Novel peptides and peptide combinations for use in immunotherapy for esophageal and other cancers

Abstract

To provide peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods.SOLUTION: Provided is a peptide or a pharmaceutically acceptable salt thereof that consists of a specific amino acid sequence, has the ability to bind to an MHC class I molecule, and can be recognized by CD8 T cells when bound to the MHC class I molecule. In one aspect, the peptide or a pharmaceutically acceptable salt thereof includes a modified and / or non-peptide bonds and constitutes a fusion protein comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptides, proteins, nucleic acids, and cells for use in immunotherapy. In particular, the present invention relates to cancer immunotherapy. The present invention also relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides, which can serve as active pharmaceutical ingredients in vaccine compositions, for example, to stimulate anti-tumor immune responses or to stimulate T cells ex vivo and transfer them into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or the peptides themselves, can also be targeted by antibodies, soluble T-cell receptors, and other binding molecules.

[0002] The present invention relates to several novel peptide sequences and their variants derived from HLA class I molecules of human tumor cells, which can be used in vaccine compositions to elicit anti-tumor immune responses or as targets for the development of pharmacologically / immunologically active compounds and cells. [Background technology]

[0003] Esophageal cancer is the eighth most common cancer worldwide, with a 5-year prevalence of 464,063 in 2012. Mortality rates are very similar to incidence rates (400,169 vs. 455,784 in 2012), indicating a high mortality rate for esophageal cancer (World Cancer Report, 2014; Ferlay et al., 2013; Bray et al., 2013).

[0004] Squamous cell carcinoma and adenocarcinoma are the two most common subtypes of esophageal cancer. Both subtypes are more common in men than women, but they have different geographic distributions. Squamous cell carcinoma is more common in low-resource areas, with particularly high incidence rates in the Islamic Republic of Iran, parts of China, and Zimbabwe. Adenocarcinoma is the most common type of esophageal cancer in white and high-socioeconomic status populations, led by the United Kingdom, Australia, the Netherlands, and the United States. The strongest risk factors for the development of esophageal squamous cell carcinoma include alcohol and tobacco consumption, while esophageal adenocarcinoma is primarily associated with obesity and gastroesophageal reflux disease. The incidence of esophageal adenocarcinoma has been steadily increasing in high-income countries, which may be due to increased incidence of obesity and gastroesophageal reflux disease as well as changes in the classification of gastroesophageal junction tumors. Neuroendocrine carcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, mucinous epithelioid carcinoma, mixed adenoendocrine carcinoma, various sarcomas, and melanoma represent rarer subtypes of esophageal cancer. (World Cancer Report, 2014).

[0005] Primary treatment strategies for esophageal cancer depend on tumor stage and location, histology, and the patient's medical condition. Except for a small subgroup of patients with squamous cell carcinoma, surgery alone is insufficient. Generally, surgery should be combined with preoperative or definitive postoperative chemotherapy or preoperative chemoradiation, while preoperative or postoperative radiation alone has not been shown to confer a survival benefit. Chemotherapeutic regimens include oxaliplatin and fluorouracil, carboplatin and paclitaxel, cisplatin and fluorouracil, FOLFOX, and cisplatin and irinotecan. Because randomized data on targeted therapy in esophageal cancer are very limited, patients with HER2-positive tumors should be treated according to guidelines for gastric cancer using a combination of cisplatin, fluorouracil, and trastuzumab (Stahl et al., 2013; Leitlinie Magenkarzinom, 2012).

[0006] In general, most types of esophageal cancer are well manageable when patients have early-stage tumors, but treatment success is very limited at later stages. Therefore, the development of new screening protocols could be very effective in reducing esophageal cancer-related mortality (World Cancer Report, 2014).

[0007] Immunotherapy may be a promising novel approach for treating advanced esophageal cancer. Several cancer-related genes and cancer-testis antigens, including the distinct MAGE genes NY-ESO-1 and EpCAM, have been shown to be overexpressed in esophageal cancer (Kimura et al., 2007; Liang et al., 2005B; Inoue et al., 1995; Bujas et al., 2011; Tanaka et al., 1997; Quillien et al., 1997). These genes are highly attractive targets for immunotherapy, and the majority of them are being investigated for the treatment of other malignancies (ClinicalTrials.gov, 2015). Furthermore, upregulation of PD-L1 and PD-L2 has been reported in esophageal cancer and correlated with a poorer prognosis. Therefore, esophageal cancer patients with PD-L1-positive tumors may benefit from anti-PD-L1 immunotherapy (Ohigashi et al., 2005).

[0008] Clinical data on immunotherapeutic approaches in esophageal cancer are currently relatively scarce, due to the very limited number of completed early-stage clinical trials (Toomey et al., 2013). A three-peptide vaccine derived from three different cancer-testis antigens (TTK protein kinase, lymphocyte antigen 6 complex locus K, and insulin-like growth factor (IGF)-II mRNA-binding protein 3) was administered to patients with advanced esophageal cancer in a phase I trial with moderate success (Kono et al., 2009). Intratumoral injection of activated T cells after ex vivo challenge with autologous malignant cells and interleukin-2 induced complete or partial tumor responses in four of two patients in a phase I / II trial (Toh et al., 2000; Toh et al., 2002). Additional clinical trials are currently being conducted to evaluate the impact of different immunotherapies on esophageal cancer, including adoptive cell therapy (NCT01691625, NCT01691664, NCT01795976, NCT02096614, NCT02457650), vaccination strategies (NCT01143545, NCT01522820), and anti-PD-L1 therapy (NCT02340975) (ClinicalTrials.gov, 2015). Summary of the Invention [Problem to be solved by the invention]

[0009] Given the severe side effects and costs associated with cancer treatment, there is a need to identify factors that can be used in the treatment of cancer in general, and esophageal cancer in particular. There is also a need to identify factors that represent biomarkers for cancer in general, and esophageal cancer in particular, that can lead to better cancer diagnosis, assessment of prognosis, and prediction of treatment success.

[0010] Cancer immunotherapy represents an option that specifically targets cancer cells while minimizing side effects. Cancer immunotherapy takes advantage of the presence of tumor-associated antigens.

[0011] The current classification of tumor-associated antigens (TAA) comprises the following major groups: a) Cancer-Testis Antigens: The first identified TAAs capable of being recognized by T cells belonged to this class and were originally called cancer-testis (CT) antigens because their members are expressed in histologically distinct human tumors and are present only in testicular spermatocytes / spermatogonia in normal tissues and occasionally in the placenta. Because testicular cells do not express class I and II HLA molecules, these antigens cannot be recognized by T cells in normal tissues and are therefore considered immunologically tumor-specific. Well-known examples of CT antigens are MAGE family members and NY-ESO-1. b) Differentiation antigens: These TAAs are shared between tumors and the normal tissues from which they arise. Most of the known differentiation antigens are found in melanomas and normal melanocytes. Many of these melanocyte-related proteins are involved in melanin biosynthesis and are therefore not tumor-specific, but are nevertheless widely utilized for cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan-A / MART-1 for melanoma, or PSA for prostate cancer. c) Overexpressed TAAs: Genes encoding ubiquitously expressed TAAs have been detected in histologically distinct tumor types and also in many normal tissues, generally at lower expression levels. While many of the epitopes processed and potentially presented by normal tissues may be below the threshold level for T cell recognition, their overexpression in tumor cells can trigger anti-cancer responses by breaking previously established immune tolerance. Prominent examples of this class of TAAs are Her-2 / neu, survivin, telomerase, or WT1. d) Tumor-specific antigens: These unique TAAs arise from mutations in normal genes (e.g., β-catenin, CDK4). Some of these molecular alterations are associated with neoplastic transformation and / or progression. Tumor-specific antigens can usually induce a strong immune response without the risk of an autoimmune reaction against normal tissue. On the other hand, these TAAs are most often associated only with the very tumor in which they were identified and are usually not shared among many individual tumors. For proteins with tumor-specific (associated) isoforms, tumor specificity (or association) of peptides may also occur if the peptide is derived from a tumor (associated) exon. e) TAAs resulting from aberrant post-translational modifications: Such TAAs may arise from proteins that are neither specific nor overexpressed in tumors, but nevertheless become tumor-associated through post-translational processes that are primarily active in tumors. Examples of this class arise from events such as altered glycosylation patterns that result in novel epitopes in tumors, such as MUC1, or protein splicing during degradation, which may or may not be tumor-specific. f) Oncoviral proteins: These TAAs are viral proteins that may play an important role in the carcinogenic process and, because they are foreign (not of human origin), can induce T cell responses. Examples of such proteins are the human papillomavirus type 16 proteins E6 and E7, which are expressed in cervical cancer.

[0012] T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by molecules of the major histocompatibility complex (MHC). The antigens, i.e., the epitopes, recognized by tumor-specific T lymphocytes can be molecules derived from all protein classes, such as enzymes, receptors, and transcription factors, which are expressed in the respective tumor cells and are usually upregulated compared to unmodified cells of the same origin.

[0013] There are two classes of MHC molecules: MHC class I and MHC class II. MHC class I molecules are composed of α heavy chains and β2 microglobulin, while MHC class II molecules are composed of α and β chains. Their three-dimensional structure provides a binding groove, which is used for non-covalent interactions with peptides.

[0014] MHC class I molecules are found on most nucleated cells. They present peptides derived primarily from endogenous proteins, defective ribosomal products (DRIPs), and proteolytic cleavage of larger peptides. However, peptides derived from endosomal compartments or exogenous sources are also frequently found on MHC class I molecules. This non-classical mode of class I presentation is referred to in the literature as cross-presentation (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules are found primarily on professional antigen-presenting cells (APCs) and primarily present peptides from exogenous or transmembrane proteins that are subsequently processed and incorporated into APCs during endocytosis.

[0015] It is well known that peptide-MHC class I complexes are recognized by CD8-positive T cells bearing the appropriate T cell receptor (TCR), while peptide-MHC class II complexes are recognized by CD4-positive helper T cells bearing the appropriate TCR. As a result, TCR, peptide, and MHC exist in a 1:1:1 stoichiometric ratio.

[0016] CD4+ helper T cells play a key role in inducing and maintaining effective responses by CD8+ cytotoxic T cells. Identification of CD4+ T cell epitopes derived from tumor-associated antigens (TAAs) is crucial for the development of drugs that trigger antitumor immune responses (Gnjatic et al., 2003). At tumor sites, T helper cells maintain a cytotoxic T cell (CTL)-friendly cytokine environment (Mortara et al., 2006) and attract effector cells, such as CTLs, natural killer (NK) cells, macrophages, and granulocytes (Hwang et al., 2007).

[0017] In the absence of inflammation, expression of MHC class II molecules is primarily restricted to cells of the immune system, particularly professional antigen-presenting cells (APCs), such as monocytes, monocyte-derived cells, macrophages, and dendritic cells. In cancer patients, tumor cells have been found to express MHC class II molecules (Dengjel et al., 2006).

[0018] Extended (longer) peptides of the invention can act as MHC class II active epitopes.

[0019] T helper cells activated by MHC class II epitopes play an important role in orchestrating the effector functions of CTLs in anti-tumor immunity. T helper cell epitopes that initiate TH1-type T helper cell responses support the effector functions of CD8+ killer T cells, including cytotoxicity directed against tumor cells that display tumor-associated peptide / MHC complexes on their cell surface. In this way, tumor-associated T helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses.

[0020] For example, in mammalian models such as mice, CD4+ T cells have been shown to be sufficient to inhibit tumor development through the inhibition of angiogenesis by secreting interferon-γ (IFNγ), even in the absence of CD8+ T lymphocytes (Beatty and Paterson, 2001; Mumberg et al., 1999). There is evidence that CD4+ T cells are direct antitumor effectors (Braumuller et al., 2013; Tran et al., 2014).

[0021] Because constitutive expression of HLA class II molecules is usually restricted to immune cells, it was previously thought that it might be possible to isolate class II peptides directly from primary tumors. However, Dengjel et al. successfully identified several MHC class II epitopes directly from tumors (WO 2007 / 028574, EP 1760088 B1).

[0022] Because both CD8- and CD4-dependent responses synergistically contribute to antitumor effects, the identification and characterization of tumor-associated antigens recognized by either CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4-positive T helper cells (ligand: MHC class II molecule + peptide epitope) is important for the development of tumor vaccines.

[0023] For an MHC class I peptide to initiate (elicit) a cellular immune response, it must also bind to an MHC molecule. This process depends on the MHC molecule allele and specific polymorphisms in the peptide's amino acid sequence. MHC class I-binding peptides are usually 8-12 amino acid residues long and typically contain two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele possesses a "binding motif" that determines which peptides can specifically bind to the binding groove.

[0024] In an MHC class I-dependent immune response, peptides must not only be able to bind to specific MHC class I molecules expressed by tumor cells, but they must also be subsequently recognized by T cells bearing specific T cell receptors (TCRs).

[0025] For a protein to be recognized by T lymphocytes as a tumor-specific or tumor-associated antigen and utilized therapeutically, certain requirements must be met. The antigen should be expressed primarily by tumor cells and not expressed or expressed in relatively low amounts by healthy tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells compared to healthy tissues. It is desirable that each antigen not only be present in certain tumors but also be present at high concentrations (i.e., the number of copies of each peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins directly involved in the transformation of normal cells into tumor cells, for example, due to their function in cell cycle regulation or apoptosis suppression. Furthermore, downstream targets of proteins directly responsible for transformation may be upregulated and thus indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets for vaccination approaches (Singh-Jasuja et al., 2004). To ensure that such peptides ("immunogenic peptides") are derived from tumor-associated antigens and elicit in vitro or in vivo T cell responses, the presence of epitopes within the amino acid sequence of the antigen is essential.

[0026] Essentially, any peptide capable of binding to an MHC molecule may serve as a T cell epitope. A prerequisite for the induction of a T cell response in vitro or in vivo is the presence of T cells bearing the corresponding TCR and the absence of immune tolerance to this particular epitope.

[0027] Therefore, TAAs are the starting point for the development of T cell-based therapeutics, including but not limited to tumor vaccines. Methods for identifying and characterizing TAAs are usually based on the use of T cells isolated from patients or healthy individuals, or they are based on the generation of differential transcriptional profiles or differential peptide expression patterns between tumor and normal tissues. However, the identification of genes that are overexpressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not provide accurate information regarding the use of antigens transcribed from these genes in immunotherapy. This is because only individual subsets of epitopes of these antigens are suitable for such use, because T cells with corresponding TCRs must exist, and immune tolerance to this particular epitope must be absent or minimal. Therefore, in a highly preferred embodiment of the present invention, it is important to select only peptides that are over- or selectively presented and that functional and / or proliferative T cells can be found against. Such functional T cells are defined as T cells that are capable of clonally expanding upon stimulation with a specific antigen and capable of performing effector functions ("effector T cells").

[0028] When targeting peptide-MHC with a specific TCR (e.g., a soluble TCR) and antibody or other binding molecule (scaffold) according to the present invention, the immunogenicity of the underlying peptide is secondary; in these cases, presentation is the determining factor. [Means for solving the problem]

[0029] In a first aspect, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 93, wherein said variant binds to MHC and / or induces cross-reactivity of T cells with said peptide or a pharmaceutically acceptable salt thereof, and wherein said peptide is not the underlying full-length polypeptide.

[0030] The present invention further relates to a peptide of the present invention comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 93, wherein said peptide or variant thereof has an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0031] The following tables show peptides according to the invention, their respective SEQ ID NOs, and the predicted genes of origin (basic) of those peptides. All peptides in Tables 1 and 2 bind to HLA-A*02. The peptides in Table 2 have previously been disclosed in large lists as a result of high-throughput screening with high error rates or calculated using algorithms, but have not previously been associated with cancer. The peptides in Table 3 are additional peptides that may be useful in combination with other peptides of the invention. The peptides in Table 4 are further useful in the diagnosis and / or treatment of various other malignancies involving overexpression or over-representation of the respective underlying polypeptides.

[0032] Table 1: Peptides according to the present invention [Table 1-1] [Table 1-2] [Table 1-3]

[0033] Table 2: Additional peptides according to the present invention with no previously known cancer association [Table 2]

[0034] Table 3: Peptides useful, for example, in personalized cancer therapy [Table 3]

[0035] The present invention further relates generally to peptides according to the invention for use in the treatment of proliferative diseases such as, for example, lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular carcinoma, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancer, and leukemia.

[0036] Particularly preferred are peptides according to the invention, alone or in combination, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93. More preferred are peptides, alone or in combination, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 76 (see Table 1) and their use in the immunotherapy of esophageal cancer, lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular carcinoma, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancer, and leukemia, preferably esophageal cancer.

[0037] Particularly preferred are peptides according to the invention, alone or in combination, selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 25, 26, 30, 32, 34, 37, 40, 51, 55, 57, 58, 59, 62, 81 and 82, and their use in the immunotherapy of esophageal cancer, lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular carcinoma, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancer and leukemia, preferably esophageal cancer. Even more particularly preferred is the peptide set forth in SEQ ID NO: 9.

[0038] As shown in Table 4A below, many of the peptides according to the invention are also found on other tumor types and therefore may also be used in immunotherapy for other indications. See also FIG. 1 and Example 1.

[0039] Table 4A: Peptides according to the present invention and their specific uses in other proliferative disorders, particularly other cancerous disorders. The table shows additional tumor types in which selected peptides were found, either over-represented in more than 5% of measured tumor samples or presented in more than 5% of measured tumor samples at a tumor-to-normal tissue geometric mean ratio of greater than 3. Over-representation is defined as higher presentation in tumor samples compared to the normal sample with maximal presentation. Normal tissues tested for over-representation were adipose tissue, adrenal gland, artery, bone marrow, brain, central nervous system, colon, duodenum, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear white blood cell, pancreas, peripheral nerve, peritoneum, pituitary gland, pleura, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid, trachea, ureter, bladder, and vein. [Table 4A-1] [Table 4A-2]

[0040] Table 4B: Peptides according to the invention and their specific uses in other proliferative disorders, particularly other cancerous disorders (modification of Table 4). The table shows additional tumor types in which selected peptides, as in Table 4A, were found, showing over-representation in more than 5% of measured tumor samples or showing a tumor-to-normal tissue geometric mean ratio of greater than 3 in more than 5% of measured tumor samples. Over-representation is defined as higher presentation in tumor samples compared to the normal sample with maximal presentation. The normal tissues tested for over-representation were adipose tissue, adrenal gland, artery, bone marrow, brain, central nervous system, colon, duodenum, esophagus, eye, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear white blood cell, pancreas, parathyroid gland, peripheral nerve, peritoneum, pituitary gland, pleura, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid, trachea, ureter, bladder, and vein. [Table 4B-1] [Table 4B-2] [Table 4B-3]

[0041] Therefore, another aspect of the present invention relates, in a preferred embodiment, to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 7, 8, 9, 11, 13, 17, 40, 57, 58, 62, 67, 72, 76, 77, 80, 82, 88, 92 and 94 for combination therapy of non-small cell lung cancer.

[0042] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 18, 19, 25, 29, 55, 58, 62, 68, 75, 76, 77, 79, 81, 84, 86, 90 and 92 for the combination therapy of lymphoma.

[0043] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 22, 55, 58, 62, 57, 61, 76, 79 and 80 for the combination therapy of small cell lung cancer.

[0044] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 17, 56, 76, 82 and 54 for the combination therapy of renal cell carcinoma.

[0045] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 16, 22, 66, 67, 80, 81, 83, 86, 87 and 89 for the combination therapy of brain cancer.

[0046] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 31, 33, 35, 36, 37, 38, 39, 41, 42, 45, 46, 48, 50, 52, 53, 54, 55, 63, 68, 70, 75 and 71 for the combination therapy of gastric cancer.

[0047] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention according to any one of SEQ ID NOs: 26, 34, 40, 74, 80, 88 and 92 for the combination therapy of colorectal cancer.

[0048] Therefore, another aspect of the present invention relates, in a preferred embodiment, to the use of at least one peptide according to the present invention as set forth in any one of SEQ ID NOs: 15, 17, 22, 35, 49, 62, 67, 70, 72, 74, 75, 80, 82 and 92 for the combination therapy of hepatocellular carcinoma.

[0049] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 37, 40, 68, 69, 71, 78, 79, 87 and 91 for the combination therapy of pancreatic cancer.

[0050] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 79 and 91 for the combination therapy of prostate cancer.

[0051] Therefore, another aspect of the present invention relates, in a preferred embodiment, to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 4, 28, 58, 61, 72, 78, 79, 80, 82, 84, 86, 88, 92 and 85 for the combination therapy of leukemia.

[0052] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 22, 28, 29, 30, 40, 56, 57, 62, 73, 74, 75, 76, 79, 80, 82, 88, 92 and 89 for the combination therapy of breast cancer.

[0053] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 1, 13, 14, 11, 16, 18, 19, 23, 28, 30, 40, 55, 57, 58, 62, 66, 70, 72, 75, 76, 80, 84, 86, 89, 92 and 83 for the combination therapy of melanoma.

[0054] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 8, 62, 70, 78, 79, 80 and 87 for the combination therapy of ovarian cancer.

[0055] Therefore, another aspect of the present invention relates, in a preferred embodiment, to the use of at least one peptide according to the present invention as set forth in any one of SEQ ID NOs: 2, 3, 4, 7, 8, 10, 12, 13, 15, 17, 30, 32, 34, 40, 47, 53, 57, 58, 62, 66, 72, 74, 75, 77, 78, 79, 83, 86, 87, and 89 for the combination therapy of bladder cancer.

[0056] Therefore, another aspect of the present invention relates, in a preferred embodiment, to the use of at least one peptide according to the present invention as set forth in any one of SEQ ID NOs: 13, 15, 29, 30, 40, 56, 57, 58, 80, 81, 83, 84 and 88 for the combination therapy of uterine cancer.

[0057] Therefore, another aspect of the present invention relates, in a preferred embodiment, to the use of at least one peptide according to the invention as set forth in any one of SEQ ID NOs: 4, 7, 11, 15, 16, 25, 30, 32, 40, 51, 56, 62, 67, 72, 75, 76, 80, 86, 89, and 92 for the combined treatment of gallbladder and bile duct cancer.

[0058] Therefore, another aspect of the present invention relates in a preferred embodiment to the use of at least one peptide according to the invention according to any one of SEQ ID No. SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, 16, 18, 19, 20, 25, 30, 32, 34, 40, 42, 57, 58, 59, 66, 67, 69, 72, 74, 75, 77, 78, 80, 84, 86, 87, 88 and 90 for combination therapy of HNSCC.

[0059] Therefore, another aspect of the present invention relates to the use of a peptide according to the invention for the combined therapy of proliferative diseases, preferably selected from the group of esophageal cancer, lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular carcinoma, renal cell, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancer, and leukemia.

[0060] The present invention further relates to peptides according to the invention which have the ability to bind to molecules of the human major histocompatibility complex (MHC) class I, or in extended forms such as length variants, have the ability to bind to MHC class II.

[0061] The present invention further relates to peptides according to the invention, said peptides consisting of or consisting essentially of the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 93 (respectively).

[0062] The present invention further relates to a peptide according to the invention, said peptide being modified and / or comprising a non-peptide bond.

[0063] The present invention further relates to a peptide according to the invention, said peptide being part of a fusion protein, in particular fused to the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii) or fused to (or in the sequence of) an antibody, e.g. an antibody specific for dendritic cells.

[0064] The present invention further relates to a nucleic acid encoding a peptide according to the invention. The present invention further relates to a nucleic acid according to the invention which is DNA, cDNA, PNA, RNA or a combination thereof.

[0065] The present invention further relates to an expression vector capable of expressing and / or which expresses a nucleic acid according to the invention.

[0066] The present invention further relates to a peptide according to the invention, a nucleic acid according to the invention or an expression vector according to the invention for use in disease treatment and medicine, in particular in the treatment of cancer.

[0067] The present invention further relates to antibodies specific for the peptides according to the invention or for complexes of said peptides according to the invention with MHC, and to methods for their production.

[0068] The present invention further relates to T cell receptors (TCRs), in particular soluble TCRs (sTCRs) and cloned TCRs integrated into autologous or allogeneic T cells; methods for producing these; and methods for producing NK cells or other cells bearing or cross-reacting with said TCRs.

[0069] Antibodies and TCRs are further embodiments of immunotherapeutic uses of the peptides according to the invention.

[0070] The present invention further relates to a host cell comprising a nucleic acid or an expression vector according to the invention as described above.The present invention further relates to a host cell according to the invention which is an antigen-presenting cell, preferably a dendritic cell.

[0071] The present invention further relates to a method for producing a peptide according to the invention, comprising the steps of culturing a host cell according to the invention and isolating the peptide from the host cell or its culture medium.

[0072] The present invention further relates to a method according to the invention, in which a sufficient amount of antigen is contacted with an antigen-presenting cell, thereby loading the antigen onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell.

[0073] The present invention further relates to a method according to the invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing or expressing said peptide containing SEQ ID NO: 1 to SEQ ID NO: 93, preferably containing SEQ ID NO: 1 to SEQ ID NO: 76 or a variant amino acid sequence.

[0074] The present invention further relates to activated T cells produced by the method according to the invention, said T cells selectively recognizing cells expressing a polypeptide comprising an amino acid sequence according to the invention.

[0075] The present invention further relates to a method of killing target cells in a patient which aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, comprising the step of administering to the patient an effective number of T cells produced according to the present invention.

[0076] The present invention further relates to the use of any of the described peptides, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention, activated T lymphocytes, T cell receptors or antibodies or other peptide- and / or peptide-MHC-binding molecules according to the invention as a medicament or in the manufacture of a medicament, preferably said medicament being effective against cancer.

[0077] Preferably, the agent is a soluble TCR or antibody-based cell therapy, vaccine or protein.

[0078] The present invention further relates to a use according to the present invention, wherein said cancer cells are esophageal cancer, lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular carcinoma, renal cell, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancer, and leukemia, preferably esophageal cancer cells.

[0079] The present invention further relates to a peptide-based biomarker according to the present invention, herein referred to as a "target," which can be used in the diagnosis of cancer, preferably esophageal cancer. The marker can be over-presentation of the peptide itself or over-expression of the corresponding gene. The marker can also be used to predict the probability of success of a treatment, preferably an immunotherapy, most preferably an immunotherapy targeting the same target identified by the biomarker. For example, tumor sections can be stained using antibodies or soluble TCR to detect the presence of the peptide of interest complexed with MHC.

[0080] Optionally, the antibody possesses additional effector functions, such as an immunostimulatory domain or a toxin.

[0081] The present invention also relates to the use of these novel targets in the context of cancer therapy.

[0082] ABHD11 antisense RNA1 (ABHD11-AS1) has been described as a long non-coding RNA that is upregulated in gastric cancer and has been shown to be associated with differentiation and Lauren histological classification. Therefore, ABHD11-AS1 may be a potential biomarker for the diagnosis of gastric cancer (Lin et al., 2014). ABHD11 activity has been shown to be associated with the development of distant metastasis in lung adenocarcinoma and may therefore be a potential novel biomarker (Wiedl et al., 2011).

[0083] ADAMTS2 was shown to be dysregulated in patients with mixed T / myeloid phenotype acute leukemia (Tota et al., 2014). ADAMTS2 was described as being associated with the JNK pathway upon upregulation through IL-6 in osteosarcoma cells (Alper and Kockar, 2014). ADAMTS2 may be a potential diagnostic marker for follicular thyroid cancer (Fontaine et al., 2009). ADAMTS2 was described as a potential marker for metastasis in tongue squamous cell carcinoma (Carinci et al., 2005). ADAMTS2 was shown to be upregulated in renal cell carcinoma and associated with shorter patient survival (Roemer et al., 2004). ADAMTS2 was shown to be regulated by transforming growth factor-β1-associated cell proliferation (Wang et al., 2003).

[0084] AHNAK2 encodes the scaffold protein AHNAK nuclear protein 2 (Marg et al., 2010). AHNAK2 is a key component of the non-classical secretory pathway of fibroblast growth factor 1 (FGF1), a factor involved in tumor growth and invasion (Kirov et al., 2015).

[0085] ANO1 encodes anoctamin 1, a calcium-activated chloride channel associated with small intestinal sarcoma and oral cancer (RefSeq, 2002). ANO1 is amplified in esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumor (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, and breast cancer (Qu et al., 2014).

