molecule of the present invention or the protein or peptide of the present invention can be used, in combination with one or more additional compounds in a sample obtained from a subject, for the diagnosis of tumors and / or determination of tumor malignancy and / or prediction of tumor prognosis. An enormous number of markers are known in the art for the diagnosis of tumors and / or determination of tumor malignancy and / or prediction of tumor prognosis and can be used in combination with the nucleic acid molecule of the present invention or the protein or peptide of the present invention. Some tumor markers are indicative of specific tumors such as breast cancer or colon cancer. Tumor markers are listed, for example, in the National Cancer Institute (https: / / www.cancer.gov / about-cancer / diagnosis-staging / diagnosis / tumor-markers-fact-sheet) or in the integrated database of cancer genes and markers CGMD (http: / / cgmd.in / ). The use of one or more additional markers generally increases the reliability of diagnosis, determination of malignancy, or prediction of prognosis.

[0149] In a fourth aspect, the present invention relates to a method for diagnosing a tumor, comprising detecting the presence of a nucleic acid molecule as defined in item (I)(g) of the first aspect of the present invention and / or a protein or peptide defined in relation to the first aspect of the present invention in a sample obtained from a subject, wherein the presence of the nucleic acid molecule of item (I)(g) of the first aspect of the present invention and / or the protein or peptide defined in relation to the first aspect of the present invention indicates a tumor in the subject.

[0150] In a fifth aspect, the present invention relates to a method for determining the malignancy of a tumor and / or for tumor prognosis, comprising determining the level of a nucleic acid molecule as defined in item (I)(g) of the first aspect of the present invention and / or a protein or peptide defined in relation to the first aspect of the present invention in a sample obtained from a subject, wherein an increase in the level of the nucleic acid molecule as defined in item (I)(g) of the first aspect of the present invention and / or the protein or peptide defined in relation to the first aspect of the present invention, compared to a control, correlates with a higher determination of tumor malignancy and / or a poor tumor prognosis diagnosis.

[0151] The methods of the fourth and fifth aspects of the present invention carry out the use of the third aspect of the present invention in the format of the method. Accordingly, the definitions and preferred embodiments provided hereinabove in relation to the third aspect of the present invention are equally applicable to the fourth and fifth aspects of the present invention.

[0152] In a sixth aspect, the present invention relates to a kit for diagnosing a tumor and / or for determining the malignancy of a tumor and / or for prognostic diagnosis of a tumor, comprising (a) a method for detecting and / or quantifying a nucleic acid molecule as defined in item (I)(g) of the first aspect of the present invention and / or a protein or peptide defined in relation to the first aspect of the present invention in a sample obtained from a subject, and (b) instructions for use of the kit.

[0153] The kit of the sixth aspect of the present invention implements, in the kit format, the means necessary to carry out the use of the third aspect of the present invention. For this reason, the definitions and preferred embodiments provided above in this specification in relation to the third aspect of the present invention are equally applicable to the kit of the sixth aspect of the present invention.

[0154] The means for detection and / or quantification of the nucleic acid molecule of the first aspect is preferably one or more of the primers and probes for HLA-H as shown in Table 1 of the examples, used in RT-qPCR, and more preferably one of the specific primer pairs for HLA-H that can optionally be used in relation to each probe. The method for detection of the protein or peptide of the present invention is preferably an antibody and / or antibody mimetic as described above in this specification. For detection and / or quantification, the antibody and / or antibody mimetic can be labeled, for example, with a fluorescent dye or a radioactive label. Examples of fluorescent dyes and radioactive labels are also described above in this specification.

[0155] The various components of the kit may be packaged in one or more containers, such as one or more vials. The vial may contain a preservative or buffer for storage in addition to the above components. The kit may preferably include instructions on how to use the components of the kit for diagnosing a tumor and / or determining the malignancy of a tumor and / or prognosticating a tumor.

[0156] Also, as discussed above in this specification, the examples show that HLA-H is expressed in tumors at various stages and is used by tumors to escape the immune system. High expression levels of HLA-H are associated with poor prognosis. In Example 4, it is further shown that the increase in HLA-H expression is positively correlated with the advanced tumor stage. Thus, the detection and / or quantification by the methods and kits described above in a sample obtained from a subject of the nucleic acid molecule defined in item (I)(g) of the first aspect of the invention and / or the protein or peptide defined in relation to the first aspect of the invention is a method for diagnosing a tumor and / or for grading a tumor and / or for tumor prognosis.

[0157] In a seventh aspect of the invention, a method for monitoring the ineffectiveness of tumor treatment in a subject having a tumor, comprising: (a) determining the amount of the nucleic acid molecule defined as item (I)(g) of the first aspect of the invention and / or the amount of the protein or peptide defined in relation to the first aspect of the invention in a sample obtained from the subject before the start of treatment; and (b) determining, one or more times after the start of treatment, the amount of the nucleic acid molecule defined as item (I)(g) of the first aspect of the invention and / or the amount of the protein or peptide defined in relation to the first aspect of the invention in a sample obtained from the subject, wherein an increase in the amount in (b) compared to (a) indicates the ineffectiveness of tumor treatment and / or a decrease in the amount in (b) compared to (a) indicates the effectiveness of tumor treatment.

