Method for diagnosing effectiveness of anti-tumor therapy

Abstract

This invention relates to a method for predicting whether a subject with a tumor will respond to a tumor treatment selected from (i) immunotherapy, (ii) chemotherapy, (iii) anti-hormonal therapy, and (iv) anti-tyrosine kinase therapy, wherein the method comprises (A) determining the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from the subject, wherein the at least one nucleic acid molecule is selected from: (a) nucleic acid molecules encoding a polypeptide comprising or consisting of any amino acid sequence of SEQ ID NO: 1 to 6, (b) nucleic acid molecules of SEQ ID NO: 1 to 6, etc. NO: A nucleic acid molecule consisting of any nucleotide sequence from 7 to 12, (c) a nucleic acid molecule encoding a polypeptide whose amino acid sequence is at least 85% identical, preferably at least 90% identical, and most preferably at least 95% identical to that of (a), (d) a nucleic acid molecule consisting of a nucleotide sequence whose nucleotide sequence is at least 95% identical, preferably at least 96% identical, and most preferably at least 98% identical to that of (b), (e) a nucleic acid molecule consisting of a degenerate nucleotide sequence relative to (d), (f) a nucleic acid molecule consisting of a fragment of any nucleic acid molecule from (a) to (e), said fragment comprising at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides, and (g) a nucleic acid molecule corresponding to any nucleic acid molecule from (a) to (f) wherein T is replaced by U, wherein said at least one protein or peptide is selected from the protein or peptide encoded by any nucleic acid molecule from (a) to (g); and (B) the level of (A) is compared with that of (i) (i) to (iii) compare the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who have responded to one or more treatments in (i) to (iii) with a corresponding predetermined standard, wherein an increase in the level of (A) compared to the level of (B) or the predetermined standard indicates that the subject has not responded to the tumor treatment, and a substantially the same level or a decrease in the level of (A) compared to the level of (B) indicates that the subject has responded to the tumor treatment; or (B') compare the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who have not responded to one or more treatments in (i) to (iii) with a corresponding predetermined standard, wherein a decrease in the level of (A) compared to the level of (B') or the predetermined standard indicates that the subject has responded to the tumor treatment, and a substantially the same level or an increase in the level of (A) compared to the level of (B') indicates that the subject has not responded to the tumor treatment.

Description

[0001] This invention relates to a method for predicting whether a subject with a tumor will respond to a tumor treatment selected from (i) immunotherapy, (ii) chemotherapy, (iii) anti-hormonal therapy, and (iv) anti-tyrosine kinase therapy, wherein the method comprises: (A) determining the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from the subject, wherein the at least one nucleic acid molecule is selected from: (a) nucleic acid molecules encoding a polypeptide comprising or consisting of any amino acid sequence of SEQ ID NO: 1 to 6, (b) nucleic acid molecules comprising or consisting of any amino acid sequence of SEQ ID NO: 1 to 6, and (c) nucleic acid molecules encoding a polypeptide comprising or consisting of any amino acid sequence of SEQ ID NO: 1 to 6. NO: A nucleic acid molecule consisting of any nucleotide sequence from 7 to 12, (c) a nucleic acid molecule encoding a polypeptide whose amino acid sequence is at least 85% identical, preferably at least 90% identical, and most preferably at least 95% identical to that of (a), (d) a nucleic acid molecule consisting of a nucleotide sequence whose nucleotide sequence is at least 95% identical, preferably at least 96% identical, and most preferably at least 98% identical to that of (b), (e) a nucleic acid molecule consisting of a degenerate nucleotide sequence relative to (d), (f) a nucleic acid molecule consisting of a fragment of any nucleic acid molecule from (a) to (e), said fragment comprising at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides, and (g) a nucleic acid molecule corresponding to any nucleic acid molecule from (a) to (f) wherein T is replaced by U, wherein said at least one protein or peptide is selected from the protein or peptide encoded by any nucleic acid molecule from (a) to (g); and (B) the level of (A) is compared with that of (i) (i) to (iii) compare the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who have responded to one or more treatments in (i) to (iii) with a corresponding predetermined standard, wherein an increase in the level of (A) compared to the level of (B) or the predetermined standard indicates that the subject has not responded to the tumor treatment, and a substantially the same level or a decrease in the level of (A) compared to the level of (B) indicates that the subject has responded to the tumor treatment; or (B') compare the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who have not responded to one or more treatments in (i) to (iii) with a corresponding predetermined standard, wherein a decrease in the level of (A) compared to the level of (B') or the predetermined standard indicates that the subject has responded to the tumor treatment, and a substantially the same level or an increase in the level of (A) compared to the level of (B') indicates that the subject has not responded to the tumor treatment.

[0002] Numerous documents, including patent applications and manufacturer's manuals, are referenced in this specification. While the disclosures in these documents are not considered relevant to the patentability of this invention, their entire contents are incorporated herein by reference. More specifically, all references are incorporated by reference to the same extent that each individual document is specifically and individually indicated to be incorporated by reference.

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

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

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

[0006] The proteins produced by MHC class III genes have several distinct functions; they are involved in inflammation and other immune system activities. The functions of some MHC genes remain unclear.

[0007] HLA genes have many possible variations, allowing each person's immune system to respond to a variety of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each with a specific number (such as HLA-B27). Closely related alleles are grouped together; for example, there are at least 40 very similar alleles that are subtypes of HLA-B27. These subtypes are named HLA-B*2701 to HLA-B*2743.

[0008] More than 100 diseases have been linked to 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 involving immune dysfunction and certain forms of cancer are also associated with specific HLA alleles. However, the role of HLA genes in the risk of developing these diseases is often unclear.

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

[0010] Further reports indicate that the HLA class I region also includes class I pseudogenes (Hughes, Mol Biol Evol. 1995 Mar; 12(2):247-58) and gene fragments. For example, HLA-H, HLA-J, and HLA-L are classified as class I pseudogenes, while HLA-N, HLA-S, and HLA-X are classified as gene fragments. In particular, Messer et al., J Immunol. 1992 Jun 15; 148(12):4043-53 reported that HLA-J is a pseudogene caused by a detrimental mutation that terminates translation in exon 2 or exon 4. Therefore, the human leukocyte antigen (HLA) gene has a long history of research as an important target for biomedical, diagnostic, and therapeutic purposes.

[0011] Furthermore, cancer is the second leading cause of death worldwide, with an estimated 9.6 million people dying from it in 2018. Globally, approximately one in six deaths are caused by cancer. Cancer incidence is currently increasing, especially as people age. Early detection and treatment of cases can reduce cancer mortality. Without early diagnosis, patients are diagnosed at a late stage, at which point curative treatment may no longer be an option. However, even when cancer is diagnosed early, tumor heterogeneity often makes finding effective treatments for specific patients difficult. This is because large tumors may comprise a variety of cells with different molecular characteristics and varying sensitivities to treatment. This heterogeneity can lead to uneven distribution of genetically distinct tumor cell subpopulations between and within disease sites (spatial heterogeneity) or temporal variations in the molecular composition of cancer cells (temporal heterogeneity). Heterogeneity provides impetus for tumor resistance to certain treatment regimens. Therefore, there is an urgent need to predict in advance whether individuals with tumors will respond to specific tumor treatments. There is also an urgent need for new tumor treatments. This invention addresses these needs.

[0012] Therefore, in a first aspect, the present invention relates to a method for predicting whether a subject with a tumor will respond to a tumor treatment selected from (i) immunotherapy, (ii) chemotherapy, (iii) anti-hormonal therapy, and (iv) anti-tyrosine kinase therapy, wherein the method comprises: (A) determining the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from the subject, wherein the at least one nucleic acid molecule is selected from: (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of any amino acid sequence of SEQ ID NO: 1 to 6, (b) a nucleic acid molecule of SEQ ID NO: 1 to 6. NO: A nucleic acid molecule consisting of any nucleotide sequence from 7 to 12, (c) a nucleic acid molecule encoding a polypeptide whose amino acid sequence is at least 85% identical, preferably at least 90% identical, and most preferably at least 95% identical to that of (a), (d) a nucleic acid molecule consisting of a nucleotide sequence whose nucleotide sequence is at least 95% identical, preferably at least 96% identical, and most preferably at least 98% identical to that of (b), (e) a nucleic acid molecule consisting of a degenerate nucleotide sequence relative to (d), (f) a nucleic acid molecule consisting of a fragment of any nucleic acid molecule from (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, and (g) a nucleic acid molecule corresponding to any nucleic acid molecule from (a) to (f) wherein T is replaced by U, wherein said at least one protein or peptide is selected from the protein or peptide encoded by any nucleic acid molecule from (a) to (g); and (B) the level of (A) is compared with that of (i) (i) to (iii) compare the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who have responded to one or more treatments in (i) to (iii) with a corresponding predetermined standard, wherein an increase in the level of (A) compared to the level of (B) or the predetermined standard indicates that the subject has not responded to the tumor treatment, and a substantially the same level or a decrease in the level of (A) compared to the level of (B) indicates that the subject has responded to the tumor treatment; or (B') compare the level of (A) with the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from one or more subjects who have not responded to one or more treatments in (i) to (iii) with a corresponding predetermined standard, wherein a decrease in the level of (A) compared to the level of (B') or the predetermined standard indicates that the subject has responded to the tumor treatment, and a substantially the same level or an increase in the level of (A) compared to the level of (B') indicates that the subject has not responded to the tumor treatment.

[0013] According to the invention, the term "object" refers to a mammal, preferably a domestic animal or pet such as a horse, cow, pig, sheep, goat, dog or cat, and most preferably a human.

[0014] A tumor is an abnormal, benign or malignant new growth of tissue that lacks physiological function and is caused by uncontrolled, usually rapid, cell proliferation. A tumor is preferably cancer. Cancer is an abnormal, malignant new growth of tissue that lacks physiological function and is caused by uncontrolled, usually rapid, cell proliferation. Cancers are preferably selected from 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 cancers, colon cancer, colorectal cancer, prostate cancer, head and neck squamous cell carcinoma, cervical cancer, glioblastoma, malignant ascites, lymphoma, and leukemia. Preferred cancers will be defined below.

[0015] The tumor or cancer is preferably a solid tumor or cancer. A solid tumor or cancer is an abnormal mass of tissue that, unlike non-solid tumors (such as leukemia), typically does not contain cysts or fluid-filled areas.

[0016] While cancer treatment can also be, for example, surgery, the cancer treatments discussed in this article are selected from (i) immunotherapy, (ii) chemotherapy, (iii) anti-hormonal therapy, and (iv) anti-tyrosine kinase therapy. Among these cancer treatments, immunotherapy is preferred.

[0017] Immunotherapy treats disease by activating or suppressing the immune system. According to the present invention, immunotherapy is the treatment of tumors; therefore, immunotherapy is tumor immunotherapy, preferably cancer immunotherapy. Tumor immunotherapy generally involves artificially stimulating the immune system to treat tumors, improving the system's natural ability to fight tumors. Immunotherapy can be classified as active, passive, or a combination of both (active and passive). Active immunotherapy guides the immune system to attack tumor cells by targeting tumor antigens. Passive immunotherapy enhances existing anti-tumor responses, including, for example, the use of monoclonal antibodies, lymphocytes, and cytokines.

[0018] Immunotherapy preferably includes the application of immune checkpoint inhibitors, and immunotherapy is therefore preferably immune checkpoint inhibitor therapy. Immune checkpoint inhibitors (also simply checkpoint inhibitors) are drugs that help the immune system respond more strongly to tumors. For example, these drugs work by releasing "brakes" to keep T cells (a type of white blood cell that is part of the immune system) from killing tumor cells. These drugs do not directly target the tumor. Instead, they interfere with the ability of tumor cells to avoid being attacked by the immune system.

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

[0020] Chemotherapy is a cancer treatment that uses drugs called cell inhibitors to stop tumor cells from continuing to divide uncontrollably. Cell inhibitors are usually administered intravenously by infusion, but some can also be taken as tablets. Chemotherapy may be given for curative purposes (almost always involving a combination of drugs) or to prolong life or alleviate symptoms (palliative chemotherapy). Cell inhibitors can work, for example, by inhibiting nucleic acid synthesis, destroying nucleic acids, or altering microtubule proteins (spindle toxins) or disrupting cell membranes. Chemotherapy is often combined with radiation therapy—this is known as chemoradiotherapy. Chemotherapy as used in this article can be adjuvant or neoadjuvant chemotherapy, preferably neoadjuvant chemotherapy. In neoadjuvant (also known as preoperative or primary) chemotherapy, the drug treatment occurs before the tumor is surgically removed. This contrasts with adjuvant chemotherapy, which is the drug treatment after surgery. The efficacy of chemotherapy agents can release tumor antigens through cell destruction, which are then presented to the immune system. This can ultimately lead to increased recognition by the immune system, thereby enhancing the effectiveness of immunotherapeutic agents such as immune checkpoint inhibitors or activators.

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

[0022] Antityrosine kinase therapy utilizes tyrosine kinase inhibitors (TKIs) as drugs that inhibit tyrosine kinases. Tyrosine kinases are enzymes responsible for activating many proteins through signal transduction cascades. Proteins are activated by the addition of a phosphate group (phosphorylation), a step inhibited by TKIs. TKIs are used as anticancer drugs. TKIs function through four different mechanisms: they can compete with adenosine triphosphate (ATP) for phosphorylation, substrates, or both, or they can act allosterically, binding to sites outside the active site and affecting their activity through conformational changes. As part of this invention, the interaction between receptor tyrosine kinases and HLA factors has been investigated.

[0023] The nucleic acid sequences of SEQ ID NO:7 to 12 are, respectively, the human HLA gene membrane-bound HLA-G, HLA-L, soluble HLA-G, HLA-H, HLA-J, and HLA-L genes. Furthermore, membrane-bound isotypes can be released through proteolytic activity, thereby increasing the soluble fractions of HLA-G and HLA-L. Preferably, the nucleic acid molecule according to the invention is genomic DNA or mRNA. In the case of mRNA, the nucleic acid molecule may additionally contain a poly-A tail.

[0024] As surprisingly discovered and illustrated in the examples below, HLA-G is expressed as a full-length transcript and a spliced ​​form containing only exons 1 through 5 of HLA-G. While full-length HLA-G contains a transmembrane domain and is therefore membrane-bound, soluble HLA-G lacks this transmembrane domain. The examples further demonstrate that high levels of expression of mRNA encoding full-length HLA-G (i.e., as indicated by measured high levels of exons 5 and 8, or exon 8 only) and high levels of mRNA encoding the soluble form (i.e., as indicated by measured high levels of exon 5 and low levels of exon 8, or exon 5 only) are associated with tumor patients who do not respond to tumor treatments as defined above. As mentioned above, membrane-bound HLA isoforms can also be released via post-translational proteolytic cleavage, resulting in the release of soluble HLA fragments.

[0025] The gene encoding HLA-L also contains a sequence encoding a transmembrane domain. Therefore, it is believed that HLA-L can exist in tumors in both a full-length membrane-bound form (SEQ ID NO:2) and a soluble form (SEQ ID NO:8). Full-length HLA-L may also be released through post-translational proteolytic cleavage, leading to the release of soluble HLA fragments.

[0026] On the other hand, the genes encoding HLA-H and HLA-J (SEQ ID NO: 11 and 12) do not contain open reading frames encoding transmembrane domains. The examples below demonstrate that HLA-H and HLA-J are soluble. The examples below also demonstrate that high expression of mRNA encoding such soluble HLAs is associated with cancer patients who are unresponsive to the cancer therapies defined above.

[0027] SEQ ID NO:1 to 6 are the amino acid sequences of the human HLA genes HLA-G, HLA-L, soluble HLA-G, HLA-H, HLA-J and HLA-L proteins, respectively.

[0028] The term "nucleic acid sequence" or "nucleic acid molecule" in this invention includes DNA, such as cDNA, or double-stranded or single-stranded genomic DNA and RNA. In this respect, "DNA" (deoxyribonucleic acid) refers to any chain or sequence of chemical structural units called nucleotide bases linked together on a deoxyribose backbone, namely adenine (A), guanine (G), cytosine (C), and thymine (T). DNA may have a single nucleotide base chain or may form two complementary strands in a double helix structure. "RNA" (ribonucleic acid) refers to any chain or sequence of chemical structural units called nucleotide bases called adenine (A), guanine (G), cytosine (C), and uracil (U) linked together on a ribose backbone. RNA typically has a single nucleotide base chain, such as mRNA. Single-stranded and double-stranded hybrid molecules are also included, namely DNA-DNA, DNA-RNA, and RNA-RNA. Nucleic acid molecules can also be modified by many methods known in the art. Non-limiting examples of such modifications include methylation, “capping,” substitution of one or more naturally occurring nucleotides with analogs, and internucleotide modifications, such as modifications involving uncharged bonds (e.g., methylphosphonates, phosphate triesters, aminophosphates, carbamates, etc.) and charged bonds (e.g., thiophosphates, dithiophosphates, etc.). Nucleic acid molecules, also referred to hereinafter as polynucleotides, may contain one or more additional covalently linked moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents (e.g., metals, radioactive metals, iron, metal oxides, etc.), and alkylating agents. Polynucleotides can be derivatized by forming methyl or ethyl phosphate triester or alkyl aminophosphate bonds. Further examples include nucleic acid mimics known in the art, such as synthetic or semi-synthetic derivatives and mixed polymers of DNA or RNA. Nucleic acid mimics or derivatives according to the present invention include phosphate-thiocyanate nucleic acids, aminophosphate nucleic acids, 2'-O-methoxyethyl ribonucleic acid, morpholinonucleotides, hexitol nucleic acids (HNA), peptide nucleic acids (PNA), and locked nucleic acids (LNA) (see Braasch and Corey, Chem Biol 2001, 8:1). LNA is an RNA derivative in which the ribose ring is restricted by a methylene bond between the 2'-oxygen and 4'-carbon. Nucleic acids containing modified bases, such as thiouracil, thioguanine, and fluorouracil, are also included. Nucleic acid molecules typically carry genetic information, including information used by cellular structures to produce proteins and / or polypeptides. Nucleic acid molecules may additionally include promoters, enhancers, response elements, signal sequences, polyadenylated sequences, introns, 5'-uncoding regions, and 3'-uncoding regions, etc.