[0086] ARHGDIA has been shown to be downregulated in hepatocellular carcinoma and during breast cancer development (Liang et al., 2014; Bozza et al., 2015). ARHGDIA has been shown to be associated with tumor invasion, metastasis, overall survival, and time to recurrence in hepatocellular carcinoma. Therefore, ARHGDIA may provide a potential therapeutic target for hepatocellular carcinoma (Liang et al., 2014). ARHGDIA was also shown to be downregulated in the lung cancer cell line A549 upon periplonsin treatment. Therefore, periplonsin-inhibited proliferation of lung cancer cells may be related to ARHGDIA (Lu et al., 2014). ARHGDIA knockdown was associated with increased apoptosis in normal and cultured tumor cells of lung origin. Therefore, ARHGDIA has been described as a negative regulator of apoptosis, which may represent a potential therapeutic target (Gordon et al., 2011). ARHGDIA has been described to be associated with the stage of ovarian clear cell and high-grade serous carcinoma (Canet et al., 2011). ARHGDIA was described as an apoptosis pathway-related gene that was shown to be deregulated in fibrosarcoma HT1080 cells during TRAIL-mediated apoptosis (Daigeler et al., 2008). ARHGDIA was shown to be upregulated in the oxaliplatin-resistant colon cancer cell line THC8307 / L-OHP and described as a gene involved in anti-apoptosis. Therefore, ARHGDIA may be a potential marker associated with oxaliplatin sensitivity (Tang et al., 2007). Overexpression of ARHGDIA was shown to be regulated by the putative tumor suppressor ACVR2, a member of the cancer-related TGFBR2 family, in wild-type ACVR2-transfected MSI-H colon cancer cell lines harboring an ACVR2 frameshift mutation (Deacu et al., 2004). ARHGDIA was described as a key regulator of Rho GTPases.ARHGDIA depletion has been shown to induce constitutive activation of the Rho GTPase and COX-2 pathways, which are associated with breast cancer progression in breast cancer xenograft animal models (Bozza et al., 2015). ARHGDIA signaling has been shown to be deregulated in colorectal cancer (Sethi et al., 2015). ARHGDIA has been shown to target MEK1 / 2-Erk for sumoylation, which is associated with the inhibition of c-Jun / AP-1, cyclin d1 transcription, and cell cycle progression. Therefore, ARHGDIA is associated with the suppression of cancer cell proliferation (Cao et al., 2014). ARHGDIA has been described as a novel suppressor in prostate cancer, which may play an important role in regulating androgen receptor signaling and prostate cancer growth and progression (Zhu et al., 2013b).

[0087] ATIC was described as a potential gene fusion partner of the cancer-associated anaplastic lymphoma kinase in anaplastic large cell lymphoma (Cheuk and Chan, 2001). ATIC was shown to present as a chimeric fusion with ALK in inflammatory myofibroblastic tumors of the bladder (Debiec-Rychter et al., 2003). Inhibition of the aminoimidazole carboxamide ribonucleotide transformylase (AICAR) activity of ATIC in model breast cancer cell lines was shown to result in a dose-dependent decrease in cell number and cell division rate. Therefore, ATIC may be a potential target in cancer therapy (Spurr et al., 2012).

[0088] CAPZB was reported to be overexpressed in human papillomavirus 18-positive oral squamous cell carcinoma and was identified as a prostate cancer susceptibility locus ( Lo et al., 2007 ; Nwosu et al., 2001 ).

[0089] COL6A1 is upregulated in the reactive stroma of castration-resistant prostate cancer and promotes tumor growth (Zhu et al., 2015). COL6A1 is overexpressed in CD166- pancreatic cancer cells, which have stronger invasive and migratory activity than CD166+ cancer cells (Fujiwara et al., 2014). COL6A1 is highly expressed in bone metastases (Blanco et al., 2012). COL6A1 has been found to be upregulated in cervical and ovarian cancers (Zhao et al., 2011; Parker et al., 2009). COL6A1 is differentially expressed in astrocytomas and glioblastomas (Fujita et al., 2008).

[0090] COL6A2 has been associated with poor overall survival in cervical cancer, high-grade serous ovarian cancer, B-precursor acute lymphoblastic leukemia, hepatocellular carcinoma, primary and metastatic brain tumors, squamous cell carcinoma of the lung, and squamous cell carcinoma of the head and neck, and has been described as a potential DNA methylator in cervical cancer ( Cheon et al., 2014 ; Chen et al., 2014d ; Vachani et al., 2007 ; Liu et al., 2010 ; Seong et al., 2012 ; Hogan et al., 2011 ).

[0091] CYFIP1 has been shown to be downregulated during invasion of epithelial tumors (Silva et al., 2009), and downregulation of CYFIP1 is associated with poor prognosis in epithelial tumors (Silva et al., 2009).

[0092] CYP2S1 has been shown to regulate colorectal cancer growth in the cell line HCT116 through its association with PGE2-mediated activation of S-catenin signaling (Yang et al., 2015b). CYP2S1 has been described as upregulated in multiple epithelial-derived cancers and hypoxic tumor cells (Nishida et al., 2010; Madanayake et al., 2013). CYP2S1 depletion in a bronchial epithelial cell line has been shown to result in altered regulation of key pathways involved in cell proliferation and migration, such as the mTOR signaling pathway (Madanayake et al., 2013). CYP2S1 depletion has been shown to be associated with drug sensitivity in colorectal and breast cancer (Tan et al., 2011). CYP2S1 has been shown to correlate with survival in breast cancer and associated with poor prognosis in colorectal cancer (Murray et al., 2010; Kumarakulasingham et al., 2005). CYP2S1 has been shown to metabolize BaP-7,8-diol to the highly mutagenic and carcinogenic benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide and may therefore play an important role in benzo[a]pyrene-induced carcinogenesis (Bui et al., 2009). CYP2S1 has been shown to be significantly upregulated in ovarian cancer metastases compared to primary ovarian cancers (Downie et al., 2005).

[0093] DES expression in the stroma of colorectal cancer has been shown to correlate with advanced stage disease (Arentz et al., 2011). DES has been shown to be upregulated in colorectal cancer (Ma et al., 2009). DES has been shown to be associated with the severity and differentiation of colorectal cancer and reduced survival (Ma et al., 2009). DES has been described as a potential oncofetal serum tumor marker for colorectal cancer (Ma et al., 2009). DES has been shown to be a specific marker for rhabdomyosarcoma (Altmannsberger et al., 1985). DES has been described as one of three components of a protein panel that may be potentially useful for staging bladder cancer using immunohistochemistry (Council and Hameed, 2009).

[0094] DIS3 has been shown to be frequently mutated in multiple myeloma and recurrently mutated in acute myeloid leukemia (Ding et al., 2012; Lohr et al., 2014). DIS3-mutated multiple myeloma has been shown to be associated with shorter median overall survival. Mutations in minor subclones have been shown to be associated with a poorer response to treatment compared with patients with DIS3 mutations in major subclones (Weissbach et al., 2015). DIS3 has been shown to be upregulated by gain of 13q in colorectal cancer. Silencing of DIS3 has been shown to affect important tumorigenesis characteristics, such as viability, migration, and invasion. Therefore, DIS3 may be a novel candidate oncogene contributing to colorectal cancer progression (de Groen et al., 2014). DIS3 has been described as part of a gene panel that may be used in combination with plasma protein-based biomarkers to enable early diagnosis of epithelial ovarian cancer (Pils et al., 2013). DIS3 may be a potential candidate gene for breast cancer susceptibility, as numerous polymorphisms have been detected during mutation screening in breast cancer families (Rozenblum et al., 2002).

[0095] EEF1A1 has been shown to be upregulated in various cancer entities, including colorectal cancer, ovarian cancer, gastric cancer, prostate cancer, glioblastoma, and squamous cell carcinoma, and has been described as a potential serum biomarker for prostate cancer (Lim et al., 2011; Qi et al., 2005; Matassa et al., 2013; Vui-Kee et al., 2012; Kuramitsu et al., 2010; Kido et al., 2010; Scrideli et al., 2008; Rehman et al., 2012). Mechanistically, EEF1A1 inhibits apoptosis through interactions with p53 and p73, promotes proliferation by suppressing the transcription of the cell cycle inhibitor p21, and is involved in the regulation of epithelial-mesenchymal transition (EMT) (Blanch et al., 2013; Choi et al., 2009; Hussey et al., 2011).

[0096] EEF1A2 has been described as upregulated in breast, ovarian, lung, pancreatic, gastric, and prostate cancers, as well as TFE3-translocated renal cell carcinoma (Pflueger et al., 2013; Sun et al., 2014; Yang et al., 2015c; Zang et al., 2015; Abbas et al., 2015). EEF1A2 has been shown to be associated with poor prognosis in ovarian, gastric, pancreatic, and lung adenocarcinomas (Duanmin et al., 2013; Yang et al., 2015c; Li et al., 2006; Lee and Surh, 2009). EEF1A2 has been described as being related to carcinogenesis because it stimulates phospholipid signaling, ultimately activating Akt-dependent cell migration and actin remodeling, which are favorable for tumor development (Abbas et al., 2015). EEF1A2 has been described to inhibit p53 function in hepatocellular carcinoma (HCC) through PI3K / AKT / mTOR-dependent stabilization of MDM4. Strong activation of the EEF1A2 / PI3K / AKT / mTOR / MDM4 signaling pathway has been shown to be associated with shorter survival in HCC and may therefore be a therapeutic target in a subset of patients (Longerich, 2014). EEF1A2 has been shown to be associated with TNM stage, invasiveness, and survival in pancreatic cancer patients. Therefore, EEF1A2 may be a potential target for the treatment of pancreatic cancer (Zang et al., 2015). EEF1A2 has also been shown to be associated with prostate cancer development through promoting proliferation and inhibiting apoptosis and may therefore serve as a potential therapeutic target in prostate cancer (Sun et al., 2014). EEF1A2 has been shown to interact with the tumor suppressor protein p16, which leads to downregulation of EEF1A2 and is associated with the inhibition of cancer cell proliferation (Lee et al., 2013). EEF1A2 has been shown to be associated with nodal metastasis and perineural invasion in pancreatic ductal adenocarcinoma (Duanmin et al., 2013). EEF1A2 has been shown to be associated with survival in breast cancer (Kulkarni et al., 2007).EEF1A2 was described as a putative oncogene and tumor suppressor gene in lung adenocarcinoma cell lines and ovarian cancer ( Lee, 2003 ; Zhu et al., 2007a ).

[0097] Upon phosphorylation, EIF2S1 has been described as a promoter of tumor progression and therapeutic resistance. However, it has also been described that EIF2S1 is involved in suppressive effects on tumorigenesis (Zheng et al., 2014). EIF2S1 has been described as a downstream effector of mTOR, which reduces cancer cell viability upon hyperphosphorylation and may therefore serve as a target for drug development (Tuval-Kochen et al., 2013).

[0098] EIF4G2 was described as one of a set of core genes associated with the elimination of tumorigenesis in pediatric glioma CD133+ cells (Baxter et al., 2014). EIF4G2 was shown to be associated with the suppression of diffuse large B-cell lymphoma development upon downregulation through miR-520C-3p (Mazan-Mamczarz et al., 2014). EIF4G2 was shown to promote protein synthesis and cell proliferation by regulating the synthesis of cell cycle proteins (Lee and McCormick, 2006). EIF4G2 was downregulated in bladder tumors, and downregulation was associated with invasive tumors (Buim et al., 2005). EIF4G2 was described as involved in MycN / IFNγ-induced apoptosis and both the viability and death of neuroblastoma cells (Wittke et al., 2001).

[0099] F7 complexed with tissue factor has been described as aberrantly expressed on the surface of cancer cells, including ovarian cancer. This complex has been further described as being involved in the induction of malignant phenotypes in ovarian cancer (Koizume and Miyagi, 2015). The F7-tissue factor complex pathway has been described as a mediator of breast cancer progression, potentially stimulating the expression of multiple malignant phenotypes in breast cancer cells. Therefore, the F7-tissue factor pathway is a potentially attractive target for breast cancer therapy (Koizume and Miyagi, 2014). F7 has been shown to be regulated by the androgen receptor in breast cancer (Naderi, 2015). F7 has been shown to be involved in the regulation of autophagy through mTOR signaling in hepatocellular carcinoma cell lines (Chen et al., 2014a). F7 has been shown to be involved in tumor invasion and metastasis in colorectal and ovarian cancer (Tang et al., 2010; Koizume et al., 2006). F7 has been shown to be ectopically upregulated in colorectal cancer (Tang et al., 2009), and F7 complexed with tissue factor has been shown to be associated with chemotherapy resistance in neuroblastoma (Fang et al., 2008a).

[0100] FAM115C is upregulated during hypoxia in non-small cell lung cancer ( Leithner et al., 2014 ).

[0101] FAM83A was described as a potential biomarker for lung cancer (Li et al., 2005). It was also described as a marker gene that could be used in panels with NPY1R and KRT19 to detect circulating cancer cells in breast cancer patients (Liu et al., 2014d). Ablation of FAM83A from breast cancer cells was shown to result in reduced MAPK signaling, accompanied by significant growth suppression in vitro and tumorigenicity in vivo (Cipriano et al., 2014). Furthermore, the FAM83 protein family has been described as a novel family of oncogenes that regulate MAPK signaling in cancer and is therefore suitable for the development of cancer therapies aimed at inhibiting MAPK signaling (Cipriano et al., 2014). FAM83A was shown to be associated with trastuzumab resistance in HER2-positive breast cancer cell lines (Boyer et al., 2013). In general, FAM83A was shown to be a candidate gene associated with EGFR-tyrosine kinase inhibitor resistance in breast cancer (Lee et al., 2012). FAM83A was described as being associated with poor prognosis in breast cancer (Lee et al., 2012). FAM83A was shown to be upregulated in non-small cell lung cancer (NSCLC) (Qu et al., 2010). FAM83A was shown to be a potential specific and sensitive marker for detecting circulating tumor cells in the peripheral blood of NSCLC patients (Qu et al., 2010).

[0102] Upregulation of FAM83D affects the proliferation and invasion of hepatocellular carcinoma cells (Wang et al., 2015a; Liao et al., 2015b). FAM83D is significantly elevated in breast cancer cell lines and primary human breast cancers (Wang et al., 2013b).

[0103] FAT1 has been described as significantly mutated in squamous cell carcinoma of the head and neck, frequently mutated in cervical adenocarcinoma, bladder cancer, early T-cell precursor acute lymphoblastic leukemia, fludarabine-refractory chronic lymphocytic leukemia, glioblastoma, and colorectal cancer, and mutated in esophageal squamous cell carcinoma (Gao et al., 2014; Neumann et al., 2013; Morris et al., 2013; Messina et al., 2014; Mountzios et al., 2014; Cazier et al., 2014; Chung et al., 2015). FAT1 has been described as repressed in oral cancer and preferentially downregulated in invasive breast cancer (Katoh, 2012). FAT1 was previously described as upregulated in leukemia and associated with poor prognosis in preB acute lymphoblastic leukemia (Katoh, 2012). FAT1 was also shown to be upregulated in pancreatic adenocarcinoma and hepatocellular carcinoma (Valletta et al., 2014; Wojtalewicz et al., 2014). FAT1 was described to suppress tumor growth through activation of Hippo signaling and promote tumor migration through induction of actin polymerization (Katoh, 2012). FAT1 was shown to be a candidate cancer driver gene in cutaneous squamous cell carcinoma (Pickering et al., 2014). FAT1 was described as a tumor suppressor associated with Wnt signaling and tumorigenesis (Morris et al., 2013).

[0104] Depending on its subcellular localization, filamin A plays a dual role in cancer: in addition to functioning in various growth signaling pathways, filamin A is involved in cell migration and adhesion pathways in the cytoplasm. Therefore, its overexpression has tumor-promoting effects. In contrast to full-length filamin A, the C-terminal fragment released upon proteolysis of the protein localizes to the nucleus, where it interacts with transcription factors, thereby suppressing tumor growth and metastasis (Savoy and Ghosh, 2013).

[0105] Tumor-specific C-terminal truncations of GBP5 have been described as potentially involved in GBP5 dysregulation in lymphoma cells (Wehner and Herrmann, 2010). GBP5 has been described to have potential cancer-related functions due to the restricted expression patterns of three GBP5 splice variants in cutaneous T-cell lymphoma tumor tissues and cell lines and melanoma cell lines (Fellenberg et al., 2004).

[0106] GJB5 has been shown to be downregulated in non-small cell lung cancer cell lines, laryngeal cancer, and head and neck squamous cell carcinoma (Zhang et al., 2012; Broghammer et al., 2004; AlMoustafa et al., 2002). GJB5 has been described to act as a tumor suppressor in non-small cell lung cancer cell lines through inhibition of cell proliferation and metastasis (Zhang et al., 2012). GJB5 has been shown to be upregulated in basal serrated adenoma / polyp, a precancerous lesion that may account for 20-30% of colon cancers (Delker et al., 2014). GJB5 expression has been described as significantly altered during the promotion and progression of skin tumors in mouse models (Slaga et al., 1996).

[0107] GLS has been described as being indirectly regulated by the MYC oncogene to increase glutamine metabolism in cancer cells (Dang et al., 2009). GLS has been shown to be repressed by the tumor suppressor NDRG2 in colorectal cancer (Xu et al., 2015). GLS has also been described as being upregulated in pancreatic ductal adenocarcinoma, triple-negative breast cancer, hepatocellular carcinoma, oral squamous cell carcinoma, colorectal cancer, and malignant glial-derived tumors (van Geldermalsen et al., 2015; Szeliga et al., 2014; Huang et al., 2014a; Cetindis et al., 2015; Yu et al., 2015a; Chakrabarti et al., 2015). GLS has been shown to be associated with survival in hepatocellular carcinoma (HCC) and has also been described as a sensitive and specific biomarker for the pathological diagnosis and prognosis of HCC (Yu et al., 2015a). Loss of one copy of GLS was shown to slow tumor progression in an immunocompetent MYC-mediated mouse model of HCC (Xiang et al., 2015). GLS has been shown to be required for tumor development, and tumor-specific GLS inhibition has been described as a potential approach for cancer treatment (Xiang et al., 2015). GLS has been shown to be associated with taxol resistance in breast cancer (Fu et al., 2015a). GLS overexpression has been shown to be highly correlated with tumor stage and progression in prostate cancer patients (Pan et al., 2015). GLS expression has been shown to be associated with deeper tumor invasion and pathological patterns of tubular adenocarcinoma in colon cancer tumorigenesis. GLS may serve as a target for colorectal cancer therapy (Huang et al., 2014a). Silencing of the GLS isoenzyme KGA was shown to result in lower survival rates in the glioma cell lines SFxL and LN229 (Martin-Rufian et al., 2014).Silencing GLS in the glioma cell lines SFxL and LN229 has also been shown to induce apoptosis by inducing lower c-myc and bcl-2 expression and higher proapoptotic bid expression (Martin-Rufian et al., 2014). ErbB2 activation has been shown to upregulate GLS expression through the NF-kB pathway, which promotes breast cancer cell proliferation (Qie et al., 2014). Knockdown or inhibition of GLS in breast cancer cells with high GLS levels has been shown to result in a significant decrease in proliferation (Qie et al., 2014).

[0108] GNA15 has been shown to be upregulated in primary and metastatic small intestinal neuroendocrine neoplasms (Zanini et al., 2015). Increased expression of GNA15 has been shown to be associated with poorer survival, suggesting that GNA15 may have a pathobiological role in small intestinal neuroendocrine neoplasms and may therefore be a potential therapeutic target (Zanini et al., 2015). GNA15 has been described as downregulated in many non-small cell lung cancer cell lines (Avasarala et al., 2013). High expression of GNA15 in normal karyotype acute myeloid leukemia has been shown to be associated with significantly poorer overall survival (de Jonge et al., 2011). GNA15 has been shown to be a key downstream effector of non-canonical Wnt signaling and a regulator of non-small cell lung cancer cell proliferation and anchorage-independent cell growth. Therefore, GNA15 is a potential therapeutic target for the treatment of non-small cell lung cancer (Avasarala et al., 2013). GNA15 has been shown to be involved in tumorigenic signaling in pancreatic cancer (Giovinazzo et al., 2013). GNA15 has been shown to stimulate STAT3 through c-Src / JAK- and ERK-dependent mechanisms upon constitutive activation in human embryonic kidney 293 cells (Lo et al., 2003).

[0109] HAS3 underexpression has been shown to be associated with advanced tumor stage, nodal metastasis, vascular invasion, and poorer disease-specific and metastasis-free survival in urothelial carcinoma of the upper urinary tract and bladder (Chang et al., 2015). Therefore, HAS3 may serve as a prognostic biomarker and novel therapeutic target in urothelial carcinoma (Chang et al., 2015). HAS3 has been shown to support pancreatic cancer growth through hyaluronan accumulation (Kultti et al., 2014). HAS3 inhibition has been shown to reduce viability in the colorectal adenocarcinoma cell line SW620 (Heffler et al., 2013). HAS3 inhibition has been associated with differential expression of several genes involved in regulating SW620 colorectal tumor cell survival (Heffler et al., 2013). HAS3 has been shown to mediate colon cancer growth by inhibiting apoptosis (Teng et al., 2011). HAS3 has been shown to be upregulated in esophageal squamous cell carcinoma, adenocarcinoma of the lung and squamous cell carcinoma, and nodular basal cell carcinoma (Tzellos et al., 2011; Twarock et al., 2011; deSa et al., 2013). HAS3 expression in stromal cells of breast cancer patients was shown to correlate with high recurrence rates and short overall survival, and HAS3 has been described as an independent prognostic factor in breast cancer (Auvinen et al., 2014). HAS3 has also been shown to be associated with serous ovarian cancer, clear cell carcinoma of the kidney, endometrioid endometrial carcinoma, and osteosarcoma (Nykopp et al., 2010; Weiss et al., 2012; Cai et al., 2011; Tofuku et al., 2006).

[0110] HIF1A has been shown to be associated with tumor necrosis in invasive endometrial cancer. HIF1A was further described as a potential target for the treatment of this disease (Bredholt et al., 2015). HIF1A has been shown to be associated with liver cancer development, sarcoma metastasis, and nasopharyngeal carcinoma (Chen et al., 2014c; El-Naggar et al., 2015; Li et al., 2015b). Single-nucleotide polymorphisms in HIF1A were shown to be significantly associated with clinical outcomes in patients with invasive hepatocellular carcinoma after surgery (Guo et al., 2015). Aberrant HIF1A activity, along with aberrant STAT3 activity, was shown to drive tumor progression in malignant peripheral nerve sheath tumor cell lines. Therefore, inhibition of the STAT3 / HIF1A / VEGF-A signaling axis was described as a viable therapeutic strategy (Rad et al., 2015). HIF1A has been described as a key target for hypoxia-driven drug resistance in multiple myeloma (Maiso et al., 2015). HIF1A was asymmetrically expressed in three different cell lines corresponding to the disease stages of multiple myeloma, suggesting that HIF1A is involved in tumorigenesis and metastasis of multiple myeloma (Zhao et al., 2014b). Long noncoding HIF1A antisense RNA-2 has been described as upregulated in nonpapillary clear cell renal carcinoma and gastric cancer and is associated with tumor cell proliferation and poor prognosis in gastric cancer (Chen et al., 2015b). Deregulation of the PI3K / AKT / mTOR pathway through HIF1A has been described as important for the quiescence, maintenance, and survival of prostate cancer stem cells (Marhold et al., 2015). HIF1A was described as one gene in a four-gene classifier that is prognostic for stage I lung adenocarcinoma (Okayama et al., 2014). Polymorphisms in HIF1A were shown to be associated with increased susceptibility to gastrointestinal cancer in Asian populations (Xu et al., 2014). HIF1A was described as a prognostic marker in sporadic male breast cancer (Deb et al., 2014).

[0111] The activity of intracellular HYOU1 protein has been shown to provide a survival advantage to cancer cells during tumor progression or metastasis. Extracellular HYOU1 protein plays an important role in the generation of antitumor immune responses by promoting the delivery of tumor antigens for cross-presentation (Fu and Lee, 2006; Wang et al., 2014). HYOU1 protein has been introduced into cancer immunotherapy and has shown positive immunomodulatory effects (Yu et al., 2013; Chen et al., 2013; Yuan et al., 2012; Wang and Subjeck, 2013).

[0112] Studies have shown that IGHG1 is overexpressed in human pancreatic cancer tissues compared with adjacent non-cancerous tissues. In contrast, IGHG1 protein was downregulated in invasive ductal carcinoma tissues (Kabbage et al., 2008; Li et al., 2011). siRNA-targeted silencing of IGHG1 could inhibit cell survival and promote apoptosis (Pan et al., 2013).

[0113] Researchers have observed IGHG3 expression in Saudi Arabian women with breast cancer. Similarly, increased copy number and levels of IGHG3 have been detected in African-American men with prostate cancer. Other reports have shown that IGHG3 expression is found in squamous non-small cell lung cancer, malignant mesothelioma, and sporadically in MALT lymphoma on tumor cells that show a tendency toward plasma cell differentiation (Remmelink et al., 2005; BinAmer et al., 2008; Ledet et al., 2013; Zhang et al., 2013; Sugimoto et al., 2014).

[0114] IGHG4 encodes the immunoglobulin heavy chain constant γ4 (G4m marker) and is located on chromosome 14q32.33 (RefSeq, 2002). A recent study detected rearrangements involving IGHG4 in primary testicular diffuse large B-cell lymphoma (Twa et al., 2015).

[0115] IGHM encodes the immunoglobulin heavy chain constant mu (RefSeq, 2002). Studies have observed downregulation of IGHM in Chinese patients with rhabdomyosarcoma. Other researchers have detected IGHM expression in diffuse large B-cell lymphoma. Another group found that in diffuse large B-cell lymphoma, the IGHM gene is conserved only on productive IGH alleles in most IgM tumors. Furthermore, epithelioid angiomyolipoma samples did not show any reactivity to IGHM enhancer 3 or transcription factor binding to the transcription factor EB (Kato et al., 2009; Blenk et al., 2007; Ruminy et al., 2011; Liu et al., 2014b).

[0116] IL36RN was described as a marker that could significantly distinguish stage III lung adenocarcinoma from stages I and II ( Liang et al., 2015 ).

[0117] Decreased INA expression has been shown to be associated with metastasis, recurrence, and shorter overall survival in pancreatic neuroendocrine tumors. Therefore, INA may be a useful prognostic biomarker for pancreatic neuroendocrine tumor invasiveness (Liu et al., 2014a). INA has been described as upregulated in oligodendroglial phenotype gliomas, and INA expression has been shown to correlate with progression-free survival in oligodendrogliomas and glioblastomas (Suh et al., 2013). INA has been described as a useful neuroblastoma marker for the differential diagnosis of pediatric small round cell tumors (Willoughby et al., 2008).

[0118] ITGA6 expression is upregulated in various cancer entities, including breast, prostate, colon, and gastric cancer, and is associated with tumor progression and cell invasion (Mimori et al., 1997; Lo et al., 2012; Haraguchi et al., 2013; Rabinovitz et al., 1995; Rabinovitz and Mercurio, 1996). The proliferative effects of ITGA6 Abeta4 mutants appear to be mediated through the Wnt / β-catenin pathway (Groulx et al., 2014). ITGA6 Abeta4 mutants result in VEGF-dependent activation of the PI3K / Akt / mTOR pathway, which plays a key role in metastatic cancer cell survival (Chung et al., 2002).

[0119] KRT14 was highly expressed in various squamous cell carcinomas, including those of the esophagus, lung, larynx, and cervix, as well as in adenomatous odontogenic tumors. However, it was absent in small cell carcinoma of the bladder and weakly expressed in lung adenocarcinoma, gastric adenocarcinoma, colorectal adenocarcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, breast invasive ductal adenocarcinoma, papillary thyroid carcinoma, and uterine endometrioid adenocarcinoma (Xue et al., 2010; Terada, 2012; Vasca et al., 2014; Hammam et al., 2014; Shruthi et al., 2014). In bladder cancer, KRT14 expression was strongly associated with poor survival (Volkmer et al., 2012).

[0120] Overexpression of KRT16 has been found in basal-like breast cancer cell lines and in carcinoma in situ. Other researchers have found no significant difference in the immunohistochemical expression of KRT16 between nonrecurrent and recurrent ameloblastomas (Joosse et al., 2012; Ida-Yonemochi et al., 2012; Safadi et al., 2016). Furthermore, computational analysis showed a correlation between KRT16 expression and shorter recurrence-free survival in metastatic breast cancer (Joosse et al., 2012).