[0158] Similarly, in a ninth aspect, the invention relates to a method for monitoring the ineffectiveness of immunosuppressive therapy in a subject in need of such therapy, comprising: (a) determining the amount of a nucleic acid molecule as defined in item (I)(g) of the first aspect of the invention and / or the amount of a protein or peptide defined in relation to the first aspect of the invention in a sample obtained from the subject before the start of treatment; and (b) determining the amount of a nucleic acid molecule as defined in item (I)(g) of the first aspect of the invention and / or the amount of a protein or peptide defined in relation to the first aspect of the invention in a sample obtained from the subject one or more times after the start of treatment, wherein a decrease in the amount in (b) as compared to (a) indicates ineffectiveness of the immunosuppressive therapy and / or an increase in the amount in (b) as compared to (a) indicates effectiveness of the immunosuppressive therapy.

[0159] The definitions and preferred embodiments provided above herein in connection with other aspects of the invention are equally applicable to the eighth and ninth aspects of the invention. For example, the means and methods for determining the amount of a nucleic acid molecule of the invention and / or the amount of a protein or peptide of the invention are described above herein in connection with the third aspect of the invention. These means and methods can be used similarly in connection with the eighth and ninth aspects of the invention.

[0160] Tumor treatment can be any tumor treatment, such as surgery, radiotherapy or chemotherapy. Tumor treatment is preferably chemotherapy. Chemotherapy involves the administration of chemotherapeutic agents. Chemotherapeutic agents that can be used according to the present invention include cell growth inhibitory compounds and cytotoxic compounds. Traditional chemotherapeutic agents act by killing rapidly dividing cells, which is one of the major characteristics of most tumor cells. Chemotherapeutic agents include, but are not limited to, taxanes, nucleoside analogs, camptothecin analogs, anthracyclines and anthracycline analogs, etoposide, bleomycin, vinorelbine, cyclophosphamide, antimetabolites, antimitotic agents, and alkylating agents. Chemotherapy can also be platinum-based, i.e., it involves the administration of platinum-based compounds (e.g., cisplatin). Chemotherapeutic agents are often used in combinations and are usually administered for 3 to 6 months. One of the most common treatments involves the administration of cyclophosphamide + doxorubicin (adriamycin; belonging to the group of anthracyclines and anthracycline analogs), known as AC. Sometimes, taxane agents such as docetaxel are added, and the regimen is sometimes called CAT. Taxanes attack the microtubules of cancer cells. Another common treatment that produces equivalent results involves the administration of cyclophosphamide, the antimetabolite methotrexate, and fluorouracil (CMF), a nucleoside analog. Another standard chemotherapy treatment involves the administration of fluorouracil, epirubicin, and cyclophosphamide (FEC), which can be supplemented with taxanes (e.g., docetaxel) or vinorelbine.

[0161] The tumor is according to the eighth aspect, preferably a non-luminal tumor. A non-luminal tumor is a tumor that is negative for hormone receptors (estrogen receptor and / or progesterone receptor) or expresses low levels of hormone receptors (estrogen receptor and / or progesterone receptor). In the case of breast tumors, luminal A tumors are hormone receptor positive, Her2 negative, and Ki-67 low-expressing, and luminal B tumors are (i) hormone receptor positive, Her2 negative, and Ki-67 high-expressing, or (ii) estrogen receptor positive, progesterone receptor negative, Her2 negative, and Ki-67 low-expressing. Non-luminal breast tumors can be divided into HER2-positive tumors and TNBC (triple-negative breast cancer), which are HER2 negative and negative for hormone receptors (estrogen receptor and / or progesterone receptor).

[0162] Similarly, immunosuppressive therapy can be any immunosuppressive therapy. For example, immunosuppressive therapy may include the administration of one or more immunosuppressive agents selected from, for example, glucocorticoids, cytostatic agents, and antibodies. According to the fifteenth aspect, the subject has received a transplanted organ or tissue (e.g., bone marrow, heart, kidney, liver) or is most likely to have an autoimmune disease or a disease of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, psoriasis, vitiligo, systemic lupus erythematosus, sarcoidosis, focal segmental glomerulosclerosis, Crohn's disease, Behcet's disease, pemphigus, ankylosing spondylitis, and ulcerative colitis), or another non-autoimmune inflammatory disease (e.g., long-term allergic asthma control, or ankylosing spondylitis).

[0163] As shown in Example 4, the increase in HLA-H expression is positively correlated with a higher tumor stage. This indicates that more clinically aggressive tumors become resistant to chemotherapy by increasing HLA-H expression. Therefore, determining the amount of the nucleic acid molecule defined in item (I)(g) of the first aspect of the present invention and / or the protein or peptide defined in relation to the first aspect of the present invention can be used to determine the ineffectiveness of tumor treatment in a subject. Since HLA-H helps tumors escape from anti-tumor treatment by its immunosuppressive function, the nucleic acid molecule defined in item (I)(g) of the first aspect of the present invention and / or the protein or peptide defined in relation to the first aspect of the present invention can likewise be used to monitor the ineffectiveness of immunosuppressive therapy.

[0164] According to a preferred embodiment of all aspects regarding the tumors described above in the present specification, the tumor is cancer.

[0165] Cancer is an abnormal malignant neoplasm of tissues without physiological function, arising from uncontrolled and usually rapid cell proliferation.

[0166] According to a more preferred embodiment of all aspects of the present invention regarding the tumors described above in the specification, the cancer is selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, cancer related to the upper digestive tract, colon cancer, colorectal cancer, prostate cancer, squamous cell carcinoma of the head and neck, cervical cancer, glioblastoma, malignant ascites, lymphoma and leukemia.

[0167] According to a more preferred embodiment of all aspects of the present invention regarding the tumors as described above in the present specification, the cancer is bladder cancer or gynecological cancer.