[0029] The term "protein" as used herein is used interchangeably with the term "polypeptide," which describes a linear molecular chain of amino acids, including single-chain proteins or fragments thereof containing at least 50 amino acids. The term "peptide" as used herein describes a group of molecules consisting of up to 49 amino acids, while the term "polypeptide" (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids. The term "peptide" as used herein describes a group of molecules, preferably increased to at least 15, at least 20, at least 25, and at least 40 amino acids. The groups of peptides and groups of 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 polymers are accordingly called homodimers or heterodimers, homotrimers or heterotrimers, etc. For example, HLA proteins contain cysteine ​​residues and therefore contain potential dimerization sites. Furthermore, peptide analogs of such proteins / (poly)peptides in which amino acids and / or peptide bonds have been replaced by functional analogs are also included in this invention. Such functional analogs include all known amino acids except for the 20 genetically encoded amino acids, such as selenocysteine. The terms "(poly)peptide" and "protein" also refer to naturally modified (poly)peptides and proteins, wherein said modifications are achieved, for example, by glycosylation, acetylation, phosphorylation, and similar modifications known in the art.

[0030] According to the present invention, the term "sequence identity percentage (%)" describes the ratio of the number of identical nucleotide / amino acid matches ("hits") between two or more aligned nucleic acid or amino acid sequences to the number of nucleotide or amino acid residues constituting the total length of the template nucleic acid or amino acid sequence. In other words, by aligning two or more sequences or subsequences, the percentage of identical amino acid residues or nucleotides (e.g., 80%, 85%, 90%, or 95% identity) can be determined when comparing and aligning sequences based on the maximum correspondence within a comparison window or a specified region using sequence comparison algorithms known in the art or through manual alignment and visual inspection. This definition also applies to complementary sequences of any sequence to be aligned.

[0031] Nucleotide and amino acid sequence analysis and alignment related to this invention are preferably performed using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A.). 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). Technicians know of other suitable procedures for aligning nucleic acid sequences.

[0032] As defined herein, the present invention relates to sequence identity with at least 85% similarity, preferably at least 90% similarity, and most preferably at least 95% similarity. However, the present invention also relates to sequence identity with increased preference having at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% similarity.

[0033] The sample can be a bodily fluid from the subject or a tissue sample from the subject's organ. Non-limiting examples of bodily fluids include whole blood, plasma, serum, urine, peritoneal and pleural fluid, cerebrospinal fluid, tears, or cells derived from the above solutions. Non-limiting examples of tissues include the colon, liver, breast, ovary, and testis. Tissue samples can be obtained by aspiration or puncture, excision, or any other surgical method that results in a biopsy or removal of cellular material. The sample can be a processed sample, such as one that has been frozen, fixed, embedded, etc. A preferred sample type is formalin-fixed paraffin-embedded (FFPE) samples. The preparation of FFPE samples is standard medical practice, and these samples can be preserved for a long time.

[0034] Methods for obtaining the level of nucleic acid molecules, proteins, or peptides in the context of the present invention are known in the art.

[0035] For example, the level of nucleic acid molecules can be obtained by real-time quantitative PCR (RT-qPCR), electrophoresis, 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" refers to a gene that has a relatively constant expression level at the RNA transcript / mRNA level in the system under examination, i.e., a tumor. Such a gene may be referred to as a housekeeping gene. Non-limiting examples of reference genes are CALM2, B2M, RPL37A, GUSB, HPRT1, and GAPDH, with CALM2 and / or B2M being preferred. Other suitable reference genes are known to those skilled in the art.

[0036] RT-qPCR is performed in a thermal cycler, which can illuminate each sample with a light beam of at least one specific wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler can also rapidly heat and cool the sample, thus utilizing the physicochemical properties of nucleic acids and DNA polymerases. Two common methods for detecting PCR products in real-time qPCR are: (1) non-specific fluorescent dyes inserted into any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides labeled with a fluorescent reporter molecule, which allow detection only after hybridization with their complementary sequence (e.g., TaqMan probes). The probes are typically fluorescently labeled. Preferably, the fluorescently labeled probes consist of oligonucleotides labeled with both a fluorescent reporter dye and a quencher dye (= dual-labeled probes). Suitable fluorescent reporter and quencher dyes / parts are known to those skilled in the art and include, but are not limited to, reporter dyes / parts 6-FAM™, JOE™, etc. And quenching dyes / partial dabcyl, TAMRATM, BHQTM-1, -2 or -3. Preferably, primers used for purposes according to the invention have a length of 15 to 30 nucleotides, and in particular deoxyribonucleotides. In one embodiment, primers are designed to (1) be specific to HLA genes or target mRNA sequences derived therefrom, (2) provide an amplicon size of less than 120 bp (preferably less than 100 bp), (3) be mRNA specific (considering exons / introns; preferably without amplification of genomic DNA), (4) have no tendency to dimerize and / or (5) have a melting temperature T in the range of 58°C to 62°C. m (Preferred T) m (Approximately 60℃). As mentioned above, RT-qPCR in (2) requires a probe, but in the case of RT-qPCR in (1), the probe can be replaced by an intercalating dye such as SYBR Green.

[0037] As an alternative to qPCR, electrophoresis can also be used, or, as another alternative, DNA microarrays can be used to obtain the nucleic acid molecules of the first aspect of this invention. The conventional method for mRNA identification and quantification is a combination of gel electrophoresis, which provides information on size and sequence-specific detection. Northern blotting is the most commonly used technique in this latter category. Ribonuclease protection assays (RPA) have been developed as a more sensitive and less labor-intensive alternative to Northern blotting. Hybridization is performed in solution with labeled ribonucleotide probes, and then 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 quantitative tool, also indicating the size of the probe hybridization region. For both Northern blotting and RPA, the accuracy and precision of quantification depend on the detection method and the reference or standard used. Most commonly, the probes are radiolabeled with 32P or 33P, in which case the final gel is exposed to X-ray film or a phosphorescent screen, and the intensity of each band is quantified separately using a densitometer or phosphorescent imager. In both cases, the exposure time can be adjusted to suit the desired sensitivity, but phosphor-based techniques are generally more sensitive and have a wider dynamic range. As an alternative to using radioactivity, probes can be labeled with antigens or haptens and then bound to antibodies conjugated with horseradish peroxidase or alkaline phosphatase, quantified via chemiluminescence or fluorescence imaging on film after substrate addition. In all these imaging applications, background from adjacent areas of the probe-free gel should be subtracted. A major advantage of gel form is that any reference standard can be imaged simultaneously with the sample. Similarly, housekeeping gene detection is performed under the same conditions on all samples.

[0038] Furthermore, next-generation sequencing (NGS) can be used. NGS is an RNA or DNA sequencing technology that has revolutionized genome research. Using NGS, the entire human genome can be sequenced in a single day. In contrast, Sanger sequencing, previously used to decipher the human genome, took over a decade to deliver a final draft. In light of this invention, NGS can be used for quantification in an open configuration (genome-wide exome sequencing) or as a focused group containing the corresponding HLA genes and isotypes disclosed in this application.

[0039] Two techniques have emerged for constructing DNA microarrays. Typically, in each case, the starting point for array design is a set of sequences corresponding to the gene to be probed or a putative gene. In the first approach, oligonucleotide probes are chemically synthesized on a glass substrate. Because oligonucleotides hybridize to cDNA probes with varying efficiencies, multiple oligonucleotide probes are synthesized, each complementary to the gene of interest. Furthermore, for each perfectly complementary oligonucleotide on the array, mismatched oligonucleotides at individual nucleotide positions are constructed and used for normalization. Oligonucleotide arrays are typically constructed in groups of approximately 10... 4 -10 6 probes / cm 2 The second major technique for constructing DNA microarrays involves the automated printing of cDNA probes directly onto glass slides or other suitable substrates. DNA clones, purification, and amplification of each gene of interest are obtained from a universal vector via PCR using universal primers. The probes are automatically deposited as dots of 50–200 μm in size. At this spacing, densities of approximately 10-1 can be achieved. 3 probes / cm 2 The density.

[0040] The level of a protein or peptide can be determined, for example, by using “molecules that bind to a protein or peptide,” and preferably “molecules that specifically bind to a protein or peptide.” A molecule that binds to a protein or peptide is one that binds primarily to a protein or peptide under known conditions. “Molecules that bind to a protein or peptide” can be one of the binding molecules described herein, preferably inhibitors of the protein or peptide, such as antibodies, aptamers, etc. The level of a protein or peptide can also be obtained by using Western blot analysis, mass spectrometry, FACS analysis, ELISA, and immunohistochemistry. These techniques are non-limiting examples of methods that can be used for the qualitative, semi-quantitative, and / or quantitative detection of proteins or peptides.

[0041] Western blot analysis is a widely used and well-known analytical technique for detecting specific proteins or peptides in a given sample (such as tissue homogenate or body extract). It uses gel electrophoresis to separate native or denatured proteins or peptides based on the length of the (poly)peptide (denaturing conditions) or 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 detected using an antibody specific to the target protein.

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

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

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

[0045] Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the selective identification of antigens (proteins) in tissue sections by utilizing the principle of specific binding of antibodies to antigens in biological tissues. Combined with specific devices, IHC can be used for quantitative in situ assessment of protein expression (see Cregger et al. (2006) Arch Pathol Lab Med, 130:1026-1030 for a review). Quantitative IHC utilizes the fact that staining intensity is correlated with absolute protein levels.

[0046] Methods for determining whether a subject responds to one or more cancer treatments and for determining whether a subject does not respond to one or more cancer treatments are well known in the art. Generally, a cancer patient responds to treatment if the tumor shrinks (in the case of a solid tumor), or if the number of tumor cells in a non-solid tumor (e.g., blood cancer) or the symptoms induced by the cancer disease decreases or remains unchanged (“stable”). Generally, a cancer patient does not respond if the tumor worsens during treatment (e.g., increases in size, increases in cell number, or worsens the symptoms induced by the cancer disease). Regarding response, tumor shrinkage is preferred.

[0047] Conclusive evidence of treatment effectiveness is improvement in clinical symptoms and survival, while conclusive evidence of treatment ineffectiveness is worsening of clinical symptoms and eventual death. As part of this invention, disease-specific survival is frequently used, defined as the period from the start of the investigated treatment option until cancer-specific death. Imaging, particularly of the tumor lesion, is often used to assess treatment efficacy earlier. Current response assessments are primarily based on changes in tumor size measured by CT (computed tomography) or other anatomical imaging modalities, where a reduction in tumor size indicates a response. Additionally, PET (positron emission tomography) and glucose analogues are used. 18 Imaging tumor metabolism with F-FDG represents an attractive method for objectively and quantitatively assessing treatment efficacy.

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

[0049] The number of responding or non-responding subjects is increased by at least 2, at least 5, at least 10, at least 25, and at least 50 subjects, respectively. Using more than one subject helps to account for bias in the differences in levels between responding and non-responding patients.

[0050] Predetermined criteria refer to values ​​previously obtained from one or more subjects who responded to one or more cancer treatments or who did not respond to one or more cancer treatments.

[0051] The increase levels of (B) and (B') are preferably at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, or 4 times higher than the level of (A). The decrease levels of (B) and (B') are preferably at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, or 4 times lower than the level of (A). The substantially identical levels of (B) and (B') preferably differ from the control or predetermined standard (i.e., higher or lower) by less than 10%, more preferably less than 5%. For example, if the level in (A) is set to 100%, the substantially identical levels can be between less than 110% and greater than 90% of the 100% control level.

[0052] As can be seen from the examples below, it was surprising to find that high expression levels of membrane-bound HLA-G (exon 8 probe), soluble or membrane-bound HLA-G (exon 3 probe), membrane-bound HLA-L (exon 7 probe), soluble HLA-H (exon 2 / 3 probe), and soluble HLA-J (exon 4 / 5 probe) were negatively correlated with survival in patients with bladder cancer who received immune checkpoint therapy (anti-PD-1 or anti-PDL-1). Higher expression levels of these HLA genes were associated with a greater likelihood of death from cancer within 2 years. It should be considered that post-transcriptional events may affect membrane-bound HLA isotypes. Therefore, membrane-bound HLA-G mRNA isotypes quantified by exon 8 may ultimately yield biologically active soluble fragments after proteolytic cleavage events following translation into protein structures. However, HLA mRNA expression levels were measured in tumor tissue samples obtained from bladder cancer patients prior to the initiation of immune checkpoint therapy. Therefore, the data in the examples demonstrate that the expression levels of HLA-G, HLA-L, HLA-H, and HLA-J genes or proteins in subjects can be used to predict whether subjects are likely to benefit from treatment before the initiation of immune checkpoint therapy. While low expression levels are associated with better disease-specific survival, high expression levels are associated with poorer disease-specific survival.

[0053] The predictive value of HLA-G, HLA-L, HLA-H, and HLA-J expression levels shown in the believed examples for survival in bladder cancer patients under immune checkpoint therapy is also applicable to other tumors and anti-tumor therapies, such as general immunotherapy, chemotherapy, anti-hormonal therapy, and anti-tyrosine therapy. This is because it can be hypothesized that high HLA-G, HLA-L, HLA-H, and HLA-J expression levels help tumor cells or tumor cell subsets evade anti-tumor therapy, since any effective anti-cancer treatment leads to the destruction of tumor cells and exposure of antigens to the immune system, thus preventing the tumor from being masked. Therefore, cellular strategies that reduce immune recognition conferred by HLA-G, HLA-L, HLA-H, and HLA-J expression are important not only for immunotherapy but also for chemotherapy and / or anti-hormonal and / or tyrosine kinase inhibitory therapy or any combination thereof.

[0054] Regarding the sequences of membrane-bound human HLA-L and soluble HLA-H, HLA-J, and HLA-L, it is further noteworthy that this paper surprisingly finds that HLA-L, HLA-H, and HLA-J are incorrectly labeled as pseudogenes in the art. In fact, these genes encode proteins, and as shown in the accompanying examples, the expression of HLA-L, HLA-H, and HLA-J can be detected in various cancers. Because HLA-L, HLA-H, and HLA-J are incorrectly labeled in the art, they can be collectively referred to as a new HLA group. Furthermore, the examples below demonstrate that high expression levels of HLA-L, HLA-H, and HLA-J in patients with bladder cancer are negatively correlated with these patients' survival rates. The higher the expression levels of these HLA genes, the greater the likelihood that the patient will die from cancer within 2 years. This evidence suggests that the expression of HLA forms L, H, and J is likely used by tumors as a mechanism to evade the immune system of cancer patients. These genes and the proteins they encode are functional and are not pseudogenes that do not encode any functional proteins.

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

[0056] SEQ ID NO:9 and 10 are nucleic acid sequences encoding membrane-bound HLA-G and HLA-L in soluble HLA forms, while SEQ ID NO:11 and 12 are soluble HLA-H and HLA-J. SEQ ID NO:3 to 6 are the corresponding amino acid sequences.

[0057] The data in the examples, based on HLA categories G, H, L, and J, demonstrate that HLA genes and proteins can predict cancer patients' responses to cancer treatments as defined herein.

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

[0059] With regard to this preferred embodiment, it should be understood that what needs to be determined is the mRNA expression level or protein level in the subject, and then compared with a corresponding control or predetermined standard, which is derived from known responders or non-responders and / or known survivors or non-survivors, as explained above in conjunction with HLA genes.

[0060] The mRNA expression level or protein level of one or more of ErbB2, EGFR, CD20, CTLA4, IDO1, LAG3, TIM3, TIM-4, CXCL9, CXCL13, TIGIT, BTLA, CD137, OX40, VISTA, B7-H7, CD27, GITR, TGF-β signaling pathway, IL-15, PD-1, and PD-L1, preferably PD-1 or PD-L1, alone does not have sufficient predictive value to determine whether a subject is likely to respond to or not respond to tumor therapies as defined herein, particularly immunotherapy, and even more particularly checkpoint therapy. They can be used in conjunction with the methods of the present invention. Therefore, additional analysis of one or more of these levels is expected to further improve the predictive value of the methods of the present invention.

[0061] PD-1 (programmed cell death protein 1, also known as CD279) is a cell surface protein that regulates the immune system’s response to human cells. This function is achieved by downregulating the immune system and promoting self-tolerance by inhibiting the inflammatory activity of T cells.

[0062] PD-L1 (programmed death-ligand 1, also known as CD274 or B7-H1) is a 40 kDa type I transmembrane protein presumably playing a major role in suppressing the immune system during specific events such as pregnancy, tissue allogeneic transplantation, autoimmune diseases, and other disease states such as hepatitis. Upregulation of PD-L1 can allow cancer to evade the host's immune system. Importantly, PD-L1 can be expressed by both tumor and non-tumor cells, such as macrophages.

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

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

[0065] CD20 is an activated glycosylated phosphoprotein expressed on the surface of all B cells, with concentrations gradually increasing from the pro-B phase (CD45R+, CD117+) until maturity. CD20 is a target of monoclonal antibodies such as rituximab, olibituzumab, olbirutuzumab, oflamumab, teimomab, tocetuzumab, and unirituximab, all of which are active agents for the treatment of all B-cell lymphomas, leukemias, and B-cell-mediated autoimmune diseases.

[0066] CTLA4 (cytotoxic T-lymphocyte-associated protein 4, also known as CD152) is a protein receptor that acts as an immune checkpoint (or checkpoint inhibitor) to downregulate the immune response. CTLA4 is constitutively expressed in regulatory T cells, but is upregulated only upon activation in regular T cells—a phenomenon particularly pronounced in cancer.