[0121] KRT5 has been shown to be upregulated in breast cancer in young women (Johnson et al., 2015). KRT5 has been shown to be associated with poor disease-free survival in young women and unfavorable clinical outcomes in premenopausal patients with hormone receptor-positive breast cancer (Johnson et al., 2015; Sato et al., 2014). KRT5 has been shown to be regulated by the tumor suppressor BRCA1 in breast cancer cell lines HCC1937 and T47D (Gorski et al., 2010). KRT5 has been shown to be deregulated in malignant pleural mesothelioma (Melaiu et al., 2015). KRT5 has been described as a diagnostic mesothelial marker for malignant mesothelioma (Arif and Husain, 2015). KRT5 has been shown to correlate with endometrial cancer progression (Zhao et al., 2013). KRT5 was shown to be mutated and downregulated in invasive tumor regions of patients with verrucous carcinoma (Schumann et al., 2012). KRT5 was shown to be part of a four-protein panel differentially expressed in colorectal cancer biopsies compared with normal tissue samples (Yang et al., 2012). KRT5 and three other proteins in the four-protein panel were described as novel markers and potential targets for the treatment of colorectal cancer (Yang et al., 2012). KRT5 was described as associated with basal cell carcinoma (Depianto et al., 2010). KRT5 was described as a candidate for identifying urothelial cancer stem cells (Hatina and Schulz, 2012).

[0122] Activation of the kynurenine pathway, involving KYNU, was shown to be significantly higher in glioblastoma, suggesting its involvement in glioma pathophysiology (Adams et al., 2014). KYNU was described as a cancer-related gene whose expression changed upon aryl hydrocarbon receptor knockdown in MDA-MB-231 breast cancer cell lines (Goode et al., 2014). KNYU was shown to be differentially expressed in highly invasive and non-invasive osteosarcoma cell lines, suggesting that it may play an important role in osteosarcoma tumorigenesis. Therefore, KYNU may also represent a potential therapeutic target (Lauvrak et al., 2013). KYNU was shown to be associated with tumorigenic re-expression in non-tumorigenic HeLa and human dermal fibroblast hybrid cells. Therefore, KYNU may provide an interesting candidate for modulating tumorigenesis (Tsujimoto et al., 1999).

[0123] Transcriptional analysis of LAMB3 in combination with two other genes has been shown to be useful in diagnosing papillary thyroid cancer and predicting the risk of lymph node metastasis (Barros-Filho et al., 2015). LAMB3 has been shown to be associated with the following cancer entities: oral squamous cell carcinoma, prostate cancer, gastric cancer, colorectal cancer, Ewing family tumors, lung cancer, breast cancer, and ovarian cancer (Volpi et al., 2011; Ii et al., 2011; Reis et al., 2013; Stull et al., 2005; Irifune et al., 2005; Tanis et al., 2014). LAMB3 has been shown to be upregulated in cervical squamous cell carcinoma, lung cancer, gastric cancer, nasopharyngeal cancer, and esophageal squamous cell carcinoma (Kwon et al., 2011; Wang et al., 2013a; Yamamoto et al., 2013; Kita et al., 2009; Fang et al., 2008b). LAMB3 has been described as a protein known to affect cell differentiation, migration, adhesion, proliferation, and survival, and it functions as an oncogene in cervical squamous cell carcinoma (Yamamoto et al., 2013). Knockdown of LAMB3 has been shown to suppress lung cancer cell invasion and metastasis in vitro and in vivo. Therefore, LAMB3 is a key gene that plays an important role in lung cancer development and metastasis (Wang et al., 2013a). LAMB3 has been shown to be regulated by the tumor suppressor miR-218 in head and neck squamous cell carcinoma (Kinoshita et al., 2012). Silencing of LAMB3 in head and neck squamous cell carcinoma has been shown to result in inhibition of cell migration and invasion (Kinoshita et al., 2012). LAMB3 expression has been shown to correlate with the depth of invasion and venous invasion in esophageal squamous cell carcinoma (Kita et al., 2009). LAMB3 methylation has been shown to correlate with several parameters of poor prognosis in bladder cancer (Sathyanarayana et al., 2004).

[0124] Inhibition of LAP3 has been shown to suppress invasion in the ovarian cancer cell line ES-2 through downregulation of fascin and MMP-2 / 9. Therefore, LAP3 may serve as a potential anti-metastatic therapeutic target (Wang et al., 2015d). High expression of LAP3 has been shown to correlate with malignancy and poor prognosis in glioma patients (He et al., 2015). LAP3 has been shown to promote glioma progression by regulating cell proliferation, migration, and invasion, and may therefore be a novel prognostic factor (He et al., 2015). Frameshift mutations in genes involved in amino acid metabolism, including LAP3, have been detected in microsatellite-high gastric and colorectal cancers (Oh et al., 2014). LAP3 has been shown to be upregulated in hepatocellular carcinoma, esophageal squamous cell carcinoma, and prostate cancer (Zhang et al., 2014; Tian et al., 2014; Lexander et al., 2005). LAP3 has been shown to promote hepatocellular carcinoma cell proliferation by regulating the G1 / S checkpoint in the cell cycle and advanced cell migration (Tian et al., 2014). LAP3 expression has further been shown to correlate with the prognosis and malignant progression of hepatocellular carcinoma (HCC) (Tian et al., 2014). Silencing of LAP3 in the esophageal squamous cell carcinoma cell line ECA109 has been shown to reduce cell proliferation and colony formation, while LAP3 knockdown resulted in cell cycle arrest (Zhang et al., 2014). Overexpression of LAP3 in the esophageal squamous cell carcinoma cell line TE1 has been shown to support cell proliferation and invasiveness (Zhang et al., 2014). Thus, LAP3 was shown to play a role in the malignant progression of esophageal squamous cell carcinoma ( Zhang et al., 2014 ).

[0125] Researchers have reported M6PR expression in colon cancer cell lines and choriocarcinoma cells (Braulke et al., 1992; O'Gorman et al., 2002). In breast cancer, low levels of M6PR expression were associated with poor patient prognosis (Esseghir et al., 2006). Furthermore, overexpression of M6PR resulted in decreased cell proliferation rates in vitro and decreased tumor growth in nude mice (O'Gorman et al., 2002).

[0126] MAPK6 has been shown to play a role in regulating cell morphology and migration in the breast cancer cell line MDA-MB-231 (Al-Mahdi et al., 2015). MAPK6 is part of the cancer-related MAPK signaling pathway and has been described as being related to BRAF and MEK1 / 2 signaling in melanoma (Lei et al., 2014; Hoeflich et al., 2006). MAPK6 has been shown to be upregulated in lung, gastric, and oral cancers (Long et al., 2012; Rai et al., 2004; Liang et al., 2005a). MAPK6 has been shown to promote lung cancer cell invasiveness by phosphorylating the oncogenic gene SRC-3. Therefore, MAPK6 may be an attractive target for therapeutic treatment of invasive lung cancer (Long et al., 2012). MAPK6 has been described as a potential target for the development of anticancer drugs against drug-resistant breast cancer cells (Yang et al., 2010). Overexpression of MAPK6 in gastric cancer has been shown to correlate with TNM stage, serosal invasion, and lymph node involvement (Liang et al., 2005a). MAPK6 has been shown to be a binding partner of the core cell cycle machinery component cyclin D3, suggesting that MAPK6 has potential activity in cell proliferation (Sun et al., 2006).

[0127] MNAT1 has been shown to be associated with poor prognosis in estrogen receptor-positive / HER2-negative breast cancer (Santarpia et al., 2013). Loss of endogenous fragmentation of MNAT1 during granulopoiesis has been shown to promote proliferation and metastasis of leukemic myeloblasts (Lou et al., 2013). MNAT1 was shown to be dysregulated in the ovarian cancer cell line OAW42 upon knockdown of the putative oncogene ADRM1 (Fejzo et al., 2013). siRNA-mediated MNAT1 gene silencing was shown to suppress cell proliferation of the pancreatic cancer cell line BxPC3 in vitro and achieved significant antitumor effects against subcutaneously implanted pancreatic tumors in vivo (Liu et al., 2007a). Genetic mutations in MNAT1 have been described to be associated with lung cancer susceptibility (Li et al., 2007). Infection of the pancreatic cancer cell line BxPC3 with a recombinant adenovirus encoding antisense MNAT1 resulted in decreased MNAT1 expression and an increased G0 / G1 cell ratio, suggesting that MNAT1 plays an important role in regulating the G1-to-S transition in the cell cycle in the pancreatic cancer cell line BxPC3 (Zhang et al., 2005). MNAT1-regulated cyclin-dependent kinase-activating kinase activity has been shown to cross-regulate neuroblastoma cell G1 arrest and is important for the switch from proliferation to differentiation in neuroblastoma cells (Zhang et al., 2004).

[0128] DNA methylation-associated silencing of NEFH in breast cancer has been shown to be frequent and cancer-specific, correlating with clinical features of disease progression (Calmon et al., 2015). NEFH has also been described to be inactivated through DNA methylation in pancreatic, gastric, and colon cancers, and thus may also contribute to the progression of these malignant lesions (Calmon et al., 2015). NEFH CpG island methylation has been shown to be associated with advanced disease, distant metastasis, and prognosis in renal cell carcinoma (Dubrowinskaja et al., 2014). Therefore, NEFH methylation may be a candidate epigenetic marker for prognosis in renal cell carcinoma (Dubrowinskaja et al., 2014). NEFH has been shown to be upregulated in extraskeletal myxoid chondrosarcoma of the vulva (Dotlic et al., 2014). Overexpression of NEFH in hepatocyte cell lines has been shown to reduce cell proliferation, while knockdown of NEFH promoted cell invasion and migration in vitro and enhanced the ability to form tumors in mice. Thus, NEFH functions as a tumor suppressor in hepatocellular carcinoma (Revill et al., 2013). NEFH has been shown to be frequently methylated in Ewing sarcoma and may therefore be involved in tumorigenesis (Alholle et al., 2013).

[0129] DNA methylation-mediated silencing of NEFL has been shown to be a frequent event in breast cancer, which may contribute to the progression of breast cancer and possibly other malignancies such as pancreatic, gastric, and colon cancer (Calmon et al., 2015). NEFL has been described as a potential tumor suppressor gene associated with cancers of several organs (Huang et al., 2014c). NEFL has been described to play a potential role in cancer cell apoptosis and invasion in head and neck squamous cell carcinoma cell lines (Huang et al., 2014c). NEFL methylation has been described as a novel mechanism conferring cisplatin chemoresistance in head and neck cancer cell lines through interaction with the mTOR signaling pathway (Chen et al., 2012). NEFL has been described as a candidate biomarker for predicting chemotherapy response and survival in patients with head and neck cancer (Chen et al., 2012). High NEFL expression has been shown to correlate with better clinical outcomes in supratentorial ependymomas (Hagel et al., 2013). NEFL is ectopically expressed in breast cancer and is reduced in primary breast cancers with lymph node metastasis compared with cancers with negative lymph nodes (Li et al., 2012). Low NEFL expression has been shown to suggest poor 5-year disease-free survival in patients with early-stage breast cancer and may therefore be a potential prognostic factor for these patients (Li et al., 2012). NEFL has also been shown to be downregulated in glioblastoma multiforme (Khalil, 2007). Allelic deletions of chromosome 8p21-23, where NEFL is located, have been described as early and frequent events in the carcinogenesis and development of lung cancer and have also been associated with breast cancer, prostate cancer, and hepatitis B virus-positive hepatocellular carcinoma ( Seitz et al., 2000 ; Becker et al., 1996 ; Haggman et al., 1997 ; Kurimoto et al., 2001 ).

[0130] NEFM has been described as a gene associated with tumor progression and processes involved in metastasis (Singh et al., 2015). NEFM has been shown to be hypomethylated and upregulated in esophageal cancer (Singh et al., 2015). NEFM has been described as a candidate tumor suppressor gene that is frequently downregulated in glioblastoma (Lee et al., 2015a). DNA methylation-associated silencing of NEFM in breast cancer has been shown to be frequent, cancer-specific, and correlated with clinical features of disease progression (Calmon et al., 2015). NEFM has also been described to be inactivated through DNA methylation in pancreatic, gastric, and colon cancers, and thus may also contribute to the progression of these malignant lesions (Calmon et al., 2015). NEFM has been shown to be associated with prostate cancer and astrocytoma (Wu et al., 2010; Penney et al., 2015). NEFM was described as a novel candidate tumor suppressor gene that was shown to be methylated in renal cell carcinoma (Ricketts et al., 2013). NEFM methylation was shown to be associated with prognosis in renal cell carcinoma (Ricketts et al., 2013). NEFM was described as a potential diagnostic marker that was shown to be differentially expressed in neuroendocrine tumor cell lines compared to non-neuroendocrine tumor cell lines (Hofsli et al., 2008).

[0131] NUP155 was described as a potential epigenetic biomarker in leukocyte DNA associated with breast cancer predisposition (Khakpour et al., 2015). NUP155 is strictly required for the proliferation and survival of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia cells and therefore constitutes a potential drug target in this disease (De et al., 2014).

[0132] OAS2 has been shown to be associated with impaired CD3-ζ chain expression through caspase-3 activation. Defects in the CD3-ζ chain have been described as frequently observed in oral cancer (Dar et al., 2015). OAS2 has been described as involved in the alternative pathway of innate immune and inflammatory pathways associated with advanced prostate cancer risk (Kazma et al., 2012). Alternative pathway analysis revealed that OAS2 is nominally associated with advanced prostate cancer risk (Kazma et al., 2012).

[0133] Lower PABPN1 expression in non-small cell lung cancer (NSCLC) has been shown to correlate with poor prognosis (Ichinose et al., 2014). Loss of PABPN1 has been described to potentially promote tumor invasiveness by freeing cancer cells from microRNA-mediated gene regulation in NSCLC (Ichinose et al., 2014). An N-terminal polyalanine expansion mutant of PABPN1 has been shown to be associated with the induction of apoptosis through the p53 pathway in HeLa and HEK-293 cell lines (Bhattacharjee et al., 2012).

[0134] PCBP1 has been described as central to the enrichment and functionality of cancer stem cells in prostate cancer cells (Chen et al., 2015a). PCBP1 has been described as an inhibitor of gastric carcinogenesis, and its downregulation is associated with a malignant phenotype in both cultured and xenografted gastric cancer cells (Zhang et al., 2015e). PCBP1 was suggested to play a role in the pathophysiology of ovarian cancer based on its differential expression between benign and malignant serum and tissue samples from patients with ovarian serous adenocarcinoma (Wegdam et al., 2014). PCBP1 was shown to be a key mediator of TGF-β-induced epithelial-mesenchymal transition (EMT), a prerequisite for tumor metastasis, in the gallbladder cancer cell line GBC-SD (Zhang and Dou, 2014). PCBP1 expression levels were shown to regulate the ability of the gallbladder cancer cell line GBC-SD to migrate and invade in vitro (Zhang and Dou, 2014). Therefore, PCBP1 may be a potential prognostic marker for gallbladder cancer metastasis (Zhang and Dou, 2014). PCBP1 downregulation has been described as potentially involved in cervical carcinogenesis (Pathak et al., 2014). PCBP1 has been described as a regulator of tumor suppression associated with the transcription factor p63 (Cho et al., 2013). High expression of PCBP1 in complete hydatidiform moles has been shown to be associated with a lower risk of progression to gestational trophoblastic neoplasia, while PCBP1 expression was significantly lower in malignant transformed moles (Shi et al., 2012). Therefore, PCBP1 has been suggested to play an important role in the pathogenesis of gestational trophoblastic neoplasia (Shi et al., 2012). Overexpression of PCBP1 was shown to result in the suppression of metastasis-associated PRL-3 protein translation and inactivation of AKT, whereas knockdown of PCBP1 was shown to cause AKT activation and promotion of tumorigenesis (Wang et al., 2010). PCBP1 was described to play a negative role in tumor invasion in the hepatocarcinoma cell line HepG2 (Zhang et al., 2010).It has been described that loss of PCBP1 in human liver tumors contributes to the formation of a metastatic phenotype ( Zhang et al., 2010 ).

[0135] PDPN has been described as upregulated in squamous cell carcinoma, mesothelioma, glioblastoma, and osteosarcoma (Fujita and Takagi, 2012). PDPN has been described as a regulator of tumor invasion and metastasis because it is associated with several pathways involved in epithelial-mesenchymal transition, collective cell migration, platelet activation, aggregation, and lymphangiogenesis (Dang et al., 2014). PDPN has been described as a marker in oral carcinogenesis and epithelioid mesothelioma (Swain et al., 2014; Ordonez, 2005). PDPN upregulation has been associated with lymph node metastasis and poor prognosis in squamous cell carcinoma of the upper aerodigestive tract (Chuang et al., 2013). PDPN has been described to be expressed in vascular tumors, malignant mesotheliomas, tumors of the central nervous system, germ cell tumors, squamous cell carcinomas, and aggressive tumors with higher invasive and metastatic potential (Raica et al., 2008). Therefore, PDPN may be considered as an attractive therapeutic target for tumor cells (Raica et al., 2008).

[0136] PHTF2 was shown to be downregulated in tongue squamous cell carcinoma ( Huang et al., 2007 ).

[0137] PKM2 has been shown to be important for cancer cell proliferation and tumor growth (Chen et al., 2014b; Li et al., 2014; DeLaBarre et al., 2014). N-myc acts as a transcriptional regulator of PKM2 in medulloblastoma (Tech et al., 2015). PKM2 appears to play a role in liver carcinogenesis, epithelial-mesenchymal transition, and angiogenesis (Nakao et al., 2014). PKM2 is one of two key factors in the Warburg effect (Tamada et al., 2012; Warner et al., 2014; Ng et al., 2015). PKM2 expression is upregulated in cancer cells (Chaneton and Gottlieb, 2012; Luo and Semenza, 2012; Wu and Le, 2013). In malignant cells, PKM2 functions in glycolysis, as a transcriptional coactivator, and as a protein kinase. In the latter function, it translocates to the nucleus and phosphorylates histone 3, ultimately triggering cell cycle progression in glioblastoma (Semenza, 2011; Luo and Semenza, 2012; Tamada et al., 2012; Venneti and Thompson, 2013; Yang and Lu, 2013; Gupta et al., 2014; Iqbal et al., 2014; Chen et al., 2014b; Warner et al., 2014). Hypoactive dimeric PKM2 may play a role in cancer instead of the active tetrameric form (Mazurek, 2011; Wong et al., 2015; Iqbal et al., 2014; Mazurek, 2007).

[0138] PKP1 has been shown to be downregulated in prostate cancer and esophageal adenocarcinoma (Kaz et al., 2012; Yang et al., 2015a). Knockdown of PKP1 in the non-neoplastic prostate BPH-1 cell line resulted in decreased apoptosis and differential expression of genes such as the prostate cancer-associated SPOCK1 gene (Yang et al., 2015a). Collectively, altered expression of PKP1 and SPOCK1 is a frequent and significant event in prostate cancer, suggesting that PKP1 has a tumor suppressor function (Yang et al., 2015a). Decreased expression of PKP1 was significantly associated with a shorter time to the development of distant metastasis in oral squamous cell carcinoma (Harris et al., 2015). Loss of PKP1 through promoter methylation has been described to be associated with the progression of Barrett's esophagus to esophageal adenocarcinoma (Kaz et al., 2012). PKP1 has been shown to be upregulated in non-small cell lung cancer and may be a good marker for identifying squamous cell carcinoma samples (Sanchez-Palencia et al., 2011). PKP1 was also shown to be upregulated in the well-differentiated liposarcoma cell line GOT3 (Persson et al., 2008). Decreased expression of PKP1 was described to promote increased motility in head and neck squamous cell carcinoma cells (Sobolik-Delmaire et al., 2007). PKP1 loss was associated with cervical carcinogenesis (Schmitt-Graeff et al., 2007). PKP1 was associated with local recurrence or metastasis and poor survival in patients with oropharyngeal squamous cell carcinoma (Papagerakis et al., 2003).

[0139] Increased PKP3 mRNA can be used as a biomarker and predictor of disease outcome (Valladares-Ayerbes et al., 2010). PKP3 overexpression correlates with poor outcomes in breast, lung, and prostate cancer, while downregulation in bladder cancer is associated with invasive behavior (Furukawa et al., 2005; Breuninger et al., 2010; Demirag et al., 2012; Takahashi et al., 2012). Loss of PKP3 leads to increased protein levels of MMP7 and PRL3, which are required for cell migration and tumorigenesis (Khapare et al., 2012; Basu et al., 2015).

[0140] Knockdown of PPP4R1 suppressed cell proliferation in the breast cancer cell line ZR-75-30 (Qi et al., 2015). Therefore, PPP4R1 may promote breast cancer cell proliferation and play an essential role in breast cancer development (Qi et al., 2015). Knockdown of PPP4R1 in the hepatocellular carcinoma cell line HepG2 resulted in reduced cell proliferation, colony formation, and G2 / M cell cycle arrest (Wu et al., 2015). Knockdown of PPP4R1 resulted in inactivation of p38 and c-Jun N-terminal kinase signaling cascades in HepG2 cells, indicating that PPP4R1 can promote cell proliferation (Wu et al., 2015). Therefore, PPP4R1 plays an important role in promoting hepatocellular carcinoma cell proliferation (Wu et al., 2015). PPP4R1 has been described as a negative regulator of inhibitors of NF-κB kinase activity in lymphocytes, and its downregulation promotes oncogenic NF-κB signaling in a subgroup of T-cell lymphomas ( Brechmann et al., 2012 ).

[0141] PRC1 has been described to be associated with radioresistance in cervical cancer, as cervical cancer tissues showed high differential expression of PRC1 after irradiation (Fu et al., 2015b). A locus in intron 14 of PRC1 has been described to be associated with breast cancer susceptibility (Cai et al., 2014). PRC1 has been described as one gene in a five-gene signature, which may be proposed as a prognostic indicator of disease-free survival in breast cancer patients (Mustacchi et al., 2013). PRC1 has been shown to be upregulated in ovarian, cervical, and bladder cancers (Espinosa et al., 2013; Ehrlichova et al., 2013; Kanehira et al., 2007). PRC1 was shown to be upregulated during 4-hydroxyestradiol-mediated malignant transformation of the breast epithelial cell line MCF-10A (Okoh et al., 2013). PRC1 has been described as a gene with important biological significance in tumor pathogenesis and can be used in gene sets to predict the prognosis of resectable patients with non-small cell lung cancer during adjuvant chemotherapy (Tang et al., 2013). PRC1 was suggested to be negatively regulated by the cell cycle-related kinase Plk1 (Hu et al., 2012). Knockdown of PRC1 in the bladder cancer cell line NIH3T3 was shown to result in a significant increase in multinucleated cells and subsequent cell death (Kanehira et al., 2007). Furthermore, PRC1 has been shown to interact with the novel cancer-testis antigen MPHOSPH1 in bladder cancer cells, suggesting that the MPHOSPH1 / PRC1 complex plays an important role in bladder carcinogenesis and may be a novel therapeutic target (Kanehira et al., 2007). PRC1 has been shown to be regulated by p53 (Li et al., 2004).

[0142] Expressed sequence tag profiling identified PRDM15 as an upregulated gene in lymphoma (Giallourakis et al., 2013).PRDM15 was described as a candidate tumor suppressor gene that may contribute to the development or progression of pancreatic cancer (Bashyam et al., 2005).

[0143] Distinct polymorphisms in PTHLH have been shown to be associated with lung cancer risk and prognosis (Manenti et al., 2000). Upregulation of PTHLH in a C57BL / 6 mouse-derived model of spontaneously metastatic breast adenocarcinoma was described as potentially involved in metastatic dissemination of breast cancer (Johnstone et al., 2015). PTHLH has also been shown to be upregulated in oral squamous cell carcinoma, chondrocyte neoplasia, adult T-cell leukemia / lymphoma, and renal clear cell carcinoma (Bellon et al., 2013; Yang et al., 2013a; Yao et al., 2014; Lv et al., 2014). PTHLH upregulation has been associated with poor pathological differentiation and poor prognosis in patients with head and neck squamous cell carcinoma (Lv et al., 2014). PTHLH has been shown to be upregulated by p38 MAPK signaling, which contributes to the extravasation of colon cancer cells in the lung through caspase-independent death in endothelial cells of the pulmonary microvasculature (Urosevic et al., 2014). PTHLH was shown to be significantly differentially expressed in squamous cell carcinoma compared with normal skin (Prasad et al., 2014). PTHLH was described as part of a four-gene signature associated with patient survival in early-stage non-small cell lung cancer (Chang et al., 2012). Disruption of the antiproliferative function of PTHLH due to a frameshift mutation was described to contribute to the development of early-stage colorectal cancer in patients with hereditary nonpolyposis colorectal cancer (Yamaguchi et al., 2006). PTHLH upregulation has been shown to be associated with poor outcomes in both overall and disease-free survival in patients with renal clear cell carcinoma who underwent nephrectomy ( Yao et al., 2014 ). PTHLH has been shown to positively regulate cell cycle progression and alter the expression of proteins involved in cell cycle regulation through the ERK1 / 2, p38, MAPK, and PI3K signaling pathways in the colorectal adenocarcinoma cell line Caco-2 ( Calvo et al., 2014 ).

[0144] RAP1GDS1 has been shown to promote the proliferation of pancreatic cancer cells (Schuld et al., 2014). Simultaneous loss of two splice variants of RAP1GDS1 in xenografts of the non-small cell lung cancer cell line NCI-H1703 in mice resulted in reduced tumor initiation (Schuld et al., 2014). RAP1GDS1 has been shown to promote cell cycle progression in multiple cancer types, making it a valuable target for cancer therapeutics (Schuld et al., 2014). RAP1GDS1 has been shown to be upregulated in breast cancer, prostate cancer, and non-small cell lung cancer (Hauser et al., 2014; Tew et al., 2008; Zhi et al., 2009). The SmgGDS-558 splice variant of RAP1GDS1 has been shown to be a unique promoter of RhoA and NF-κB activity, playing a functional role in breast cancer malignancy (Hauser et al., 2014). High RAP1GDS1 expression has been shown to be associated with poorer clinical outcomes in breast cancer (Hauser et al., 2014). RAP1GDS1 has been shown to regulate cell proliferation, migration, and NF-κB transcriptional activity in non-small cell lung cancer, thus promoting the malignant phenotype of this disease. Therefore, RAP1GDS1 is an interesting therapeutic target in non-small cell lung cancer (Tew et al., 2008). RAP1GDS1 has been shown to be fused to NUP98 in T-cell acute lymphoblastic leukemia (Romana et al., 2006).

[0145] RNPEP activity has been shown to be upregulated in colorectal adenoma, papillary thyroid carcinoma, breast cancer, and renal clear cell carcinoma ( Ramirez-Exposito et al., 2012 ; Larrinaga et al., 2013 ; Perez et al., 2015 ; Varona et al., 2007 ). RNPEP has been shown to be associated with tumor growth of rat C6 gliomas implanted in the subcutaneous region ( Mayas et al., 2012 ).

[0146] RORA has been described as a potential lung cancer oncogene (Wang et al., 2015e). RORA has been shown to be associated with the expression of the potential tumor suppressor gene OPCML in colon cancer (Li et al., 2015a). Two single nucleotide polymorphisms in RORA have been shown to be associated with breast cancer (Truong et al., 2014). RORA has been described as a potential tumor suppressor and therapeutic target for breast cancer (Du and Xu, 2012). RORA has been shown to be downregulated in colorectal adenocarcinoma and breast cancer (Kottorou et al., 2012; Du and Xu, 2012). Stable overexpression of RORA in the liver tumor cell line HepG2 has been shown to affect the expression of genes involved in glucose metabolism and hepatocarcinogenesis, suggesting a link between RORA and carcinogenesis in cells of liver origin (Chauvet et al., 2011). RORA was shown to be differentially methylated in gastric cancer compared with normal gastric mucosa (Watanabe et al., 2009). RORA was described to be associated with the regulation of cell proliferation and differentiation, and the suppression of metastatic behavior in the androgen-independent prostate cancer cell line DU145 (Moretti et al., 2002).

[0147] RPS17 has been shown to be differentially expressed in metastatic uveal melanoma and in normal whole blood and tissues susceptible to metastatic complications from uveal melanoma, suggesting that RPS17 may play a role in the tropism of uveal melanoma metastasis (Demirci et al., 2013).RPS17 has been shown to be upregulated in hepatocellular carcinoma (Liu et al., 2007b).

[0148] Knockdown of RPS26 was shown to induce p53 stabilization and activation, resulting in p53-dependent cell growth inhibition (Cui et al., 2014). RPS26 was further shown to play a role in DNA damage response by directly affecting p53 transcriptional activity (Cui et al., 2014).