[0168] According to a further more preferred embodiment of all aspects regarding the tumors as described above in the present specification, the cancer is breast cancer or ovarian cancer.

[0169] Since it is being examined in Example 4, ovarian cancer is preferred. According to a preferred embodiment of all aspects of the invention as described above in this specification, the sample is a body fluid or a tissue sample from an organ.

[0170] With respect to the embodiments characterized in this specification, particularly in the claims, it is intended that each embodiment referred to in a dependent claim be combined with each embodiment (independent or dependent) of each claim. With respect to the embodiments characterized in this specification, particularly in the claims, it is intended that each embodiment referred to in a dependent claim 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 options D, E, and F, and claim 3 is dependent on claims 1 and 2 and recites three alternatives G, H, and I, then this specification, unless otherwise specifically mentioned, clearly 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.

[0171] Similarly, even when the independent claims and / or dependent claims do not recite alternatives, when a dependent claim refers back to multiple preceding claims, it is understood that the combinations of subject matters thereby covered are considered to be explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 that refers back to claim 1, and dependent claim 3 that refers back to both claims 2 and 1, the combination of the subject matters of claims 3 and 1 will be as clearly and unambiguously disclosed as the combination of the subject matters of claims 3, 2, and 1. If there is a further dependent claim 4 that refers to any one of claims 1 to 3, the combinations of the subject matters 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 Description of the Drawings

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[0192] The present invention will be described by way of examples Example 1: Spearman correlation between HLA-H expression and bladder cancer candidate genes The initial cohort included 407 cases assembled by the 19 institutions of the Cancer Genome Atlas (TCGA) Research Network (Network CGAR. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature. 2014;507(7492):315-22. doi: 10.1038 / nature12965 PubMed PMID: 24476821; PubMed Central PMCID: PMCPMC3962515). RNA-Seq (HiSeq) was used for whole-genome analysis in tumor samples as previously described. In this way, four molecular subtypes have been defined based on the mRNA expression pattern (i.e., the TCGA subtypes): subtypes I and II are luminal-like, subtype I is defined by FGFR3 alterations and increased FGFR3 expression, while subtype II is characterized by ERBB2 mutations and estrogen receptor β (ESR2) enrichment. Subtypes III and IV are described as basal-like, defined by increased expression of epithelial lineage genes and stem / progenitor cell keratins. Clinical and molecular data are publicly available on the cBioPortal for Cancer Genomics website (http: / / www.cbioportal.org / study?id=blca_tcga#clinical)). The dataset was downloaded and verified. Since most patients in the TCGA cohort had muscle-invasive bladder cancer (MIBC), patients in stages T0 and T1 were excluded from the analysis. Except for 10 patients who received radiotherapy, the exact treatment method was not documented. Therefore, the authors used the documented pN status as a surrogate for surgical treatment. All patients with pNX were excluded from the analysis, similar to those who received neoadjuvant therapy, radical radiotherapy, or had confirmed metastases.The association between selected candidate markers for relevant tumor biological motifs such as molecular subtypes (e.g., KRT5, KRT20), hormone axes (e.g., ESR1, ESR2), adhesion motifs (e.g., CDH1, CDH2, CDH11), cell cycle genes (e.g., CCND1, CCNE2), subtype-specific target genes (e.g., ERBB and FGFR), etc. and HLA gene expression was analyzed.

[0193] As shown in Figure 1, the substantial amount of HLA-H "pseudogene" mRNA could be determined by RNA seq to reach amounts similar to those of well-defined genes such as ERBB2, CDH1, and FGFR2 and FGFR3.

[0194] Next, it was analyzed whether HLA-H expression was associated with other bladder cancer candidate genes for subtyping and drug targeting. As shown in Figure 2, the mRNA expression of HLA-H showed a negative correlation with the luminal subtype markers ESR2, ERBB2, ERBB3, CDH1, and KRT 20 (p<0.0001), but a positive correlation with the basal subtype marker KRT5 (Spearman rho 0.2953; p<0.0001) and the epithelial-mesenchymal transition (EMT) markers SNAI1-3. Furthermore, the mRNA expression was negatively associated with FGFR2, FGFR3, and FGFR4, showing moderate correlation coefficients between Spearman rho -0.1813 and -0.2404 (p = 0.0002, p<0.0001, and p<0.0001 respectively).

[0195] Next, the association between HLA-H mRNA expression in MIBC and tumor stage, tumor malignancy according to lymph node status, age, gender, and histological subtype (papillary vs. non-papillary) was analyzed. As shown in Figure 3, HLA-H mRNA expression was significantly associated with the non-papillary histological subtype (p = 0.0029). No significant associations were observed for other clinical variables such as gender, age, and lymph node status.

[0196] Example 2: Measurement of HLA mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR) in a cohort of muscle-invasive bladder cancer patients treated with checkpoint inhibitor drugs when progression occurred after chemotherapy became ineffective after cystectomy

[0197] Paraffin-embedded tumor tissue samples from radical cystectomy and corresponding transurethral resection surgical specimens were obtained from 78 patients suffering from advanced urothelial cancer mainly with confirmed MIBC (pT2-T4). The patients were treated with therapeutic antibodies targeting the immune checkpoint targets PD-1 or PD-L1. Ethical approval was obtained from all participating facilities and informed consent was obtained from all patients. For RNA extraction from FFPE tissues, a single 10-μm curl was processed according to a commercially available bead-based extraction method (XTRAKT kit; STRATIFYER Molecular Pathology GmbH, Cologne, Germany). Briefly, FFPE tissue sections were liquefied using lysis buffer while paraffin melting was performed in a thermomixer. Tissue lysis was achieved using proteinase K solution. The lysate was then mixed with germanium-coated magnetic particles in the presence of a special buffer promoting nucleic acid binding. Purification was performed by consecutive cycles of mixing, magnetization, centrifugation, and removal of contaminants. RNA was eluted with 100 μl of elution buffer and the RNA eluate was then stored at -80 °C until use. All extracts were tested for sufficient high-quality RNA content by quantification by real-time PCR (RT-qPCR) of the constitutively expressed gene calmodulin 2 gene (CALM2), known as a stable reference / housekeeping gene. Specimens with low CALM2 expression were excluded.