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

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

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

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

[0071] CXCL9 (chemokine (CXC motif) ligand 9) is a small cytokine belonging to the CXC chemokine family, also known as interferon-gamma induced monokine (MIG). CXCL9 is a T cell chemokine induced by IFN-γ.

[0072] CXCL13 (chemokine (CXC motif) ligand 1, also known as B lymphocyte chemokine (BLC) or B cell attractant chemokine 1 (BCA-1)) is a small chemokine belonging to the CXC chemokine family. As its name suggests, this chemokine exhibits selective chemotaxis towards B cells belonging to the B-1 and B-2 subsets, and its effects are induced through interaction with the chemokine receptor CXCR5.

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

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

[0075] CD137, also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9) or 4-1BB, is induced by lymphocyte activation (ILA). The most characteristic activity of CD137 is its co-stimulatory activity on activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolysis. Furthermore, it can enhance immune activity to eliminate tumors.

[0076] Ox40 (also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4) and CD134) is a secondary costimulatory immune checkpoint molecule expressed 24 to 72 hours after activation; its ligand OX40L is not expressed on resting antigen-presenting cells, but is expressed upon cell activation. OX40 expression depends on complete T cell activation; in the absence of CD28, OX40 expression is delayed and its level is reduced fourfold.

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

[0078] B7-H7 (also known as human endogenous retrovirus-H long terminal repeat-associated 2 (HHLA2)) is a member of the B7 family that regulates human T cell function. Previously known as having an undetermined function, B7-H7 has been identified as a specific ligand for human CD28H. The B7-H7-CD28H pathway strongly promotes CD4+ T cell proliferation and cytokine production via an AKT-dependent signaling cascade in the presence of TCR signaling, suggesting that B7-H7 contains a novel co-stimulatory pathway. The first IgV domain of B7-H7 likely binds to a presumed receptor, exhibiting the highest homology with other members of the B7 family.

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

[0080] GITR (a glucocorticoid-induced TNFR-related protein, also known as tumor necrosis factor receptor superfamily member 18 (TNFRSF18) and activation-inducible TNFR family receptor (AITR)) has been shown to increase in expression upon T cell activation and is believed to play a key role in dominant immune self-tolerance maintained by CD25+ / CD4+ regulatory T cells. Mouse knockout studies have also shown that this receptor regulates CD3-driven T cell activation and programmed cell death.

[0081] The transforming growth factor β (TGFβ) signaling pathway is involved in many cellular processes in adult organisms and developing embryos, including cell growth, cell differentiation, apoptosis, cell homeostasis, and other cellular functions. Although the range of cellular processes regulated by the TGFβ signaling pathway is broad, the process itself is relatively simple. TGFβ superfamily ligands bind to type II receptors, recruiting and phosphorylating type I receptors. The type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs), which can now bind to coSMAD / SMAD4. The R-SMAD / coSMAD complex accumulates in the cell nucleus, where it acts as a transcription factor and participates in the regulation of target gene expression.

[0082] IL-15 (interleukin-15) is a cytokine structurally similar to interleukin-2 (IL-2). Like IL-2, IL-15 binds to a complex consisting of the IL-2 / IL-15 receptor β chain (CD122) and common γ chains (γ-C, CD132), and signals through this complex. IL-15 is secreted by monocytes (and some other cells) after viral infection. This cytokine induces the proliferation of natural killer cells; these are cells of the innate immune system whose primary function is to kill virus-infected cells.

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

[0084] The definitions provided above, together with the first aspect of the invention, are applicable to the second aspect of the invention.

[0085] Inhibitors of nucleic acid molecules, preferably those defined in the first aspect of the invention, are preferably selected from small molecules, aptamers, siRNA, shRNA, miRNA, ribozymes, antisense nucleic acid molecules, CRISPR-Cas9-based constructs, CRISPR-Cpf1-based constructs, broad-spectrum nucleases, zinc finger nucleases, and transcription activator-like (TAL) effector (TALE) nucleases. Further details regarding these categories will be provided below.

[0086] The inhibitor of the HLA protein according to the present invention is preferably selected from small molecules, antibodies or antibody mimics and aptamers, wherein the antibody mimics are preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofibrins, affiliins, Kunitz domain peptides, etc. Three specific binding molecules and proantibodies.

[0087] As used herein, the term "antibody mimic" refers to a compound that, like an antibody, can specifically bind to an antigen, such as the HLA proteins of SEQ ID NO: 1 to 6 in this case, but is structurally unrelated to antibodies. Antibody mimics are typically synthetic peptides or proteins with a molar mass of approximately 3 to 20 kDa. For example, antibody mimics may be selected from affibodies, adnectins, antiicalins, DARPins, avimers, nanofibrins, affiliins, Kunitz domain peptides, and... These polypeptides are well known in the art and are described in further detail below.

[0088] As used herein, the term "affibody" refers to a family of antibody mimics derived from the Z domain of staphylococcal protein A. Structurally, the affibody molecule is based on a triple-helix bundle domain, which can also be incorporated into fusion proteins. The affibody itself has a molecular weight of approximately 6 kDa and is stable under high temperature and acidic or alkaline conditions. Target specificity is obtained by randomizing 13 amino acids located in the two α-helices associated with the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).

[0089] As used herein, the term “adnectin” (also known as “monobody”) refers to a molecule based on the 10th extracellular domain (10Fn3) of human fibronectin III, which employs a 94-residue Ig-like β-sandwich fold with 2 to 3 exposed loops but lacks a central disulfide bond (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectin with desired target specificity (i.e., targeting HLA proteins) can be genetically engineered by introducing modifications into specific loops of the protein.

[0090] As used herein, the term “anticalin” refers to engineered proteins derived from lipid carriers (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci US A. 96(5): 1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13: 245-255). Anticalins possess an eight-chain β-barrel that forms a highly conserved core unit in lipid carriers and naturally forms ligand binding sites through four structurally variable loops at the open ends. Although heterologous to the IgG superfamily, anticalins exhibit characteristics that have been considered typical of antibody binding sites to date: (i) high structural plasticity due to sequence variations and (ii) enhanced conformational flexibility, allowing them to be induced to adapt to targets of different shapes.

[0091] As used herein, the term "DARPin" refers to a designed ankyrin repeat domain (166 residues) that provides a rigid interface generated by β-turns of a typical triplet. DARPins typically carry three repeats corresponding to an artificially congruent sequence, with six positions in each repeat being randomized. Therefore, DARPins lack structural flexibility (Gebauer and Skerra, 2009).

[0092] As used herein, the term "avimer" refers to a class of antibody mimics consisting of two or more peptide sequences, each having 30 to 35 amino acids, derived from the A domains of various membrane receptors and linked by adaptor peptides. Binding to the target molecule occurs through the A domains, and the domains with the desired binding specificity against HLA proteins can be selected, for example, by phage display technology. The binding specificity of different A domains contained in an avimer can be the same but is not required to be the same (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

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

[0094] As used herein, the term "affilin" refers to an antibody mimic developed by using γ-B crystals or ubiquitin as a backbone and by modifying the amino acids on the surface of these proteins through random mutagenesis. The selection of affilins with the desired target specificity, i.e., against the HLA gene of this invention, is achieved, for example, through phage display or ribosome display techniques. Depending on the backbone, the molecular weight of affilins is approximately 10 or 20 kDa. As used herein, the term affilin also refers to a dimer or polymeric form of affilin (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

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

[0096] As used in this article, terminology This refers to non-immunoglobulin-derived binding peptides derived from the human Fyn SH3 domain. Fyn SH3-derived peptides are well-known in the field and described 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.

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

[0098] As used herein, the term "probody" refers to an antibody prodrug that is activatable by a protease. A probody consists of a true IgG heavy chain and a modified light chain. A masking peptide is fused to the light chain via a peptide linker that can be cleaved by tumor-specific proteases. The masking peptide prevents the probody from binding to healthy tissue, thereby minimizing toxic side effects. For example, in a probody, a small molecule, antibody, or protein drug or aptamer can bind to the masking peptide, which restricts or prevents binding to the HLA protein of the present invention, and this masking peptide is cleavable by a protease. A protease is an enzyme that digests proteins into smaller fragments by cleaving specific amino acid sequences called substrates. In normal healthy tissue, protease activity is tightly controlled. In cancer cells, protease activity is upregulated. In healthy tissue or cells, protease activity is regulated and is minimal, so the target binding region of the probody remains masked and therefore cannot bind. On the other hand, in diseased tissue or cells, protease activity is upregulated, and the target binding region of the probody is exposed, thus enabling binding and / or inhibition.

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

[0100] Compounds that inhibit the expression of nucleic acid molecules and / or proteins of the present invention are compounds that, according to the present invention (i) reduce or prevent transcription of genes encoding nucleic acid molecules and / or proteins of the present invention, or (ii) reduce or prevent translation of mRNA encoding proteins of the present invention. Compounds of (i) include compounds that interfere with transcription mechanisms and / or their interaction with the promoter of said gene and / or with expression control elements, such as enhancers, located remotely from the promoter. Compounds of (ii) include compounds that interfere with translation mechanisms. Compounds that inhibit the expression of nucleic acid molecules and / or proteins of the present invention specifically inhibit the expression of nucleic acid molecules and / or proteins of the present invention, for example, by specifically interfering with promoter regions controlling expression. Preferably, the transcription of nucleic acid molecules and / or proteins of the present invention or the translation of proteins of the present invention is preferably increased by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, for example at least 90% or 95%, at least 98%, and most preferably about 100% (e.g., compared to the same experimental setup without said compound).

[0101] According to the present invention, compounds that inhibit the activity of nucleic acid molecules, peptides, and / or proteins of the present invention cause said nucleic acid molecules, peptides, and / or proteins to perform their functions with reduced efficiency. Compounds that inhibit the activity of nucleic acid molecules, peptides, and / or proteins of the present invention specifically inhibit the activity of said nucleic acid molecules, peptides, and / or proteins. As will be further detailed below, compounds that inhibit the activity of nucleic acid molecules, peptides, and / or proteins of the present invention can specifically inhibit the activity of said nucleic acid molecules, peptides, and / or proteins by interacting with said nucleic acid molecules, peptides, and / or proteins themselves, or by specifically inhibiting (preferably killing) the cells that produce said nucleic acid molecules, peptides, and / or proteins and / or cells that bind to said peptides or proteins. Preferably, the activity of nucleic acid molecules, peptides, and / or proteins 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%, and most preferably about 100% (e.g., compared to the same experimental setup without the compound).

[0102] Alternatively, compounds that inhibit the activity of the nucleic acid molecules, peptides, and / or proteins described in this invention also include nucleic acids or analogues thereof for vaccinating patients against specific HLA allotypes. Vaccination methods may be based on the RNA, protein, or peptide level and require additional modifications for stabilization in vivo. This approach can be employed from personalized mutant vaccination methods (Sahin U. Personalized RNA vaccines mobilizes poly-specific therapeutic immunity against cancer. Nature 2017).

[0103] As a further option, compounds that inhibit the activity of the nucleic acid molecules, peptides and / or proteins described in this invention also include naturally occurring autoantibodies isolated against the corresponding HLA genes, isotypes and fragments, or cells that produce naturally occurring autoantibodies, which can be modified or multiplied before reintroduction into the corresponding patient.

[0104] The activity of the nucleic acid molecules, peptides, and / or proteins of the present invention is preferably their ability to induce resistance to tumor treatments as defined above in cancer patients. Means and methods for determining such activity have been established in the art and are described in the examples below. According to the medical aspects of the present invention, this activity of the nucleic acid molecules and / or proteins of the present invention is therefore inhibited.

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

[0106] Compounds that inhibit the expression and / or activity of nucleic acid molecules and / or proteins of the present invention can be formulated into vesicles, such as liposomes or exosomals. From a drug delivery perspective, liposomes have attracted considerable interest due to their specificity and duration of action. Liposome cellular delivery systems have been used to efficiently deliver nucleic acids, such as siRNA, into cells in vivo (Zimmermann et al. (2006) Nature, 441:111-114). Liposomes are monolayered or multilayered vesicles with a membrane formed of lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes, while unable to fuse effectively with the cell wall, are phagocytosed by macrophages and other cells in vivo. Exosomes are lipid packages that can carry a variety of different molecules, including RNA (Alexander et al. (2015), Nat Commun; 6:7321). Exosomes containing the molecules contained therein can be taken up by recipient cells. Therefore, exosomes are important mediators of intercellular communication and regulators of cellular niches. Exosomes can be used for diagnostic and therapeutic purposes because they can be used as delivery carriers, such as for contrast agents or drugs.

[0107] Compounds that inhibit the expression and / or activity of nucleic acid molecules, peptides, and / or proteins of the present invention can be administered to the subject at appropriate doses and / or therapeutically effective amounts. The therapeutically effective amount for a given situation will be readily determined through routine experiments and within the skill and judgment of a general clinician or physician. Typically, the routine dosing regimen for the pharmaceutical composition should be in the range of 1 μg to 5 g units per day. However, a more preferred dose may be 0.01 mg to 100 mg per day, even more preferably 0.01 mg to 50 mg, and most preferably 0.01 mg to 10 mg. Furthermore, if, for example, the compound is an iRNA reagent, such as siRNA, the total pharmaceutically effective amount of the administered pharmaceutical composition will generally be less than about 75 mg / kg 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 / kg body weight. More preferably, the amount of iRNA reagent per kg body weight will be less than 2000 nmol (e.g., about 4.4 × 10⁻⁶). 16(Number of copies), for example, 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 iRNA reagent / kg body weight. The treatment time required to observe changes and the interval for post-treatment response vary depending on the desired effect. The specific amount can be determined by routine tests known to those skilled in the art. Suitable tests are, for example, described in Tamhane and Logan (2002), “Multiple Test Procedures for Identifying the Minimum Effective and Maximum Safe Doses of a Drug”, Journal of the American Statistical Association, 97(457):1-9.

[0108] Compounds that inhibit the expression and / or activity of nucleic acid molecules, peptides, and / or proteins of the present invention are preferably mixed with pharmaceutically acceptable carriers or excipients to form pharmaceutical compositions. According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient. The pharmaceutical compositions of the present invention comprise the aforementioned compounds. It may optionally comprise other molecules capable of altering the properties of the compounds of the present invention, thereby, for example, stabilizing, modulating, and / or activating their functions. The composition may be in solid, liquid, or gaseous form, particularly in the form of powder, tablet, solution, or aerosol. The pharmaceutical compositions of the present invention may optionally and additionally comprise pharmaceutically acceptable carriers. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO, etc. Compositions comprising these carriers can be formulated using known conventional methods. The means and methods for preparing pharmaceutical compositions are described, for example, in Formulation tools for Pharmaceutical Development (2005), ISBN-10:1907568999 or Handbook of Pharmaceutical Manufacturing Formulations, ISBN-10:9781420081169.

[0109] The pharmaceutical composition can be administered via any suitable route. For example, the actual route to be chosen depends on the physical and chemical properties of the drug, the desired site of action, the extent of drug absorption from different routes, drug metabolism, and the patient's condition. Examples of routes of administration are enteral / gastrointestinal, local, and parenteral. Furthermore, in cases of bladder cancer or its neoplastic lesions, the pharmaceutical composition can be applied to the bladder as an infusion therapy. Administration as an infusion therapy is considered part of this invention, particularly for combinations of immunological, chemotherapeutic, anti-hormonal, or anti-tyrosine kinase compounds with anti-HLA agents as part of this application.

[0110] These pharmaceutical compositions can be administered to the subject at an appropriate dosage. The dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any given patient depends on many factors, including the patient's body size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, and other medications being taken concurrently.

[0111] As described above, the data in the examples indicate that high expression levels of these HLAs are associated with poorer disease-specific survival. Furthermore, it can be inferred that high expression levels of HLA-G, HLA-L, HLA-H, and HLA-J may help tumor cells or tumor cell subsets evade antitumor therapy.

[0112] Therefore, combination therapy is also hypothesized, in which classic antitumor therapies (e.g., (i) immunotherapy; (ii) chemotherapy; (iii) antihormonal therapy; and / or (iv) antityrosine kinase therapy) are combined with HLA-G, HLA-L, HLA-H, or HLA-J inhibitors to further improve antitumor treatment. Such combination antitumor therapy can be administered as a preventative measure, particularly in patients initially diagnosed with high levels of HLA-G, HLA-L, HLA-H, and / or HLA-J expression using the methods of this invention. In these patients, treatment failure may translate into treatment success.

[0113] In a preferred embodiment of the second aspect of the invention, the subject has been predicted to be unresponsive to (i) immunotherapy; (ii) chemotherapy; (iii) anti-hormone therapy; and / or (iv) anti-tyrosine kinase therapy performed by the methods of the first aspect of the invention.

[0114] The fact that the subject was previously diagnosed as unresponsive to (i) immunotherapy; (ii) chemotherapy; (iii) anti-hormone therapy; and / or (iv) anti-tyrosine kinase therapy indicates the need for additional treatment with the binding molecule, preferably the inhibitor of the present invention.

[0115] This is because the HLA gene expression discussed above is believed to protect malignant cells in the subject from (i) immunotherapy; (ii) chemotherapy; (iii) anti-hormone therapy; and / or (iv) anti-tyrosine kinase therapy, thereby combining the molecule, preferably the inhibitor of the present invention, with (i) immunotherapy; (ii) chemotherapy; (iii) anti-hormone therapy; and / or (iv) anti-tyrosine kinase therapy can turn (expected) treatment failure into treatment success.

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

[0117] The term "small molecule" as used herein is preferably an organic molecule. Organic molecules refer to or belong to a class of compounds with a carbon basis, meaning carbon atoms are linked together by carbon-carbon bonds. The initial definition of the term "organic" relates to the source of the compound; organic compounds are carbon-containing compounds obtained from plant, animal, or microbial sources, while inorganic compounds are obtained from mineral sources. Organic compounds can be natural or synthetic. Organic molecules are preferably aromatic molecules, more preferably heteroaromatic molecules. In organic chemistry, the term aromaticity is used to describe cyclic (ringed) or planar (flattened) molecules with resonant rings that are more stable than other geometric or interconnected arrangements with the same atomic group. Aromatic molecules are very stable and do not readily decompose or react with other substances. In heteroaromatic molecules, at least one atom in the aromatic ring is an atom other than carbon, such as N, S, or O. For all the above-mentioned 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.