[0149] S100A2 has been shown to be associated with non-small cell lung cancer and has been described as a predictive marker of poor overall survival in patients with lung squamous cell carcinoma (Hountis et al., 2014; Zhang et al., 2015d). S100A2 has been described as a downstream target of the oncogenic KRAS gene and a promoter of tumor progression in lung cancer (Woo et al., 2015). S100A2 has been described as a promising marker for predicting overall survival in pancreatic ductal adenocarcinoma (Jamieson et al., 2011). Altered expression of S100A2 by the nitrosamine N-nitrosopyrrolidone has been described as a potential reason for tumor progression in esophageal squamous cell carcinoma in black South Africans (Pillay et al., 2015). S100A2 has been shown to be upregulated in the early stages of non-small cell lung cancer, plasma from nasopharyngeal carcinoma patients, laryngeal carcinoma, gastric carcinoma, and epidermal tumors (Zhu et al., 2013a; Lin et al., 2013; Zhang et al., 2015a; Zha et al., 2015; Wang et al., 2015c). Methylation-associated inactivation of S100A2 has been shown to be frequent in head and neck and bladder cancers and may therefore be a critical event in the tumorigenesis of these diseases (Lee et al., 2015c). In oral squamous cell carcinoma, cytoplasmic expression of S100A2 has been shown to be upregulated, while nuclear expression is downregulated (Kumar et al., 2015). Cytoplasmic upregulation of S100A2 has been shown to be a potential predictor of recurrence risk in patients with oral squamous cell carcinoma (Kumar et al., 2015). S100A2 has been described to play a role in breast cancer metastasis (Naba et al., 2014). S100A2 has been shown to be a BRCA1 / p63 co-regulated tumor suppressor gene, which plays a role in regulating mutant p53 stability by regulating the binding of mutant p53 to HSP90 (Buckley et al., 2014). S100A2 has been described as a candidate tumor suppressor gene, which is downregulated in recurrent NPC and may therefore play an important role in the development of recurrent NPC (Huang et al., 2014b).S100A2 has been shown to be downregulated in gastric cancer, and its downregulation has been shown to be associated with advanced depth of invasion, lymph node metastasis, reduced recurrence-free probability, and reduced overall survival (Liu et al., 2014e). Therefore, S100A2 downregulation may be an independent negative prognostic biomarker for gastric cancer (Liu et al., 2014e). S100A2 has also been shown to negatively regulate the MEK / ERK signaling pathway in MGC-803 cancer cells (Liu et al., 2014e). Overexpression of S100A2 has been shown to induce epithelial-mesenchymal transition in A549 lung cancer cells in immunodeficient mice, followed by increased invasion, enhanced Akt phosphorylation, and increased tumor growth (Naz et al., 2014). The tumorigenicity of S100A2 has been further described as being related to its regulation of PI3 / Akt signaling and its functional interaction with the TGFβ signaling protein Smad3 (Naz et al., 2014). S100A2 expression has been shown to correlate with histological grade, lymph node metastasis, clinical stage, and poor survival in patients with perihilar and extrahepatic cholangiocarcinoma (Sato et al., 2013). Therefore, S100A2 may function as a prognostic marker in patients with cholangiocarcinoma (Sato et al., 2013).

[0150] S100A8 has been described as a key mediator in acute and chronic inflammation, interacting with myeloid-derived suppressor cells in a positive feedback loop to promote tumor development and metastasis (Zheng et al., 2015). S100A8 has been described as a potential diagnostic biomarker, prognostic indicator, and therapeutic target in non-small cell lung cancer (NSCLC) (Lim and Thomas, 2013). Overexpression of S100A8 has been shown to be associated with advanced disease stage, invasion, metastasis, and poor survival in bladder cancer (Yao et al., 2007). S100A8 has been shown to be a diagnostic marker for invasive bladder cancer (Ismail et al., 2015). S100A8 has been shown to be upregulated in anaplastic thyroid cancer, giant cell tumor of bone, and colorectal cancer (Reeb et al., 2015; Zhang et al., 2015b; Liao et al., 2015a). In vivo analysis in mice using anaplastic thyroid cancer cells with S100A8 knockdown revealed reduced tumor growth and lung metastasis, as well as significantly prolonged animal survival (Reeb et al., 2015). S100A8 has been shown to promote anaplastic thyroid cancer cell proliferation through its interaction with RAGE, which activates p38, ERK1 / 2, and JNK signaling pathways in tumor cells (Reeb et al., 2015). Therefore, S100A8 may represent a valid therapeutic target in anaplastic thyroid cancer (Reeb et al., 2015). S100A8 has been shown to be associated with high-risk chronic lymphocytic leukemia (Alsagaby et al., 2014). S100A8 has been associated with kidney cancer progression and has been described as a prospective biomarker and therapeutic target for kidney cancer (Mirza et al., 2014). S100A8 has been described as part of calprotectin, a heterodimer required for non-inflammation-driven liver tumor progression and may represent a therapeutic target for hepatocellular carcinoma ( De et al., 2015 ). S100A8 has been shown to regulate colon cancer cell cycle and proliferation by inhibiting p21 while inducing Id3 expression ( Zhang et al., 2015b ).

[0151] SERPINH1 encodes the serine proteinase inhibitor, serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (collagen-binding protein 1), which functions as a collagen-specific molecular chaperone in the endoplasmic reticulum (ER) (RefSeq, 2002). SERPINH1 is overexpressed in many human cancers, including gastric cancer, lung cancer, pancreatic ductal adenocarcinoma, glioma, and ulcerative colitis-associated cancer (Zhao et al., 2014a). SERPINH1 has also been shown to be upregulated in hepatocellular carcinoma, esophageal squamous cell carcinoma, cholangiocarcinoma, gastric cancer, lung cancer, pancreatic ductal adenocarcinoma, ulcerative colitis-associated cancer, and glioma (Zhao et al., 2014a; Padden et al., 2014; Lee et al., 2015b; Naboulsi et al., 2015). Overexpression of SERPINH1 has been shown to be associated with poor prognosis in patients with esophageal squamous cell carcinoma, and the levels of SERPINH1 immunostaining and pathological stage were significantly correlated with overall and recurrence-free survival (Lee et al., 2015b). Therefore, SERPINH1 may be a potential prognostic biomarker for esophageal squamous cell carcinoma (Lee et al., 2015b). SERPINH1 knockdown in glioma cells has been shown to inhibit glioma cell proliferation, migration, and invasion in vitro, while SERPINH1 knockdown in vivo has been shown to inhibit tumor growth and induce apoptosis (Zhao et al., 2014a). Therefore, SERPINH1 may be a therapeutic target for the treatment of glioma (Zhao et al., 2014a). SERPINH1 has been shown to be downregulated in metastases compared to primary oral squamous cell carcinoma tumors with multiple lymph node metastases, suggesting that SERPINH1 may be related to the metastatic potential of these tumors (Nikitakis et al., 2003). SLC7A11 has been shown to be downregulated in drug-resistant variants of the W1 ovarian cancer cell line and may therefore play a role in cancer cell drug resistance (Januchowski et al., 2013).SLC7A11 has been described to regulate the tumor microenvironment and confer a growth advantage to cancer (Savaskan and Eyupoglu, 2010). SLC7A11 has been reported to be involved in the neurodegenerative process in glioma, making SLC7A11 a potential prime target for cancer therapy (Savaskan et al., 2015). SLC7A11 has been shown to be suppressed by p53 in the context of ferroptosis, and the p53-SLC7A11 axis has been described as conserved in p53(3KR) mutants, contributing to its ability to suppress tumor development in the absence of classical tumor suppressor mechanisms (Jiang et al., 2015). SLC7A11 has been described as a functional subunit of system Xc, and its function is increased in aggressive breast cancer cells (Linher-Melville et al., 2015). High membrane staining of SLC7A11 in cisplatin-resistant bladder cancer has been shown to correlate with poorer clinical outcomes, and SLC7A11 inhibition has been described as a promising therapeutic approach for the treatment of this disease (Drayton et al., 2014). SLC7A11 has been shown to be differentially expressed in the human promyelocytic leukemia cell line HL-60 exposed to benzene and its metabolites, highlighting the potential link between SLC7A11 and leukemogenesis (Sarma et al., 2011). Disruption of SLC7A11 has been described to result in growth inhibition of various carcinomas, including lymphoma, glioma, prostate cancer, and breast cancer (Chen et al., 2009). Inhibition of SLC7A11 has been shown to inhibit cell invasion of the esophageal cancer cell line KYSE150 in vitro and its experimental metastasis in nude mice, thus establishing a role for SLC7A11 in tumor metastasis ( Chen et al., 2009 ).

[0152] SRPR has been shown to be amplified in cases of acute myeloid leukemia with double minute chromosomes (Crossen et al., 1999).

[0153] The human ortholog of SSR4 has been shown to be differentially expressed in the opossum melanoma cell lines TD6b and TD15L2 and upregulated in advanced tumors, suggesting the involvement of SSR4 as a candidate gene with potential functions that may be related to UV-induced melanoma development and metastasis (Wang and VandeBerg, 2004). SSR4 mRNA levels have been shown to be enriched in osteosarcoma cell lines OHS, SaOS-2, and KPDXM compared with normal osteoblasts (Olstad et al., 2003).

[0154] Dysregulated expression of STK17A has been associated with various cancer types. Its reduced expression in cervical and colorectal cancers is associated with the pro-apoptotic properties of STK17A, which are associated with tumor progression. STK17A is overexpressed in a grade-dependent manner in glioblastoma and head and neck cancer through its effects on other tumor-related pathways, such as TGF-β (Mao et al., 2013; Thomas et al., 2013; Park et al., 2015; Bandres et al., 2004). STK17A is a direct target of the tumor suppressor gene p53 and a modulator of reactive oxygen species (ROS) (Kerley-Hamilton et al., 2005; Mao et al., 2011).

[0155] SYK has been described as a modulator of tumorigenesis; in some cells, it acts as a tumor promoter by providing survival functions, and in others, it acts as a tumor suppressor by limiting epithelial-mesenchymal transition and inhibiting migration (Krisenko and Geahlen, 2015). SYK has been described as being associated with B-cell receptor (BCR) activation in B-cell lymphoma (Seda and Mraz, 2015). Inhibition of key kinases in the BCR pathway, such as SYK, has been shown to reduce chronic lymphocytic leukemia cell viability in preclinical models (Davids and Brown, 2012). SYK has been shown to be upregulated in chronic lymphocytic leukemia (Feng and Wang, 2014). SYK has been described as being associated with the pathogenesis of chronic lymphocytic leukemia and may be valuable in assessing the therapeutic efficacy and prognosis of this disease (Feng and Wang, 2014). SYK has been described as a potential tumor suppressor in breast cancer, and its absence in primary breast cancer correlates with poor outcome (Navara, 2004). SYK has been shown to play an important role in paclitaxel resistance in ovarian cancer (Yu et al., 2015b). SYK downregulation has been described as associated with the development of various cancers, including colorectal cancer (Peng et al., 2015). A distinct polymorphism in the SYK promoter has been shown to be an independent risk factor for colorectal cancer development in the Han population of southern China (Peng et al., 2015). SYK is frequently methylated in hepatocellular carcinoma, and SYK methylation has been demonstrated to identify a subset of hepatocellular carcinoma cases with poor prognosis (Shin et al., 2014).

[0156] TP63 translocations have been described as an event in a subset of anaplastic lymphoma kinase-positive anaplastic large cell lymphomas, which are associated with the aggressive course of the disease (Hapgood and Savage, 2015). TP63 has been described to play a complex role in cancer due to its involvement in epithelial differentiation, cell cycle arrest, and apoptosis (Lin et al., 2015). The TP63 isoform TAp63 has been described to be overexpressed in hematologic malignancies, while TP63 missense mutations have been reported in squamous cell carcinomas and TP63 translocations in lymphomas and some lung adenocarcinomas (Orzol et al., 2015). Aberrant splicing leading to overexpression of the TP63 isoform DeltaNp63 is frequently found in human cancers, such as squamous cell carcinoma of the skin, where it supports tumor initiation and progression (Missero and Antonini, 2014; Inoue and Fry, 2014).

[0157] TPM1 has been shown to be downregulated in renal cell carcinoma, esophageal squamous cell carcinoma cell lines, metastatic canine mammary carcinoma, and neuroblastoma cell lines (Klopfleisch et al., 2010; Yager et al., 2003; Zare et al., 2012; Wang et al., 2015b). TPM1 expression has been shown to be associated with tumor size, Fuhrman grade, and prognosis in renal cell carcinoma patients. TPM1 transfection of renal cell carcinoma cell lines OSRC-2 and 786-O was shown to enhance apoptosis while reducing migration and invasion capabilities (Wang et al., 2015b). Thus, TPM1 has been described as overexpressed in renal cell carcinoma cells, while exhibiting characteristics of a tumor suppressor gene (Wang et al., 2015b). The RAS / PI3K / AKT and RAS / MEK / ERK signaling pathways have been described to be involved in TPM1 regulation and repression in the intrahepatic cholangiocarcinoma cell line HuCCT1 and esophageal squamous cell carcinoma cell lines (Zare et al., 2012; Yang et al., 2013b). TPM1 has been described as a tumor suppressor whose overexpression in the breast cancer cell line MCF-7 inhibits anchorage-independent cell growth (Zhu et al., 2007b). Epigenetic repression of TPM1 has been described to be associated with altered TGF-β tumor suppressor function and may contribute to the metastatic properties of tumor cells (Varga et al., 2005).

[0158] Tryptase has been shown to be upregulated in certain patients with acute myeloid leukemia (Jin et al., 2014). Tryptase expression has been described to be regulated by SCF / C-Kit signaling through the ERK1 / 2 and p38 MAPK pathways (Jin et al., 2014). Mast cell tryptase has been described to be involved in colorectal cancer angiogenesis and has been shown to be more highly expressed in the serum of colorectal cancer patients before radical surgical resection than after resection (Ammendola et al., 2014).

[0159] TSHZ3 was shown to be downregulated in the oral squamous cell carcinoma cell line SCC-9 compared with the non-tumorigenic cell line OKF6-TERT1R (Marcinkiewicz and Gudas, 2014).TSHZ3 was described as a transcriptional regulator gene found to be recurrently rearranged in some cases of high-grade serous ovarian cancer (McBride et al., 2012).TSHZ3 was described as a candidate tumor suppressor gene with downregulated expression in breast and prostate cancer (Yamamoto et al., 2011).

[0160] TSPAN10 has been shown to be a differentially expressed gene between metastatic melanoma and normal skin samples, and it may be a potential biomarker for metastatic melanoma treatment (Liu et al., 2014c). Among several genes, TSPAN10 in particular has been shown to be upregulated in uterine leiomyosarcoma metastases compared to primary leiomyosarcoma, thus contributing to the differentiation of these conditions and aiding in the understanding of tumor progression in this cancer (Davidson et al., 2014).

[0161] TTPAL was described as a candidate oncogene that exhibits mutations in microsatellite unstable colorectal cancer ( Tuupanen et al., 2014 ).

[0162] TUBGCP2 was shown to be upregulated in taxol-resistant ovarian cancer cell lines and was described to be associated with the sensitization of the non-small cell lung cancer cell line NCI-H1155 to taxol (Huang and Chao, 2015). TUBGCP2 was shown to be upregulated in glioblastoma, where its overexpression antagonizes the inhibitory effect of CDK5 regulatory subunit-associated tumor suppressor protein 3 on DNA damage G2 / M checkpoint activity (Draberova et al., 2015).

[0163] VIM has been described as a downstream target of STAT3, which is associated with breast cancer progression upon STAT3 deregulation (Banerjee and Resat, 2015). VIM has been described as a potential nasopharyngeal carcinoma-associated protein (Chen et al., 2015c). Negative vimentin methylation status has been shown to predict improved prognosis in pancreatic cancer patients (Zhou et al., 2014). VIM has been shown to be upregulated through C6orf106 in non-small cell lung cancer and subsequently associated with enhanced cancer cell invasion (Zhang et al., 2015c). VIM has been described as an independent predictor of overall survival in patients with squamous cell lung cancer (Che et al., 2015). VIM has been described as a biomarker that can potentially distinguish melanoma subtypes and may predict melanoma aggressiveness in different subgroups of melanoma (Qendro et al., 2014). VIM was shown to be upregulated in renal clear cell carcinoma (Shi et al., 2015). High VIM expression was described as an independent prognostic indicator of renal clear cell carcinoma (Shi et al., 2015). VIM was shown to function as a scaffold, recruiting Slug to ERK and promoting Slug phosphorylation, which was described as a requirement for the initiation of epithelial-mesenchymal transition (EMT), a developmental process adopted during tumorigenesis (Virtakoivu et al., 2015).

[0164] WDR1 has been shown to be upregulated in interstitial fluid from ovarian cancer and in high-grade canine cutaneous mast cell tumors with poor prognosis compared with low-grade mast cell tumors with favorable prognosis (Schlieben et al., 2012; Haslene-Hox et al., 2013). WDR1 has been shown to be downregulated in chemotherapy-resistant advanced serous epithelial ovarian cancer (Kim et al., 2011). WDR1 downregulation in chemotherapy-resistant advanced serous epithelial ovarian cancer has been shown to correlate with poor overall survival (Kim et al., 2011). WDR1 has been shown to be upregulated in breast cancer at the region between the invasive tumor front and normal tissue (interface region) and may therefore be related to breast cancer progression and metastasis (Kang et al., 2010).

[0165] Fusion of YWHAE with NUTM2B / NUTM2E was described as an event observed in a small number of clear cell sarcomas of the kidney (Karlsson et al., 2015). YWHAE-NUTM2 fusion was described as a frequent event in high-grade endometrial stromal sarcoma (Ali et al., 2014). High-grade endometrial stromal sarcoma with YWHAE-NUTM2 fusion was described as a subset of endometrial stromal sarcoma with aggressive clinical behavior and poor prognosis (Kruse et al., 2014). Breaks in three loci, including YWHAE, were described as potentially contributing to the development of uterine angiosarcoma (Suzuki et al., 2014). YWHAE has been shown to be downregulated in gastric cancer, and reduced YWHAE levels are associated with diffuse gastric cancer and the early onset of this pathology, suggesting that YWHAE may play a role in gastric carcinogenesis (Leal et al., 2012). YWHAE has been shown to be differentially expressed in tissues from breast cancer patients, regardless of recurrence, and has been shown to be associated with both disease-free survival and overall survival (Cimino et al., 2008). Therefore, YWHAE may be used as an independent prognostic marker and potential drug target for breast cancer (Cimino et al., 2008).

[0166] Alterations in ZNF292 have been described as a driver of chronic lymphocytic leukemia (Puente et al., 2015). ZNF292 has been described as a tumor suppressor gene in colorectal cancer (Takeda et al., 2015). ZNF292 has been described as an immunogenic antigen with clinical relevance in head and neck squamous cell carcinoma (Heubeck et al., 2013). DETAILED DESCRIPTION OF THE INVENTION

[0167] Stimulation of an immune response depends on the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor-associated antigens has raised the possibility of using the host's immune system to intervene in tumor growth. Various mechanisms that utilize both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

[0168] Certain elements of the cellular immune response can specifically recognize and destroy tumor cells. Isolation of T cells from tumor-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in the innate immune defense against cancer. In particular, CD8+ T cells play a key role in this response, recognizing class I molecules of the major histocompatibility complex (MHC) bearing peptides, usually 8–10 amino acid residues long, derived from proteins or defective ribosomal products (DRIPS) located in the cytosol. In humans, MHC molecules are also called human leukocyte antigens (HLA).

[0169] As used herein, unless otherwise specified, all terms are defined as set forth below.

[0170] The term "T cell response" refers to the specific proliferation and activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, the effector functions may be lysis of peptide-pulsed, peptide precursor-pulsed, or native peptide-presenting target cells; secretion of cytokines, preferably interferon-γ, TNF-α, or IL-2, induced by the peptide; secretion of effector molecules, preferably granzymes or perforins, induced by the peptide; or degranulation.

[0171] The term "peptide" is used herein to designate a series of amino acid residues that are typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. Peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids in length, 10, 11, 12, 13, or 14 or more amino acids in length, and in the case of MHC class II peptides (extended variants of the peptides of the invention), they can be 15, 16, 17, 18, 19, or 20 or more amino acids in length.

[0172] Furthermore, the term "peptide" is intended to include salts of a series of amino acid residues that are typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, such as a chloride salt or acetate (trifluoroacetate). It should be noted that since peptides are not salts in vivo, the salts of the peptides according to the present invention have substantially different in vivo states from peptides.

[0173] The term "peptide" is also intended to include "oligopeptides." The term "oligopeptide" is used herein to designate a series of amino acid residues typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, provided the correct epitope or epitopes are retained therein. Oligopeptides are typically less than about 30 amino acid residues in length and more than about 15 amino acids in length.

[0174] The term "polypeptide" refers to a series of amino acid residues typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, provided the correct epitope is retained. In contrast to the terms peptide or oligopeptide, the term polypeptide is intended to refer to a molecule containing more than about 30 amino acid residues.

[0175] A peptide, oligopeptide, protein, or polynucleotide encoding such a molecule is "immunogenic" if it is capable of inducing an immune response (and thus is an "immunogen" in the context of the present invention). For the purposes of the present invention, immunogenicity is more specifically defined as the ability to induce a T-cell response. An "immunogen" is therefore a molecule capable of inducing an immune response, and in the context of the present invention, a molecule capable of inducing a T-cell response. In another embodiment, the immunogen can be a peptide, a peptide-MHC complex, an oligopeptide, and / or a protein used to raise specific antibodies or TCRs thereagainst.

[0176] A class I T cell "epitope" requires a short peptide bound to a class I MHC receptor, forming a ternary complex (MHC class I α chain, β-2-microglobulin, and peptide) that can be recognized by a T cell bearing a compatible T cell receptor that binds the MHC / peptide complex with appropriate affinity. Peptides that bind to MHC class I molecules are typically 8-14 amino acids in length, most typically 9 amino acids in length.

[0177] In humans, there are three different genetic loci that encode MHC class I molecules (human MHC molecules also called human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different MHC class I alleles that can be expressed from these loci.

[0178] Table 5: Frequency F of HLA-A*5 and HLA-A*24 and the most frequent HLA-DR serotypes. Frequencies are estimated from haplotype frequencies Gf in the US population, adapted from Mori et al. (1997), using the Hardy-Weinberg formula, F = 1-(1-Gf)². Due to linkage disequilibrium, the combination of A*02 or A*24 with a particular HLA-DR allele may be more abundant or less frequent than predicted from their single frequencies. For more details, see Chanock et al. (2004). [Table 5-1] [Table 5-2]

[0179] The peptides of the present invention preferably bind to A*02 when included in the vaccines of the present invention described herein. The vaccines may also contain pan-binding MHC class II peptides. Thus, the vaccines of the present invention can be used to treat cancer in A*02-positive patients, while the pan-binding properties of these peptides do not require the selection of MHC class II allotypes.

[0180] When the A*02 peptide of the present invention is combined with a peptide that binds to another allele, such as A*24, it can treat a higher proportion of any patient population compared to treating any MHC class I allele alone.While in the majority of populations, any one allele alone can treat less than 50% of patients, a vaccine comprising HLA-A*24 and HLA-A*02 epitopes can treat at least 60% of patients in any reasonable population.Specifically, in various regions, the following percentages of patients are positive for at least one of these alleles: 61% in the United States, 62% in Western Europe, 75% in China, 77% in South Korea, and 86% in Japan (calculated from www.allelefrequencies.net).

[0181] In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

[0182] Nucleotide sequences encoding particular peptides, oligopeptides, or polypeptides may be of natural origin, or they may be synthetically constructed. Generally, DNA fragments encoding the peptides, polypeptides, and proteins of the invention are assembled from cDNA fragments and short oligonucleotide linkers, or from stretches of oligonucleotides, to provide synthetic genes that can be expressed in recombinant transcription units comprising regulatory elements derived from microbial or viral operons.

[0183] As used herein, the term "nucleotide encoding a peptide" refers to a nucleotide sequence that encodes a peptide containing artificial start and stop codons that are compatible with the biological system in which the sequence is expressed, for example, by a dendritic cell or another cell line useful for producing a TCR.

[0184] As used herein, a reference to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acids. Thus, for example, a specific sequence refers to the single-stranded DNA of such a sequence, the duplex of such a sequence and its complement (double-stranded DNA), and the complement of such a sequence, unless the context clearly indicates otherwise.

[0185] The term "coding region" refers to that portion of a gene that naturally or normally encodes an expression product of the gene in its natural genomic environment, i.e., the region that encodes the natural expression product of the gene in vivo.

[0186] The coding region may be derived from a non-mutated ("normal"), mutated or modified gene, or may even be derived from a DNA sequence or gene that is entirely synthesized in the laboratory using methods well known to those skilled in the art of DNA synthesis.

[0187] The term "expression product" refers to a polypeptide or protein that is the natural translation product of a gene and of any nucleic acid sequence that encodes an equivalent due to the degeneracy of the genetic code and therefore encodes the same amino acid.

[0188] The term "fragment," when referring to a coding sequence, means a portion of DNA comprising less than the entire coding region, the expression product of which retains essentially the same biological function or activity as the expression product of the entire coding region.

[0189] The term "DNA fragment" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, that is derived from DNA that has been isolated at least once in a substantially pure form, i.e., free from contaminating endogenous material, in an amount or concentration that allows the fragment and its constituent nucleotide sequences to be identified, manipulated, and recovered by standard biochemical methods, for example, using a cloning vector. Such fragments are provided in the form of an open reading frame uninterrupted by internal untranslated sequences, or introns, typically present in eukaryotic genes. Non-translated DNA sequences may be present downstream of the open reading frame, where they do not interfere with the manipulation or expression of the coding region.

[0190] The term "primer" means a short nucleic acid sequence that can pair with a single strand of DNA and provide a free 3'-OH end at which DNA polymerase initiates deoxyribonucleotide chain synthesis.

[0191] The term "promoter" refers to a region of DNA involved in RNA polymerase binding to initiate transcription.

[0192] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is of natural origin). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but is still isolated in the sense that such a vector or composition is not part of its natural environment.

[0193] The polynucleotides and recombinant or immunogenic polypeptides disclosed by the present invention may be in "purified" form. The term "purified" does not require complete purity; rather, it is intended as a relative definition and can include highly purified or only partially purified preparations, as these terms are understood by those of skill in the art. For example, individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. Purification of the starting material or natural substance to at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude, is expressly contemplated. Furthermore, claimed polypeptides having a purity of preferably 99.999%, or at least 99.99% or 99.9%, by weight; even more desirably 99% or greater, are expressly encompassed.

[0194] The nucleic acid and polypeptide expression products disclosed by the present invention, as well as expression vectors containing such nucleic acids and / or such polypeptides, may be in "enriched form." As used herein, the term "enriched" refers to a concentration of a substance that is (for example) at least about 2, 5, 10, 100, or 1000 times its natural concentration, advantageously 0.01% by weight, preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The sequences, constructs, vectors, clones, and other substances comprising the present invention may advantageously be in enriched or isolated form. The term "active fragment" generally refers to a fragment of a peptide, polypeptide, or nucleic acid sequence that generates an immune response (i.e., has immunogenicity) when administered alone, optionally with a suitable adjuvant, or in a vector, to an animal, such as a mammal, including, for example, a rabbit or mouse, and also humans, where such immune response takes the form of stimulating a T-cell response in the recipient animal, such as a human. Alternatively, the "active fragments" may also be used to induce T cell responses in vitro.

[0195] As used herein, when used in reference to a polypeptide, the terms "portion," "segment," and "fragment" refer to a sequence of consecutive residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide is treated with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptide resulting from such treatment will correspond to a portion, segment, or fragment of the starting polypeptide. When used in reference to a polynucleotide, these terms refer to the product resulting from treatment of said polynucleotide with any endonuclease.

[0196] According to the present invention, when referring to a sequence, the term "percent identity" or "percent identical" means that the sequence is compared to the claimed or described sequence ("reference sequence") after alignment of the sequence being compared ("comparison sequence") with the described or claimed sequence. The percent identity is then determined according to the following formula: Percent identity=100[1-(C / R)] where C is the number of differences between the reference sequence and the compared sequence over the alignment length between the reference sequence and the compared sequence; (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence; and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence that differs from an aligned base or amino acid in the comparison sequence constitutes a difference; (iiii) the alignment must start at position 1 of the aligned sequence; R is the number of bases or amino acids in the reference sequence over the alignment length with the comparison sequence, and any gaps that occur in the reference sequence are also counted as bases or amino acids.

[0197] If there is an alignment between a comparison sequence and a reference sequence for which the percent identity is calculated as above that is about or exceeds a particular minimum percent identity, then the comparison sequence has a particular minimum percent identity with the reference sequence, even if there is an alignment therein whose percent identity, as calculated as above, is less than the particular percent identity.

[0198] Thus, as noted above, the present invention provides a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that cross-reacts T cells with said peptide. The peptides of the present invention have the ability to bind human major histocompatibility complex (MHC) class I molecules or extended versions of said peptides to class II.

[0199] In the present invention, the term "homologous" refers to the degree of identity between two amino acid sequences, i.e., peptide or polypeptide sequences (see percentage identity above). The aforementioned "homology" is determined by comparing two sequences aligned under optimal conditions across the sequences being compared. Such sequence homology can be calculated, for example, by creating an alignment using the ClustalW algorithm. Publicly available sequence analysis software, more specifically Vector NTI, GENETYX, or other tools, are provided by public databases.