[0198] For detailed analysis of gene expression by RT-qPCR, primers adjacent to the target site and a fluorescently labeled probe that hybridizes 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 make the RNA-specific measurement possible by positioning the primer / probe sequences across exon / exon boundaries. Furthermore, primers / probes were selected so as not to bind to sequence regions having known polymorphisms (SNPs). When multiple isoforms of the same gene exist, primers were selected to appropriately amplify all relevant or selected splice variants. All primer pairs were checked for specificity by conventional PCR reactions. After further optimizing the primers / probes, the primers and probes listed in Table 1 gave 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. TaqMan (registered trademark) validation experiments were performed, showing that the amplification efficiencies of the target and control were almost equal, which is a prerequisite for relative quantification of gene expression by the comparative ΔCT method.

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Table 1

[0200] The final cohort consisted of 55 bladder resection specimens from primary tumor tissues. Comparative analysis was performed using TUR biopsies and FFPE samples from metastatic lesions or, if available, synchronous upper urinary tract tumors (UTUC).

[0201] A gene-specific TaqMan-based primer / probe set was designed for the evaluation of HLA-H mRNA expression and tested for sensitivity and specificity. Immunohistochemical staining of CK5, CK20, GATA3, FOXA1, and CD44 was performed to examine whether HLA-H expression was associated with the subtypes of bladder cancer defined by the international classification consensus. Representative FFPE blocks with at least 50% or more tumor content (minimum tumor diameter 5x5 mm), having a distinct invasive border, and no necrotic areas or granulomatous inflammation were selected. All IHC staining was performed and read for all whole-slide sections as follows. Immunohistochemical staining was performed on 4-μm tissue sections using an automated Ventana Benchmark Ultra stainer (Ventana, Tucson, Arizona, USA).

[0202] Briefly, tissue sections were deparaffinized, antigen retrieval was performed by heat treatment in Tris / borate / EDTA solution pH 8.4 (Ventana), and endogenous peroxidase was blocked with 1% H2O2. PDL1 immunostaining was performed using a commercially available assay kit from DAKO that was adapted and validated for the Ventana platform (DAKO 28-8, DAKO, USA). CK5 (clone EBM26, monoclonal mouse, DiagnosticBioSystems®, diluted 1:50), CK20 (clone EK20.8, mouse monoclonal, DAKO®, diluted 1:50), GATA3 (clone E50-823, mouse, monoclonal, DCS®, diluted 1:100), CD44 (clone EDF1485, mouse, monoclonal, Dako®, diluted 1:50), and FOXA1 (clone EAB55178, mouse, monoclonal, Abcam, diluted 1:2000) were immunostained using the Benchmark Ultra autostainer (Ventana, USA) according to a standardized staining protocol. Development was performed using the ultraVIEW TM DAB system (Ventana). All tissue sections were counterstained with hematoxylin II / Mayer hematoxylin (Ventana).

[0203] As shown in Figure 5, when determining bladder cancer subtypes in this cohort of advanced chemotherapy-resistant tumors by IHC, the mRNA expression of the stem cell marker CD44 (Spearman rho 0.3349; p = 0.0125), which is highly frequent in KRT5-positive basal bladder cancer, was particularly associated with HLA-H.

[0204] Molecular subtyping of bladder cancer has become one of the major means to explore the stratification of tumors into hormone-dependent luminal tumors with less immune cell infiltration and basal or inflammatory subtypes with a higher frequency of tumor infiltration, which affects the survival rate in non-IO treatment conditions of muscle-invasive bladder cancer. (Pfannstiel C, Strissel PL, Chiappinelli KB, Sikic D, Wach S, Wirtz RM, Wullweber A, Taubert H, Breyer J, Otto W, Worst T, Burger M, Wullich B, Bolenz C, Fuhrich N, Geppert CI, Weyerer V, Stoehr R, Bertz S, Keck B, Erlmeier F, Erben P, Hartmann A, Strick R, Eckstein M; BRIDGE Consortium, Germany; BRIDGE Consortium, Germany; BRIDGE Consortium, Germany; BRIDGE Consortium, Germany. The Tumor Immune Microenvironment Drives a Prognostic Relevance That Correlates with Bladder Cancer Subtypes.Cancer Immunol Res.2019 Jun;7(6):923-938. doi: 10.1158 / 2326-6066.CIR-18-0758.Epub 2019 Apr 15.).

[0205] Therefore, it was of great interest to determine the correlation of HLA-H mRNA expression based on molecular subtyping and targeting of immune checkpoint targets. Furthermore, different primer-probe sets were designed at different exon / exon boundaries to further elucidate the potential effects of splicing events occurring with respect to this previously predicted "pseudogene".