[0118] Alternatively, the "small molecule" of this invention can be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds that do not contain carbon atoms (except carbon dioxide, carbon monoxide, and carbonates). Preferably, the molecular weight of the small inorganic molecule is less than about 2000 Da, or less than about 1000 Da, for example less than about 500 Da, and even more preferably less than about 250 Da. The size of the small molecule can be determined by methods known in the art, such as mass spectrometry. The small molecule can be designed, for example, based on the crystal structure of the target molecule, where sites that may be responsible for biological activity can be identified and verified in vivo in assays such as high-throughput screening (HTS) assays.

[0119] Nucleotide-based inhibitors comprise or consist of nucleic acid sequences. Nucleotide-based inhibitors may comprise or consist of RNA, DNA, or both. The nucleotide-based or nucleotide-analog-based inhibitors of the present invention are molecules that specifically bind to the HLA genes of SEQ ID NO:7 to 12 and also inhibit the activity of HLA encoded by said genes. As used herein, specific binding means that the inhibitor specifically targets HLA and, in particular, produces substantially no off-target inhibition of other cellular nucleic acid molecules.

[0120] The amino acid-based inhibitor comprises or consists of an amino acid sequence and preferably an amino acid sequence of at least 25, more preferably at least 50 amino acids. The amino acid-based inhibitor of the present invention is a molecule that specifically binds to HLAs selected from SEQ ID NO:1 to 6 and also inhibits the activity of said HLAs. The amino acid-based inhibitor preferably comprises natural amino acids, but may also comprise non-natural amino acids. Preferably, the amino acid-based inhibitor is selected or designed to specifically bind to an amino acid sequence selected from SEQ ID NO:1 to 6.

[0121] In a second aspect of the invention, the conjugate molecule, preferably the inhibitor, can also be a cell such as a T cell, wherein the T cell is preferably a CAR-T cell.

[0122] Cells typically carry binding molecules on their surface, preferably inhibitors of at least one nucleic acid molecule or at least one protein or peptide of the present invention. In the case of T cells, the binding molecule, preferably the inhibitor, is a naturally occurring or chimeric T cell receptor that specifically targets at least one protein or peptide of the present invention. Chimeric antigen receptor T cells (also known as CAR-T cells) are genetically modified T cells that produce artificial T cell receptors for use in immunotherapy.

[0123] Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T-cell receptors, or artificial T-cell receptors) are therefore receptor proteins that have been engineered to endow T cells with the novel ability to specifically target at least one protein or peptide of the present invention. These receptors are chimeric because they combine antigen binding and T-cell activation functions into a single receptor.

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

[0125] Aptamers are nucleic acid or peptide molecules that bind to specific target molecules. Aptamers are typically created by selecting them from large random sequence libraries, but naturally occurring aptamers also exist in riboswitches. Aptamers can be used as macromolecular drugs for basic research and clinical purposes. Aptamers can bind to ribozymes and undergo self-cleavage in the presence of their target molecules. These compounds also have other research, industrial, and clinical applications.

[0126] Nucleic acid aptamers are a class of nucleic acids that are typically composed of (usually short) oligonucleotide chains. Typically, they have been engineered through repeated rounds of in vitro selection or the equivalent SELEX (evolved through an exponentially enriched ligand system) to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms.

[0127] Peptide aptamers are typically peptides or proteins designed to interfere with interactions between other proteins within the cell. They consist of variable peptide rings attached to a protein scaffold at both ends. This dual-structure constraint significantly increases the binding affinity of aptamers, achieving levels comparable to antibodies (nanomolar range). The variable peptide ring typically contains 10 to 20 amino acids, and the scaffold can be any protein with good solubility. Currently, the bacterial protein thioredoxin-A is the most commonly used scaffold protein, with the variable peptide ring inserted into a redox active site, specifically the -Cys-Gly-Pro-Cys- ring (SEQ ID NO:13) in the wild-type protein, where the two cysteine ​​side chains can form disulfide bonds. Peptide aptamer selection can be performed using various systems, but the yeast two-hybrid system is currently the most widely used.

[0128] Aptamers are useful in biotechnology and therapeutic applications because they offer molecular recognition properties comparable to those of commonly used biomolecules, particularly antibodies. Beyond their discriminative recognition, aptamers offer advantages over antibodies because they can be engineered entirely in vitro, are easily produced through chemical synthesis, have ideal storage properties, and cause little to no immunogenicity in therapeutic applications. Unmodified aptamers are rapidly cleared from the bloodstream with half-lives ranging from minutes to hours, primarily due to nuclease degradation and clearance from the body via the kidneys, a consequence of their inherently low molecular weight. Applications of unmodified aptamers currently focus on treating transient conditions such as blood clotting, or treating organs that can be locally delivered (e.g., the eye). This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. Various modifications are available to scientists, such as 2'-fluorosubstituted pyrimidines, polyethylene glycol (PEG) linkages, and fusion with albumin or other half-life-extending proteins, which can extend the half-life of aptamers to days or even weeks.

[0129] Ribozymes (derived from ribonucleases, also known as RNases or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze the cleavage of themselves or other RNAs, but they have also been found to catalyze transaminase activity on ribosomes. Well-characterized, non-restrictive examples of small self-cleaving RNAs are hammerhead, hairpin, hepatitis D virus, and in vitro selected lead-dependent ribozymes, while group I introns are an example of larger ribozymes. The principles of catalytic self-cleavage have matured in recent years. Among RNA molecules with ribozymatic activity, hammerhead ribozymes are best characterized. Since it has been shown that hammerhead structures can integrate into heterologous RNA sequences, thereby transferring ribozymatic activity to these molecules, it appears possible to create catalytic antisense sequences against almost any target sequence, provided the target sequence contains a potentially matching cleavage site. The basic principle for constructing hammerhead ribozymes is as follows: Select a region of interest in RNA containing a GUC (or CUC) triplet. Take two oligonucleotide chains, each typically with 6 to 8 nucleotides, and insert the catalytic hammerhead sequence between them. The best results are usually obtained with short ribozymes and target sequences.

[0130] A recent development of this invention can also be used to recognize combinations of aptamers for small compounds with hammerhead ribozymes. The conformational changes induced in the aptamer after binding to the target molecule can modulate the catalytic function of the ribozyme.

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

[0132] Naturally found siRNAs have a well-defined structure: short double-stranded RNA (dsRNA) with a 3' overhang of 2 nucleotides at either end. 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 long dsRNA or small hairpin RNA into siRNA). siRNAs can also be exogenously (artificially) introduced into cells to achieve specific knockdown of genes of interest. Therefore, based on sequence complementarity with appropriately tailored siRNAs, virtually any gene with a known sequence can be targeted. Double-stranded RNA molecules or their metabolic products can mediate target-specific nucleic acid modifications, particularly RNA interference and / or DNA methylation. Exogenously introduced siRNAs may lack overhangs at their 3' and 5' ends; however, preferably at least one RNA strand has a 5'- and / or 3'-overhang. Preferably, one end of the double strand has a 3'-overhang of 1 to 5 nucleotides, more preferably 1 to 3 nucleotides, and most preferably 2 nucleotides. The other end can be blunt or have a 3'-protrusion of up to 6 nucleotides. Generally, this invention relates to any RNA molecule suitable as a target for the present invention. The most effective silencing to date has been achieved using a duplex of siRNA consisting of a 21-nucleotide sense strand and a 21-nucleotide antisense strand paired in a manner with a 2-nucleotide 3'-protrusion. The sequence of the 2-nucleotide 3'-protrusion contributes little to the specificity of target recognition limited to unpaired nucleotides adjacent to the first base pair. The 2'-deoxynucleotide in the 3'-protrusion is as effective as a ribonucleotide but is generally less expensive to synthesize and may be more nuclease-resistant. siRNA delivery can be accomplished using any method known in the art, such as by combining siRNA with saline and administering the combination intravenously or intranasally, or by formulating siRNA in glucose (e.g., 5% glucose), or cationic lipids and polymers can be used for in vivo delivery of siRNA via a systemic route via intravenous (IV) or intraperitoneal (IP) (Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol.437: Drug Delivery Systems–Chapter 3: Delivering Small Interfering RNA for Novel Therapeutics).

[0133] Short hairpin RNA (shRNA) is an RNA sequence that produces a tight hairpin turn, which can silence gene expression through RNA interference. shRNA is introduced into cells using a vector and the U6 promoter ensures its consistent expression. This vector is typically passed on to daughter cells, thus allowing gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular apparatus into siRNA, which then binds to an RNA-induced silencing complex (RISC). This complex binds to and cleaves the mRNA that matches the bound siRNA. The si / shRNA used in this invention is preferably chemically synthesized using appropriately protected ribonucleoside phosphoramide and a conventional DNA / RNA synthesizer. Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). Most conveniently, siRNA or shRNA is obtained from commercial RNA oligonucleotide synthesis suppliers, who sell RNA synthesis products of varying qualities and prices. Generally, the RNA suitable for this invention is conventionally synthesized and readily available in a quality suitable for RNAi.

[0134] Other molecules that enable RNAi include, for example, microRNAs (miRNAs). These RNAs are single-stranded RNA molecules. Endogenous miRNA molecules regulate gene expression by binding to complementary mRNA transcripts and triggering the degradation of those transcripts through a process similar to RNA interference. Therefore, exogenous miRNAs, after being introduced into the appropriate cells, can be used as inhibitors of the HLA genes of this invention.

[0135] As used herein, the term "antisense nucleic acid molecule" refers to a nucleic acid complementary to a target nucleic acid. The antisense molecule according to the invention is capable of interacting with the target nucleic acid, and more specifically, it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene and / or translation of the target mRNA is reduced or blocked. Standard methods involving antisense technology have been described (see, for example, Melani et al., Cancer Res. (1991) 51:2897-2901).

[0136] CRISPR / Cas9 and CRISPR-Cpf1 technologies are applicable to almost all cells / model organisms and can be used for knockout mutations, chromosome deletions, DNA sequence editing, and gene expression regulation. Gene expression regulation can be manipulated using a catalytic death Cas9 enzyme (dCas9), which is conjugated to a transcriptional repressor to repress the transcription of a specific gene, in this case, the HLA gene according to the invention. Similarly, a catalytically inactive “dead” Cpf1 nuclease (CRISPR from Prevotella and Francisella-1) can be fused with a synthetic transcriptional repressor or activator to downregulate endogenous promoters, such as those controlling HLA gene expression. Alternatively, the DNA-binding domain of a zinc finger nuclease (ZFN) or a transcriptional activator-like effector nuclease (TALEN) can be engineered to specifically recognize the HLA gene or its promoter region or its 5'-UTR, thereby repressing HLA expression.

[0137] This article also relates to the provided inhibitors of nucleic acid molecules that target HLA genes or regulatory molecules involved in HLA expression. These molecules that reduce or eliminate the expression of target HLA or regulatory molecules include, but are not limited to, a wide range of nucleases, zinc finger nucleases, and transcription activator-like (TAL) effector (TALE) nucleases. This approach is described in Silva et al., CurrGene Ther. 2011; 11(1):11-27; Miller et al., Nature biotechnology. 2011; 29(2):143-148, and Klug, Annual review of biochemistry. 2010; 79:213-231.

[0138] The term "antibody" as used in this invention includes, for example, polyclonal or monoclonal antibodies from any species and their humanized forms. Furthermore, derivatives or fragments thereof that retain binding specificity to targets such as HLA proteins of SEQ ID NO:1 to 6 are also included in the term "antibody". Antibody fragments or derivatives particularly include Fab or Fab' fragments, Fd, F(ab')2, Fv or scFv fragments, single-domain VH or V-like domains such as VhH or V-NAR-domains, and multimeric forms such as microantibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically conjugated 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). The multimeric form specifically includes bispecific antibodies that can bind to two different types of antigens simultaneously. The first antigen can be found on the HLA protein of the present invention. The second antigen can be, for example, a tumor marker specifically expressed on cancer cells or a certain type of cancer cell. Non-limiting examples of bispecific antibody forms are Biclonics (bispecific, full-length human IgG antibody), DART (dual-affinity retargeting antibody), and BiTE (composed of two single-chain variable fragments (scFv) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).

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

[0140] Various techniques for generating antibodies are well known in the art and described, for example, in Harlow and Lane (1988) and (1999) and Altshuler et al., 2010, loc. cit. Thus, polyclonal antibodies can be obtained from the blood of an animal after immunization with a mixture of antigen, additives, and adjuvants, while monoclonal antibodies can be generated by any technique that provides antibodies produced from continuous cell line cultures. Examples of these techniques are described, for example, in Harlow E and Lane D, ColdSpring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, and include those originally created by… Hybridoma techniques, including the three-cell hybridoma technique, described in Milstein, 1975, and human B-cell hybridoma techniques (see, for example, Kozbor D, 1983, Immunology Today, vol. 4:7; Li J, et al., 2006, PNAS, vol. 103(10), 3557), as well as EBV hybridoma techniques for producing human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc., 77-96). Furthermore, recombinant antibodies can be obtained from monoclonal antibodies or prepared de novo using various display methods such as phage, ribosome, mRNA, or cell display. Suitable systems for expressing recombinant (humanized) antibodies can be selected from, for example, 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). Furthermore, the described techniques for producing single-chain antibodies (see especially U.S. Patent 4,946,778) are applicable to the production of single-chain antibodies specific to epitopes of the HLA genes of this invention. Surface plasmon resonance employed in the BIAcore system can be used to improve the efficiency of phage antibodies.

[0141] As used herein, the term "protein drug" refers to a protein or peptide that, when administered to a subject, exhibits a therapeutic (cure or preventative) effect. Examples of protein drug categories will be discussed below.

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

[0143] Small molecule, antibody, or protein drug and aptamer can also be generated in the form of drug conjugates. In this case, the small molecule, antibody, or protein drug and aptamer themselves may not have inhibitory activity; the inhibitory activity is solely imparted by the drug. The small molecule, antibody, or protein drug and aptamer impart site-specific binding of the drug to cells that produce and / or bind to the HLA protein of the present invention. The drug is preferably capable of killing cells that produce and / or bind to the HLA protein of the present invention. Thus, by combining the targeting ability of the molecule that binds to the HLA protein of the present invention with the cellular killing ability of the drug, the drug conjugate becomes an inhibitor capable of distinguishing between healthy and diseased tissues and cells. Cleavable and non-cleavable linkers for designing drug conjugates are known in the art. Non-limiting examples of drugs capable of killing cells are cell-inhibiting drugs and radioactive isotopes that deliver radiation directly to cancer cells.

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

[0145] In a more preferred embodiment of the second aspect of the invention, the protein drug is an antibody mimic, preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affiliins, Kunitz domain peptides, etc. Three specific binding molecules and proantibodies.

[0146] In another preferred embodiment of the second aspect of the invention, the nucleotide-based inhibitor comprises (a) a nucleic acid sequence comprising or consisting of a nucleic acid sequence complementary to at least 12 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NO: 7 to 12 or a sequence at least 80% identical thereto, (b) a nucleic acid sequence comprising or consisting of a nucleic acid sequence comprising at least 80% identical to a complementary strand of one or more nucleic acid sequences selected from SEQ ID NO: 7 to 12, (c) a nucleic acid sequence comprising or consisting of the nucleic acid sequence of (a) or (b), wherein the nucleic acid sequence is DNA or RNA, (d) an expression vector expressing the nucleic acid sequence defined in any one of (a) to (c), preferably under the control of a tumor-specific promoter, or (e) a host comprising the expression vector of (d).

[0147] The nucleic acid sequences defined in (a) to (c) of this preferred embodiment comprise or consist of sequences complementary to the nucleotides of one or more HLA genes defined in SEQ ID NO:7 to 12. Therefore, the nucleic acid sequences defined in (a) to (c) comprise or are antisense nucleic acid sequences.

[0148] According to a further preferred embodiment of the present invention, the nucleic acid sequence of item (a) comprises or consists of a sequence that is preferably additionally complementary to at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides selected from one or more sequences of SEQ ID NO: 7 to 12. These at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides are preferably consecutive portions of one or more sequences selected from SEQ ID NO: 7 to 12, i.e., the nucleotides are consecutive in their respective SEQ ID NOs. The form of the nucleic acid sequence of item (a) is not particularly limited, as long as it comprises or consists of at least 12 consecutive nucleotides complementary to the nucleic acid sequences selected from SEQ ID NO: 7 to 12. The nucleic acid sequence of item (a) comprises or consists of antisense oligonucleotides. Therefore, the nucleic acid sequence in (a) reflects the basic principle of the antisense technology described above, namely, using oligonucleotides to silence selected target RNA through fine specificity based on complementary pairing. Therefore, it should be understood that the nucleic acid sequence in (a) is preferably in the form of antisense oligonucleotides or forms part of siRNA or shRNA as defined above. The antisense oligonucleotides are preferably LNA-GapmeRs, AntagonomiRs, or antimiRs.

[0149] The nucleic acid sequence of item (b) needs to have at least 70% identity with the complementary strand of one or more nucleic acid sequences selected from SEQ ID NO:7 to 12, and is typically much longer than the nucleic acid sequence of item (a), which contains an antisense oligonucleotide and at least 12 consecutive nucleotides selected from nucleic acid sequences of SEQ ID NO:7 to 12. The nucleic acid sequence of item (b) according to the preferred embodiment of the present invention is capable of interacting with the target HLA gene, and more specifically, capable of hybridizing with the target HLA gene. By forming a hybrid, HLA function is reduced or blocked.