[0200] One skilled in the art would be able to assess whether T cells induced by variants of a particular peptide are capable of cross-reacting with the peptide itself (Appay et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997).

[0201] By "variant" of a given amino acid sequence, we mean that, for example, the side chains of one or two amino acid residues are altered (e.g., by replacing them with the side chain of another naturally occurring amino acid residue, or with other side chains) so that the peptide can still bind to an HLA molecule substantially similarly to a peptide consisting of the given amino acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 93. For example, the peptide may be modified to at least maintain, if not improve, its ability to interact with and bind to the binding groove of an appropriate MHC molecule, such as HLA-A*02 or -DR, and thus it at least maintains, if not improves, its ability to bind to the TCR of an activated CTL.

[0202] These T cells can subsequently cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptides defined in embodiments of the present invention. As can be deduced from academic literature and databases (Rammensee et al., 1999; Godkin et al., 1997), specific positions of HLA-binding peptides are typically anchor residues, forming core sequences that fit the binding motif of HLA receptors, defined by the polar, electrophysical, hydrophobic, and spatial properties of the polypeptide chain that constitutes the binding groove. Thus, one skilled in the art could modify the amino acid sequences set forth in SEQ ID NOs: 1-93 by retaining the known anchor residues and determine whether such variants maintain the ability to bind to MHC class I or II molecules. The variants of the present invention maintain the ability to bind to the TCR of activated T cells, which can subsequently cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptides defined in embodiments of the present invention.

[0203] The original (unmodified) peptides disclosed herein may be modified by substitution of one or more residues at different, possibly selective, positions within the peptide chain, unless otherwise specified. Preferably, these substitutions are located at the ends of the amino acid chain. Such substitutions may be conservative in nature, e.g., an amino acid is replaced by an amino acid with a similar structure and characteristics, such as a hydrophobic amino acid being replaced by another hydrophobic amino acid. Even more conservative substitutions are those with amino acids of identical or similar size and chemical properties, such as the replacement of leucine with isoleucine. In studies of sequence diversity in naturally occurring homologous protein families, certain amino acid substitutions are often more tolerated than others, and these often correlate with the similarity in size, charge, polarity, and hydrophobicity between the original amino acid and its substitute, which is the basis for the definition of a "conservative substitution."

[0204] Conservative substitutions are defined herein as exchanges within one of the following five groups: Group 1 - small aliphatic, non-polar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2 - polar negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3 - polar positively charged residues (His, Arg, Lys); Group 4 - large aliphatic non-polar residues (Met, Leu, Ile, Val, Cys); and Group 5 - large aromatic residues (Phe, Tyr, Trp).

[0205] A less conservative substitution might involve the substitution of another amino acid with similar characteristics but somewhat different in size, such as the substitution of an alanine with an isoleucine residue. A highly non-conservative substitution might involve the substitution of a polar amino acid for another, or a basic amino acid for an acidic amino acid. However, such "radical" substitutions cannot be dismissed as potentially ineffective, because chemical effects are not completely predictable and radical substitutions can produce serendipitous effects that cannot be predicted from simple chemical principles.

[0206] Of course, such substitutions may involve structures other than conventional L-amino acids. Thus, D-amino acids may be substituted for L-amino acids normally found in the antigenic peptides of the invention and still be encompassed by the disclosure herein. Furthermore, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the invention.

[0207] If substitutions at two or more positions are found to result in a peptide with substantially equal or greater antigenic activity, as defined below, combinations of these substitutions are tested to determine whether the combination of substitutions results in an additive or synergistic effect on the antigenicity of the peptide. At most, no more than four positions within a peptide are substituted simultaneously.

[0208] Peptides consisting essentially of amino acid sequences as set forth herein may have one or two non-anchor amino acids (see below for anchor motifs) exchanged without substantially altering or adversely affecting their ability to bind to human major histocompatibility complex (MHC) class I or II molecules, compared to the unmodified peptide. In another embodiment, in peptides consisting essentially of amino acid sequences as set forth herein, one or two amino acids may be exchanged with their conservative exchange partners (see below), without substantially altering or adversely affecting their ability to bind to human major histocompatibility complex (MHC) class I or II molecules, compared to the unmodified peptide. In another embodiment, in peptides consisting essentially of amino acid sequences as set forth herein, one or two amino acids may be exchanged with their conservative exchange partners (see below), without substantially altering or adversely affecting their ability to bind to human major histocompatibility complex (MHC) class I or II molecules, compared to the unmodified peptide.

[0209] Amino acid residues that do not substantially contribute to interaction with the T cell receptor may be modified by substitution with other amino acids whose incorporation does not substantially affect T cell reactivity or eliminate binding to the relevant MHC. Thus, except for the provisos given, the peptides of the invention may be any peptide (we include by that term oligopeptide or polypeptide) comprising the amino acid sequence as given or a portion or variant thereof.

[0210] Table 6: Variants and motifs of peptides set forth in SEQ ID NOs: 4, 9, and 18 [Table 6-1] [Table 6-2]

[0211] Longer (extended) peptides may also be suitable. MHC class I epitopes are typically 8-11 amino acids in length, but can be generated by peptide processing from longer peptides or proteins containing the actual epitope. Preferably, the residues flanking the actual epitope are those that do not substantially affect the proteolytic cleavage required to expose the actual epitope during processing.

[0212] The peptides of the present invention can be extended by up to four amino acids, i.e., 1, 2, 3 or 4 amino acids can be added to either end in any combination between 4:0 and 0:4. Extension combinations according to the present invention are listed in Table 7.

[0213] Table 7: Extension combinations of peptides of the invention [Table 7]

[0214] The amino acids for extension / elongation can be the peptide of the original protein sequence or any other amino acid. Extension can be used to increase the stability or solubility of the peptide.

[0215] Thus, epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes, or may include epitopes that differ from the reference peptide by no more than four residues, so long as they have substantially the same antigenic activity.

[0216] In alternative embodiments, the peptide is extended on one or both sides by more than four amino acids, preferably up to a total length of 30 amino acids. This may result in an MHC class II binding peptide. MHC class II binding can be tested by methods known in the art.

[0217] Thus, the present invention provides peptides and variants of MHC class I epitopes, wherein the peptides or variants have an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14, i.e., 8, 9, 10, 11, 12, 13, 14 amino acids, and for extended class II binding peptides, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

[0218] Of course, the peptides or variants according to the invention have the ability to bind to molecules of the human major histocompatibility complex (MHC) class I or II. The binding of peptides or variants to MHC complexes may be tested by methods known in the art.

[0219] Preferably, when T cells specific for a peptide according to the invention are tested with a substituted peptide, the peptide concentration at which the substituted peptide achieves half-maximal increase in lysis over background is about 1 mM or less, preferably about 1 μM or less, more preferably about 1 nM or less, even more preferably about 100 pM or less, and most preferably about 10 pM or less. It is also preferred that the substituted peptide be recognized by T cells from more than one individual, at least two, and more preferably three individuals.

[0220] In a particularly preferred embodiment of the invention, the peptide consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 93.

[0221] "Consisting essentially of" is intended to mean that the peptide according to the present invention contains, in addition to the sequence set forth in any of SEQ ID NOs: 1 to 93 or a variant thereof, an additional N- and / or C-terminally positioned sequence of amino acids that do not necessarily constitute part of the peptide that functions as an epitope for an MHC molecule epitope.

[0222] Nevertheless, these sequences may be important for providing efficient introduction of the peptides according to the invention into cells. In one embodiment of the invention, the peptide is part of a fusion protein comprising the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33, hereinafter "Ii"), e.g., from NCBI, GenBank accession number X00497. In other fusions, the peptides of the invention may be fused to antibodies, or functional parts thereof, as described herein, in particular to antibody sequences, so as to be specifically targeted by said antibodies, or may be fused to or within, for example, antibodies specific for dendritic cells, as described herein.

[0223] Furthermore, the peptides or variants may be further modified to improve stability and / or binding to MHC molecules in order to elicit a stronger immune response. Methods for such optimization of peptide sequences are well known in the art and include, for example, the introduction of reverse peptide or non-peptide bonds.

[0224] In reverse peptide bond, amino acid residues are not linked by peptide (-CO-NH-) bond, but peptide bond is reversed. Such retro-inverso peptidomimetics can be produced using methods known in the art, such as those described in Meziere et al. (1997) (Meziere et al., 1997), which is incorporated herein by reference. This approach involves the generation of pseudopeptides that contain changes that involve the main chain rather than the direction of the side chain. Meziere et al. (Meziere et al., 1997) demonstrate that these pseudopeptides are useful for MHC binding and T helper cell response. Retro-inverse peptides that contain NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis.

[0225] Examples of non-peptide bonds are -CH-NH, -CHS-, -CHCH-, -CH=CH-, -COCH-, -CH(OH)CH-, and -CHSO-. U.S. Pat. No. 4,897,445 provides a method for solid-phase synthesis of non-peptide bonds (-CH-NH) in polypeptide chains, involving polypeptides synthesized by standard procedures and non-peptide bonds synthesized by reacting amino acids with amino aldehydes in the presence of NaCNBH.

[0226] Peptides comprising the above sequences may be synthesized with additional chemical groups at their amino and / or carboxy termini to enhance the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the amino termini of the peptides. Similarly, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the amino terminus of the peptides. Furthermore, hydrophobic groups, t-butyloxycarbonyl, or amide groups may be added to the carboxy terminus of the peptides.

[0227] Furthermore, the peptides of the present invention may be synthesized to alter their configuration. For example, the D-isomer of one or more amino acid residues of the peptide may be used instead of the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the present invention may be substituted with one of the known non-naturally occurring amino acid residues. Changes such as these may serve to increase the stability, bioavailability, and / or binding activity of the peptides of the present invention.

[0228] Similarly, the peptides or variants of the present invention may be chemically modified by reacting specific amino acids either before or after peptide synthesis. Examples of such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which is incorporated herein by reference. Chemical modifications of amino acids include, but are not limited to, acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide and sulfhydryl modification of carboxyl groups by oxidation of cysteine ​​to cysteic acid with performic acid, mercury derivatization, mixed disulfide formation with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanate at alkaline pH. In this regard, those skilled in the art are referred to Chapter 15 of *Current Protocols in Protein Science*, Eds. Coligan et al. (John Wiley and Sons, NY 1995-2000) (Coligan et al., 1995) for more detailed procedures regarding the chemical modification of proteins.

[0229] Briefly, for example, modification of arginyl residues in proteins is often based on the reaction of vicinal dicarbonyl compounds, such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione, to form adducts. Another example is the reaction of arginine residues with methylglyoxal. Cysteines can be modified without the simultaneous modification of other nucleophilic sites, such as lysines and histidines. Consequently, numerous reagents are available for cysteine ​​modification. Company websites, such as Sigma-Aldrich (http: / / www.sigma-aldrich.com), provide information on specific reagents.

[0230] Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during heat treatment of biopharmaceuticals. Specific glutamic acid residues can be modified using Woodward's reagent K. N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide can be used to form intramolecular crosslinks between lysine and glutamic acid residues. For example, diethylpyrocarbonate is a reagent for modifying histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. Reactants of lysine residues and other α-amino groups are useful, for example, in binding peptides to surfaces or protein / peptide crosslinking. Lysine is the attachment site for poly(ethylene)glycol and is the primary modification site for protein glycosylation. Methionine residues in proteins can be modified, for example, with iodoacetamide, bromoethylamine, and chloramine T.

[0231] Tyrosyl residues can be modified using tetranitromethane and N-acetylimidazole. Cross-linking through the formation of dityrosine can be achieved with hydrogen peroxide / copper ions.

[0232] Recent studies on the modification of tryptophan have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

[0233] Successful modification of therapeutic proteins and peptides with PEG is often associated with increased circulatory half-life, while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used for hydrogel preparation. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

[0234] Peptides or variants in which the peptide is modified or contains non-peptide bonds are preferred embodiments of the present invention. Generally, peptides and variants (at least those containing peptide bonds between amino acid residues) may be synthesized by the Fmoc-polyamide solid-phase peptide synthesis method disclosed by Lukas et al. (Lukas et al., 1981) and the references cited therein. Temporary N-amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is carried out using 20% ​​piperidine in N,N-dimethylformamide. Side chain functional groups may be protected as their butyl ethers (for serine, threonine, and tyrosine), butyl esters (for glutamic acid and aspartic acid), butyloxycarbonyl derivatives (for lysine and histidine), trityl derivatives (for cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (for arginine). When glutamine or asparagine is the C-terminal residue, a 4,4'-dimethoxybenzhydryl group is utilized to protect the side-chain amide functionality. The solid-phase support is based on a polydimethyl-acrylamide polymer composed of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and acryloylsarcosine methyl ester (functionalizer). The peptide-to-resin cleavable linker used is an acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives, except for asparagine and glutamine, which are added using a reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isatin test procedures. Upon completion of synthesis, the peptide is cleaved from the resin support and the side chain protecting groups are simultaneously removed by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture.Commonly used scavengers include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the constituent amino acids of the peptide being synthesized. A combination of solid-phase and solution-phase methods for the synthesis of peptides is also possible (see, for example, (Bruckdorfer et al., 2004) and references cited therein).

[0235] Trifluoroacetic acid is removed by evaporation under vacuum, followed by trituration with diethyl ether to yield the crude peptide. Any scavengers present are removed by a simple extraction procedure, which, upon lyophilization of the aqueous phase, gives the scavenger-free crude peptide. Reagents for peptide synthesis are commonly available, for example, from Calbiochem-Novabiochem (Nottingham, UK).

[0236] Purification may be achieved by any one or combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reversed-phase high performance liquid chromatography using, for example, an acetonitrile / water gradient separation.

[0237] Analysis of peptides may be carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, by fast atom bombardment (FAB) mass spectrometry, and by MALDI and ESI-Q-TOF mass spectrometry.

[0238] To select over-represented peptides, a representation profile is calculated, which shows the median sample representation and the variation of repeated tests. The profile juxtaposes the target tumor entity sample to the baseline of normal samples. Then, each of these profiles can be combined into an over-representation score by calculating the p-value of a linear mixed-effects model (Pinheiro et al., 2015) and correcting for multiple tests by false discovery rate (Benjamini and Hochberg, 1995) (see Example 1).

[0239] For identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock-frozen samples were purified and HLA-associated peptides were isolated. The isolated peptides were separated and sequenced by online nanoelectrospray ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequences were confirmed by comparing the fragmentation patterns of natural tumor-associated peptides (TUMAPs) recorded from esophageal cancer samples (N = 16 A*02 positive samples) with those of corresponding synthetic standard peptides of the same sequence. Because the peptides were directly identified as ligands of HLA molecules in primary tumors, these results provide direct evidence of the native processing and presentation of the identified peptides on primary cancer tissues obtained from 16 esophageal cancer patients.

[0240] The discovery pipeline XPRESIDENT® v2.1 (see, e.g., U.S. Patent No. 2013-0096016, the entire contents of which are incorporated herein by reference) enables the identification and selection of plausible over-presented peptide vaccine candidates based on direct relative quantification of HLA-restricted peptide levels on cancer tissues compared to several different non-cancerous tissues and organs. This was achieved through the development of label-free differential quantification that combines algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, charge state deconvolution, and normalization using acquired LC-MS data processed through a proprietary data analysis pipeline.

[0241] Presentation levels were established, including error estimates for each peptide and sample. Peptides that are exclusively presented on tumor tissues and peptides that are over-presented in tumors compared to non-cancerous tissues and organs were identified.

[0242] HLA-peptide complexes from esophageal cancer tissue samples were purified to isolate HLA-binding peptides and analyzed by LC-MS (see Examples). All TUMAPs included in this application were identified using this approach on primary esophageal cancer samples, and their presentation on primary esophageal cancer was confirmed.

[0243] TUMAPs identified in multiple esophageal cancer and normal tissues were quantified using ion counting of label-free LC-MS data. The method assumes that the LC-MS signal area of ​​a peptide correlates with its abundance in the sample. All quantitative signals of a peptide from various LC-MS experiments were normalized based on central tendency, averaged per sample, and merged into a bar graph called a presentation profile. The presentation profile integrates different analytical methods, such as protein database searching, spectral clustering, charge state deconvolution, and retention time alignment and normalization.

[0244] In addition to over-representation of peptides, mRNA expression of underlying genes was also examined. Through RNASeq analysis of normal tissues and cancer tissues, mRNA data was obtained (see Example 2). Another source of normal tissue data was a publicly available database of RNA expression data from approximately 3000 normal tissue samples (Lonsdale, 2013). The peptides derived from proteins whose mRNAs are highly expressed in cancer tissues but are very low or absent in vital normal tissues are preferably included in the present invention.

[0245] Furthermore, the discovery pipeline XPRESIDENT® v2.0 allows for the direct absolute quantification of MHC-restricted, preferably HLA-restricted, peptide levels in cancer or other infected tissues. Briefly, total cell number was calculated from the total DNA content of the analyzed tissue sample. The total peptide amount of TUMAP in the tissue sample was measured by nanoLC-MS / MS as the ratio between natural TUMAP and a known amount of an isotope-labeled version of TUMAP, a so-called internal standard. The efficiency of TUMAP isolation was determined by spiking all selected TUMAP peptide:MHC complexes into the tissue lysate as early as possible in the TUMAP isolation procedure and detecting them by nanoLC-MS / MS following completion of the peptide isolation procedure. Total cell number and total peptide amount were calculated from triplicate measurements per tissue sample. Peptide isolation efficiency was calculated as the average from 10 spike experiments, each measured in triplicate (see Example 6 and Table 12).

[0246] The present invention provides peptides useful for treating cancer / tumors, preferably esophageal cancer, that overly or exclusively present the peptides of the present invention. These peptides have been shown by mass spectrometry to be naturally presented by HLA molecules on primary human esophageal cancer samples.

[0247] Many of the original genes / proteins (also referred to as "full-length proteins" or "basal proteins") from which the peptides are derived have been shown to be highly overexpressed in cancer compared to normal tissues, demonstrating the high tumor relevance of the original genes, where "normal tissue" in the context of the present invention shall mean either healthy esophageal cells or other normal tissue cells (see Example 2). Furthermore, the peptides themselves are strongly over-represented on tumor tissues, but not on normal tissues, and "tumor tissue" in the context of the present invention shall mean samples derived from patients suffering from esophageal cancer (see Example 1).

[0248] HLA-bound peptides can be recognized by the immune system, particularly by T lymphocytes, which can destroy cells that present the recognized HLA / peptide complex, such as esophageal cancer cells that present the derived peptide.

[0249] The peptides of the present invention have been shown to be capable of stimulating T cell responses and / or being over-presented, and therefore can be used in accordance with the present invention to produce antibodies and / or TCRs, such as soluble TCRs (see Examples 3 and 4). Furthermore, when complexed with the respective MHC, the peptides can also be used to produce antibodies and / or TCRs, particularly TCRs, according to the present invention. The respective methods are well known to those skilled in the art and can also be found in the respective references. Thus, the peptides of the present invention are useful for generating an immune response in patients, by which tumor cells can be destroyed. An immune response in patients can be induced by directly administering the described peptides, or appropriate precursors (e.g., extended peptides, proteins, or nucleic acids encoding these peptides), to the patient, ideally in combination with an agent (i.e., an adjuvant) that enhances immunogenicity. Because the target peptides of the present invention are not presented in equivalent copy numbers on normal tissues, the immune response resulting from such therapeutic vaccination can be expected to be highly specific to tumor cells, preventing the risk of unwanted autoimmune reactions against the patient's normal cells.

[0250] The present specification further relates to a T cell receptor (TCR) comprising an α chain and aβ chain ("α / β TCR"). Also provided are HAVCR1-001 peptides that are capable of binding to TCRs and antibodies when presented by MHC molecules. The present specification also relates to nucleic acids, vectors, and host cells for expressing the TCRs and peptides of the present specification; and methods of using the same.

[0251] The term "T cell receptor" (abbreviated as TCR) refers to a heterodimeric molecule comprising an α polypeptide chain (α chain) and aβ polypeptide chain (β chain), which heterodimeric receptor can bind to peptide antigens presented by HLA molecules. The term also includes so-called γ / δ TCRs.

[0252] In one embodiment, the description provides a method of producing a TCR as described herein, the method comprising culturing a host cell capable of expressing the TCR under conditions suitable to promote expression of the TCR.

[0253] In another embodiment, the description relates to a method described herein in which a sufficient amount of antigen is contacted with an antigen-presenting cell to load the antigen onto a class I or II MHC molecule expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell, or in which the antigen is loaded onto a class I or II MHC tetramer by tetramerizing an antigen / class I or II MHC complex monomer.

[0254] The α and β chains of α / β TCRs, and the γ and δ chains of γ / δ TCRs, are generally each considered to have two "regions", a variable and a constant region. The variable region consists of joining variable regions (V) and joining regions (J). The variable region may also contain a leader region (L). The β and δ chains may also contain a diversity region (D). The α and β constant regions may also contain a C-terminal transmembrane (TM) region that anchors the α and β chains to the cell membrane.

[0255] With respect to gamma / delta TCRs, the term "TCRgamma variable region" as used herein refers to the junction of the TCRgamma V (TRGV) region and the TCRgamma J (TRGJ) region without the leader region (L), and the term TCRgamma constant region refers to the extracellular TRGC region or a C-terminally truncated TRGC sequence. Similarly, the term "TCRdelta variable region" refers to the junction of the TCRdelta V (TRDV) region and the TCRdelta D / J (TRDD / TRDJ) region without the leader region (L), and the term "TCRdelta constant region" refers to the extracellular TRDC region or a C-terminally truncated TRDC sequence.

[0256] The TCRs herein preferably bind to the HAVCR1-001 peptide-HLA molecule complex with a binding affinity (KD) of about 1 μM or less, about 0.1 μM or less, about 25 μM or less, or about 10 μM or less. High-affinity TCRs with binding affinities of about 1 μM or less, about 100 nM or less, about 50 nM or less, or about 25 nM or less are more preferred. Non-limiting examples of preferred binding affinity ranges for the TCRs of the present invention include about 1 nM to about 10 nM; about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; and about 90 nM to about 100 nM.

[0257] As used herein, in the context of the TCRs herein, "specific binding" and grammatical variants thereof are used to mean a TCR that has a binding affinity (KD) of 1 μM or less for the HAVCR1-001 peptide-HLA molecule complex.

[0258] The α / β heterodimeric TCRs herein may have a disulfide bond introduced between their constant regions. Preferred TCRs of this type include those having a TRAC constant region sequence and a TRBC1 or TRBC2 constant region sequence, with Thr48 of TRAC and Ser57 of TRBC1 or TRBC2 replaced by cysteine ​​residues that form a disulfide bond between the TRAC constant region sequence and the TRBC1 or TRBC2 constant region sequence of the TCR.

[0259] In the presence or absence of the above-described introduced interchain bond, the α / β heterodimeric TCR of the present specification may have a TRAC constant region sequence and a TRBC1 or TRBC2 constant region sequence, and the TRAC constant region sequence of the TCR and the TRBC1 or TRBC2 constant region sequence may be linked by a natural disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

[0260] The TCRs herein may comprise a detectable label selected from the group consisting of a radionuclide, a fluorophore, and biotin. The TCRs herein may be conjugated to a therapeutically active agent such as a radionuclide, a chemotherapeutic agent, or a toxin.

[0261] In one embodiment, the TCRs herein having at least one mutation in the alpha chain and / or having at least one mutation in the beta chain have modified glycosylation compared to the non-mutated TCR.

[0262] In one embodiment, a TCR comprising at least one mutation in the TCR α chain and / or TCR β chain has at least twice the binding affinity and / or binding half-life for the HAVCR1-001 peptide-HLA molecule complex as a TCR comprising an unmutated TCR α chain and / or an unmutated TCR β chain. The affinity enhancement and utilization of tumor-specific TCRs depend on the existence of a window of optimal TCR affinity. The existence of such a window is based on the observation that TCRs specific for HLA-A2-restricted pathogens generally have KD values ​​approximately 10-fold lower than TCRs specific for HLA-A2-restricted tumor-associated self-antigens. While tumor antigens have the potential to be immunogenic, because tumors arise from an individual's own cells, it is now known that only mutant proteins or proteins with altered translational processing are perceived as foreign by the immune system. Upregulated or overexpressed antigens (so-called self-antigens) do not necessarily induce a functional immune response against tumors. T cells expressing highly reactive TCRs against these antigens are negatively selected in the thymus by a process known as central immune tolerance, i.e., the persistence of only T cells with low-affinity TCRs against self-antigens. Thus, the affinity of the TCRs or variants herein for HAVCR1-001 can be increased by methods known in the art.

[0263] The present specification further relates to a method for identifying and isolating TCRs according to the present specification, comprising the steps of incubating PBMCs from an HLA-A*02-negative healthy donor with A2 / HAVCR1-001A2, incubating the PBMCs with tetrameric phycoerythrin (PE), and isolating high avidity T cells by fluorescence-activated cell sorting (FACS) Calibur analysis.

[0264] The present specification further relates to a method for identifying and isolating TCRs according to the present specification, comprising the steps of obtaining a transgenic mouse carrying the entire human TCRαβ gene locus (1.1 and 0.7 Mb) whose T cells express a diverse human TCR repertoire that compensates for the mouse TCR deficiency, immunizing the mouse with HAVCR1-001, incubating PBMCs obtained from the transgenic mouse with tetrameric phycoerythrin (PE), and isolating high avidity T cells by fluorescence-activated cell sorting (FACS) Calibur analysis.

[0265] In one embodiment, to obtain T cells expressing the TCRs of the present invention, nucleic acids encoding the TCR-α and / or TCR-β chains of the present invention are cloned into an expression vector, such as a gamma retrovirus or lentivirus. Recombinant viruses are generated and then tested for functionality, such as antigen specificity and functional binding activity. An aliquot of the final product is then used to transduce a target T cell population (generally purified from the patient's PBMCs), which is expanded before infusion into the patient.

[0266] In another embodiment, to obtain T cells expressing the TCRs herein, TCR RNA is synthesized by techniques known in the art, such as, for example, an in vitro transcription system. The in vitro synthesized TCR RNA is then introduced by electroporation into primary CD8+ T cells obtained from a healthy donor, resulting in re-expression of the tumor-specific TCR-α and / or TCR-β chains.

[0267] To increase expression, the nucleic acids encoding the TCRs herein may be operably linked to strong promoters such as retroviral long terminal repeat (LTR), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin, ubiquitin, and simian virus 40 (SV40) / CD43 composite promoter, elongation factor (EF)-1a, and spleen focus forming virus (SFFV) promoter. In preferred embodiments, the promoter is heterologous to the nucleic acid to be expressed.

[0268] In addition to a strong promoter, the TCR expression cassettes herein may contain additional elements that can enhance transgene expression, including a central polypurine tract (cPPT), which facilitates nuclear translocation of the lentiviral construct (Follenzi et al., 2000), and a woodchuck hepatitis virus posttranscriptional regulatory element (wPRE), which increases RNA stability and thereby increases the level of transgene expression (Zufferey et al., 1999).

[0269] The α and β chains of the TCR of the present invention may be encoded by nucleic acids on separate vectors or may be encoded by polynucleotides on the same vector.

[0270] Achieving high levels of TCR surface expression requires that both the TCR-α and TCR-β chains of the introduced TCR be transcribed at high levels. To achieve this, the TCR-α and TCR-β chains herein may be cloned into a bicistronic construct within a single vector, which has been shown to overcome this obstacle. Because the TCR-α and TCR-β chains are generated from a single transcript that splits into two proteins during translation, ensuring the generation of equimolar ratios of the TCR-α and TCR-β chains, the use of a viral internal ribosome entry site between the TCR-α and TCR-β chains results in coordinated expression of both chains (Schmitt et al. 2009).

[0271] The nucleic acids encoding the TCRs herein may be codon-optimized to increase expression from host cells. Redundancy in the genetic code allows some amino acids to be coded for by more than one codon, but certain codons are less "optimal" than others due to the relative availability of compatible tRNAs as well as other factors (Gustafsson et al., 2004). Modifying the TCR-α and TCR-β gene sequences so that each amino acid is coded for by the optimal codon for mammalian gene expression, as well as removing mRNA instability motifs or potential splice sites, has been shown to significantly enhance TCR-α and TCR-β gene expression (Scholten et al., 2006).

[0272] Furthermore, mispairing between the introduced and endogenous TCR chains can lead to specificity gain, which poses a significant risk of autoimmunity. For example, the formation of mixed TCR dimers can reduce the number of CD3 molecules available to form properly paired TCR complexes, which can significantly reduce the functional avidity of cells expressing the introduced TCR (Kuball et al., 2007).