[0206] As shown in Figure 6, in the mRNA expression of standard markers for molecular subtyping within the cohort of chemotherapy-resistant muscle-invasive bladder cancer, it was revealed that PD1-positive immune cell infiltration was dominant in the basal KRT5-positive subtype. However, no negative correlation between KRT5 and KRT20, which indicates that this selected population of treatment-resistant tumors is heterogeneous, was observed. Interestingly, quantification of the exon / exon boundary of exon 2 / 3 encoding the extracellular portion of HLA-H including the exon 2 / 3 boundary was strongly associated with high PD-L1 expression (Spearman rho 0.5053; p = 0.0044), but not with PD1 expression, and was associated in the KRT5 & KRT20-negative subpopulation of bladder cancer.

[0207] From the correlation between HLA-H splice variants and the mRNA expression of FGFR1, FGFR2, FGFR3 and FGFR4, an isoform including exon 2 / 3 that was not associated with FGFR mRNA expression was revealed (see Figure 7). Furthermore, FGFR family expression predicts survival in progressive or metastatic bladder cancer, FGFR2 is associated with good outcomes, while mutations, fusions or overexpression of FGFR3 are associated with adverse outcomes despite immunooncology treatment with checkpoint inhibitor drugs, as has been filed elsewhere (EP19168923.1; applicant STRATIFYER Molecular Pathology GmbH).

[0208] Therefore, it was decided to examine whether the interaction between HLA-H exon 2 / exon 3 mRNA expression, which is associated with PD-L1 mRNA expression, affects the survival rate of FGFR2-negative tumors with a high risk of cancer-specific survival despite anti-PD-L1 and PD-1 therapies. As shown in Figure 8, progressive or metastatic urothelial cancer patients showed significantly poor disease-specific survival when primary FGFR2-negative tumors expressed HLA-H as determined by RNA-specific RT-qPCR, as determined from the initiation of primary, secondary, or tertiary treatment with an immunomodulatory checkpoint inhibitor such as atezolizumab, pembrolizumab, or nivolumab. The probability of survival of patients with FGFR2-negative tumors expressing high levels of HLA-H Ex2 / 3 was 10% after 2 years, while the probability of survival of patients with FGFR2-negative tumors not expressing HLA-H Ex2 / 3 was 65% after 2 years (p = 0.0013).

[0209] To clarify the impact of HLA-H expression on postoperative survival rate, an analysis was conducted taking into account the primary metastasis site. This is based on the initial finding that IO treatment has a differential effect depending on the metastasis site, for example, the effect is low with visceral metastasis to the liver. This is probably due to the exclusion of PD1-positive T cells from the liver in metastatic urothelial cancer patients independent of the classical checkpoint mechanism (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 according to the first symptoms of metastasis with local progression, and local regional lymph nodes or extraregional retroperitoneal lymph nodes were classified as 0 or 0.5 respectively, while bone, liver, lung, lung and bone or seeding to the lung and liver were classified with increasing indices (1, 2, 3, 4, 5 respectively). For this analysis, 55 datasets from primary tumor tissues with sufficient clinical data and primary tumor tissue material were available, 20 patients had local progression or lymph node metastasis, while 18 patients had metastases to bone or liver first, 17 patients had metastases with lung lesions as a single site or in combination with bone or liver lesions, while all of them had been treated more (74%) with IO drugs and mainly primary therapy.

[0210] Interestingly, the adverse results for HLA-H expression were particularly prominent in the metastatic situation. As shown in Figure 9, when the HLA-H exon 2 / 3 mRNA expression was at a high level (>= 29.95), it was associated with poor disease-specific survival rate with only 30% survival rate one year later in 6 HLA-H exon 2 / 3 positive patients, while in 11 HLA-H exon 2 / 3 negative patients, the survival rate one year later was 80%, which was only a significant trend due to the early crossing of the curves, disturbing the log-rank test.

[0211] Taking FGFR2 expression into account, the predicted value could be increased. As shown in Figure 10, high HLA-H exon 2 / 3 mRNA expression (>=29.95) was associated with poor disease-specific survival in HLA-H exon 2 / 3 positive patients with a 1-year survival probability of only 0% in FGFR-negative patients, while HLA-H exon 2 / 3 & FGFR2-negative patients had a 1-year survival probability of 70%. Metastatic bladder cancer patients with high FGFR2 expression had a 1-year disease-specific survival rate of 100% (p = 0.0012).

[0212] As described above, it was found that HLA-H exon 2 / exon 3 boundary mRNA expression was associated with PD-L1 mRNA expression (Spearman rho 0.5053; p = 0.0044). Therefore, it seemed reasonable to stratify the entire patient cohort (advanced and metastatic; n = 55) according to the underlying principle of the applied immune checkpoint therapy (i.e., which of PD-1 or PD-L1 therapy was applied). Pembrolizumab and nivolumab specifically affect the checkpoint by binding to PD-1 positive T cells, while atezolizumab binds to PD-L1 present on tumor cells or macrophages and additional cell sources, thereby potentially having a more multifaceted effect. Importantly, looking at patients treated with atezolizumab, the importance of HLA-H exon 2 / exon 3 boundary expression became particularly evident. As shown in Figure 11, high HLA-H exon 2 / 3 mRNA expression (>=29.89) was associated with poor disease-specific survival in HLA-H exon 2 / 3 positive patients with a 1-year survival probability of only 10%, while HLA-H exon 2 / 3 negative patients had a 1-year survival probability of 80% (p = 0.0240).