[0150] (b) The sequence identity of the molecule with the sequences selected from SEQ ID NO:7 to 12 is preferably increased to at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99%, and 100%. Sequence identity with each of SEQ ID NO:7 to 12 may be selected individually. The means and methods for determining sequence identity are known in the art. Preferably, a BLAST (Basic Local Alignment Search Tool) procedure is used to determine sequence identity for one or more sequences in SEQ ID NO:7 to 12.

[0151] In the nucleic acid sequence of item (c), the nucleotide sequence can be RNA or DNA. RNA or DNA encompasses chemically modified RNA nucleotides or DNA nucleotides. It is well known that RNA contains nucleotide U, while DNA contains nucleotide T.

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

[0153] Expression vectors can be plasmids used to introduce specific transcripts into target cells. Once the expression vector enters the cell, the protein encoded by the gene is produced by the ribosome complex, the cell's transcription and translation apparatus. Plasmids are typically designed to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the transcript.

[0154] Non-limiting examples of expression vectors include prokaryotic plasmid vectors such as the pUC series, pBluescript (Stratagene), pET series expression vectors (Novagen), or pCRTOPO (Invitrogen), and mammalian cell-compatible vectors 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 from Intvitrogen). For the formulation of pharmaceutical compositions, a suitable vector should be selected according to Good Manufacturing Practices. Such vectors are known in the art, for example from Ausubel et al., Hum Gene Ther. 2011 Apr; 22(4):489-97 or Alley et al., Hum Gene Ther. May 2011; 22(5):595–604.

[0155] Typical mammalian expression vectors contain promoter elements that mediate the initiation of mRNA transcription, protein-coding sequences, and signals required for transcription termination and polyadenylation of transcripts. They may also include elements such as origin of replication, antibiotic resistance genes, and regulators (as part of an inducible promoter). The lac promoter is a typical inducible promoter used in prokaryotic cells and can be induced using the lactose analog isopropylthiol-bD-galactopyranoside (“IPTG”). For recombinant expression and secretion, the polynucleotide of interest can be linked, for example, between a PelB leader signal that guides the recombinant protein to the periplasm and gene III in a phage particle called pHEN4 (described in Ghahroudi et al., 1997, FEBS Letters 414:521-526). Other elements may include enhancers, Kozak sequences, and intercalation sequences flanked by donor and acceptor sites for RNA splicing. Efficient transcription can be achieved using early and late promoters of SV40, long terminal repeats (LTRs) of retroviruses (e.g., RSV, HTLV, HIVI), and early promoters of cytomegalovirus (CMV). However, cellular elements (e.g., human actin promoters) can also be used. Suitable expression vectors for carrying out the uses of this invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), and pBC12MI (ATCC 67109). Alternatively, recombinant (poly)peptides can be expressed in stable cell lines containing gene constructs integrated into the chromosome. Co-transfection with selection markers such as dhfr, gpt, neomycin, and hygromycin can identify and isolate transfected cells. Transfected nucleic acids can also be amplified to express large amounts of the encoded (poly)peptide. DHFR (dihydrofolate reductase) markers can be used to develop cell lines carrying hundreds or even thousands of copies of the gene of interest. Another useful selection marker is glutamine synthase (GS) (Murphy et al. 1991, Biochem J. 227:277-279; Bebbington et al. 1992, Bio / Technology 10:169-175). Using these markers, mammalian cells are grown in selective media, and cells with the highest resistance are selected. As mentioned above, the expression vector will preferably include at least one selection marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culture in Escherichia coli and other bacteria. For information on vector modification techniques, see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, 3 Vol.Vectors typically contain one or more origins of replication (ori), a genetic system for cloning or expression, one or more markers for selection in the host, such as antibiotic resistance, and one or more expression cassettes. Suitable origins of replication (ori) include, for example, ColE1, the SV40 viral origin of replication, and the M13 origin of replication.

[0156] The sequence to be inserted into the vector can be synthesized, for example, by standard methods, or isolated from natural sources. The linking of the coding sequence to transcriptional regulatory elements and / or other amino acid coding sequences can be performed using established methods. Transcriptional regulatory elements (part of the expression cassette) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements include regulatory sequences ensuring transcription initiation (e.g., translation start codons, promoters, enhancers, and / or insulators), internal ribosome entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476), and optional poly-A signals ensuring transcription termination and transcript stability. Additional regulatory elements may include transcriptional and translational enhancers, and / or naturally associated or heterologous promoter regions. Preferably, the nucleotide sequence defined in (a) of the preferred embodiment of the invention described above is operatively linked to such expression control sequences that allow expression in prokaryotes or eukaryotic cells.

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

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

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

[0160] According to a more preferred embodiment of the first and second aspects of the present invention, the immune checkpoint inhibitor is selected from the group consisting of: trastuzumab, cetuximab, rituximab, nivolumab, pembrolizumab, cimiprimab, atezolizumab, durvalumab, avelumab, ipilimumab, renalidomide, LY3321367, MBF453, TSR-022, urerucumab, PFZ-05082566, 1-7F9(IPH2101) GSK2831781, MEDI16469, MEDI16383, MOXR0916, varilurumab, TRX518, NKG2D ligand-anti-tumor Fv fusion complex (preclinical development), Galunisertib, ALT-803 (IL-15-IL-15alpha-Sushi-Fc fusion complex), icardostab, IMP321, and NJ-63723283.

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

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

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

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

[0165] Atezolizumab is an antibody against programmed cell death ligand 1 (PD-L1) and is used in cancer immunotherapy. Devallumab and avilumab are other anti-PD-L1 antibodies used to treat cancer.

[0166] Ipilimumab is an anti-CTLA-4 monoclonal antibody. It is used to treat cancers, particularly melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, and metastatic hormone-resistant prostate cancer.

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

[0168] LY3321367, MBF453, and TSR-022 are anti-HAVCR2 monoclonal antibodies used to treat cancer.

[0169] Utomimulab (BMS-663513 or anti-4-1BB antibody) and Utomimulab (PF-05082566) are anti-CD137 antibodies. More specifically, they specifically bind to and activate immune cells expressing CD137, thereby stimulating an immune response, particularly a cytotoxic T-cell response against tumor cells.

[0170] IPH2101 is a human monoclonal antibody against KIR (1-7F9) that has been developed for the treatment of acute myeloid leukemia.

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

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

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

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

[0175] Varilurumab specifically binds to CD27. It is used to treat cancers such as advanced breast cancer or ovarian cancer.

[0176] TRX518 is an antibody that blocks glucocorticoid-induced interactions with the TNF superfamily receptor (GITR). This antibody may be used to treat tumors.

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

[0178] ALT-803 (IL-15-IL-15alpha-Sushi-Fc fusion complex) is an IL-15 hyperagonist complex comprising an IL-15 mutant (IL-15N72D) fused to the IL-15 receptor α / IgG1 Fc fusion protein. ALT-803 can trigger antigen-specific antitumor responses.

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

[0180] IMP321 (Eftilagimod alpha) is a soluble version of LAG3 used to enhance the immune response against tumors.

[0181] JNJ-63723283 is a monoclonal antibody targeting the negatively immunomodulatory human cell surface receptor programmed cell death 1 protein (PD-1, PCDC-1), possessing potential immune checkpoint inhibitory and antitumor activities. Upon administration, the anti-PD-1 monoclonal antibody JNJ-63723283 binds to PD-1 and inhibits its interaction with its ligands, programmed cell death 1 ligand 1 (PD-L1, PD-1L1) and PD-1 ligand 2 (PD-L2, PD-1L2). Inhibition of ligand binding blocks PD-1-mediated signaling, leading to T cell activation and the induction of T cell-mediated antitumor cellular immune responses.

[0182] According to another preferred embodiment of the first and second aspects of the present invention, the anti-hormone treatment comprises anti-estrogen treatment and / or anti-progesterone and / or anti-androgen treatment.

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

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

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

[0186] In cases of bladder cancer or its neoplastic lesions, intravesical infusion therapy is preferred. Administration as intravesical infusion therapy is considered part of this invention, particularly combinations of immunological, chemotherapeutic, anti-hormonal, or anti-tyrosine kinase compounds with the aforementioned anti-HLA agents.

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

[0188] The kit to be prepared utilizes the tools required to implement the invention in kit form. Therefore, the definitions and preferred embodiments provided above in conjunction with the first aspect of the invention also apply to the kits of the invention.

[0189] As an example of this invention, tools for detecting and / or quantifying nucleic acid molecules may be one or more primers and probes as shown in Table 1 below. However, any detection module capable of quantifying nucleic acids, such as arrays, NGS, or other molecular systems, is suitable as part of this invention. Tools for detecting proteins or peptides are preferably antibodies and / or protein binders and / or peptide binders as described above. For detection and / or quantification, the antibodies and / or protein binders and / or peptide binders may be labeled using, for example, fluorescent dyes or radioactive labels. Examples of fluorescent dyes and radioactive labels have also been described above.

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

[0191] In a preferred embodiment of the third aspect of the invention, the tool comprises primer pairs and optionally primer or probe detection means comprising hydrolysis probes or other labeled primers or probes known to those skilled in the art for quantifying target sequences, such as scorpion primers, FRET probes, or molecular beacons, for sequence-specific detection of at least one nucleic acid molecule as defined above.

[0192] Primer pairs and optionally hydrolysis probes are typically used to specifically detect at least one nucleic acid molecule as defined above in the real-time quantitative PCR described above. Preferred primer pairs and hydrolysis probes are shown in Table 1 below.

[0193] A hydrolysis probe is a sequence-specific DNA probe, composed of oligonucleotides labeled with a fluorescent reporter molecule, which is only detectable after hybridization with its complementary sequence (e.g., TaqMan probes). More specifically, a hydrolysis probe is a dual-labeled oligonucleotide. The 5' end of the oligonucleotide is labeled with a fluorescent reporter molecule, while the 3' end is labeled with a quencher molecule. The probe sequence is specific to the region of interest in the amplified target molecule. The hydrolysis probe is designed with a sequence length such that the 5' fluorophore and the 3' quencher are sufficiently close to suppress fluorescence. During the extension phase of a PCR cycle, DNA polymerase synthesizes the complementary strand downstream of the PCR primer. When the extension reaches the bound hydrolysis probe, the 5'-3' exonuclease activity of the DNA polymerase degrades the hydrolysis probe. The cleavage of the hydrolysis probe separates the fluorescent reporter molecule from the rest of the probe, causing the reporter molecule to fluoresce.

[0194] With regard to the embodiments characterized in this specification, particularly in the claims, each embodiment mentioned in the dependent claims is intended to be combined with each embodiment of each claim (independent or dependent) to which the dependent claim is to be. For example, if independent claim 1 refers to three alternatives A, B, and C, dependent claim 2 refers to three alternatives D, E, and F, and claim 3, which is dependent on claims 1 and 2, refers to three alternatives G, H, and I, it should be understood that the specification expressly discloses embodiments corresponding to the combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless otherwise specified.

[0195] Similarly, and where independent and / or dependent claims do not refer to alternatives, it should be understood that if a dependent claim refers back to multiple preceding claims, any combination of the subject matter thereby covered is considered explicitly disclosed. For example, where independent claim 1, dependent claim 2 refers to claim 1, and dependent claim 3 refers to both claims 2 and 1, the combination of the subject matter of claims 3 and 1 is explicitly and explicitly disclosed, just as the combination of the subject matter of claims 3, 2, and 1 is. If there is a further dependent claim 4 relating to any one of claims 1 to 3, then the combination of the subject matter of claims 4 and 1, claims 4, 2 and 1, claims 4, 3 and 1, and claims 4, 3, 2, and 1 is explicitly and explicitly disclosed. Attached Figure Description

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

[0197] Figure 2 Data distribution of luminal and basal subtype markers, checkpoint target genes, and FGFR1 to 4 gene expression determined by RT-qPCR in tissues of patients with muscle-invasive bladder cancer.

[0198] Figure 3The expression of HLA-G, HLA-H, HLA-J, HLA-L, and HLA-V mRNA in different exon regions was quantified by RT-qPCR. Relative mRNA expression was determined using the 40-DCT method with CALM2 as a reference gene. A higher 40-DCT value indicates higher gene expression.

[0199] Figure 4 Spearman correlations between genes identified by RT-qPCR in luminal and basal subtype markers, checkpoint target genes, FGFR1 to 4 genes, and HLA-G exon 8 mRNA expression analysis from muscle-invasive bladder cancer tissues.

[0200] Figure 5 Spearman correlations (n=61) were analyzed by RT-qPCR of luminal and basal subtype markers, checkpoint target genes, FGFR1 to 4 genes, and HLA-G exon 3 to 6 mRNA expression in tissues of patients with muscle-invasive bladder cancer.

[0201] Figure 6 The correlation between HLA-H mRNA expression and FGFR receptor, PD-1, PD-L1, and basal and luminal cell type markers in patients with urothelial carcinoma.

[0202] Figure 7 Cluster analysis of HLA genes and urothelial markers by immunohistochemical and molecular assessment. High gene expression is highlighted in red, while low gene expression is represented in blue. Gene descriptions are on the left side of the cluster analysis. Each column represents a UBC sample from a patient who underwent cystectomy.

[0203] Figure 8 Cluster analysis of the FGF receptor gene with PD-1, PD-L1, and basal and luminal markers. High gene expression is highlighted in red, while low gene expression is represented in blue. Gene descriptions are on the left side of the cluster analysis. Each column represents a UBC sample from a patient who underwent cystectomy.

[0204] Figure 9 Kaplan Meier plots show the disease-specific survival (DSS) probability in patients with muscle-invasive bladder cancer, based on HLA-G exon 8 expression stratification quantified by RT-qPCR. Relative mRNA expression was determined using a 40-DCT method with CALM2 as a reference gene.

[0205] Figure 10Kaplan Meier plots show the disease-specific survival (DSS) probability of 57 patients with locally advanced or metastatic muscle-invasive bladder cancer (UBC), based on stratification of HLA-G exon 8 expression quantified by RT-qPCR. Relative mRNA expression was determined using a 40-DCT method with CALM2 as a reference gene.

[0206] Figure 11 Kaplan Meier plots show the disease-specific survival (DSS) probability of 57 patients with locally advanced or metastatic muscle-invasive bladder cancer (UBC), based on stratification of HLA-G exon 3 expression quantified by RT-qPCR. Relative mRNA expression was determined using the 40-DCT method with CALM2 as a reference gene.

[0207] Figure 12 Kaplan Meier plots show the disease-specific survival (DSS) probability of 57 patients with locally advanced or metastatic muscle-invasive bladder cancer (UBC), based on HLA-J exon 4 / 5 expression stratification quantified by RT-qPCR. Relative mRNA expression was determined using a 40-DCT method with CALM2 as a reference gene.

[0208] Figure 13 Kaplan Meier plots show the disease-specific survival (DSS) probability of 20 patients with locally advanced or lymph node-positive bladder cancer (UBC), based on stratification of HLA-G exon 8 expression quantified by RT-qPCR. Relative mRNA expression was determined using a 40-DCT method with CALM2 as a reference gene.

[0209] Figure 14 Kaplan Meier plots show the disease-specific survival (DSS) probability of 20 patients with locally advanced or lymph node-positive bladder cancer (UBC), based on stratification of HLA-G exon 3 expression quantified by RT-qPCR. Relative mRNA expression was determined using a 40-DCT method with CALM2 as a reference gene.

[0210] Figure 15 Kaplan Meier plots show the disease-specific survival (DSS) probability of 19 patients with locally advanced or lymph node-positive bladder cancer (UBC), based on stratification of HLA-L exon 7 expression quantified by RT-qPCR. Relative mRNA expression was determined using the 40-DCT method with CALM2 as a reference gene.

[0211] Figure 16 Kaplan Meier plots show the disease-specific survival (DSS) probability of 17 patients with locally advanced or lymph node-positive bladder cancer (UBC), based on stratification of HLA-L exon 7 expression quantified by RT-qPCR. Relative mRNA expression was determined using a 40-DCT method with CALM2 as a reference gene.

[0212] Figure 17 Kaplan Meier plot showing the disease-specific survival (DSS) probability of 17 patients with muscle-invasive bladder cancer that has metastasized to the lungs and bones or liver, based on HLA-H exon 2 / 3 expression stratification quantified by RT-qPCR. Relative mRNA expression was determined using the 40-DCT method with CALM2 as a reference gene.

[0213] Examples illustrate the present invention.

[0214] Example 1 Example

[0215] Example 1: HLA analysis in advanced, chemotherapy-refractory urothelial carcinoma

[0216] HLA expression was analyzed in urethral resection (TUR) biopsy and cystectomy samples from primary tumors that were refractory to chemotherapy and subsequently treated with PD-1 and PD-L1 checkpoint inhibitors (i.e., atezolizumab, nivolumab, and pembrolizumab) as first- or second-line immuno-oncology (“IO”) therapy, and was correlated with histopathological and molecular parameters, as well as response to IO therapy and disease-specific survival after IO.

[0217] Between 2016 and 2018, 72 newly diagnosed patients with histologically confirmed urothelial carcinoma (including bladder and upper urothelial carcinoma) were enrolled in the study. Nivolumab, pembrolizumab, and atezolizumab were administered as first-, second-, and third-line monotherapy according to the approved prescriptions. All hematoxylin-eosin (HE) stained tumor tissue sections from the cohort samples were evaluated and classified by two urological pathologists according to the UICC TNM classification (2017). Rare histological variants were classified according to the World Health Organization (WHO 2016) classification of tumors of the urogenital system. Following central histopathological review, 18 tissues were excluded due to insufficient tumor material or not being urothelial carcinoma. Five patients had only lymph node tissue available and were therefore excluded from the primary analysis of the prognostic and / or predictive role of HLA gene expression (see [link to study]). Figure 1 (CONSORT flowchart). The database of clinical data was closed on October 16, 2018, along with the parallel submission to the FDA.