[0273] To reduce mispairing, the C-terminal regions of the introduced TCR chains herein may be modified to increase interchain affinity while decreasing the ability of the introduced chains to pair with endogenous TCRs. These strategies may include replacing the C-terminal regions of human TCR-α and TCR-β with their murine counterparts (murinized C-terminal regions); introducing a second cysteine ​​residue into both the TCR-α and TCR-β chains of the introduced TCR to create a second interchain disulfide bond in the C-terminal regions (cysteine ​​modification); exchanging interacting residues within the C-terminal regions of the TCR-α and TCR-β chains ("knob-in-hole"); and fusing the variable regions of the TCR-α and TCR-β chains directly to CD3ζ (CD3ζ fusion) (Schmitt et al. 2009).

[0274] In one embodiment, the host cell is genetically engineered to express a TCR of the present disclosure. In a preferred embodiment, the host cell is a human T cell or T cell progenitor cell. In some embodiments, the T cell or T cell progenitor cell is obtained from a cancer patient. In other embodiments, the T cell or T cell progenitor cell is obtained from a healthy donor. The host cell herein can be allogeneic or autologous with respect to the patient being treated. In one embodiment, the host is a gamma / delta T cell transformed to express an alpha / beta TCR.

[0275] A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. Preferably, pharmaceutical compositions are sterile and manufactured in accordance with GMP guidelines.

[0276] Pharmaceutical compositions comprise peptides in either free form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, "pharmaceutically acceptable salt" refers to derivatives of the disclosed peptides, wherein the peptide is modified by forming an acid or base salt of the drug. For example, acid salts are prepared from the free base by reaction with a suitable acid (typically, the neutral form of the drug has a neutral NH group). Suitable acids for preparing acid salts include both organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid, as well as inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, and nitric acid and phosphoric acid. Conversely, basic salts of acidic moieties which may be present on the peptide are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine and the like.

[0277] In one particularly preferred embodiment, the pharmaceutical composition comprises the peptide as a salt of acetic acid (acetate), trifluoroacetic acid or hydrochloric acid (chloride).

[0278] Preferably, the agent of the present invention is an immunotherapeutic agent such as a vaccine. It can be administered to a patient directly, to an affected organ, or systemically via id, im, sc, ip, and iv routes; or it can be applied ex vivo to cells derived from the patient or a human cell line, which are subsequently administered to the patient; or it can be used ex vivo to select a subpopulation of immune cells derived from the patient, which are then re-administered to the patient. When nucleic acids are administered ex vivo to cells, it may be useful to transfect the cells to co-express an immunostimulatory cytokine, such as interleukin-2. The peptides may be substantially pure, combined with an immunostimulatory adjuvant (see below), used in combination with an immunostimulatory cytokine, or administered via a suitable delivery system, such as liposomes. The peptides may also be conjugated to a suitable carrier, such as keyhole limpet hemocyanin (KLH) or mannan (see WO 95 / 18145 and (Longenecker et al., 1993)). The peptides may also be labeled, be fusion proteins, or be hybrid molecules. The peptides whose sequences are described in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of help provided by CD4 T helper cells. Therefore, for MHC class I epitopes that stimulate CD8 T cells, the fusion partner or section of the hybrid molecule suitably provides an epitope that stimulates CD4-positive T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those identified in the present invention.

[0279] In one embodiment, the vaccine comprises at least one peptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 93 and at least one additional peptide, preferably 2 to 50, more preferably 2 to 25, even more preferably 2 to 20, and most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 peptides. The peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

[0280] A further aspect of the present invention provides nucleic acids (e.g., polynucleotides) encoding the peptides or peptide variants of the present invention. The polynucleotide may be, for example, either single-stranded and / or double-stranded DNA, cDNA, PNA, RNA, or a combination thereof, or may be a native or stabilized form of polynucleotide, such as a polynucleotide having a phosphorothioate backbone, and may or may not contain introns, so long as it encodes the peptide. Of course, only peptides containing natural amino acid residues linked by naturally occurring peptide bonds may be encoded by the polynucleotide. A still further aspect of the present invention provides expression vectors capable of expressing the polypeptides according to the present invention.

[0281] A variety of methods have been developed for linking polynucleotides, particularly DNA, to vectors, for example, via complementary cohesive ends. For example, complementary homopolymer sequences can be added to the DNA fragment to be inserted into the vector DNA. The vector and DNA fragment are then linked by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0282] Synthetic linkers containing one or more restriction enzyme recognition sites provide an alternative method for joining DNA fragments to vectors. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from several sources, including International Biotechnologies Inc. New Haven, CN, USA.

[0283] A desirable method for modifying DNA encoding a polypeptide of the present invention is to use the polymerase chain reaction as disclosed in Saiki RK, et al. (Saiki et al., 1988). This method may be used to introduce the DNA into a suitable vector, for example, by modifying appropriate restriction enzyme recognition sites, or it may be used to modify the DNA in other useful ways known in the art. If a viral vector is used, a poxvirus or adenovirus vector is preferred.

[0284] The DNA (or RNA, in the case of retroviral vectors) may then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the invention. Thus, DNA encoding the peptide or variant of the invention may be used to construct expression vectors, which are then used to transform suitable host cells for the expression and production of the polypeptide of the invention, according to known techniques, appropriately modified in light of the teachings contained herein. Such techniques include, for example, those disclosed in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

[0285] The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend on the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

[0286] Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate direction and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, and such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all hosts will be transformed by the vector. Therefore, it is necessary to select transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence with any necessary regulatory elements that encodes a selectable trait in transformed cells, such as antibiotic resistance.

[0287] Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

[0288] Host cells transformed with the recombinant DNA of the present invention are then cultured under appropriate conditions known to those of skill in the art and for a period of time sufficient to express the polypeptide, which may then be recovered, in light of the teachings disclosed herein.

[0289] Numerous expression systems are known, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. Preferably, the expression system may be mammalian cells, such as CHO cells, available from the ATCC Cell Biology Collection.

[0290] Typical mammalian cell vector plasmids for constitutive expression comprise a CMV or SV40 promoter with an appropriate poly(A) tail and a resistance marker such as neomycin. One example is pSVL, available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector, pMSG, is also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, typically available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405, and pRS406 are Yeast Integrating plasmids (YIps) that incorporate the yeast selectable markers HIS3, TRP1, LEU2, and URA3. Plasmid pRS413-416 is a Yeast Centromeric Plasmid (Ycps). CMV promoter-based vectors (e.g., from Sigma-Aldrich) offer transient or stable expression, cytoplasmic or secreted expression, and N- or C-terminal tagging with various combinations of FRAG, 3xFLAG, c-myc, or MAT. These fusion proteins allow recombinant proteins to be detected, purified, and analyzed. Dual-tagged fusions offer versatility in detection.

[0291] The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg / L in COS cells. In less potent cell lines, protein levels are typically about 0.1 mg / L. The presence of the SV40 origin of replication results in high levels of DNA replication in SV40-permissive COS cells. CMV vectors can contain, for example, the pMB1 (a derivative of pBR322) origin of replication in bacterial cells, a b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and an f1 origin. Vectors containing a preprotrypsin leader (PPT) sequence can direct secretion of FRAG fusion proteins into the culture medium for purification using anti-FRAG antibodies, resins, and plates. Other vectors and expression systems for use in a variety of host cells are well known in the art.

[0292] In another embodiment, two or more peptides or peptide variants of the invention are encoded and thus expressed sequentially (similar to a "beads and beads" construct), whereby the peptides or peptide variants may be linked or fused together by a stretch of linker amino acids, e.g., LLLLLL, or may be linked without any additional peptide between them. These constructs may also be used for cancer therapy and may induce immune responses involving both MHC I and MHC II.

[0293] The present invention also relates to host cells transformed with the polynucleotide vector constructs of the present invention. Host cells can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some circumstances, typically E. coli strains such as E. coli DH5, available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 (ATCC No. 31343), available from the American Type Culture Collection (ATCC), Rockville, MD, USA. Preferred eukaryotic host cells include yeast, insect, and mammalian cells, preferably vertebrate cells such as those derived from mouse, rat, monkey, or human fibroblastic and colonic cell lines. Yeast host cells include YPH499, YPH500, and YPH501, publicly available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryonic cells NIH / 3T3 available from the ATCC as CRL1658, monkey kidney-derived COS-1 cells available from the ATCC as CRL1650, and human embryonic kidney cells 293. Preferred insect cells are Sf9 cells, which can be transfected with baculovirus expression vectors. Reviews regarding the selection of appropriate host cells for expression can be found, for example, in the textbook "Methods in Molecular Biology: Recombinant Gene Expression, Reviews and Protocols," Part One, Second Edition, ISBN 978-1-58829-262-9 by Paulina Balbas and Argelia Lorence, and other references known to those skilled in the art.

[0294] Transformation of suitable cell hosts with the DNA constructs of the present invention is typically accomplished by well-known methods, which depend on the type of vector used. For the transformation of prokaryotic host cells, see, for example, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook, 2012). The transformation of yeast cells is described in Sherman et al. (Sherman et al., 1986). The method of Beggs (Beggs, 1978) is also useful. For vertebrate cells, reagents useful for transfecting such cells, such as calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation is also useful for transforming and / or transfecting cells and is well known in the art for transforming yeast, bacterial, insect, and vertebrate cells.

[0295] Successfully transformed cells, i.e., cells containing the DNA construct of the present invention, can be identified by well-known techniques such as PCR. Alternatively, antibodies can be used to detect the presence of proteins in the supernatant.

[0296] It will be understood that certain host cells of the present invention, such as bacteria, yeast, and insect cells, are useful in preparing the peptides of the present invention. However, other host cells may be useful in certain therapeutic methods. For example, antigen-presenting cells such as dendritic cells may be usefully used to express the peptides of the present invention so that they may be loaded into appropriate MHC molecules. Thus, the present invention provides host cells comprising a nucleic acid or expression vector according to the present invention.

[0297] In a preferred embodiment, the host cell is an antigen-presenting cell, particularly a dendritic cell or an antigen-presenting cell. APC loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) was approved by the US Food and Drug Administration (FDA) on April 20, 2010 (sipuleucel-T) for the treatment of asymptomatic or minimally symptomatic metastatic HRPC (Rini et al., 2006; Small et al., 2006).

[0298] A further aspect of the invention provides a method for producing a peptide or variant thereof, comprising culturing a host cell and isolating the peptide from the host cell or its culture medium.

[0299] In another embodiment, the peptides, nucleic acids, or expression vectors of the present invention are used in medical treatment. For example, the peptides or their variants may be formulated for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Preferred methods of peptide injection include sc, id, ip, im, and iv. Preferred methods of DNA injection include id, im, sc, ip, and iv. For example, a dose of 50 μg to 1.5 mg, preferably 125 μg to 500 μg, of peptide or DNA may be administered, depending on the peptide or DNA. Doses in this range have been successfully used in previous clinical trials (Walter et al., 2012).

[0300] The polynucleotides used for active vaccination may be substantially pure or contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for designing and introducing such nucleic acids are well known in the art. A review is provided, for example, by Teufel et al. (Teufel et al., 2005). Polynucleotide vaccines are easy to prepare, but the mechanism of action of these vectors in inducing immune responses is not fully understood. Suitable vectors and delivery systems include viral DNA and / or RNA, such as adenovirus, vaccinia virus, retrovirus, herpesvirus, adeno-associated virus, or hybrid-based systems containing components of two or more viruses. Non-viral delivery systems include cationic lipids and cationic polymers and are well known in the DNA delivery art. Physical delivery, such as via a "gene gun," may also be used. The peptide or peptides encoded by the nucleic acid may be a fusion protein with an epitope that stimulates T cells of the respective reverse CDRs, for example, as described above.

[0301] The medicaments of the present invention may also contain one or more adjuvants. Adjuvants are substances that nonspecifically promote or enhance immune responses (e.g., immune responses to antigens mediated by CD8-positive T cells and helper T (TH) cells) and are therefore considered useful in the medicaments of the present invention. Suitable adjuvants include 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), resiquimod, ImuFact®, and the like. IMP321, interleukins such as IL-2, IL-13 and IL-21, interferon-α or -β or their PEGylated derivatives, IS patch, ISS, ISCOMATRIX, ISCOM, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, water-in-oil and Adjuvants include, but are not limited to, oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactide-co-glycolide) [PLG]-based and dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants specific for dendritic cells and their preparations (e.g., MF59) have been previously described (Allison and Krummel, 1995). Cytokines may also be used.Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, the entire contents of which are specifically incorporated herein by reference), and acting as immune enhancers (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al., 1996).

[0302] CpG immunostimulatory oligonucleotides have also been reported to enhance adjuvant effects in vaccine settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced TH1 cell activation and potent cytotoxic T lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which is particularly necessary for inducing a strong response when the antigen is relatively weak. They also accelerate immune responses, allowing for a nearly two-order reduction in antigen dose in some experiments with antibody responses equivalent to those of the total vaccine dose without CpG (Krieg, 2006). U.S. Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) manufactured by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, may also be used.

[0303] Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera); dsRNA analogs such as poly(I:C) and their derivatives (e.g., AmpliGen®, Hiltonol®, poly(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA; and cyclophosphamide, sunitinib, bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Immunoactive small molecules and antibodies, such as Trap, ZD2171, AZD2171, anti-CTLA4, and other antibodies that target critical structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor), and SC58175, may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation.

[0304] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon alpha, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, and PLG or virosome microparticle formulations.

[0305] In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon alpha.

[0306] In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is selected from the group consisting of colony-stimulating factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is cyclophosphamide, imiquimod, or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 20, Montanide ISA 50V, Montanide ISA-51, poly ICLC (Hiltonol®), and anti-CD40 mAB, or a combination thereof.

[0307] The composition is used for parenteral administration, such as subcutaneous, intradermal, or intramuscular administration, or for oral administration. For this purpose, the peptide and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. The composition may further contain excipients such as buffers, binders, blasting agents, diluents, flavors, lubricants, etc. The peptide may also be administered together with immune stimulants, such as cytokines. A detailed list of excipients that can be used in such compositions can be found, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for the prevention, prophylaxis, and / or treatment of adenomatous or cancerous diseases. Exemplary formulations are found, for example, in EP 2112253.

[0308] It is important to understand that the immune response elicited by the vaccine of the present invention attacks cancers at different cell division stages and different developmental stages. Furthermore, different cancer-associated signaling pathways are attacked. This is an advantage over vaccines that address only one or a few targets, sometimes leading to tumors easily adapting to the attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Therefore, combining several tumor-associated peptides ensures that every possible tumor has at least some of the targets. The composition is designed with the expectation that each tumor will express several antigens, covering several independent pathways necessary for tumor growth and maintenance. Therefore, the vaccine can be easily used "off the shelf" for a larger patient population. This means that pre-selection of patients to be treated with the vaccine can be limited to HLA typing and does not require any additional biomarker assessment of antigen expression, yet it is still certain that several targets are simultaneously attacked by the induced immune response, which is important for efficacy (Banchereau et al., 2001; Walter et al., 2012).

[0309] As used herein, the term "scaffold" refers to a molecule that specifically binds to a (e.g., antigenic) determinant. In one embodiment, the scaffold can also direct the entity to which it is attached (e.g., a (second) antigen-binding moiety) to a target site, such as a specific tumor cell or tumor stroma bearing an antigenic determinant (e.g., a peptide-MHC complex described herein). In another embodiment, the scaffold can activate signaling through its target antigen, such as a T-cell receptor complex antigen. Scaffolds include, but are not limited to, antibodies and fragments thereof, antibody antigen-binding domains comprising an antibody heavy chain variable region and an antibody light chain variable region, binding proteins comprising at least one ankyrin repeat motif and a single-domain antigen-binding (SDAB) molecule, aptamers, (soluble) TCRs, and (engineered) cells such as allogeneic or autologous T cells. Binding assays can be performed to assess whether a molecule is a scaffold that binds to a target.

[0310] "Specific" binding means that the scaffold binds to the target peptide-MHC complex better than other natural peptide-MHC complexes, so that a scaffold equipped with an active molecule capable of killing cells bearing a specific target cannot kill other cells that do not have a specific target but present other peptide-MHC complexes. If the peptide of the cross-reactive peptide-MHC is not naturally occurring, i.e., not derived from the human HLA-peptidome, binding to other peptide-MHC complexes is irrelevant. Tests to evaluate target cell killing are well known in the art. They should be performed using target cells (primary cells or cell lines) with unmodified peptide-MHC presentation, or cells loaded with peptide to reach naturally occurring peptide-MHC levels.

[0311] Each scaffold may comprise a label, which allows the binding scaffold to be detected by determining the presence or absence of a signal provided by the label. For example, the scaffold may be labeled with a fluorescent dye or any other applicable cell marker molecule. Such marker molecules are well known in the art. For example, the fluorescent label provided by a fluorescent dye may allow the binding aptamer to be visualized by fluorescence or laser scanning microscopy or flow cytometry.

[0312] Each scaffold can be conjugated to a second active molecule, such as, for example, IL-21, anti-CD3, anti-CD28, etc.

[0313] For more information regarding polypeptide scaffolds, see, e.g., the background section of WO 2014 / 071978 A1 and the references cited therein.

[0314] The present invention further relates to aptamer.Aptamer (see, for example, International Publication No. 2014 / 191359 and the documents cited therein) is a short single-stranded nucleic acid molecule, which can fold into a predetermined three-dimensional structure and recognize specific target structure.They have been considered as a suitable alternative for developing targeted therapy.Aptamer has been shown to selectively bind with various complex targets with high affinity and specificity.

[0315] Aptamers that recognize molecules located on cell surfaces have been identified within the past decade, providing a means for developing diagnostic and therapeutic approaches. Aptamers have been shown to be nearly non-toxic and immunogenic, making them promising candidates for biomedical applications. Indeed, aptamers, such as those recognizing prostate-specific membrane antigen (PSMA), have been successfully used for targeted therapy and have been shown to function in xenograft in vivo models. Furthermore, aptamers that recognize specific tumor cell lines have been identified.

[0316] DNA aptamers can be selected to exhibit broad-spectrum recognition properties against various cancer cells, particularly those derived from solid tumors, while not recognizing non-tumorigenic and primary healthy cells. If an identified aptamer not only recognizes a specific tumor subtype but also interacts with a range of tumors, this makes the aptamer applicable as a so-called broad-spectrum diagnostic and therapeutic agent.

[0317] Furthermore, investigation of cell binding behavior by flow cytometry showed that the aptamer exhibited very good apparent affinity in the nanomolar concentration range.

[0318] Aptamers are useful for diagnostic and therapeutic purposes. Furthermore, it has been shown that some aptamers are taken up by tumor cells and can therefore function as molecular vehicles for the targeted delivery of anti-cancer drugs, such as siRNA, into tumor cells.

[0319] Aptamers can be selected using cell-SELEX (extraneous evolution of proteins) technology against complex targets such as cells and tissues, and against peptide complexes comprising, preferably consisting of, any of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 93 according to the present invention and an MHC molecule.

[0320] Using the peptides of the present invention, specific antibodies against MHC / peptide complexes can be generated and developed. These can be used for therapeutic purposes to target toxins or radioactive substances to diseased tissues. Another use of these antibodies can be to target radionuclides to diseased tissues for imaging purposes such as PET. This application can help detect small metastases or determine the size and precise location of diseased tissues.

[0321] Therefore, it is a further aspect of the present invention to provide a method for producing a recombinant antibody that specifically binds to human major histocompatibility complex (MHC) class I or II complexed with an HLA-restricted antigen, the method comprising the steps of: immunizing a genetically engineered non-human mammal comprising cells expressing said human major histocompatibility complex (MHC) class I or II with a soluble form of an MHC class I or II molecule complexed with said HLA-restricted antigen; isolating mRNA molecules from antibody-producing cells of said non-human mammal; producing a phage display library that displays protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, wherein said at least one phage displays said antibody that specifically binds to said human major histocompatibility complex (MHC) class I or II complexed with said HLA-restricted antigen.

[0322] It is a further aspect of the present invention to provide antibodies that specifically bind to human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen, wherein the antibodies are preferably polyclonal, monoclonal, bispecific and / or chimeric antibodies.

[0323] Respective methods for producing such antibodies and single-chain class I major histocompatibility complexes, as well as other tools for producing these antibodies, are disclosed in WO 03 / 068201, WO 2004 / 084798, WO 01 / 72768, WO 03 / 070752, and in the literature (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), the contents of which are all expressly incorporated by reference in their entirety for the purposes of the present invention.

[0324] Preferably, the antibody binds to the complex with a binding affinity of less than 20 nanomolar, preferably less than 10 nanomolar, which is also considered "specific" in the context of the present invention.

[0325] The present invention relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that cross-reacts T cells with said peptide, wherein said peptide is not the underlying full-length polypeptide.

[0326] The present invention further relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 93, said peptide or variant having an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0327] The present invention further relates to a peptide according to the invention, which has the ability to bind to a molecule of the human major histocompatibility complex (MHC) class I or II.

[0328] The present invention further relates to a peptide according to the invention, wherein the peptide consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 93.

[0329] The present invention further relates to a peptide according to the invention, wherein the peptide is (chemically) modified and / or comprises non-peptide bonds.

[0330] The present invention further relates to a peptide according to the invention, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii), or the peptide is fused to (or into) an antibody, e.g., a dendritic cell-specific antibody.

[0331] Another embodiment of the present invention relates to non-naturally occurring peptides, wherein the peptides consist of or consist essentially of the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:48 and are synthetically produced (e.g., synthesized) as pharmaceutically acceptable salts. Methods for synthetically producing peptides are well known in the art. Because peptides produced in vivo are not salts, salts of peptides according to the present invention are substantially different from the in vivo state of the peptides. The non-naturally occurring salt forms of the peptides mediate the solubility of the peptides, particularly in the context of pharmaceutical compositions comprising the peptides, such as the peptide vaccines disclosed herein. Sufficient, or at least substantial, solubility of the peptide is necessary to efficiently deliver the peptide to the subject being treated. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. These salts according to the present invention include PO4 as the anion. 3- , SO4 2- , CH3COO - , Cl - , Br - , NO 3- , ClO 4- , I - , SCN - , and NH as the cation 4+ , Rb + , K. + , Na + , Cs + , Li + , Zn 2+ , Mg 2+ , Ca 2+ , Mn 2+ , Cu 2+ and Ba 2+In particular, the salts include alkali and alkaline earth salts such as the Hofmeister series of salts comprising (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CH3COO, NH4Cl, NH4Br, NH4NO3, NH4CIO4, NHI, NHI SCN, Rb3PO4, Rb2HPO4, RbH2PO4, Rb2SO4, Rb4CH3COO, Rb4Cl, Rb4Br, Rb4NO3, Rb4CIO4, Rb4I, Rb4SCN, K3PO4, K2HPO4, KH2PO4, K2SO4, KCH3COO, KCl, KBr, KNO3, KClO4, KI, KSCN, Na3PO4, Na2HPO4, NaH2PO4, Na2SO4, NaCH3COO, NaCl, NaB r, NaNO3, NaCIO4, NaI, NaSCN, ZnCI2Cs3PO4, Cs2HPO4, CsH2PO4, Cs2SO4, CsCH3COO, CsCl, CsBr, CsNO3, CsCIO4, CsI , CsSCN, Li3PO4, Li2HPO4, LiH2PO4, Li2SO4, LiCH3COO, LiCl, LiBr, LiNO3, LiClO4, LiI, LiSCN, Cu2SO4, Mg3(PO4) 2, Mg2HPO4, Mg(H2PO4)2, Mg2SO4, Mg(CH3COO)2, MgCl2, MgBr2, Mg(NO3)2, Mg(ClO4)2, MgI2, Mg(SCN)2, MnCl2, Ca3( PO4), Ca2HPO4, Ca(H2PO4), CaSO4, Ca(CH3COO), CaCl2, CaBr2, Ca(NO3), Ca(ClO4), CaI2, Ca(SCN), Ba3(PO4), Ba2HPO4, Ba(H2PO4), BaSO4, Ba(CH3COO), BaCl2, BaBr2, Ba(NO3), Ba(ClO4), BaI2, and Ba(SCN). Particularly preferred are NH acetates, such as chlorides or acetates (trifluoroacetates), MgCl2, KH2PO4, Na2SO4, KCl, NaCl, and CaCl2.

[0332] In general, peptides and variants (at least those containing peptide bonds between amino acid residues) may be synthesized by the Fmoc-polyamide solid-phase peptide synthesis method disclosed by Lukas et al. (Lukas et al., 1981) and the references cited therein. Temporary N-amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is carried out using 20% ​​piperidine in N,N-dimethylformamide. Side chain functional groups may be protected as their butyl ethers (for serine, threonine, and tyrosine), butyl esters (for glutamic acid and aspartic acid), butyloxycarbonyl derivatives (for lysine and histidine), trityl derivatives (for cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (for arginine). When glutamine or asparagine is the C-terminal residue, a 4,4'-dimethoxybenzhydryl group is utilized to protect the side-chain amide functionality. The solid-phase support is based on a polydimethyl-acrylamide polymer composed of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and acryloylsarcosine methyl ester (functionalizer). The peptide-to-resin cleavable linker used is an acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives, except for asparagine and glutamine, which are added using a reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isatin test procedures. Upon completion of synthesis, the peptide is cleaved from the resin support and the side chain protecting groups are simultaneously removed by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture.Commonly used scavengers include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the constituent amino acids of the peptide being synthesized. A combination of solid-phase and solution-phase methods for the synthesis of peptides is also possible (see, for example, (Bruckdorfer et al., 2004) and references cited therein).

[0333] Trifluoroacetic acid is removed by evaporation under vacuum, followed by trituration with diethyl ether to yield the crude peptide. Any scavengers present are removed by a simple extraction procedure, which, upon lyophilization of the aqueous phase, gives the scavenger-free crude peptide. Reagents for peptide synthesis are commonly available, for example, from Calbiochem-Novabiochem (Nottingham, UK).

[0334] Purification may be achieved by any one or combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reversed-phase high performance liquid chromatography using, for example, an acetonitrile / water gradient separation.

[0335] The present invention further relates to nucleic acids encoding peptides according to the invention, with the proviso that the peptide is not a complete (full-length) human protein.

[0336] The present invention further relates to a nucleic acid according to the invention which is DNA, cDNA, PNA, RNA or a combination thereof.

[0337] The present invention further relates to an expression vector capable of expressing a nucleic acid according to the invention.

[0338] The present invention further relates to a peptide according to the invention, a nucleic acid according to the invention or an expression vector according to the invention for use in medicine, in particular in the treatment of esophageal cancer.

[0339] The present invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector according to the invention.

[0340] The present invention further relates to a host cell according to the invention which is an antigen-presenting cell, preferably a dendritic cell.

[0341] The present invention further relates to a method for producing a peptide according to the invention, comprising the steps of culturing a host cell according to the invention and isolating the peptide from the host cell or its culture medium.

[0342] The present invention further relates to a method according to the invention, wherein a sufficient amount of antigen is contacted with antigen-presenting cells, thereby loading the antigen onto class I or II MHC molecules expressed on the surface of appropriate antigen-presenting cells.

[0343] The present invention further relates to a method according to the present invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing the peptide containing SEQ ID NO: 1 to SEQ ID NO: 93 or the heterologous amino acid sequence.

[0344] The present invention further relates to activated T cells produced by the method according to the invention, said T cells selectively recognizing cells that aberrantly express a polypeptide comprising an amino acid sequence according to the invention.

[0345] The present invention further relates to a method of killing target cells in a patient which aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, comprising the step of administering to the patient an effective number of T cells according to the present invention.

[0346] The present invention further relates to the use of any of the described peptides, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention, or activated cytotoxic T lymphocytes according to the invention as a medicament or in the manufacture of a medicament. The present invention further relates to the use according to the invention, wherein the medicament is effective against cancer.

[0347] The present invention further relates to a use according to the present invention, wherein the medicament is a vaccine.The present invention further relates to a use according to the present invention, wherein the medicament is effective against cancer.

[0348] The present invention further relates to the use according to the invention, wherein said cancer cells are esophageal cancer cells or other solid or hematological tumor cells such as lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular carcinoma, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancer, and leukemia.

[0349] The present invention further relates to specific peptide-based labeled proteins and biomarkers according to the present invention, referred to herein as "targets," which can be used in the diagnosis and / or prognosis of esophageal cancer. The present invention also relates to the use of these novel targets for cancer therapy.

[0350] The terms "antibody" or "antibodies" are used broadly herein and include both polyclonal and monoclonal antibodies. In addition to intact or "intact" immunoglobulin molecules, the term "antibody" also includes fragments (e.g., CDRs, Fv, Fab, and Fc fragments), or polymers of these immunoglobulin molecules and humanized versions of immunoglobulin molecules, so long as they exhibit any of the desired properties according to the present invention (e.g., specific binding of an esophageal cancer marker (poly)peptide, delivery of a toxin to esophageal cancer cells expressing an elevated level of a cancer marker gene, and / or inhibition of the activity of an esophageal cancer marker polypeptide).