[0213] To further elucidate the interaction between PD-L1 expression and HLA-H function, and considering the pleiotropic effects, a cohort of patients with progressive and metastatic bladder cancer treated with immune checkpoint inhibitors was stratified based on PD-L1 protein expression on immune cells (presumably macrophages) based on an in vitro diagnostic test for PD-L1 determination. Here, a cutoff value of 5% positive immune cells was selected, excluding non-specific staining of macrophages based on basal peroxidase activity not related to the IHC detection system.

[0214] Interestingly, patients with a low frequency of PD-L1-positive tumors infiltrating immune cells showed better survival rates, while patients with a high frequency of PD-L1-positive tumors infiltrating immune cells had inferior survival rates, especially when expressing the HLA-H exon 2 / exon 3 isoform. As shown in Figure 12, high HLA-H exon 2 / 3 mRNA expression (>=29.95) was associated with poor disease-specific survival in patients with HLA-H exon 2 / 3-positive patients having PD-L1-positive immune cells exceeding 5% and a 1-year survival probability of only 10%, while HLA-H exon 2 / 3-negative patients and patients with no or low frequencies of PD-L1-positive immune cells had a 1-year survival probability of 60% (p = 0.0156).

[0215] However, knowing the overlap in tumor tissues having PD-L1-positive immune cells vs. PD-L1-positive tumor cells, the interaction between HLA-H expression and PD-L1 expression was also examined for tumor cells with a standard cutoff value of 10% for PD-L1-positive tumor cells.

[0216] Here, in PD-L1 positive patients, when the expression of HLA-H exon 2 / exon 3 was high, the survival rate was inferior compared to the case when the expression of HLA-H exon 2 / exon 3 was low. As shown in Figure 13, high HLA-H exon 2 / 3 mRNA expression (>=33.19) was associated with poor disease-specific survival of patients with patients having HLA-H exon 2 / 3 positive patients with 10% or more PD-L1 positive tumor cells and a survival probability of only 0% after 1 year. However, when the PD-L1 positive tumor cells were less than 10%, the survival probability after 1 year was 40% for HLA-H exon 2 / 3 negative patients and 65% (p = 0.0022).

[0217] Example 3: Measurement of HLA-H mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR) in a cohort of muscle-invasive bladder cancer patients who received neoadjuvant therapy with 3 cycles of gemcitabine / Cisplatinum chemotherapy Paraffin-embedded tumor tissue samples from transurethral resection before chemotherapy and corresponding radical cystectomy surgical specimens were obtained from 55 patients with progressive urothelial cancer having histologically confirmed MIBC, UICC stage II and III (cT2-3 and cN0 or cN+M0). Patients received three cycles of neoadjuvant chemotherapy with gemcitabine 1250 mg / m2 (d1; d8) and cisplatin 70 mg / m2 (d1) followed by radical cystectomy. The main selection criteria were the availability of FFPE tissue before and after treatment. The main exclusion criteria were variant histological types to obtain a homogeneous bladder cancer cohort for analysis. Ethical approval was obtained from the participating institutions and informed consent was obtained from all patients. For RNA extraction from FFPE tissue, a single 10-μm curl was processed according to a commercially available bead-based extraction method (XTRAKT kit; STRATIFYER Molecular Pathology GmbH, Cologne, Germany). Briefly, FFPE tissue sections were liquefied using lysis buffer while melting of paraffin was performed in a thermomixer. Tissue lysis was achieved using proteinase K solution. Subsequently, the lysate was mixed with germanium-coated magnetic particles in the presence of a special buffer that promotes nucleic acid binding. Purification was performed by consecutive cycles of mixing, magnetization, centrifugation and removal of contaminants. RNA was eluted with 100 μl of elution buffer and then the RNA eluate was stored at -80 °C until use. All extracts were tested for sufficient high-quality RNA content by quantification of the constitutively expressed gene calmodulin 2 gene (CALM2), known as a stable reference / housekeeping gene. Specimens with low CALM2 expression were excluded. The final cohort consisted of 52 bladder resection specimens from primary tumor tissue after excluding 6 samples due to insufficient tumor material. Comparative analysis was performed using TUR biopsies and FFPE samples from the bladder resection tissue.

[0218] To verify the importance of HLA-H expression in bladder cancer other than IO therapy, HLA-H has been quantified in a cohort of muscle-invasive bladder cancers of the same size that have not yet been treated with systemic therapy. As shown in Figure 15, a significant amount of HLA-H "pseudogene" mRNA at the exon 2 / exon 3 boundary could be determined by RT-qPCR. Furthermore, bladder cancer subtype markers (KRT5, KRT20) and targets (PD-1, PD-L1, ESR1, ERBB2, FGFR1-4) were measured.

[0219] The association between HLA-H and bladder cancer subtype markers and targets was first evaluated in TUR biopsies of the pre-operative MIBC cohort. As shown in Figure 16, HLA-H expression measured by RT-qPCR at the Exon2 / exon 3 boundary showed a positive correlation with KRT5 and FGR1 expression, both of which are associated with the basal phenotype. Furthermore, a negative association was observed between KRT20 and ESR1, and KRT20 was a classical luminal marker. Therefore, although this association did not reach statistical significance due to the limited sample size, it reflected the initial findings that most of the 400 MIBC samples that were bladder resection specimens, including T1 tumors and variant histotypes.