[0218] For mRNA detection, RNA was extracted from FFPE tissue from TUR biopsies, cystectomies, and corresponding bladder tissue mappings using a commercial kit (Xtract, Stratifyer). For each reaction, 2.5 μl of total RNA extracted from FFPE sections was mixed with 2.5 μl of analytical mixture, 2.5 μl of enzyme mixture, and 2.5 μl of water in one well of a 96-well optical reaction plate. PCR reactions were measured using a Versant kPCR Cycler (Siemens) or Light Cycler 480 (Roche) under appropriate conditions according to the manufacturer's instructions (5 min 50°C, 1 cycle; 20 sec 95°C, 1 cycle; 15 sec 95°C; 1 min 60°C, 40 cycles). Relative mRNA expression was associated with IO treatment response as determined by RECIST (Resolution for Solid Tumor Response) criteria at each site and with disease-specific survival determined from the initiation of IO treatment to cancer-specific death. We used biostatistics JMP SAS 9.0.0 (SAS, Cary, North Carolina, USA) to perform a partitioned test to assess potential differences in response to IO treatment.

[0219] To perform detailed analysis of gene expression using RT-qPCR, primers flanking the region of interest and fluorescently labeled probes hybridizing between them were used. 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 achieve RNA-specific measurements by locating primer / probe sequences across exon / exon boundaries. Furthermore, primers / probes that do not bind to sequence regions with known polymorphisms (SNPs) were selected. If multiple isotypes of the same gene are present, primers were selected to appropriately amplify all relevant or selected splice variants. The specificity of all primer pairs was checked by routine PCR reactions. After further optimization of primers / probes, the primers and probes listed in Table 1 yielded the best results. These primers / probes are superior to those known in the prior art, for example, in terms of specificity and amplification efficiency. To normalize the amount of RNA in the samples, CALM2 was selected as a reference gene because it was not differentially regulated in the analyzed samples. Validation experiments showed that the efficiency of target amplification and control amplification was roughly equal, which is a prerequisite for relative quantification of gene expression by comparing the αCT method.

[0220] Table 1: Primers and probes used for HLA mRNA quantification

[0221]

[0222]

[0223] RT-qPCR analysis of the luminal and basal subtypes in the UC cohort revealed a wide dynamic range for KRT5 and KRT20 mRNA, within a similar range of 40-DCT values ​​from 19 to 48. PD-1 and PD-L1 mRNA expression ranged from 19 to 41. The dynamic range of FGFR genes differed significantly within the FGFR family. The relative ranges for FGFR1 mRNA were 29 to 37, FGFR2 mRNA 19 to 39, FGFR3 mRNA 19 to 43, and FGFR4 mRNA 19 to 36. Figure 2 ).

[0224] In addition to luminal and basal markers, mRNA expression analysis of PD-1, PD-L1, and FGFR families, expression profiles of classical HLA and exon expression of HLA genes and pseudogenes were also performed. Figure 3 ).

[0225] Nonparametric Spearman correlations of FGFR gene 1-4, PD-1, PD-L1, basal and luminal markers, and exon 8 HLA primer sets revealed a strong and significant association of PD-1 in urothelial tumors expressing HLA-G exon 8 (spearman rho 0.2904, p = 0.0232). In addition to PD-1, high FGFR1 expression (spearman rho 0.2724, p = 0.0337) was also associated with HLA-G exon 8 expression. However, no significant associations with any HLA were observed in luminal urothelial carcinoma. Figure 4 ).

[0226] Surprisingly, the Spearman correlations between luminal and basal subtype markers, checkpoint target genes, FGFR1 to 4 genes, and the remaining HLA-G exons revealed a strong and significant association between HLA-G and the checkpoint marker PD-1, as evidenced by similar high co-expression in all HLA-G exons (PD-1 at the 3' end of exon 3: Spearman rho 0.2768, p = 0.0308; PD-1 in exon 4: Spearman rho 0.2768, p = 0.0308; PD-1 in exon 5: Spearman rho 0.3220, p = 0.0114; PD-1 in exon 6: Spearman rho 0.3805, p = 0.0025). Figure 5These interesting findings for PD-L1 were only confirmed in HLA-G exon 5 (spearman rho 0.2695, p = 0.0357). For the 3' end of exon 3, a high significant correlation was also observed for FGF receptor 3 (spearman rho 0.2990, p = 0.0193) and FGF receptor 4 (spearman rho 0.2703, p = 0.0352). This correlation for exon 4 could not be determined, although exon 4 expression was associated with high mRNA expression of the basal cell marker KRT5 (spearman rho 0.2931, p = 0.0219). The basal marker KRT5 (spearman rho 0.3526, p = 0.0053) also showed a significant correlation with HLA-G exon 6. In addition, FGF receptor 3 (spearman rho 0.2972, p = 0.0200) and FGF receptor 4 (spearman rho 0.3552, p = 0.0050) also showed significant correlation with the mRNA expression of HLA-G exon 6.

[0227] In addition, nonparametric Spearman correlation analysis was performed on HLA-H luminal and basal subtype markers, PD1, PD-L1, and FGFR1 to 4 genes. Figure 6 However, no correlation was observed between HLA-H expression and luminal or basal markers or checkpoint inhibitors.

[0228] In addition, cluster analysis was performed on the FGF receptor gene with PD-1, PD-L1, and basal and luminal markers. The analysis showed that PD-1 and PD-L1 expression occurred in the basal urothelial carcinoma subtype. Furthermore, FGFR1 mRNA was also more highly expressed in cytokeratin 5-positive tumors, while FGF receptors 2 to 4 showed higher expression in the luminal urothelial carcinoma subtype.

[0229] Cluster analysis of HLA genes using urothelial carcinoma markers assessed by immunohistochemistry revealed that HLA-G expression mainly occurred in the basal urothelial carcinoma subtype (IHC_ST_CK5), and basal urothelial carcinoma subtypes could be further classified by HLA-Am RNA expression. Some basal tumor subtypes showed high HLA-G expression. Figure 7A). Finally, cluster analysis of HLA exon 8 expression was performed using immunohistochemical cell and subtaxonomic markers (CK5, CD44, CK20, FOXA1, GATA3), PD-1, PD-L1, and HLA-H expression. The analysis showed that HLA-G exon 5 and exon 8 expression, HLA-H expression, and PD-1 and PD-L1 were more likely to be classified into the basal subtype. However, HLA-G, HLA-H, PD-1, and PD-L1 expression were also observed in luminal urothelial tumors. Figure 7 B). Furthermore, computational simulations of the promoter (silico promoter analysis) revealed several estrogen-responsive elements (EREs) and progesterone-responsive elements (PREs) in the HLA-G gene. This indicates the significant potential of HLA-G expression not only in basal carcinoma subtypes but also in luminal carcinoma subtypes. Since mRNA exon and exon / exon junction expression vary across luminal and basal carcinoma subtypes, single-exon expression and exon / exon junction analysis should be applied as stratification tools to basal and luminal urothelial carcinoma subtypes. Surprisingly, computational simulations of the HLA-H promoter region also revealed several estrogen-responsive elements. Together with cluster analysis, this highlights the important role of the pseudogene HLA-H as a tool for further stratification of urothelial carcinoma. Figure 8 As shown, the FGF receptor gene was further clustered with PD-1, PD-L1, and basal and luminal markers. The analysis demonstrated that PD-1 and PD-L1 expression occurred in the equivalent basal urothelial carcinoma subtype. Furthermore, FGFR1 mRNA expression was also higher in cytokeratin 5-positive tumors, while FGF receptors 2 to 4 showed higher expression in the equivalent luminal urothelial carcinoma subtype. This demonstrates the representativeness of the cohort analyzing HLA gene interactions.

[0230] Example 2: Exon expression of different HLA genes in urothelial carcinoma as a measure of disease-specific survival (DSS) markers

[0231] To determine the predictive value of HLA gene expression in bladder cancer tissue from patients with advanced or metastatic urothelial carcinoma who received immuno-oncology checkpoint therapy (IO therapy) (i.e., atezolizumab, nivolumab, or pembrolizumab), a detailed clinical follow-up assessment was conducted, including WHO classification, major metastatic site, IO therapy initiation, and cancer-specific death or last contact date. Immuno-oncology disease-specific survival was calculated from the start of IO therapy to cancer-specific death or last contact, and was subsequently censored.

[0232] like Figure 9As shown, the correlation between changes in HLA-G mRNA expression and disease-specific survival (DSS) in patients with urothelial carcinoma was analyzed. When all available tissues, including metastatic lymph nodes (n=60), were considered, Kaplan-Meier analysis showed that HLA-G exon 8 mRNA expression above a 40-DCT value of 28.43 indicated poorer disease-specific survival (p=0.0102).

[0233] However, to exclude the non-cancer-related effects of HLA expression in non-tumor-associated lymphocytes in lymph nodes, metastatic lymph node tissue was excluded in subsequent analyses, leaving 57 samples for further examination. Figure 1 The survival rate analysis is shown below. Figure 10 As shown, high HLA-G exon 8 mRNA expression (>=28.43) was significantly associated with poorer disease-specific survival. The 2-year survival probability of HLA-G exon 8 positive patients was 35%, while that of HLA-G exon 8 negative patients was 65% (p=0.0298).

[0234] Because the examined HLA-G-specific exon 8 region was not translated into protein, further confirmatory analysis was performed by identifying the HLA-G exon 3 region (which is part of the translation region near the HLA-G signal peptide). Figure 11 As shown, high HLA-G exon 3 mRNA expression (>=28.23) was significantly associated with poorer disease-specific survival. The 2-year survival probability of HLA-G exon 3 positive patients was 30%, while that of HLA-G exon 8 negative patients was 70% (p=0.0156).

[0235] Next, the prognostic value of other HLA genes in the overall cohort was analyzed. Particular attention was paid to "pseudogenes" in the current classification, such as HLA-J, H, V, or L. Figure 12 As shown, high HLA-J exon 4 / 5 mRNA expression (>=25.08) was associated with poorer disease-specific survival. The 2-year survival probability of 36 HLA-J exon 4 / 5 positive patients was 35%, while the 2-year survival probability of 19 HLA-J exon 4 / 5 negative patients was 70%.

[0236] To further clarify the correlation between HLA-G expression and survival after IO treatment, this analysis was further refined by considering only primary tumor tissue as well as primary metastatic sites. This was based on the initial finding that IO treatment has different effects depending on the metastatic site, for example, poorer results in visceral metastases to the liver, possibly because PD1-positive T cells are excluded from the liver in patients with metastatic urothelial carcinoma, independent of the classic 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 grouped according to the initial manifestation of metastasis: local progression, local regional lymph nodes, or extra-regional retroperitoneal lymph nodes were classified as 0 or 0.5, respectively, while spread to bone, liver, lung, lung and bone, or lung and liver was classified as an increasing index (1, 2, 3, 4, 5, respectively). For this analysis, 54 datasets of primary tumor tissue with sufficient clinical dates and primary tumor tissue material were available. Among them, 19 patients had local progression or lymph node metastasis, while 17 patients initially metastasized to the bone or liver, and 18 patients metastasized to the lungs, with single or combined bone or liver involvement. All patients had received IO drug therapy and were primary >1-line therapy (74%).

[0237] In patients with advanced or lymph node-positive urothelial bladder cancer, high HLA-G mRNA expression is associated with poorer disease-specific survival from the initiation of IO therapy to cancer-specific death. Figure 13 As shown, patients with high HLA-G exon 8 mRNA expression (>=28.545) had a significantly worse prognosis. The survival rate of 11 HLA-G exon 8 positive patients after 2 years was only 25%, while the survival rate of 9 HLA-G exon 8 negative patients after 2 years was 100% (p=0.0068).

[0238] Since the HLA-G specific exon 8 region detected was not translated into protein, further confirmatory analysis was performed by identifying the HLA-G exon 3 region, which is part of the translational region close to the HLA-G signal peptide.

[0239] like Figure 14As shown, high HLA-G exon 3 mRNA expression (>=26.535) was significantly associated with poorer disease-specific survival; the 2-year survival probability was only 15% for 10 HLA-G exon 3 positive patients, compared to 100% for 10 HLA-G exon 3 negative patients (p=0.0013). This is similar to the predictive value of HLA-G exon 8 mRNA expression and further demonstrates that HLA-G expression is associated with worse outcomes, despite treatment with checkpoint inhibitor IO drugs in advanced and lymph node-positive disease.

[0240] Next, we investigated whether other HLA genes, namely classical or non-classical genes, known genes or pseudogenes, could predict IO outcomes in urothelial bladder cancer.

[0241] As an example, an analysis was developed to quantify the HLA-L mRNA of the "pseudogene"—a region resembling the 8th region of HLA-G exon at the 3' end of the "pseudogene." Figure 15 As shown, high HLA-L exon 7 mRNA expression (>=29.89) was associated with poorer disease-specific survival, with only a 30% survival rate at 2 years for 10 HLA-L exon 7 positive patients, compared to an 80% survival rate for 10 HLA-L exon 7 negative patients. However, this association did not reach statistical significance via the log-rank test due to the crossover of survival curves. It is debatable whether this is due to the still low sample size or the fact that the log-rank test may be ineffective in this situation, as very early conditions after 1 month do indeed exaggerate the p-value, and therefore may not be the optimal choice for assessing risk.

[0242] This indicates that not only HLA-G but also other HLA genes and / or pseudogenes are associated with poorer outcomes, despite treatment with checkpoint inhibitor IO drugs. From a therapeutic perspective, this suggests that targeting not only HLA-G but also other HLA genes and / or pseudogenes should be employed to overcome or disrupt resistance to IO drugs.

[0243] Next, from a tumor biology perspective, we examined whether HLA genes also have predictive power in the most aggressive scenarios, when multiple organs, particularly the lungs, were confirmed to have metastasized via CT scans during pre-IO treatment. Figure 16 As shown, high HLA-L exon 7 mRNA expression (>=30.195) was associated with poorer disease-specific survival. The survival rate of 16 HLA-L exon 7 positive patients was only 0% after 1 year, while the survival rate of 11 HLA-L exon 7 negative patients was 70% after 1 year (p=0.0418).