[0351] Whenever possible, the antibodies of the present invention may be purchased from commercial sources. Alternatively, the antibodies of the present invention may be produced using well-known methods. Those skilled in the art will understand that either the full-length esophageal cancer marker polypeptide or a fragment thereof may be used to produce the antibodies of the present invention. The polypeptides used to produce the antibodies of the present invention may be partially or completely purified from natural sources, or may be produced using recombinant DNA technology.

[0352] For example, cDNA encoding a peptide according to the present invention, such as the peptides set forth in SEQ ID NO:1 to SEQ ID NO:93 polypeptides; or a variant or fragment thereof, can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells), and the recombinant protein can then be purified and used to produce monoclonal or polyclonal antibody preparations that specifically bind to the esophageal cancer marker polypeptides, which can be used to produce antibodies according to the present invention.

[0353] Those skilled in the art will understand that generating two or more distinct sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining antibodies with the specificity and affinity required for their intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). Antibodies are tested for their desired activity by known methods according to the purpose for which they will be used (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on antibody generation and testing, see, e.g., Greenfield, 2014). For example, antibodies may be tested in ELISA assays, Western blots, or immunohistochemical staining of formalin-fixed tumor or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested by known clinical testing methods.

[0354] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous antibody population; i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. As used herein, "monoclonal antibody" specifically includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. Pat. No. 4,816,567, the entire contents of which are incorporated herein by reference).

[0355] Monoclonal antibodies of the invention may be prepared using hybridoma technology. In the hybridoma technology, a mouse or other suitable host animal is typically immunized with an immunizing agent to produce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0356] Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

[0357] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate antibody fragments, particularly Fab fragments, can be accomplished using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment produces F(ab')2 fragments and pFc' fragments. This brings about fragmentation.

[0358] Antibody fragments, whether attached to other sequences or not, may also contain insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide additional properties, such as removing / adding amino acids capable of disulfide bonding, increasing its biological lifespan, or altering its secretion characteristics. In any case, the antibody fragment must retain biologically active properties, such as binding activity or modulation of binding in the binding region. Functional or active regions of an antibody may be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to skilled practitioners and may include site-directed mutagenesis of nucleic acids encoding the antibody fragment.

[0359] The antibodies of the present invention may further comprise humanized or human antibodies. Humanized forms, such as non-human (e.g., murine) antibodies, are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0360] Methods for humanizing non-human antibodies are well known in the art. Humanized antibodies typically have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which typically come from an "import" variable region. Humanization can essentially be performed by substituting rodent CDR(s) or CDR(s) sequences for the corresponding human antibody sequences. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable region has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0361] Transgenic animals (e.g., mice) can be used that are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of antibody heavy chain joining region genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge. Human antibodies can also be produced in phage display libraries.

[0362] The antibody of the present invention is preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmacologically acceptable carriers include saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles such as films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the antibody being administered.

[0363] The antibody can be administered to a subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular) or by other methods, such as infusion, that ensure delivery to the bloodstream in an effective form. The antibody can also be administered intratumorally or via a peritumoral route to exert a local as well as a systemic therapeutic effect. Local or intravenous injection is preferred.

[0364] Effective dosages and schedules for administering antibodies may be determined empirically, and making such determinations is within the skill of those in the art. One of ordinary skill in the art will understand that the antibody dose that must be administered will vary depending, for example, on the subject receiving the antibody, the route of administration, the particular antibody type used, and other agents being administered. A typical daily dose of an antibody used alone may range from about 1 μg / kg to up to 100 mg / kg of body weight per day or more, depending on the factors discussed above. Following antibody administration, preferably to treat esophageal cancer, the efficacy of the therapeutic antibody can be assessed by a variety of methods well known to skilled practitioners. For example, standard tumor imaging techniques may be used to monitor the size, number, and / or distribution of cancer in the treated subject. A therapeutically administered antibody that halts tumor growth, causes tumor shrinkage, and / or prevents the development of new tumors, compared to the disease course that would occur in the absence of antibody administration, is an effective antibody for cancer treatment.

[0365] A further aspect of the present invention provides a method for producing soluble T cell receptors (sTCRs) that recognize specific peptide-MHC complexes. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by targeted mutagenesis of complementarity-determining regions. Phage display can be used to select T cell receptors (US Patent Publication No. 2010 / 0113300, (Liddy et al., 2012)). To stabilize T cell receptors during phage display and in pharmaceutical applications, the α and β chains can be linked, for example, by a non-natural disulfide bond, other covalent bonds (single-chain T cell receptors), or a dimerization domain (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). T cell receptors can be linked to toxins, drugs, cytokines (see, for example, U.S. Patent No. 2013 / 0115191), effector cell recruiting domains such as anti-CD3 domains, etc. to exert specific functions on target cells. Furthermore, they can be expressed in T cells used for adoptive transfer. Further information can be found in WO 2004 / 033685 A1 and WO 2004 / 074322 A1. TCR combinations are described in WO 2012 / 056407 A1. Further manufacturing methods are disclosed in WO 2013 / 057586 A1.

[0366] Additionally, the peptides and / or TCRs or antibodies or other binding molecules of the present invention may be used to confirm a pathologist's cancer diagnosis based on a biopsy sample.

[0367] The antibody or TCR may also be used for in vivo diagnostic assay. Usually, the antibody is labeled with a radionucleotide (such as In, Tc, C, I, H, P or S) so that tumors can be localized using immunoscintigraphy. In one embodiment, the antibody or its fragment binds to the extracellular domain of two or more targets of proteins selected from the group consisting of the above-mentioned proteins, and the affinity (Kd) is less than 1 x 10 μM.

[0368] Diagnostic antibodies may be labeled with probes suitable for detection by various imaging methods. Probe detection methods include, but are not limited to, fluorescence, optical, confocal, and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin, and other fluorophores, radioisotopes, gold, gadolinium, and other lanthanides, paramagnetic iron, fluorine-18, and other positron-emitting radionuclides. Furthermore, probes may be bifunctional or multifunctional, and may be detectable by more than one of the listed methods. These antibodies may be directly or indirectly labeled with the probes. Particularly well-recognized techniques for attaching probes to antibodies include covalent coupling of the probe, incorporation of the probe into the antibody, and covalent coupling of a chelating compound for probe binding. For immunohistochemistry, diseased tissue samples may be fresh or frozen, or may be embedded in paraffin and fixed in a preservative such as formalin. Fixed or embedded sections containing the sample are contacted with labeled primary and secondary antibodies, and the antibodies are used to detect in situ protein expression.

[0369] Another aspect of the invention includes an in vitro method of producing activated T cells, the method comprising contacting ex vivo T cells with antigen-loaded human MHC molecules expressed on the surface of suitable antigen-presenting cells for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigen is used in conjunction with the antigen-presenting cells.

[0370] Preferably, the mammalian cells have no or reduced levels or function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter involved in antigen processing.

[0371] The human peptide loading-deficient cell line T2 is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, under catalog number CRL1992; the Drosophila cell line Schneider strain 2 is available from the ATCC under catalog number CRL19863; the mouse RMA-S cell line is described in Ljunggren et al. (Ljunggren and Karre, 1985).

[0372] Preferably, prior to transfer, the host cells do not substantially express MHC class I molecules. It is also preferred that the stimulator cells express molecules important for providing costimulatory signals for T cells, such as B7.1, B7.2, ICAM-1, and LFA3. Nucleic acid sequences for many MHC class I molecules and costimulatory molecules are publicly available from the GenBank and EMBL databases.

[0373] When an MHC class I epitope is used as the antigen, the T cells are CD8 positive T cells.

[0374] When antigen-presenting cells are transfected to express such epitopes, the cells preferably comprise an expression vector capable of expressing a peptide containing SEQ ID NO: 1 to SEQ ID NO: 93, or a variant amino acid sequence thereof.

[0375] Several other methods can be used to produce T cells ex vivo. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTLs. Plebanski et al. (Plebanski et al., 1995) utilized autologous peripheral blood lymphocytes (PLBs) to prepare T cells. Furthermore, autologous T cells can also be produced by pulsing dendritic cells with peptides or polypeptides or infecting them with recombinant viruses. B cells can also be used to produce autologous T cells. Furthermore, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses can be used to prepare autologous CTLs. S. Walter et al. (Walter et al., 2003) described ex vivo priming of T cells using artificial antigen-presenting cells (aAPCs), which is also a suitable method for producing T cells against selected peptides. In the present invention, aAPCs were generated by conjugating preformed MHC:peptide complexes to surface polystyrene particles (microbeads) using biotin:streptavidin biochemistry. This system allows for precise control of MHC density on aAPCs, which allows for selective elicitation of high- or low-avidity antigen-specific T cell responses from blood samples with high efficiency. In addition to MHC:peptide complexes, aAPCs should possess other proteins with costimulatory activity, such as anti-CD28 antibodies, conjugated to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, such as cytokines like interleukin-12.

[0376] Allogeneic cells may also be used in the preparation of T cells, methods being detailed in WO 97 / 26328, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present antigens, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells, etc. Plant viruses may also be used (see, e.g., Porta et al., who describe the development of cowpea mosaic virus as a high-yield system for the presentation of foreign peptides (Porta et al., 1994)).

[0377] Activated T cells directed against the peptides of the invention are useful in therapy. Thus, a further aspect of the invention provides activated T cells obtainable by the methods of the invention described above.

[0378] The activated T cells produced by the above method selectively recognize cells that abnormally express a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 93.

[0379] Preferably, the T cells recognize cells by interaction with (e.g., binding to) an HLA / peptide complex through their TCR. The T cells are useful in a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, to which an effective number of activated T cells are administered. The T cells administered to the patient may be derived from the patient and activated as described above (i.e., they are autologous T cells). Alternatively, the T cells are derived from another individual rather than the patient. Of course, it is preferred if the individual is a healthy individual. By "healthy individual," we mean that the individual is generally in good health, preferably has a competent immune system, and more preferably is not suffering from any disease that can be easily tested for and detected.

[0380] In vivo, target cells for CD8-positive T cells according to the present invention may be tumor cells (which sometimes express MHC class II) and / or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class II; (Dengjel et al., 2006)).

[0381] The T cells of the present invention may be used as an active ingredient in a therapeutic composition. Accordingly, the present invention also provides a method for killing target cells in a patient whose target cells abnormally express a polypeptide comprising an amino acid sequence of the present invention, the method comprising the step of administering to the patient an effective number of T cells as defined above.

[0382] By "aberrantly expressed," we also mean that the polypeptide is overexpressed compared to expression levels in normal (healthy) tissue, or that the gene is silent in the tissue from which the tumor originates, but is expressed in the tumor. By "overexpressed," we mean that the polypeptide is present at a level at least 1.2 times the level present in normal tissue; preferably at least 2 times, more preferably at least 5 or 10 times the level present in normal tissue.

[0383] T cells may be obtained by methods known in the art, such as those described above.

[0384] Protocols for this so-called adoptive transfer of T cells are well known in the art and are reviewed in Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

[0385] Another aspect of the invention involves the use of peptides that complex with MHC to generate T cell receptors, the nucleic acids of which are cloned and introduced into host cells, preferably T cells. These engineered T cells can then be transferred into patients for cancer treatment.

[0386] Any molecule of the invention, i.e., peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor or nucleic acid encoding same, is useful for treating disorders characterized by cells that escape the immune response. Thus, any molecule of the invention may be used as a medicament or in the manufacture of a medicament. The molecule may be used alone or in combination with other molecules of the invention. These molecules may be used in combination with known molecules.

[0387] The present invention provides (a) a container containing the above-described pharmaceutical composition in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; and (c) optionally, (i) instructions for use of the solution, or (ii) instructions for reconstitution and / or use of the lyophilized formulation.

[0013] The present invention is further directed to a kit comprising:

[0388] The kit may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. The container is preferably a bottle, vial, syringe, or test tube; it may be a multi-use container. The pharmaceutical composition is preferably lyophilized.

[0389] The kit of the present invention preferably comprises a lyophilized formulation of the present invention in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., dual-chamber syringes), and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. Preferably, the kit and / or container include instructions on or associated with the container, which indicate instructions for reconstitution and / or use. For example, the label may indicate that the lyophilized formulation is to be reconstituted to a peptide concentration as described above. The label may further indicate that the formulation is useful for or intended for subcutaneous administration.

[0390] The container containing the formulation may be a multi-use vial, which allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (e.g., sodium bicarbonate solution).

[0391] Upon mixing of the diluent and the lyophilized formulation, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg / mL / peptide (=75 μg) and preferably no more than 3 mg / mL / peptide (=1500 μg). The kit may further include other items desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions.

[0392] The kits of the present invention may have a single container containing a pharmaceutical composition formulation according to the present invention, with or without added other components (e.g., other compounds or pharmaceutical compositions of these other compounds), or may have separate containers for each component.

[0393] Preferably, the kits of the invention comprise a formulation of the invention packaged for use in conjunction with the co-administration of a second compound (such as an adjuvant (e.g., GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic factor or inhibitor, an apoptosis inducer or chelator, or a pharmaceutical composition thereof). The kit components may be premixed, or each component may be in a separate, distinct container prior to administration to a patient. The kit components may be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The kit components may also be provided as solids, which may be converted to a liquid by the addition of a suitable solvent, preferably provided in another, distinct container.

[0394] The containers of the therapeutic kit may be vials, test tubes, flasks, bottles, syringes, or any other means of enclosing a solid or liquid. Typically, when there are two or more components, the kit will contain a second vial or other container to allow for separate dosing. The kit may also contain a separate container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit will contain a device (e.g., one or more needles, syringes, eyedroppers, pipettes, etc.) to allow for administration of the agents of the invention that are components of the kit.

[0395] The formulation is suitable for administration of the peptide by any acceptable means, such as oral (enteral), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, administration is sc, and most preferably id, and may be by infusion pump.

[0396] Since the peptides of the present invention are isolated from esophageal cancer, the agents of the present invention are preferably used to treat esophageal cancer.

[0397] The present invention further relates to a method for producing a personalized medicine for an individual patient, comprising the step of producing a pharmaceutical composition comprising at least one peptide selected from a reservoir of pre-screened TUMAPs, wherein the at least one peptide used in the pharmaceutical composition is selected for suitability in the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be adapted to produce T cell clones for downstream applications such as TCR isolation, or soluble antibodies, and other therapeutic options.

[0398] "Personalized medicine" shall mean a therapy specifically tailored for an individual patient, used exclusively for the treatment of such individual patient, including proactive personalized cancer vaccines and adoptive cell therapy using autologous patient tissue.

[0399] As used herein, the term "reservoir" refers to a group or set of peptides that have been pre-screened for immunogenicity and / or over-representation in a particular tumor type. The term "reservoir" is not intended to imply that the specific peptides included in the vaccine are pre-manufactured and stored in a physical facility, although this possibility is contemplated. It is expressly contemplated that peptides may be produced de novo for each personalized vaccine produced, or may be pre-manufactured and stored. The reservoir (e.g., in the form of a database) is composed of tumor-associated peptides that are highly overexpressed in tumor tissues of esophageal cancer patients with diverse HLA-A, HLA-B, and HLA-C alleles. It may contain MHC class I and MHC class II peptides or extended MHC class I peptides. In addition to tumor-associated peptides collected from several esophageal cancer tissues, the reservoir may also contain HLA-A*02- and HLA-A*24-tagged peptides. These peptides allow quantitative comparison of the magnitude of T cell immunity induced by TUMAPs, thus allowing important conclusions to be drawn about the vaccine's ability to elicit antitumor responses. Second, they serve as important positive control peptides derived from "non-self" antigens in cases where no vaccine-induced T cell responses to TUMAPs derived from "self" antigens are observed in patients. Third, they may allow conclusions to be drawn about the patient's immunocompetence status.

[0400] TUMAPs for the reservoir are identified using an integrated functional genomics approach (XPresident®) that combines gene expression analysis, mass spectrometry, and T-cell immunology. The approach ensures that only TUMAPs that are truly present on a high percentage of tumors but not or only minimally expressed on normal tissues are selected for further analysis. For initial peptide selection, esophageal cancer samples from patients and blood from healthy donors were analyzed in a stepwise approach: 1. HLA ligands from malignant substances were identified by mass spectrometry. 2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis was used to identify gene overexpression in malignant tissue (esophageal cancer) compared with a range of normal organs and tissues. 3. The identified HLA ligands were compared with gene expression data. Preferably, peptides that were over- or selectively presented on tumor tissues, encoded by selectively expressed or over-expressed genes as detected in step 2, were considered suitable TUMAP candidates for multi-peptide vaccines. 4. A literature search was conducted to identify additional evidence supporting the validity of the identified peptides as TUMAPs. 5. The relevance of overexpression at the mRNA level was confirmed by re-detection of selected TUMAPs from step 3 on tumor tissues and their absence (or rare) detection in healthy tissues. 6. To assess whether the selected peptides could induce T cell responses in vivo, in vitro immunogenicity assays were performed using human T cells from healthy donors and esophageal cancer patients.

[0401] In one embodiment, peptides are pre-screened for immunogenicity prior to inclusion in the reservoir. As a non-limiting example, the immunogenicity of peptides included in the reservoir is determined by a method comprising ex vivo T cell priming through repeated stimulation of CD8+ T cells from healthy donors with artificial antigen-presenting cells loaded with peptide / MHC complexes and anti-CD28 antibodies.

[0402] This method is preferable for rare cancers and patients with rare expression profiles. In contrast to multiple peptide mixtures with fixed compositions, the currently developed reservoirs allow for significantly higher matching of vaccines with the actual expression of antigens in tumors. Multitargeting approaches utilize several "off-the-shelf" peptides, selected individually or in combination, for each patient. Theoretically, an approach based on the selection of, for example, five different antigenic peptides from a library of 50 antigenic peptides alone would yield approximately 17 million possible drug product (DP) compositions.

[0403] In one aspect, peptides are selected for inclusion in the vaccine based on their suitability for an individual patient based on the methods according to the invention described herein or as follows.

[0404] HLA phenotype, transcriptomic, and peptidomic data are collected from patient tumor material and blood samples to identify the most relevant peptides for each patient, containing "reservoir" and patient-specific (i.e., mutated) TUMAPs. Peptides are selected that are selectively or overexpressed in the patient's tumor and, if possible, demonstrate strong in vitro immunogenicity when tested with the patient's individual PBMCs.

[0405] Preferably, peptides to be included in the vaccine are identified by a method comprising the steps of: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; (b) comparing the peptides identified in (a) with the peptide reservoir described above; and (c) selecting at least one peptide from the reservoir (database) that is related to the tumor-associated peptides identified in the patient. For example, TUMAPs presented by tumor samples are identified by the steps of: (a1) comparing expression data from the tumor sample with expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with sequences of MHC ligands bound to MHC class I and / or class II molecules in the tumor sample to identify MHC ligands derived from proteins overexpressed or aberrantly expressed by the tumor. Preferably, the sequences of the MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor sample and sequencing the eluted ligands. Preferably, the tumor sample and normal tissue are obtained from the same patient.

[0406] In addition to, or as an alternative to, selecting peptides using a repository (database) model, TUMAPs may be identified de novo in patients and then included in vaccines. As one example, candidate TUMAPs may be identified in patients by: (a1) comparing expression data from the tumor sample with expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins overexpressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with the sequences of MHC ligands bound to MHC class I and / or class II molecules in the tumor sample to identify MHC ligands derived from proteins overexpressed or aberrantly expressed by the tumor. As another example, proteins containing mutations specific to tumor samples may be identified by comparison with normal counterpart tissues from individual patients, and TUMAPs specifically targeting the mutations may be identified. For example, the genomes of tumors and corresponding normal tissues may be sequenced by whole genome sequencing. To discover nonsynonymous mutations in the protein-coding regions of genes, genomic DNA and RNA are extracted from tumor tissue, and normal, nonmutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMCs). The applied NGS approach is limited to resequencing of the protein-coding region (exome resequencing). For this purpose, exonic DNA from human samples is captured using a supplier-provided target enrichment kit, followed by sequencing, for example, with a HiSeq2000 (Illumina) instrument. Additionally, tumor mRNA is sequenced for direct quantification of gene expression and validation of the mutated gene being expressed in the patient's tumor. The resulting millions of sequence reads are processed through a software algorithm. The output list contains mutations and gene expression. Tumor-specific somatic mutations are identified and prioritized by comparison with PBMC-derived germline diversity. The newly identified peptides can then be tested for immunogenicity as described above for the reservoir, and candidate TUMAPs that retain appropriate immunogenicity are selected for inclusion in a vaccine.

[0407] In an exemplary embodiment, peptides to be included in the vaccine are identified by the following steps: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients by the method described above (Method); (b) comparing the peptides identified in a) with a reservoir of peptides pre-screened for immunogenicity and over-presentation in tumors compared to corresponding normal tissues; (c) selecting at least one peptide from the reservoir that is related to the tumor-associated peptides identified in the patient; and (d) optionally selecting at least one newly identified peptide in (a) and confirming its immunogenicity.

[0408] In an exemplary embodiment, peptides to be included in the vaccine are identified by the steps of: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; and (b) selecting at least one newly identified peptide in (a) and confirming its immunogenicity.

[0409] Once peptides for a personalized peptide-based vaccine are selected, the vaccine is manufactured as a liquid formulation consisting of individual peptides, preferably dissolved in 20-40% DMSO, such as about 33% DMSO, preferably about 30-35% DMSO.

[0410] Each peptide included in the product is dissolved in DMSO. The concentration of the single peptide solution must be selected depending on the number of peptides included in the product. Equal amounts of the single peptide DMSO solutions are mixed to obtain a solution containing all peptides included in the product at a concentration of approximately 2.5 mg / ml per peptide. The mixed solution is then diluted 1:3 with water for injection to obtain a concentration of 0.826 mg / ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22 μm sterile filter to obtain the final bulk solution.

[0411] The final bulk solution is filled into vials and stored at -20°C until use. Each vial contains 700 μL of solution containing 0.578 mg of each peptide, of which 500 μL (approximately 400 μg per peptide) is applied for intradermal injection.

[0412] In addition to being useful for treating cancer, the peptides of the present invention are also useful as diagnostics: because the peptides were produced from esophageal cancer cells, and because these peptides were determined to be absent or present at lower levels in normal tissue, these peptides can be used to diagnose the presence of cancer.

[0413] The presence of the claimed peptides on blood samples or tissue biopsies can assist pathologists in cancer diagnosis. Detection of specific peptides by means of antibodies, mass spectrometry, or other methods known in the art can tell a pathologist that a tissue sample is malignant, inflamed, or generally diseased, or can be used as a biomarker for esophageal cancer. The presence of peptide groups can allow for classification or subclassification of diseased tissue.

[0414] The detection of peptides on diseased tissue samples allows the evaluation of the benefits of therapies involving the immune system, especially when T lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a well-described mechanism by which infected malignant cells escape immune surveillance. The presence of peptides therefore indicates that this mechanism is not being utilized by the analyzed cells.

[0415] The peptides of the present invention may be used to analyze lymphocyte responses to these peptides, such as T cell or antibody responses to peptides or peptides complexed with MHC molecules. These lymphocyte responses can be used as prognostic markers to determine further treatment steps. These responses can also be used as surrogate response markers in immunotherapy approaches that aim to induce lymphocyte responses by different means, such as vaccination with proteins, nucleic acids, or autologous materials, or adoptive transfer of lymphocytes. In the setting of gene therapy, lymphocyte responses to peptides can be taken into account in the evaluation of side effects. Monitoring lymphocyte responses may also be a useful tool for follow-up testing of transplantation therapy, for example, for detecting graft-versus-host disease and host-versus-graft disease.