[0220] Next, the expression of HLA-H exon 2 / exon 3 mRNA was correlated with the response to the above three courses of neoadjuvant chemotherapy (Gem / Cis), and a pathological complete response was defined as the absence of vital tumor cells in the surgical specimen (cystectomy) after chemotherapy. A pathological complete response was obtained in 42% of the patients. When the patients were stratified by a partition test based on relative HLA-H exon 2 / 3 mRNA expression, the responsiveness to neoadjuvant chemotherapy was reduced by 2-fold in tumors expressing high levels of HLA-H, which accounted for 60% of all tumors. The HLA-H positive tumors responded in 29% of the cases, and the HLA-H negative tumors responded in 62% of the cases. This indicates that HLA-H expression was measured in pre-chemotherapy bladder cancer biopsy specimens. In a total of 70% of the tumors with high HLA-H expression, they did not respond to neoadjuvant chemotherapy.

[0221] Next, HLA-H exon 2 / exon 3 mRNA expression was measured in bladder resection specimens after 3 cycles of neoadjuvant chemotherapy (Gem / Cis). As shown in Fig. 18, HLA-H expression was equally high in non-responsive tumors and showed almost identical separation in resistant and responsive tumors. In 70% of the tumors with high HLA-H expression, there was no response to neoadjuvant chemotherapy.

[0222] Example 4: Measurement of HLA-H mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR) in a cohort of advanced ovarian cancer patients who received neoadjuvant therapy with a chemotherapy regimen of 6 cycles of paclitaxel / cisplatin

[0223] Forty-five patients with histologically confirmed FIGO stage III-IV epithelial ovarian or peritoneal cancer, who were ineligible for optimal upfront surgery and were candidates for neoadjuvant chemotherapy (hereinafter, the cancer may also be referred to as ovarian cancer), were enrolled in this study from September 2004 to December 2007. Other study inclusion criteria were age > 18 years and sufficient hematological, renal, hepatic, and cardiac function for platinum-based chemotherapy. Exclusion criteria were Karnofsky performance status (KPS) < 70%, history of other malignancies, and surgical contraindications. The possibility of optimal debulking surgery by open laparoscopy at baseline was excluded. After excluding 9 patients with biopsy samples insufficient for microarray analysis and 1 patient who was found to be ineligible due to a diagnosis of peritoneal mesothelioma after histological revision, the initial study population of 45 patients was limited to 35. A standard regimen of carboplatin AUC5 and paclitaxel 175 mg / m2 Q3 administered over 3 hours every 3 weeks was performed for 6 cycles as neoadjuvant therapy. In 3 patients over 75 years old and 1 patient with poor general condition (KPS 70%), single-agent carboplatin was preferred over combination chemotherapy.

[0224] After surgery, surgical specimens were analyzed to evaluate histopathological response. To date, no histopathological criteria have been established to describe treatment response after neoadjuvant chemotherapy in ovarian cancer. According to the literature on the efficacy of primary chemotherapy in the ovary (Le et al. 2007, Sassen et al. 2007) and breast cancer (Ogston et al. 2003), pathologic complete response was defined as the absence of cancer cells in the surgical specimen, and very good partial response was considered to be only small clusters (<1 cm) or individual cancer cell remnants with no macroscopic residual disease detected postoperatively. Partial pathologic response was defined as a 30% to 90% reduction in tumor volume at surgery compared with the initial diagnostic laparoscopy, while stable disease was defined as either no reduction or a reduction of less than 30% in tumor volume at surgery. Only patients who achieved complete or very good partial response were considered histopathologic responders, while all other cases were considered histopathologic non-responders.

[0225] For mRNA analysis, the collected tissues were snap-frozen and stored in liquid nitrogen until analysis. Approximately 20 to 100 mg of frozen ovarian tumor tissue was pulverized in liquid nitrogen. RNA was extracted using a commercial kit (Qiagen), RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), cDNA was synthesized from 1 mg of total RNA using an Invitrogen kit (Invitrogen Corp.), and HLA-H, the subtyping markers, and the target genes were examined as described above by RT-qPCR using an RNA-specific primer-probe set. Analysis was limited to cases where pre- and post-treatment tissue samples were available, and matched-pair analysis was possible for a total of 29 patients.

[0226] Similar to the situation in the above-mentioned bladder cancer, a substantial amount of HLA-H mRNA could be detected in RNA extracts from pre-treatment biopsies and post-treatment resections of ovarian cancer patients, which reached levels equivalent to HLA-G in the same tissues. However, the median mRNA expression of HLA-H exon 2 / exon 3 was lower (40-DCT 31.22) than that of HLA-G exon 2 / exon 3 (40-DCT 35.00). There was no correlation between HLA-H and HLA-G expression.

[0227] When correlating the difference in HLA-H exon 2 / exon 3 mRNA expression before and after chemotherapy with pathological response, an inverse correlation was observed between the increase in HLA-H expression after chemotherapy and pathological response, by both Pearson correlation (r = -0.2290) and Spearman correlation (r = -0.1922). As shown in Figure 20, from the correlation with pathological stage, the increase in HLA-H expression showed a positive correlation with higher FIGO stages (Spearman r = 0.4219; p = 0.0226), indicating that more clinically aggressive tumors respond to chemotherapy by increasing HLA-H expression. Interestingly, this ability tended to be more prominent in tumors with lower malignancy (Spearman r = -0.3274; p = 0.095).