[0244] In such cases of high translocation, other "pseudogenes" are also important, such as HLA-H. Figure 17 As shown, high HLA-H exon 2 / 3 mRNA expression (>=29.95) is associated with poorer disease-specific survival. Patients with positive HLA-H exon 2 / 3 mRNA have a survival probability of only 30% after 1 year, while patients with negative HLA-H exon 2 / 3 mRNA have a survival probability of 80% after 1 year. sequence list <110> Interlex LLC <120> Methods for diagnosing the effectiveness of antitumor therapy <130> AB2938 PCT <150> EP 19 18 4681.5 <151> 2019-07-05 <160> 79 <170> BiSSAP 1.3 <210> 1 <211> 338 <212> PRT <213> Homo sapiens <220> <223> HLA-G membrane-bound <400> 1 Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala 1 5 10 15 Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe 20 25 30 Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala 35 40 45 Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser 50 55 60 Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly 65 70 75 80 Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln 85 90 95 Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser 100 105 110 Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly 115 120 125 Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly 130 135 140 Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala 145 150 155 160 Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val 165 170 175 Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu 180 185 190 His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro 195 200 205 Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr 210 215 220 Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr 225 230 235 240 Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu 245 250 255 Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val 260 265 270 Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu 275 280 285 Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser Ser Leu Pro 290 295 300 Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val Leu Ala Ala 305 310 315 320 Val Val Thr Gly Ala Ala Val Ala Ala Val Leu Trp Arg Lys Lys Ser 325 330 335 Ser Asp <210> 2 <211> 341 <212> PRT <213> Homo sapiens <220> <223> HLA-L membrane-bound <400> 2 Met Gly Val Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Leu Gly Ala 1 5 10 15 Leu Ala Leu Thr Glu Thr Trp Ala Ala Thr Pro Val Arg Gly Trp Ser 20 25 30 Gly Gly Arg Arg Gly Trp Ser Arg Arg Gly Trp Ser Ile Gly Thr Arg 35 40 45 Arg His Gly Thr Pro Arg Ala Thr Arg Arg Phe Thr Glu Thr Cys Gly 50 55 60 Pro Cys Ser Ala Ile Thr Thr Arg Ala Arg Pro Val Thr Val Arg Leu 65 70 75 80 Arg Trp Gln Gly Leu His Arg Pro Glu Arg Gly Pro Ala Leu Leu Asp 85 90 95 Arg Arg Glu His Ser Gly Ser Asp Leu Pro Ala Gln Val Gly Ser Gly 100 105 110 Gln Ile Leu Arg Ala Gly Gln Gly Leu Pro Glu Gly Lys Cys Met Glu 115 120 125 Trp Leu Arg Arg His Leu Glu Asn Gly Lys Glu Thr Leu Gln His Ala 130 135 140 Asp Pro Pro Lys Ala His Val Thr Gln His Pro Ile Ser Asp His Glu 145 150 155 160 Ala Thr Leu Arg Cys Trp Ala Leu Gly Leu Tyr Pro Ala Glu Ile Thr 165 170 175 Leu Thr Trp Gln Gln Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu 180 185 190 Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Val Ala 195 200 205 Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Met Cys His Val Gln 210 215 220 His Glu Gly Leu Pro Glu Pro Leu Thr Leu Arg Trp Glu Pro Ser Ser 225 230 235 240 Gln Pro Thr Ile Pro Ile Val Gly Ile Val Ala Gly Leu Phe Leu Leu 245 250 255 Gly Ala Val Val Thr Gly Ala Val Val Ala Ala Ala Met Trp Arg Lys 260 265 270 Lys Ser Ser Gly Ser Asn Cys Ala Gln Tyr Ser Asp Ala Ser His Asp 275 280 285 Thr Cys Lys Glu Asp Tyr Ala Cys Ser Cys Ser Gly Val Cys Val Leu 290 295 300 Ile Ser Phe Ser Pro Gly Cys Pro Ser Ser Leu Thr Ala Ala Gly Val 305 310 315 320 Ile Phe Pro Val Ile Asn Pro Thr Arg Trp Lys Ala Ala Pro Ala His 325 330 335 Arg Ser Leu Trp Tyr 340 <210> 3 <211> 319 <212> PRT <213> Homo sapiens <220> <223> HLA-G soluble <400> 3 Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala 1 5 10 15 Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe 20 25 30 Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala 35 40 45 Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser 50 55 60 Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly 65 70 75 80 Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln 85 90 95 Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser 100 105 110 Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly 115 120 125 Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly 130 135 140 Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala 145 150 155 160 Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val 165 170 175 Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu 180 185 190 His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro 195 200 205 Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr 210 215 220 Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr 225 230 235 240 Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu 245 250 255 Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val 260 265 270 Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu 275 280 285 Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Ser Lys Glu Gly Asp Gly 290 295 300 Gly Ile Met Ser Val Arg Glu Ser Arg Ser Leu Ser Glu Asp Leu 305 310 315 <210> 4 <211> 246 <212> PRT <213> Homo sapiens <220> <223> HLA-L soluble <400> 4 Met Gly Val Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Leu Gly Ala 1 5 10 15 Leu Ala Leu Thr Glu Thr Trp Ala Ala Thr Pro Val Arg Gly Trp Ser 20 25 30 Gly Gly Arg Arg Gly Trp Ser Arg Arg Gly Trp Ser Ile Gly Thr Arg 35 40 45 Arg His Gly Thr Pro Arg Ala Thr Arg Arg Phe Thr Glu Thr Cys Gly 50 55 60 Pro Cys Ser Ala Ile Thr Thr Arg Ala Arg Pro Val Thr Val Arg Leu 65 70 75 80 Arg Trp Gln Gly Leu His Arg Pro Glu Arg Gly Pro Ala Leu Leu Asp 85 90 95 Arg Arg Glu His Ser Gly Ser Asp Leu Pro Ala Gln Val Gly Ser Gly 100 105 110 Gln Ile Leu Arg Ala Gly Gln Gly Leu Pro Glu Gly Lys Cys Met Glu 115 120 125 Trp Leu Arg Arg His Leu Glu Asn Gly Lys Glu Thr Leu Gln His Ala 130 135 140 Asp Pro Pro Lys Ala His Val Thr Gln His Pro Ile Ser Asp His Glu 145 150 155 160 Ala Thr Leu Arg Cys Trp Ala Leu Gly Leu Tyr Pro Ala Glu Ile Thr 165 170 175 Leu Thr Trp Gln Gln Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu 180 185 190 Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Val Ala 195 200 205 Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Met Cys His Val Gln 210 215 220 His Glu Gly Leu Pro Glu Pro Leu Thr Leu Arg Trp Glu Pro Ser Ser 225 230 235 240 Gln Pro Thr Ile Pro Ile 245 <210> 5 <211> 219 <212> PRT <213> Homo sapiens <220> <223> HLA-J soluble <400> 5 Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ser Gly Thr Leu Ala Leu 1 5 10 15 Ala Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe Ser Thr Ala 20 25 30 Val Ser Trp Pro Gly Arg Gly Glu Pro Ser Phe Ile Ala Val Gly Tyr 35 40 45 Val Asp Asp Thr Gln Phe Val Arg Val Asp Ser Asp Ala Val Ser Leu 50 55 60 Arg Met Lys Thr Arg Ala Arg Trp Val Glu Gln Glu Gly Pro Glu Tyr 65 70 75 80 Trp Asp Leu Gln Thr Leu Gly Ala Lys Ala Gln Ala Gln Thr Asp Arg 85 90 95 Val Asn Leu Arg Thr Leu Leu Arg Tyr Tyr Asn Gln Ser Glu Ala Asp 100 105 110 Pro Pro Gln Asp Thr Arg Asp Pro Pro Pro Ser Leu Asn Met Arg His 115 120 125 Asn Glu Val Leu Gly Ser Gly Leu Leu Pro Cys Gly Asp His Ile Asp 130 135 140 Leu Ala Ala Gly Trp Gly Gly Pro Asp Pro Gly His Gly Ala Arg Gly 145 150 155 160 Asp Gln Ala His Arg Gly Trp Asn Leu Pro Glu Val Gly Gly Cys Gly 165 170 175 Ser Ala Phe Trp Arg Gly Thr Glu Ile His Met Pro Cys Ala Ala Gln 180 185 190 Gly Ala Ala Gln Ala Pro His Pro Glu Met Gly His Thr Phe Leu Glu 195 200 205 Thr Ser Arg Gly Ser Lys Thr Arg Arg Phe Leu 210 215 <210> 6 <211> 221 <212> PRT <213> Homo sapiens <220> <223> HLA-H soluble <400> 6 Met Val Leu Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ser Gly Ala 1 5 10 15 Leu Ala Leu Thr Leu Thr Gln Thr Trp Ala Arg Ser His Ser Met Arg 20 25 30 Tyr Phe Tyr Thr Thr Met Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe 35 40 45 Ile Ser Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser 50 55 60 Asp Asp Ala Ser Pro Arg Glu Glu Pro Arg Ala Pro Trp Met Glu Arg 65 70 75 80 Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr Gln Ile Cys Lys Ala Gln 85 90 95 Ala Gln Thr Glu Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr Tyr Asn 100 105 110 Gln Ser Glu Gly Gly Ser His Thr Met Gln Val Met Tyr Gly Cys Asp 115 120 125 Val Gly Pro Asp Gly Arg Phe Leu Arg Gly Tyr Glu Gln His Ala Tyr 130 135 140 Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr 145 150 155 160 Ala Ala Asp Met Ala Ala Gln Ile Thr Lys Arg Lys Trp Glu Ala Ala 165 170 175 Arg Arg Ala Glu Gln Leu Arg Ala Tyr Leu Glu Gly Glu Phe Val Glu 180 185 190 Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Ala 195 200 205 Asp Pro Pro Gln Asp Thr Tyr Asp Pro Pro Pro His Leu 210 215 220 <210> 7 <211> 1578 <212> DNA <213> Homo sapiens <220> <223> HLA-G membrane-bound <400> 7 agtgtggtac tttgtcttga ggagatgtcc tggactcaca cggaaactta gggctacgga 60 atgaagttct cactcccatt aggtgacagg ttttagaga agccaatcag cgtcgccgcg 120 gtcctggttc taaagtcctc gctcacccac ccggactcat tctccccaga cgccaaggat 180 ggtggtcatg gcgccccgaa cctcttcct gctgctctcg gggccctga ccctgaccga 240 gacctgggcg ggctcccact ccatgaggta tttcagcgcc gccgtgtccc ggcccggccg 300 cggggagccc cgcttcatcg ccatgggcta cgtggacgac acgcagttcg tgcggttcga 360 cagcgactcg gcgtgtccga ggatggagcc gcgggcgccg tgggtggagc aggaggggcc 420 ggagtattgg gaagaggaga cacggaacac caaggcccac gcacagactg acagaatgaa 480 cctgcagacc ctgcgcggct actacaacca gagcgaggcc agttctcaca ccctccagtg 540 gatgattggc tgcgacctgg ggtccgacgg acgcctcctc cgcgggtatg aacagtatgc 600 ctacgatggc aaggattacc tcgccctga cgaggacctg cgctcctgga ccgcagcgga 660 cactgcggct cagatctcca agcgcaagtg tgaggcggcc aatgtggctg aacaaaggag agcctacctg gagggcacgt gcgtggagtg gctccacaga tacctggaga acgggaagga 780 gatgctgcag cgcgcggacc cccccaagac acacgtgacc caccaccctg tctttgacta 840 tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca tactgacctg 900 gcagcgggat ggggaggacc agacccagga cgtggagctc gtggagacca ggcctgcagg 960 ggatggaacc ttccagaagt gggcagctgt ggtggtgcct tctggagagg agcagagata cacgtgccat gtgcagcatg aggggctgcc ggagcccctc atgctgagat ggagcagtc 1080. ttccctgccc accatcccca tcatgggtat cgttgctggc ctggttgtcc ttgcagctgt 1140 agtcactgga gctgcggtcg ctgctgtgct gtggagaaag aagagctcag attgaaaagg agggagctac tctcaggctg caatgtgaaa cagctgccct gtgtgggact gagtggcaag tccctttgtg acttcaagaa ccctgactcc tctttgtgca gagaccagcc cacccctgtg 1320 cccaccatga cctcttcct catgctgaac tgcattcctt ccccaatcac ctttcctgtt 1380 ccagaaaagg ggctgggatg tctccgtctc tgtctcaaat ttgtggtcca ctgagctata 1440 acttacttct gtattaaaat tagaatctga gtataaattt actttttcaa attatttcca 1500 agagagattg atgggttaat taaaggagaa gattcctgaa atttgagaga caaaataaat 1560 ggaagacatg agaacttt 1578 <210> 8 <211> 4622 <212> DNA <213> Homo sapiens <220> <223> HLA-L membrane-bound <400> 8 acgatcccgg cactacagtc ccggcgcaac cacccgcact cagattctc ccaaacgcca 60 aggatggggg tcatggctcc ccgaaccctc ctcctgctgc tcttgggggc cctggccctg 120 accgagacct gggccgcgac tccgtgagtc cgaggatgga gcggcgggcg ccgtgggtgg 180 agcaggaggg gctggagtat tgggaccagg agacacggaa cgccaagggc cacgcgcaga 240 tttaccgagt gaacctgcgg accctgctcc gctattacaa ccagagcgag gccggtatga 300 acagttcgcc tacgatggca aggattacat cgccctgaac gaggacctgc actcctggac 360 cgccgcgaac acagcggctc agatctccca gcacaagtgg gaagcggaca aatactcaga 420 gcaggtcagg gcctacctga gggcaagtgc atggagtggc tccgcagaca cctggagaac 480 gggaaggaga cgctgcagca cgcggatccc ccaaaggcac atgtgaccca gcaccccatc 540 tctgaccatg aggccaccct gaggtgctgg gccctgggcc tctaccctgc ggagatcaca 600 ctgacctggc agcaggatgg ggaggaccag acccaggaca cggagcttgt ggagaccagg 660 cctgcagggg acggaacctt ccagaagtgg gtggctgtag tggtgccttc cggagaggag 720 cagagataca tgtgccatgt gcagcatgag gggctgccag agcccctcac cctgagatgg 780 gagccgtctt ctcagcccac catccccatc gtgggcatcg ttgctggcct gtttctcctt 840 ggagctgtgg tcactggagc tgtggttgct gctgcgatgt ggaggaagaa aagctcaggc 900 agcaattgtg ctcagtactc tgatgcatct catgatactt gtaaagagga ctatgcctgt 960 tcctgttctg gtgtctgcgt tctgatctct ttctcccctg ggtgtccctc atctctgaca 1020 gcagcaggag tcatttttcc tgtcattaac cccacaaggt ggaaggcagc ccctgcacac 1080 agaagtctgt ggtattaaga gatgaatttt caagcccgtg cagcttttac cctatttcca 1140 gggctctttc ttggattgta ttttctatct tttccccaac ctttttaaag gaactagatt 1200 ctgaaattag cagagaagag ggatgccaca agttctcatc ttaggtaact ttctagtgga 1260 actcctcttc tgctcagctc tcctacccac tctcccttcc ctgagttgta gtaatcctag 1320 cactggctct aatgcaaact catggatcta taaagcaaag tctaacttag atttatattt 1380 gtttggaaat tgggattcat agtcaaagat tgttctttcc taagagggaa atataattgc 1440 atgctgcagt gtgcagaggg ttggtgtgaa ggagggatgc agggaggaag ggagggagga 1500 cacacaagca gcactgctgg gaaaagcaca ggcggcctgg atgtcagtgt gaggggacct 1560 tgtgctgtcg ttgctgcaaa accgcatttg gcctgaggct atgttaataa agatactgcc 1620 tttagaatag gaggtgctct acagtgatga ttcattcagc cgacatttgc tgtctgccag 1680 acatatgaca gaatgttttt gcatctgggg aaagtcattg aagtaaaatc agaaaaatct 1740 ctagccttgt ggagcatgtg ttccagtggg aagaggcaga cggtacatac actctaatat 1800 atgcagagta aatgaggaaa gtgttagaag gtgataagtg ctgtggaaca ggtgatcaga 1860 gtatgggttg tgggacagag aaggtagcta ttgtgccggg gttgtcagcg tgggccttgt 1920 tgggaaggtg acctttgatg aaatttga aggacataaa ggaatttgtc atgagggtat 1980 ctggaagaag ttttttctag ggagtaggaa ccttcagtgt cagtgtacca gggcaggatc 2040 atgtctgtgt gttctgggaa gaacacggga tcgggtatgg ctagagcaga gagtcactga 2100 gataaggtca ggggtttggt cagatcatgt gggcataggg ctcaagtatg tgggaaggat 2160 tttgatttg aatgagatag ttttaagcag aataaagaca tgccacaact tctcttttaa 2220 aaggatcact gtagctgctc tgctgagaac agaatccaaa ggccggcgat gagcaaggca 2280 ggtgggaaaa ctgtaggaaa tgagtgcagt atttcaggct ggagatgtcg gttacttcaa 2340 ctggggtgtg agcagtggaa atagtgggac gtgattggat tcctactatt tccaatcact 2400 ttataccgca ttttctaatg gactaaatct ggggtatgag aaagaagagt aaaggatacc 2460 aaaaatgtca gactgtgact aaaaagagtt gccatcagct gagaatgaga agactagcag 2520 gagcatatga gaggaggga cgtcgcaggc agtcactatg ggagacgtgg gatctgagat 2580 gccgctgaga aataccagtg aggtagtcgg gttggcagtt ggacagatga atctggagac 2640 atttaggaga aatagacttg ggaggtgatg tcatataaca gttatttaaa gccttgagtc 2700 tgaatgacgt ctccaaggga gtgattggct gtagagaga acaggaacaa ggactgaaca 2760 ctaggcctct gttgctaaag gatctgatca gacaacacac ctagatcaga ctgcacagtc 2820 ctgaccccac atctagaagg tacatagacc agggagttct agactttcct gtggacagga 2880 atcacctgga catcacctta agtctaagct gatctggaat cgagaatgag atttcctact 2940 tatataatgt tgctgttggc gctgatgctg ctggtcttca gatcccactt ttggtagcaa 3000 gaacacagac caggattcct aggctatgca tcagcctcgc ctgtgaggct tgttaataag 3060 caattcctgc actccatgcg caacattctg acacaggggc atctgtggag aggcctgagt 3120 attctacaac aagcccacag caaacctggt gctcagccag atttgatatc actgagatca 3180 gtagttggag aatgcccagg atggggaggg gtctcagacc cacatttaag tgttgcttta 3240 ttctgggtt tttatttatt tatttattta tttttaagga ggatgtgttt ctttaattat 3300 aagacaggat gctgagagat aaatgtcatt ttctctatca tggggtatag ccagatggaa 3360 gattgagaag tggctcacag ctcagcagaa tgaaaaaata tctgaatgct gctttctgaa 3420 actactctcc agaatgattt cacactcact ccttggagca aacaatgact tgcaaatttt 3480 tctaatttaa acataaagga gtgtacatat tggtattagt attcatttta ttttgggggaa 3540 gggcactgta ttagtccata gtccgttttc acactgccga taaagacata cccaacattg 3600 ggaagaaaaa gaggtttaat tggacttaca gttccatttg gctggggagg cctcagaatc 3660 atggtgggag gcgaaaggca cttctttacat ggtggtggca agaagaaaatg aggaagaagc 3720 aaatgccaaa acccctgata aacacattgg atctcaggag acttattcat tatcatgaga 3780 atagcatggg aaagactggc ccccatgatt caattacctc cccctgggtc cctcccacaa 3840 catgtgggaa ttctgggaga tacaattca gttgagatt gggtggggac acagccaaac 3900 cacattggac acagaaccag gtttgaagct accagccag gaacataatc cacagccacc 3960 ctaattcaga tctctcatag gaaccactgt ccctgctcct gagcacagat gctactgcat 4020 atacctctga taccctgatg gccgacactg ggccctgtgg caagactgc tatcactgct 4080 gctcctgaga actgctccac tactgctcct cagccatctt taccaaatg cagtatttac 4140 tgtcccagcc tctctgtgtc atctcatcct gattagaagc ccacatgtgg ttatctaaat 4200 tgtgcagcca aagcccttg cagtgtttaa ctgcaataat gttggggaaa gtgaattttt 4260 ctcctttgta gaaggaggta gtccctgcct tctaataaga ctctcaaca taggagaga 4320 attcagttgc tggaggtaga ggggtgagggg atggaaaag atgacaaat ttcaatcct 4380 agaatcatgt tctgagacta gaacttatc tagtacattg caggcacctg gttttggttg 4440 agtgtataat aaatgacata gttcaactta ttcccttgac agtttgtttt ggggtccagc 4500 ttttgtctac cccagttttc accacagat acgtggagaa gcattgtgtg atggtaaaat 4560 gtttacttga aagcctttttt ccctatcttt gtctcttgct aggattaaa acccgtatct gt 4622 <210> 9 <211> 1188 <212> DNA <213> Homo sapiens <220> <223> HLA-G soluble <400> 9 agtgtggtac tttgtcttga ggagatgtcc tggactcaca cggaaactta gggctacgga atgaagttct cactcccatt aggtgacagg tttttagaga agccaatcag cgtcgccgcg gtcctggttc taaagtcctc gctcacccac ccggactcat tctccccaga cgccaaggat 180 ggtggtcatg gcgccccga ccctcttcct gctgctctcg ggggccctga ccctgaccga 240 gacctgggcg ggctcccact ccatgaggta tttcagcgcc gccgtgtccc ggcccggccg 300 cggggagccc cgcttcatcg ccatgggcta cgtggacgac acgcagttcg tgcggttcga 360 cagcgactcg gcgtgtccga ggatggagcc gcggggcgccg tgggtggagc aggaggggcc 420 ggagtattgg gaagaggaga cacggaacac caaggcccac gcacagactg acagaatgaa 540. cctgcagacc ctgcgcggct actacaacca gagcgaggcc agttctcaca ccctccagtg gatgattggc tgcgacctgg ggtccgacgg acgcctcctc cgcgggtatg aacagtatgc 600 ctacgatggc aaggattacc tcgccctga cgaggacctg cgctcctgga ccgcagcgga 660 cactgcggct cagatctcca agcgcaagtg tgaggcggcc aatgtggctg aacaaaggag agcctacctg gagggcacgt gcgtggagtg gctccacaga tacctggaga acgggaagga 780 gatgctgcag cgcgcggacc cccccaagac acacgtgacc caccaccctg tctttgacta 840 tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca tactgacctg 900 gcagcgggat ggggaggacc agacccagga cgtggagctc gtggagacca ggcctgcagg 960 ggatggaacc ttccagaagt gggcagctgt ggtggtgcct tctggagagg agcagagata cacgtgccat gtgcagcatg aggggctgcc ggagcccctc atgctgagat ggagcagtc 1080. ttccctgccc accatcccca tcatgggtat cgttgctggc ctggttgtcc ttgcagctgt 1140 agtcactgga gctgcggtcg ctgctgtgct gtggagaaag aagagctc <210> 10 <211> 898 <212> DNA <213> Homo sapiens <220> <223> Soluble HLA-L <400> 10 acgatcccgg cactacagtc ccggcgcaac cacccgcact cagattctc ccaaacgcca 60 aggatggggg tcatggctcc ccgaaccctc ctcctgctgc tcttgggggc cctggccctg 120 accgagacct gggccgcgac tccgtgagtc cgaggatgga gcggcgggcg ccgtgggtgg 180 agcaggaggg gctggagtat tgggaccagg agacacggaa cgccaagggc cacgcgcaga 240 tttaccgagt gaacctgcgg accctgctcc gctattacaa ccagagcgag gccggtatga 300 acagttcgcc tacgatggca aggattacat cgccctgaac gaggacctgc actcctggac 360 cgccgcgaac acagcggctc agatctccca gcacaagtgg gaagcggaca aatactcaga 420 gcaggtcagg gcctacctga gggcaagtgc atggagtggc tccgcagaca cctggagaac 480 gggaaggaga cgctgcagca cgcggatccc ccaaaggcac atgtgaccca gcaccccatc 540 tctgaccatg aggccaccct gaggtgctgg gccctgggcc tctaccctgc ggagatcaca 600 ctgacctggc agcaggatgg ggaggaccag acccaggaca cggagcttgt ggagaccagg 660 cctgcagggg acggaacctt ccagaagtgg gtggctgtag tggtgccttc cggagaggag 720 cagagataca tgtgccatgt gcagcatgag gggctgccag agcccctcac cctgagatgg 780 gagccgtctt ctcagcccac catccccatc gtgggcatcg ttgctggcct gttctcctt 840 ggagctgtgg tcactggagc tgtggttgct gctgcgatgt ggaggaagaa aagctcag 898 <210> 11 <211> 1552 <212> DNA <213> Homo sapiens <220> <223> Soluble HLA-J <400> 11 ctatactatc tcatgcaccc aggcacaact tttccagatt taaagaaaaa gaaaaaagaa 60 ataaaagaaa aaaacctctg tctctacacc tccattccca gggagagctc cctctggc 120 accaagctcc ctggggtgag ttttcttttt gaagagtcca ggggaacagc ctgcgacggg 180 tccttcttcc tggacactca cgacgcggac ccagttctca ctcccactga gtgtcgggtt 240 ttagggaagc caatcagcgt cgcgcggccc cggttctaaa gtccccacgc acccaccggg 300 actcggagtc tccccagacg ccgacgatgg ggtcatggcg ccccgaaccc tcctcctgct 360 gctctcgggg accctggccc tggccgagac ctgggcgggc tcccactcca tgaggtattt 420 cagcaccgcc gtttcctggc cgggccgcgg ggagcccagc ttcattgccg tgggctacgt 480 ggacgacacg cagttcgtgc gggtcgacag tgacgccgtg agtctgagga tgaagacgcg 540 ggcgcggtgg gtggagcagg aggggccgga gtattgggac ctacagacac tgggcgccaa 600 ggcccaggca cagactgacc gagtgaacct gcggaccctg ctccgctact acaaccagag 660 cgaggcggac cccccccaag acacacgtga cccacccccc tctctgaaca tgaggcataa 720 cgaggtcctg ggttctgggc ttctaccctg cggagatcac attgacctgg cagcgggatg 780 gggaggacca gacccaggac atggagctcg tggagaccag gcccacaggg gatggaacct 840 tccagaagtg ggcggttgtg gtagtgcctt ctggagagga acagagatac acatgccatg 900 tgcagcacaa ggggctgccc aagcccctca tcctgagatg ggtcacacat ttctggaaac 960 ttctcaaggt tccaagacta ggaggttcct ctaggacctc atggccctgc taccttcctg 1020 gcctctcaca ggacgttttc ttcccgcaga tagaaaagga gggagctact ctcaggctgc 1080 aagcagccaa agtgcccagg gctctgatgt gtctctcacg gcttgtaaag tgtgagacag 1140 ctgccttgtg tgggactgag aggcaagatt tgttcatgcc ttccctttgt gacttcaaga 1200 acctgactt ctctttctgc aaaggcatct gaatgtgtct gtgtccctat aggcataatg 1260 tgaggtggtg gggagaccag cccacacccg tgtccaccat gaccctgttc cccacactga 1320 cctacattcc ttccccgatc acctttcctg ttccagagaa gtggtgctgg gatgtctcca 1380 tctctgtctc aacttcatgg tgcactgagc tgtaacttct tacttcccta ttaaaattag 1440 aatctgagta taaatttact ttttcaaat tatttccatg acgggttgat gggttaatta 1500 aaggagaaga ttcctaaaat ttgagagaca aaataaatgg aagacatgag aa 1552 <210> 12 <211> 1704 <212> DNA <213> Homo sapiens <220> <223> Soluble HLA-H <400> 12 agttctctt cttctcacaa cctgcgacgg gtccttcttc cttgatactc acgaagcgga 60 cacagttctc attcccacta ggtgtcgggt ttctagagaa gccaatcggt gccgccgcgg 120 tcccggttct aaagtcccca cgcacccacc gggactcaga ttctccccag acgccgagga 180 tggtgctcat ggcgccccga accctctcc tgctgctctc aggggccctg gccctgaccc 240 tgacccagac ctgggcgcgc tccactcca tgaggtattt ctacaccacc atgtcccggc 300 ccggccgcgg ggagccccgc ttcatctccg tcggctacgt ggacgatacg cagttcgtgc 360 ggttcgacag cgacgacgcg agtccgagag aggagccgcg ggcgccgtgg atggagcggg 420 aggggccaga gtattgggac cggaaacac agatctgcaa ggcccaggca cagactgaac 480 gagagaacct gcggatcgcg ctccgctact acaaccagag cgagggcggt tctcacacca 540 tgcaggtgat gtatggctgc gacgtggggc ccgacgggcg cttctcccgc gggtatgaac 600 agcacgccta cgacggcaag gattacatcg ccctgaacga ggacctgcgc tcctggaccg 660 cggcggacat ggcagctcag atcaccaagc gcaagtggga ggcggcccgt cgggcggagc 720 agctgagagc ctacctggag ggcgagttcg tggagtggct ccgcagatac ctggagaacg 780 ggaaggagac gctgcagcgc gcggaccccc cccaagacac atatgaccca ccaccccatc 840 tctgaccatg aggccaccct gaggtgctgg gccctgggct tctaccctgc ggagatcaca 900 ctgacctggc agcgggatgg ggaggaccag acccaggaca cggagctcgt ggagaccagg 960 cctgcagggg atggaacctt ccagaagtgg gcggctgtgg tggtgccttc tggagaggag 1020 cagagataca cctgccatgt gcagcatgag ggtctgcccg agcccctcac cctgagatgg 1080 gagccatctt cccagcccaa cgtccccatc gtgggcatcg ttgctggcct ggttctactt 1140 gtagctgtgg tcactggagc tgtggtcgct gctgtaatgt ggaggaagaa gagctcagat 1200 agaaaaggag ggagctactc tcaggctgca acagcaacag tgcccagggc tctgatgtgt 1260 ctctcacggc ttgaaagtgt gagacagctg ccttgtgtgg gactgagagg caagagttgt 1320 tcctgccttc cctttgtgac ttgaagaacc ctgactttct ttctacaaag gcacctgaat 1380 gtgtctgtgt tcctgtaggc ataatgtgtg gaggagggga gaccaaccca ccctcatgtc 1440 caccatgacc ctcttcccca cgctgatctg tgttccctcc ccaatcatct ttcctgttcc 1500 agagaggagg ggctgagatg tctccatctt tttctcaact ttatgtgcac tgagctgtaa 1560 cttcttactt ccctcttaaa attagaatct gagtaaacat ttactttttc aaattcttgc 1620 catgagaggt tgatgactta attaaaggag aagattccta aaatttgaga gacaaaataa 1680 atggaacaca tgagaacctt ccag 1704 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> active redox site <400> 13 Cys Gly Pro Cys 1 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-G Ex3 <400> 14 ggccgggta ttgggaaga 19 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HLA-G Ex3 probe <400> 15 caaggcccac gcacagactg aca 23 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-G Ex3 <400> 16 gcagggtctg caggttcatt 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-G Ex4 <400> 17 ctgcggctca gatctccaa 19 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-G Ex4 <400> 18 cgcaagtgtg aggcggccaa t 21 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-G Ex4 <400> 19 caggtaggct ctcctttgtt cag 23 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-G Ex5 <400> 20 caccaccctg tctttgacta tgag 24 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-G Ex5 <400> 21 accctgaggt gctgggccct g <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-G Ex5 <400> 22 ccgcagggta gaag <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-G Ex6 <400> 23 catccccatc atgggtatcg <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-G Ex <400> 24 tgctggcctg gttgtccttg approx <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-G Ex6 <400> 25 ccgcagctcc agtgactaca 20 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-G Ex8 <400> 26 gaccctcttc ctcatgctga ac 22 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-G Ex8 <400> 27 cattccttcc ccaatcacct ttcctgtt 28 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse HLA-G Ex8 <400> 28 catcccagcc ccttttctg 19 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-G Ex3-5 <400> 29 ttcatcgcca tgggctacg 19 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-G Ex3-5 <400> 30 cgacacgcag ttcgtgcggt tc 22 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-G Ex3-5 <400> 31 atcctcggac acgccgagt 19 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA- G Ex2 / 3 <400> 32 ccgaaccctc ttcctgctgc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-G Ex2 / 3 <400> 33 cgagacctgg gcgggctccc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-G Ex2 / 3 <400> 34 gcgctgaaat acctcatgga 20 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-H Ex 2 / 3 <400> 35 gagagaacct gcggatcgc 19 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-H Ex2 / 3 <400> 36 agcgagggcg gttctcacac catg 24 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-H Ex2 / 3 <400> 37 ccacgtcgca gccatacat 19 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-H <400> 38 gagagaacct gcggatcgc 19 <210> 39 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> HLA-H Probe <400> 39 accagagcga gggcggttct cacac 25 <210> 40 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> HLA-H Reverse Primer <400> 40 cgggccggga catggt 16 <210> 41 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer KRT 5 <400> 41 cgccacttac cgcaagct 18 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe KRT5 <400> 42 tggagggcga ggaatgcaga ctca 24 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> KRT5 Reverse Primer <400> 43 acagagatgt tgactggtcc aactc 25 <210> 44 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer KRT20 <400> 44 gcgactacag tgcatattac agacaa 26 <210> 45 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Probe KRT20 <400> 45 ttgaagagct gcgaagtcag attaaggatg ct 32 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer KRT20 <400> 46 cacaccgagc attttgcagt t 21 <210> 47 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer PD-1 <400> 47 ggccagcccc tgaagga 17 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe PD-1 <400> 48 acccctcagc cgtgcctgtg ttc 23 <210> 49 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer PD-1 <400> 49 ggaaatccag ctccccatag tc 22 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer PDL1 <400> 50 tggcatccaa gatacaaact caa 23 <210> 51 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe PDL1 <400> 51 caaagtgata cacatttgga ggagacgtaa 30 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer PDL1 <400> 52 ttgaagatca gaagttccaa tgct 24 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer FGFR1 <400> 53 aattcaaacc tgaccacaga attg 24 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe FGFR1 <400> 54 aggctacaag gtccgttatg ccacc 25 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer FGFR1 <400> 55 caccacagag tccattatga tgct 24 <210> 56 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer FGFR2 <400> 56 aagcaggagc atcgcattg 19 <210> 57 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Probe FGFR2 <400> 57 aggctacaag gtacgaaacc agcactgg 28 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer FGFR2 <400> 58 cagatgggac cacactttcc a 21 <210> 59 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer FGFR3 <400> 59 agcgcgtact gtgccactt 19 <210> 60 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe FGFR3 <400> 60 agtgtgcggg tgacagacgc tcc 23 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer FGFR3 <400> 61 ctccccgtct tcgtcatctc 20 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer FGFR4 <400> 62 ggatggacag gcctttcatg 20 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe FGFR4 <400> 63 cattggaggc attcggctgc g 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer FGFR4 <400> 64 cacgagactc cagtgctgat g 21 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer CALM2 <400> 65 gagcgagctg agtggttgtg 20 <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe CALM2 <400> 66 tcgcgtctcg gaaaccggta gc 22 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer CALM2 <400> 67 agtcagttgg tcagccatgc t 21 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-L Ex2 / 3 <400> 68 cctgctccgc tattacaacc a 21 <210> 69 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-L Ex2 / 3 <400> 69 cgaggccggt atgaacagtt cgccta 26 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-L Ex2 / 3 <400> 70 cgttcagggc gatgtaatcc 20 <210> 71 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-L Ex5 / 6 <400> 71 gctgtggttg ctgctgcg 18 <210> 72 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-L Ex5 / 6 <400> 72 agaaaagctc aggcagcaat tgtgctcag 29 <210> 73 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-L Ex5 / 6 <400> 73 catagtcctc tttacaagta tcatgagatg 30 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-L Ex7 <400> 74 tcctcttctg ctcagctctc cta 23 <210> 75 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-L Ex7 <400> 75 ctctcccttc cctgagttgt agtaatccta gcact 35 <210> 76 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-L Ex7 <400> 76 gctttataga tccatgagtt tgcatta 27 <210> 77 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer HLA-J Ex4 / 5 <400> 77 caaggggctg cccaagc 17 <210> 78 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe HLA-J Ex4 / 5 <400> 78 catcctgaga tgggtcacac atttctggaa 30 <210> 79 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer HLA-J Ex4 / 5 <400> 79 cctcctagtc ttggaacctt gagaagt 27