Claims

1. A nucleic acid molecule, a vector, a host cell, or a protein or peptide, or a combination thereof, for use as an immunosuppressant, as a tumor vaccine, or as a fertility enhancer, (I) A nucleic acid molecule (a) encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 54; or (b) consisting of the nucleotide sequence of SEQ ID NO:2; or (c) encoding a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 or 54; or (d) consists of a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:2; or (e) consists of a nucleotide sequence that is degenerate to the nucleic acid molecule of (d); or (f) a fragment of the nucleic acid molecule according to any one of (a) to (e), said fragment comprising at least 250 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides; or (g) a nucleic acid molecule according to any one of (a) to (f), in which T is replaced by U; (II) a vector comprising the nucleic acid molecule of (I); (III) a host cell is transformed, transduced or transfected with the vector of (II); and (IV) A protein or peptide encoded by the nucleic acid molecule of (I). A nucleic acid molecule, a vector, a host cell, or a protein or peptide, or a combination thereof.

2. An inhibitor of a nucleic acid molecule according to claim 1 and / or a binding molecule of a protein according to claim 1, preferably an inhibitor of a protein according to claim 1, for use as an immune activator, preferably for use in the treatment of tumors.

3. A binding molecule, preferably an inhibitor, according to claim 2, (i) the inhibitor of the nucleic acid molecule is selected from a small molecule, an aptamer, an siRNA, an shRNA, an miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1-based construct, a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease; and / or (ii) the binding molecule of the protein, preferably an inhibitor of the protein, is selected from a small molecule, an antibody or an antibody mimetic, an aptamer, where the antibody mimetic is preferably selected from an affibody, an adnectin, an anticalin, a DARPin, an avimer, a nanophytin, an affilin, a Kunitz domain peptide, a Fynomer®, a trispecific binding molecule and a probody; A binding molecule, preferably an inhibitor.

4. Use of a nucleic acid molecule according to claim 1 (I) (g) or a protein or peptide according to claim 1 for diagnosing a tumor, and / or grading a tumor, prognosing a tumor, and / or classifying a tumor as an HLA-H low or high expressing tumor, and / or diagnosing transplant failure in a sample obtained from a subject.

5. A method for diagnosing a tumor comprising detecting the presence of a nucleic acid molecule described in claim 1 (I) (g) and / or a protein or peptide described in claim 1 in a sample obtained from a subject, wherein the presence of a nucleic acid molecule described in claim 1 (I) (g) and / or a protein described in claim 1 indicates a tumor in the subject.

6. A method for grading and / or prognosing a tumor, comprising determining the level of a nucleic acid molecule described in claim 1 (I) (g) and / or the level of a protein or peptide described in claim 1 in a sample obtained from a subject, wherein an increase in the level of the nucleic acid molecule described in claim 1 (I) (g) and / or the level of the protein or peptide described in claim 1 compared to a control correlates with a higher tumor grading and / or an adverse tumor prognosis.

7. 1. A kit for diagnosing and / or grading and / or prognosing a tumor, comprising: (a) means for the detection and / or quantification of a nucleic acid molecule according to claim 1(I)(g) and / or a protein or peptide according to claim 1 in a sample obtained from a subject, and (b) Instructions for use of the kit. Including the kit.

8. 1. A method for monitoring ineffectiveness of a tumor treatment in a subject having a tumor, comprising: (a) determining the amount of the nucleic acid molecule according to claim 1(I)(g) and / or the amount of the protein or peptide according to claim 1 in a sample obtained from the subject before the start of the treatment; and (b) determining the amount of the nucleic acid molecule according to claim 1(I)(g) and / or the amount of the protein or peptide according to claim 1 in a sample obtained from the subject one or more times after initiation of the treatment, A method, wherein an increase in the amount in b) compared to a) indicates ineffectiveness of the tumor treatment and / or a decrease in the amount in b) compared to a) indicates effective tumor treatment.

9. 1. A method for monitoring ineffectiveness of immunosuppressive therapy in a subject in need of said treatment, comprising: (a) determining the amount of the nucleic acid molecule according to claim 1(I)(g) and / or the amount of the protein or peptide according to claim 1 in a sample obtained from the subject before the start of the treatment; and (b) determining the amount of the nucleic acid molecule according to claim 1(I)(g) and / or the amount of the protein or peptide according to claim 1 in a sample obtained from the subject at one or more times after the start of the treatment, A method, wherein a decrease in the amount in b) compared to a) indicates ineffectiveness of the immunosuppressive therapy and / or an increase in the amount in b) compared to a) indicates effectiveness of the immunosuppressive therapy.

10. The nucleic acid molecule, vector, host cell and / or protein or peptide, or a combination thereof, of claim 1, the binding molecule, preferably the inhibitor, of claim 2 or 3, the use of claim 4, the method of claim 5, 6 or 8, or the kit of claim 7, wherein the tumor is a cancer.

11. The nucleic acid molecule, vector, host cell and / or protein or peptide, or a combination thereof, binding molecule, preferably inhibitor, use, method or kit according to claim 10, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, upper gastrointestinal tract cancer, colon cancer, colorectal cancer, prostate cancer, squamous cell carcinoma of the head and neck, cervical cancer, glioblastoma, malignant ascites, lymphoma and leukemia.

12. The nucleic acid molecule, vector, host cell and / or protein or peptide, or a combination thereof, binding molecule, preferably inhibitor, use, method or kit according to claim 10, wherein the cancer is bladder cancer or gynecological cancer.

13. The nucleic acid molecule, vector, host cell and / or protein or peptide, or a combination thereof, binding molecule, preferably inhibitor, use, method or kit according to claim 10, wherein the cancer is breast cancer or ovarian cancer.

14. The nucleic acid molecule, vector, host cell and / or protein or peptide, or a combination thereof, binding molecule, preferably inhibitor, use, method or kit according to the above claims, wherein said sample is a body fluid or a tissue sample from an organ.

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