Claims

1. The use of a substance for detecting the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from a subject with urothelial carcinoma in the preparation of a kit for predicting whether said subject will respond to tumor therapy selected from anti-PD-1 or anti-PD-L1 immune checkpoint therapy. The method described herein includes: (A) Determine the level of at least one nucleic acid molecule and / or at least one protein or peptide in a sample obtained from said object. The at least one nucleic acid molecule is selected from nucleic acid molecules or fragments thereof encoding a polypeptide comprising any one of the amino acid sequences SEQ ID NO:2 and 4 to 6, said fragment comprising at least 250 nucleotides, and The at least one protein or peptide is selected from the proteins or peptides encoded by the at least one nucleic acid molecule; and (B) Compare the level of (A) with the level of the at least one nucleic acid molecule and / or the at least one protein or peptide in samples obtained from one or more subjects who have responded to the tumor treatment, or with a corresponding predetermined standard. Wherein, an increase in the level of (A) compared to the level of (B) or a predetermined standard indicates that the subject has no response to the tumor treatment, and a substantially equal or decreased level of (A) compared to the level of (B) indicates that the subject has a response to the tumor treatment; or (B') Compare the level of (A) with the level of the at least one nucleic acid molecule and / or the at least one protein or peptide in samples obtained from one or more subjects that have not responded to the tumor treatment, or with a corresponding predetermined standard. Wherein, a decrease in the level of (A) compared to the level of (B') or a predetermined standard indicates that the subject has responded to the tumor treatment, and a level of (A) that is substantially the same as or increases compared to the level of (B') indicates that the subject has not responded to the tumor treatment. in, The anti-PD-1 or anti-PD-L1 immune checkpoint therapy is the application of an immune checkpoint inhibitor selected from the following: nivolumab, pembrolizumab, and atezolizumab, and the term "substantially similar" means a difference of less than 10%.

2. The use according to claim 1, wherein the fragment comprises at least 300 nucleotides.

3. The use according to claim 1, wherein the fragment comprises at least 450 nucleotides.

4. The use according to claim 1, wherein the fragment comprises at least 600 nucleotides.

5. The use according to claim 1, wherein the polypeptide consists of any one of the amino acid sequences SEQ ID NO:2 and 4 to 6.

6. The use according to claim 1, wherein the at least one nucleic acid molecule (a) Consists of any one of the nucleotide sequences SEQ ID NO:8 and 10 to 12, or (c) consists of a degenerate nucleotide sequence relative to (a).

7. The use according to claim 6, wherein any one of the nucleotide sequences SEQ ID NO:8 and 10 to 12 is SEQ ID NO:11 or 12.

8. The use according to claim 1, wherein the T in the at least one nucleic acid molecule is replaced by U.

9. The use according to claim 1, wherein any one of the amino acid sequences SEQ ID NO:2 and 4 to 6 is SEQ ID NO:5 or 6.

10. The use according to any one of claims 1-9, wherein the method further comprises determining the mRNA expression level or protein level selected from one or both of PD-1 and PD-1L.

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