[0416] The present invention will now be described in the following examples which depict preferred embodiments thereof and with reference to the accompanying drawings, in which: For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0417] [Figure 1A]Overrepresentation of various peptides in normal tissues (white bars) and esophageal cancer (black bars) is shown. A) Gene symbol: KRT14 / KRT16, peptide STYGGGLSV (SEQ ID NO: 1). Tissues from left to right: 1 adipose tissue, 3 adrenal gland, 8 artery, 5 bone marrow, 7 brain, 5 breast, 2 cartilage, 1 central nervous system, 13 colon, 1 duodenum, 2 gallbladder, 5 heart, 14 kidney, 21 liver, 44 lung, 4 lymph node, 4 white blood cell sample, 3 ovary, 8 pancreas, 5 peripheral nerve, 1 peritoneum, 3 pituitary gland, 4 placenta, 3 pleura, 3 prostate, 6 rectus muscle, 7 salivary gland, 4 skeletal muscle, 6 skin, 2 small intestine, 4 spleen, 5 stomach, 6 testis, 3 thymus, 3 thyroid, 7 trachea, 2 ureter, 6 urinary bladder, 2 uterus, 2 veins, 6 esophagus, 16 esophageal cancer sample. The peptide was also detected in 4 / 91 lung cancer samples. Figure 1B) Gene symbol: GJB5, peptide SIFEGLLSGV (SEQ ID NO: 7). Tissues (left to right): 1 adipose tissue, 3 adrenal gland, 8 artery, 5 bone marrow, 7 brain, 5 breast, 2 cartilage, 1 central nervous system, 13 colon, 1 duodenum, 2 gallbladder, 5 heart, 14 kidney, 21 liver, 44 lung, 4 lymph node, 4 white blood cell sample, 3 ovary, 8 pancreas, 5 peripheral nerve, 1 peritoneum, 3 pituitary gland, 4 placenta, 3 pleura, 3 prostate, 6 rectus muscle, 7 salivary gland, 4 skeletal muscle, 6 skin, 2 small intestine, 4 spleen, 5 stomach, 6 testis, 3 thymus, 3 thyroid, 7 trachea, 2 ureter, 6 bladder, 2 uterus, 2 veins, 6 esophagus, 16 esophageal cancer samples. The peptide was further detected in 1 / 43 prostate cancer, 1 / 3 gallbladder cancer, 1 / 20 ovarian cancer, 5 / 91 lung cancer, and 1 / 4 bladder cancer. Figure 2C) Gene symbol: PKP3, peptide SLVSEQLEPA (SEQ ID NO: 34). Tissues (left to right): 1 adipose tissue, 3 adrenal gland, 8 artery, 5 bone marrow, 7 brain, 5 breast, 2 cartilage, 1 central nervous system, 13 colon, 1 duodenum, 2 gallbladder, 5 heart, 14 kidney, 21 liver, 44 lung, 4 lymph node, 4 white blood cell sample, 3 ovary, 8 pancreas, 5 peripheral nerve, 1 peritoneum, 3 pituitary gland, 4 placenta, 3 pleura, 3 prostate, 6 rectus muscle, 7 salivary gland, 4 skeletal muscle, 6 skin, 2 small intestine, 4 spleen, 5 stomach, 6 testis, 3 thymus, 3 thyroid, 7 trachea, 2 ureter, 6 bladder, 2 uterus, 2 veins, 6 esophagus, 16 esophageal cancer samples. The peptide was further detected in 8 / 24 colorectal cancers, 1 / 20 ovarian cancer, 1 / 46 gastric cancer, 5 / 91 lung cancer, and 2 / 4 bladder cancer. Figure 3D) Gene symbol: RNPEP, peptide YTQPFSHYGQAL (SEQ ID NO: 37).Tissues (left to right): 1 adipose tissue, 3 adrenal gland, 8 artery, 5 bone marrow, 7 brain, 5 breast, 2 cartilage, 1 central nervous system, 13 colon, 1 duodenum, 2 gallbladder, 5 heart, 14 kidney, 21 liver, 44 lung, 4 lymph node, 4 white blood cell sample, 3 ovary, 8 pancreas, 5 peripheral nerve, 1 peritoneum, 3 pituitary gland, 4 placenta, 3 pleura, 3 prostate, 6 rectus muscle, 7 salivary gland, 4 skeletal muscle, 6 skin, 2 small intestine, 4 spleen, 5 stomach, 6 testis, 3 thymus, 3 thyroid, 7 trachea, 2 ureter, 6 bladder, 2 uterus, 2 vein, 6 esophagus, 16 esophageal cancer sample. The peptide was further detected in 1 / 19 pancreatic cancer, 7 / 46 gastric cancer, and 1 / 91 lung cancer. (Figure 4E) Gene symbol: NUP155, peptide ALQEALENA (SEQ ID NO: 80). Samples from left to right: 4 cell lines (1 kidney, 1 pancreas, 1 prostate, 1 myeloid leukemia), 3 normal tissues (1 lung, 1 prostate, 1 small intestine), 47 cancer tissues (5 brain cancers, 2 breast cancers, 1 colon cancer, 2 esophageal cancers, 1 chronic leukemia, 2 liver cancers, 22 lung cancers, 7 ovarian cancers, 4 prostate cancers, 1 rectal cancer). Figure 5F) Gene symbol: KRT5, peptide SLYNLGGSKRISI (SEQ ID NO: 2). Tissues from left to right: 20 cancer tissues (9 head and neck cancers, 2 esophageal cancers, 1 esophageal and gastric cancer, 7 lung cancers, 1 bladder cancer). Figure 6G) Gene symbol: KRT5, peptide TASAITPSV (SEQ ID NO: 3). Tissues from left to right: 17 cancer tissues (2 esophageal cancers, 6 head and neck cancers, 7 lung cancers, 2 bladder cancers). Figure 7H) Gene symbol: S100A2, peptide SLDENSDQQV (SEQ ID NO: 10). Tissues from left to right: 7 cancer tissues (3 head and neck cancers, 2 esophageal cancers, 1 lung cancer, and 1 bladder cancer). Figure 8I) Gene symbol: LAMB3, peptide ALWLPTDSATV (SEQ ID NO: 11). Tissues from left to right: 12 cancer tissues (2 esophageal cancers, 1 gallbladder cancer, 8 lung cancers, and 1 skin cancer). Figure 9J) Gene symbol: IL36RN, peptide SLSPVILGV (SEQ ID NO: 13). Tissues from left to right: 26 cancer tissues (8 head and neck cancers, 3 esophageal cancers, 10 lung cancers, 3 skin cancers, 1 bladder cancer, and 1 uterine cancer). Figure 10K) Gene symbol: ANO1, peptide LLANGVYAA (SEQ ID NO: 15). Tissues from left to right: 8 cancer tissues (2 esophageal cancers, 1 gallbladder cancer, 1 liver cancer, 1 lung cancer, 1 stomach cancer, 1 bladder cancer, and 1 uterine cancer). Figure 11L) Gene symbols: F7, IGHV4-31, IGHG1, IGHG2, IGHG3, IGHG4, IGHM, peptide MISRTPEV (SEQ ID NO: 17).Tissues from left to right: 19 cancer tissues (2 esophageal cancers, 2 kidney cancers, 2 liver cancers, 9 lung cancers, 1 lymph node cancer, 1 testicular cancer, and 2 bladder cancers. Figure 12M) Gene symbol: QSER1, peptide SLNGNQVTV (SEQ ID NO: 30). Tissues from left to right: 1 cell line (1 pancreas), 14 cancer tissues (1 head and neck cancer, 1 bile duct cancer, 1 brain cancer, 1 breast cancer, 1 esophageal cancer, 1 kidney cancer, 1 lung cancer, 2 skin cancers, 3 bladder cancer, and 2 uterine cancers). Figure 13N) Gene symbol: HAS3, peptide YMLDIFHEV (SEQ ID NO: 32). Tissues from left to right: 1 normal tissue (1 uterus), 15 cancer tissues (1 brain cancer, 2 esophageal cancers, 1 gallbladder cancer, 3 head and neck cancers, 4 lung cancer, and 4 bladder cancer). Figure 14O) Gene symbol: PKP3, peptide SLVSEQLEPA (SEQ ID NO: 34). Tissues from left to right: 1 cell line (1 pancreas), 1 normal tissue (1 colon), 28 cancer tissues (6 head and neck cancers, 1 breast cancer, 1 cecum cancer, 3 colon cancers, 1 colorectal cancer, 3 esophageal cancers, 6 lung cancers, 1 ovarian cancer, 3 rectal cancers, 3 bladder cancers). Figure 15P) Gene symbol: SERPINH1, peptide GLAFSLYQA (SEQ ID NO: 40). Tissues from left to right: 3 cell lines (1 kidney, 2 pancreas), 4 normal tissues (1 adrenal gland, 1 lung, 2 placenta), 41 cancer tissues (3 head and neck cancers, 3 breast cancers, 2 colon cancers, 2 esophageal cancers, 1 gallbladder cancer, 1 liver cancer, 15 lung cancers, 1 ovarian cancer, 1 pancreatic cancer, 3 rectal cancers, 2 skin cancers, 1 stomach cancer, 4 bladder cancer, 2 uterine cancers). Figure 16Q) Gene symbol: TMEM132A, peptide ALVEVTEHV (SEQ ID NO: 56). Tissues from left to right: 7 normal tissues (5 lung, 1 thyroid, 1 trachea), 64 cancer tissues (6 head and neck cancers, 12 brain cancers, 4 breast cancers, 3 esophageal cancers, 1 gallbladder cancer, 5 kidney cancers, 21 lung cancers, 1 lymph node cancer, 7 ovarian cancers, 1 pancreatic cancer, 1 skin cancer, 2 uterine cancers). Figure 17R) Gene symbol: PRCl, peptide GLAPNTPGKA (SEQ ID NO: 57). Tissues from left to right: 14 cancer tissues (1 head and neck cancer, 1 breast cancer, 2 esophageal cancers, 6 lung cancers, 1 ovarian cancer, 1 skin cancer, 1 bladder cancer, 1 uterine cancer). Figure 18S) Gene symbol: MAPK6, peptide LILESIPVV (SEQ ID NO: 58). Tissues from left to right: 2 cell lines (1 blood cell, 1 skin), 25 cancer tissues (5 head and neck cancers, 1 colon cancer, 2 esophageal cancers, 1 leukemia, 8 lung cancers, 2 lymph node cancers, 3 skin cancers, 2 bladder cancers, 1 uterine cancer).Figure 19T) Gene symbol: PPP4R1, peptide SLLDTLREV (SEQ ID NO: 59). Tissues from left to right: 1 normal tissue (1 small intestine), 8 cancer tissues (1 head and neck cancer, 2 esophageal cancer, 4 lung cancer, 1 ovarian cancer). Figure 20U) Gene symbol: TP63, peptide VLVPYEPPQV (SEQ ID NO: 77). Tissues from left to right: 2 normal tissues (1 esophagus, 1 trachea), 47 cancer tissues (8 head and neck cancer, 4 esophageal cancer, 1 gallbladder cancer, 14 lung cancer, 7 lymph node cancer, 2 prostate cancer, 1 skin cancer, 8 bladder cancer). Figure 21V) Gene symbol: KIAA0947, peptide AVLPHVDQV (SEQ ID NO: 81). Tissues from left to right: 3 cell lines (1 blood cell, 1 pancreas), 12 cancer tissues (5 brain cancer, 2 esophageal cancer, 1 lung cancer, 3 lymph node cancer, 1 uterine cancer). [Figure 1B] Same as above [Figure 1C] Same as above [Figure 1D] Same as above [Figure 1E] Same as above [Figure 1F] Same as above [Figure 1G] Same as above [Figure 1H] Same as above [Figure 1I] Same as above [Figure 1J] Same as above [Figure 1K] Same as above [Figure 1L] Same as above [Figure 1M] Same as above [Figure 1N] Same as above [Figure 1O] Same as above [Figure 1P] Same as above [Figure 1Q] Same as above [Figure 1R] Same as above [Figure 1S] Same as above [Figure 1T] Same as above [Figure 1U] Same as above [Figure 1V] Same as above [Figure 2A]Figure 2 shows exemplary expression profiles of the genes of interest of the present invention that are highly overexpressed or exclusively expressed in esophageal cancer in a panel of normal tissues (white bars) and 11 esophageal cancer samples (black bars). Tissues from left to right: 7 arteries, 1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 4 bone marrow, 1 colon, 2 esophagus, 2 gallbladder, 1 kidney, 6 lymph nodes, 1 pancreas, 1 pituitary gland, 1 rectum, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thymus, 1 thyroid, 5 trachea, 1 bladder, 1 breast, 3 ovaries, 3 placenta, 1 prostate, 1 testis, 1 uterus, and 11 esophageal cancer samples. Figure 2A) Gene symbol: PTHLH; Figure 2B) Gene symbol: KRT14; Figure 2C) Gene symbol: FAM83A; Figure 2D) Gene symbol: PDPN. [Figure 2B] Same as above [Figure 2C] Same as above [Figure 2D] Same as above [Figure 3-1] Exemplary results of peptide-specific in vitro CD8+ T cell responses from healthy HLA-A*02+ donors are shown, i.e., exemplary immunogenicity data: flow cytometry results after peptide-specific multimer staining. Figure 3A) Gene symbol: SF3B3, peptide ELDRTPPEV (SEQ ID NO: 97); Figure 3B) Gene symbol: TNC, peptide AMTQLLAGV (SEQ ID NO: 101). CD8+ T cells were also primed with artificial APCs coated with anti-CD28 mAb and HLA-A*02 complexed with SEQ ID NO: 5 peptide (C, left panel), SEQ ID NO: 2 peptide (D, left panel), and SEQ ID NO: 77 peptide (E, left panel), respectively. After three cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A*02 / SEQ ID NO: 5 (C), A*02 / SEQ ID NO: 2 (D), or A*02 / SEQ ID NO: 77 (E). The right panels (C, D, and E) show control staining of cells stimulated with an irrelevant A*02 / peptide complex. Viable singlet cells were gated on CD8+ lymphocytes. Boolean gating helped eliminate false-positive events detected by multimers specific for different peptides. The frequency of specific multimer+ cells among CD8+ lymphocytes is shown. [Figure 3-2] Same as above [Figure 3-3] Same as above [Example]

[0418] Example 1 Identification and quantification of tumor-associated peptides displayed on the cell surface Tissue samples Patient tumor tissues were obtained from Asterand (Detroit, USA and Royston, Herts, UK); ProteoGenex Inc. (Culver City, CA, USA); Tissue Solutions Ltd. (Glasgow, UK); and University Hospital of Tubingen. Normal tissues were obtained from Asterand (Detroit, USA and Royston, Herts, UK); Bio-Options Inc. (CA, USA); BioServe (Beltsville, MD, USA); Capital BioScience Inc. (Rockville, MD, USA); Geneticist Inc. (Glendale, CA, USA); University Hospital of Geneva; University Hospital of Heidelberg; Kyoto Prefectural University of Medicine (KPUM); University Hospital Munich; ProteoGenex Inc. (Culver City, CA, USA); University Hospital of Tubingen; and Tissue Solutions Ltd. (Glasgow, UK). Informed consent was obtained from all patients before surgery or autopsy. Tissues were shock-frozen immediately after resection and stored below -70°C until TUMAP isolation.

[0419] Isolation of HLA peptides from tissue samples HLA peptide pools from shock-frozen tissue samples were obtained from solid tissues by immunoprecipitation using the HLA-A*02-specific antibody BB7.2, the HLA-A, -B, and -C-specific antibody W6 / 32, CNBr-activated Sepharose, acid treatment, and ultrafiltration according to a slightly modified protocol ( Falk et al., 1991 ; Seeger et al., 1999 ).

[0420] mass spectrometry The resulting HLA peptide pool was separated according to their hydrophobicity by reversed-phase chromatography (nanoAcquity UPL C system, Waters), and the eluted peptides were analyzed in an LTQ-velos and fusion hybrid mass spectrometer (ThermoElectron) equipped with an ESI source. The peptide pool was directly loaded onto an analytical fused silica microcapillary column (75 μm i.d. × 250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) at a flow rate of 400 nL / min. Peptides were subsequently separated using a two-step 180-min binary gradient from 10% to 33% B at a flow rate of 300 nL / min. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Gold-coated glass capillaries (PicoTip, New Objective) were used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using the TOP5 strategy. Briefly, a scan cycle began with a high-mass-accuracy full scan in the Orbitrap (R = 30,000), followed by an MS / MS scan of the five most abundant precursor ions, also in the Orbitrap (R = 7,500), with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted using SEQUEST and additional manual adjustments. Identified peptide sequences were confirmed by comparison of the generated native peptide fragmentation patterns with those of sequence-identical synthetic reference peptides.

[0421] Label-free relative LC-MS quantification was performed by ion counting, i.e., by extracting and analyzing LC-MS features (Mueller et al., 2007). The method assumes that the LC-MS signal area of ​​a peptide correlates with its abundance in the sample. The extracted features were further processed by charge-state deconvolution and retention time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to combine quantitative data from different samples and tissue-to-peptide presentation profiles. Quantitative data were normalized using a two-stage method according to central tendency, which accounts for technical and biological intra-replicate variability. In this way, each identified peptide can be correlated with quantitative data, allowing for relative quantification between samples and tissues. Furthermore, all quantitative data obtained for peptide candidates was manually inspected to ensure data consistency and confirm the accuracy of the automated analysis. Presentation profiles were calculated for each peptide, showing the average sample presentation as well as replicate variability. The profile juxtaposes the esophageal cancer samples to a baseline of normal tissue samples.

[0422] The presentation profile of an exemplary over-presented peptide is shown in Figure 1. The presentation scores of representative peptides are shown in Table 8.

[0423] Table 8: Presentation Score. The table lists peptides that are very highly over-represented in tumors compared to a normal tissue panel (+++), highly over-represented in tumors compared to a normal tissue panel (++), and over-represented in tumors compared to a normal tissue panel (+). The normal tissue panel consisted of adipose tissue, adrenal glands, arteries, veins, bone marrow, brain, central and peripheral nerves, colon, rectum, small intestine including duodenum, esophagus, gallbladder, heart, kidneys, liver, lungs, lymph nodes, mononuclear white blood cells, pancreas, peritoneum, pituitary gland, pleura, salivary gland, skeletal muscle, skin, spleen, stomach, thymus, thyroid, trachea, ureter, and bladder. [Table 8-1] [Table 8-2] [Table 8-3]

[0424] Example 2 Expression profiling of genes encoding the peptides of the present invention The over- or specific expression of peptides on tumor cells compared to normal cells is sufficient for their usefulness in immunotherapy, and some peptides are tumor-specific, even though their origin proteins are also present in normal tissues. Nevertheless, mRNA expression profiling can increase the level of safety in selecting peptide targets for immunotherapy. In particular, for therapeutic options with high safety risks, such as affinity-matured TCRs, ideal target peptides are derived from proteins that are specific to tumors and not found on normal tissues.

[0425] RNA origin and preparation Surgically removed tissue specimens were provided as described above after informed consent was obtained from each patient (see Example 1). Tumor tissue specimens were snap-frozen immediately after surgery and then homogenized using a mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI Reagent (Ambion, Darmstadt, Germany), followed by purification with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

[0426] Total RNA from tumor tissues for RNA-Seq experiments was obtained from Proteo Genex Inc. (Culver City, CA, USA); Tissue Solutions Ltd. (Glasgow, UK). Total RNA from healthy human tissues for RNA-Seq experiments was obtained from Asterand (Detroit, USA and Royston, Herts, UK); Proteo Genex Inc. (Culver City, CA, USA); Geneticist Inc. (Glendale, CA, USA); Istituto Nazionale Tumori "Pascale", Molecular Biology and Viral Oncology Unit (IRCCS) (Naples, Italy); University Hospital of Heidelberg (Germany); and BioCat GmbH (Heidelberg, Germany).

[0427] The quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip kit (Agilent).

[0428] RNAseq experiments Gene expression analysis of tumor and normal tissue RNA samples was performed by next-generation sequencing (RNAseq) at CeGaT (Tübingen, Germany). Briefly, sequencing libraries were generated using the Illumina HiSeq v4 reagent kit, including RNA fragmentation, cDNA conversion, and addition of sequencing adapters, according to the vendor's protocol (Illumina Inc, San Diego, CA, USA). Libraries from multiple samples were mixed equimolarly and sequenced on an Illumina HiSeq 2500 sequencing instrument according to the manufacturer's instructions, generating 50-bp single-end reads. Processed reads were mapped to the human genome (GRCh38) using STAR software. Expression data are provided at the transcript level as RPKM (reads per kilobase per million mapped reads, generated by the software Cufflinks) and at the exon level (total reads, generated by the software Bedtools) based on annotations in the Ensembl sequence database (Ensembl77). Exon reads are normalized for exon length and alignment size to obtain RPKM values.

[0429] Representative expression profiles of source genes of the present invention that are highly overexpressed or exclusively expressed in esophageal cancer are shown in Figure 2. Expression scores of additional exemplary genes are shown in Table 9.

[0430] Table 9: Expression Score. The table lists peptides derived from genes that are very highly overexpressed (+++) in tumors compared to a normal tissue panel, highly overexpressed (++) in tumors compared to a normal tissue panel, and overexpressed (+) in tumors compared to a normal tissue panel. Baseline scores were calculated from normal tissue measurements in adipose tissue, adrenal gland, artery, bone marrow, brain, colon, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, pancreas, pituitary gland, rectum, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid, trachea, bladder, and veins. [Table 9-1] [Table 9-2]

[0431] Example 3 In vitro immunogenicity of MHC class I-presented peptides To obtain information about the immunogenicity of the TUMAPs of the present invention, we performed studies using an in vitro T cell priming assay based on repeated stimulation of CD8+ T cells with artificial antigen-presenting cells (aAPCs) loaded with peptide / MHC complexes and anti-CD28 antibodies. In this way, we were able to demonstrate the immunogenicity of the HLA-A*0201-restricted TUMAPs of the present invention and demonstrated that these peptides are T cell epitopes against which CD8+ precursor T cells are present in humans (Table 10).

[0432] In vitro priming of CD8+ T cells To perform ex vivo stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD28 antibodies, we first isolated CD8+ T cells from fresh HLA-A*02 leukapheresis products through positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) from healthy donors obtained from the University Clinics Mannheim, Germany, after informed consent.

[0433] PBMCs and isolated CD8+ lymphocytes or PBMCs were cultured in T cell medium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U / ml penicillin / 100 μg / ml streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany), and 20 μg / ml gentamicin (Cambrex) until use. 2.5 ng / ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U / ml IL-2 (Novartis Pharma, Nurnberg, Germany) were also added to TCM at this stage.

[0434] Generation of pMHC / anti-CD28 coated beads, T cell stimulation, and readout were performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and eight different pMHC molecules per readout condition.

[0435] Purified costimulatory mouse IgG2a anti-human CD28 Ab9.3 (Jung et al., 1987) was chemically biotinylated using sulfo-N-hydroxysuccinimide biotin as recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were 5.6 μm diameter streptavidin-coated polystyrene particles (Bangs Laboratories, Illinois, USA).

[0436] The pMHC used for positive and negative control stimulation were A*0201 / MLA-001 (peptide ELAGIGILTV (sequence number 102) derived from modified Melan-A / MART-1) and A*0201 / DDX5-001 (YLLPAIVHI derived from DDX5, sequence number 103), respectively.

[0437] 800,000 beads / 200 μl were coated in a 96-well plate in the presence of 4 × 12.5 ng of different biotin pMHC, washed, and subsequently added with 600 ng of biotin anti-CD28 in a volume of 200 μl. 1 × 10 beads were cultured in 200 μl of TCM supplemented with 5 ng / ml IL-12 (PromoCell). 6 2 × 10 CD8 T cells 5 Stimulation was initiated in 96-well plates by co-incubation with 1000 washed coated beads at 37°C for 3 days. Half of the medium was then replaced with fresh TCM supplemented with 80 U / ml IL-2, and incubation continued at 37°C for 4 days. This stimulation cycle was performed a total of three times. For pMHC multimer readout using eight different pMHC molecules per condition, a two-dimensional combinatorial coding approach was used as previously described (Andersen et al., 2012) with minor modifications, including conjugation to five different fluorescent dyes. Finally, multimer analysis was performed by staining cells with Live / Dead near-infrared dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany), and fluorescent pMHC multimers. Analysis was performed using a BD LSRII SORP hemocytometer equipped with the appropriate lasers and filters. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis was evaluated using FlowJo software (Tree Star, Oregon, USA). Specific multimer+ CD8+ lymphocytes were detected by in vitro stimulation compared with negative control stimulation. Immunogenicity of a given antigen was detected if at least one evaluable in vitro stimulation well from a single healthy donor was found to contain specific CD8+ T cell lines after in vitro stimulation (i.e., the well contained at least 1% specific multimer+ among CD8+ T cells, and the percentage of specific multimer+ cells was at least 10-fold higher than the median value of the negative control stimulation).

[0438] In vitro immunogenicity of esophageal cancer peptides To test HLA class I peptides, in vitro immunogenicity could be demonstrated by generating peptide-specific T cell lines. Exemplary flow cytometry results after TUMAP-specific multimer staining for two peptides of the invention (SEQ ID NO: 97 and SEQ ID NO: 101) along with the corresponding negative control are shown in Figure 3. Results for five peptides from the invention are summarized in Table 10A.

[0439] Table 10A: In vitro immunogenicity of HLA class I peptides of the present invention Exemplary results of in vitro immunogenicity experiments of the peptides of the invention carried out by the Applicant: <20% = +; 20% - 49% = ++; 50% - 69% = +++; >= 70% = ++++ [Table 10A]

[0440] Table 10B: In vitro immunogenicity of HLA class I peptides of the present invention 1 shows exemplary results of in vitro immunogenicity experiments of the peptides of the present invention carried out by the applicant. The results of the in vitro immunogenicity experiments are shown. The percentage of positive wells and donors (within assessable ranges) is summarized as shown: <20% = +; 20% - 49% = ++; 50% - 69% = +++; >= 70% = ++++ [Table 10B]

[0441] Example 4 Peptide synthesis All peptides were synthesized using standard, well-established solid-phase peptide synthesis using the Fmoc strategy. The identity and purity of individual peptides were determined by mass spectrometry and analytical RP-HPLC. Peptides were obtained as white to off-white lyophilizates (trifluoroacetate salts) with purity >50%. All TUMAPs are preferably administered as trifluoroacetate or acetate salts, although other salt forms are also possible.

[0442] Example 5 MHC binding assay Candidate peptides for T cell-based therapy according to the present invention were further tested for their MHC binding ability (affinity). Individual peptide-MHC complexes were generated by UV-ligand exchange, and the UV-sensitive peptide was cleaved upon UV irradiation and exchanged with the peptide of interest to be analyzed. Only peptide candidates that could effectively bind and stabilize peptide-receptor MHC molecules prevented MHC complex dissociation. To determine the yield of the exchange reaction, an ELISA based on the detection of the light chain (β2m) of the stabilized MHC complex was performed. The assay was performed as generally described in Rodenko et al. (Rodenko et al., 2006).

[0443] A 96-well MAXISorp plate (NUNC) was coated with 2 μg / ml streptavidin in PBS overnight at room temperature, washed four times, and blocked for 1 hour at 37°C in 2% BSA containing blocking buffer. Refolded HLA-A*020102:01 / MLA-001 monomer served as a standard covering a range of 15-500 ng / ml. The peptide-MHC monomer for the UV exchange reaction was diluted 100-fold in blocking buffer. Samples were incubated for 1 hour at 37°C, washed four times, incubated with 2 μg / ml HRP-conjugated anti-β2m for 1 hour at 37°C, washed again, and detected with TMB solution quenched with NH2SO4. Absorbance was measured at 450 nm. For the generation and manufacture of antibodies or fragments thereof, and / or T cell receptors or fragments thereof, candidate peptides that exhibit high exchange yields (preferably greater than 50%, most preferably greater than 75%) are generally preferred, as they exhibit sufficient binding activity to MHC molecules to prevent dissociation of the MHC complex.

[0444] Table 11: MHC class I binding scores. Binding of HLA class I-restricted peptides to HLA-A*02:01 varied with peptide exchange yield: >10% = +; >20% =++; >50 = +++; >75% = ++++ [Table 11-1] [Table 11-2] [Table 11-3]

[0445] Example 6 Absolute quantification of tumor-associated peptides displayed on the cell surface The generation of binders, such as antibodies and / or TCRs, is a laborious process and may only be performed for a few select targets. For tumor-associated and specific peptides, selection criteria include, but are not limited to, the exclusivity of presentation and the density of peptides presented on the cell surface. Quantifying TUMAP copies per cell in solid tumor samples requires absolute quantification of isolated TUMAPs, the efficiency of TUMAP isolation, and cell counting of the tissue sample being analyzed.

[0446] Peptide quantification by nanoLC-MS / MS To accurately quantify peptides by mass spectrometry, a calibration curve for each peptide was constructed using an internal standard. The internal standard was a double-isotopically labeled variant of each peptide; that is, two isotopically labeled amino acids were included in the TUMAP synthesis. It differs from tumor-associated peptides only in mass but does not exhibit any other physicochemical properties (Anderson et al., 2012). The internal standard was added to each MS sample to normalize all MS signals to the internal standard's MS signal and account for potential technical variations between MS experiments.

[0447] Calibration curves were generated in at least three different matrices: HLA peptide eluates from natural samples as well as routine MS samples, and each preparation was measured in duplicate MS experiments. For evaluation, MS signals were normalized to those of the internal standard, and calibration curves were calculated by logistic regression.

[0448] For quantification of tumor-associated peptides from tissue samples, an internal standard was also added to each sample, and the MS signal was normalized to the internal standard and quantified using a peptide calibration curve.

[0449] Efficiency of peptide / MHC isolation As with any protein purification process, isolation of proteins from tissue samples involves some loss of target protein. To determine the efficiency of TUMAP isolation, peptide / MHC complexes were generated for all TUMAPs selected for absolute quantification. To distinguish these complexes from native peptide / MHC complexes, monoisotopically labeled versions of TUMAPs were used; i.e., one isotope-labeled amino acid was included in the TUMAP synthesis. These complexes were added to freshly prepared tissue lysates as early as possible in the TUMAP isolation procedure and then captured like native peptide / MHC complexes in the following affinity purification. Therefore, measuring the recovery of monoisotopically labeled TUMAPs allows conclusions regarding the isolation efficiency of individual native TUMAPs.

[0450] Isolation efficiency was analyzed in a small number of samples and was comparable between these tissue samples. In contrast, isolation efficiency varied between individual peptides. This suggests that isolation efficiency, although determined only in a limited number of tissue samples, may be extrapolated to any other tissue specimen. However, isolation efficiency may not extrapolate from one peptide to another, so each TUMAP must be analyzed individually.

[0451] Cell counting in solid-frozen tissue To measure cell numbers in tissue samples subjected to absolute peptide quantification, we applied DNA content analysis. This method is applicable to a wide range of samples of different origins, most importantly, frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, tissue samples are processed into homogenous lysates, from which small lysate aliquots are removed. The aliquots are divided into three portions, from which DNA is isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). A fluorescence-based DNA quantification assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) is used to quantify the total DNA content from each DNA isolation in at least two replicates.

[0452] To calculate cell number, a DNA standard curve was generated from aliquots of single healthy blood cells with a defined range of cell numbers. The standard curve was used to calculate total cell content from the total DNA content from each DNA isolation. Given the known volumes of the lysate aliquots and the total lysate volume, the average total cell number of the tissue samples used for peptide isolation was extrapolated.

[0453] Peptide copy number per cell Using the data from the previous experiments, we calculated the TUMAP copy number per cell by dividing the total peptide amount in the sample by the total cell number, and then dividing by the isolation efficiency. The cellular copy numbers of selected peptides are shown in Table 12.

[0454] Table 12: Absolute copy numbers. The table lists the results of absolute peptide quantification in NSCLC tumor samples. The median copy number per cell is shown for each peptide: <100 = +; >=100 =++; >=1,000 +++; >=10,000 = ++++. The number of samples for which evaluable high-quality MS data was available is shown.

Table 12

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Claims

1. A peptide or a pharmaceutically acceptable salt thereof, which consists of the amino acid sequence shown in SEQ ID NO: 1, has the ability to bind to an MHC class I molecule, and when bound to the MHC class I molecule, becomes capable of being recognized by CD8 T cells.

2. A peptide or a pharmaceutically acceptable salt thereof according to claim 1, which contains a non-peptide bond.

3. A fusion protein comprising the peptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof and 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii).

4. An antibody or antigen-binding fragment thereof that specifically binds to the peptide of claim 1 or 2, or the peptide of claim 1 or 2 bound to an MHC class I molecule; or A soluble or membrane-bound antibody or antigen-binding fragment thereof that specifically binds to a peptide described in claim 1 or 2, or a peptide described in claim 1 or 2 bound to an MHC class I molecule, the antibody or antigen-binding fragment thereof being (i) a monoclonal antibody and / or a humanized antibody or antigen-binding fragment thereof, or (ii) a bispecific antibody and / or a chimeric antibody or antigen-binding fragment thereof.

5. A T cell receptor reactive with an HLA ligand bound to an MHC class I molecule, said ligand being a peptide according to claim 1. a T cell receptor, wherein the T cell receptor is a soluble or membrane-bound T cell receptor, or The T cell receptor is provided as a soluble molecule and possesses additional effector functions, including an immunostimulatory domain or a toxin.

6. A nucleic acid encoding the peptide or pharmaceutically acceptable salt thereof of claim 1, the antibody or antigen-binding fragment thereof of claim 4, or the T cell receptor of claim 5, wherein the nucleic acid is linked to a heterologous promoter sequence.

7. An expression vector containing the nucleic acid of claim 6.

8. A recombinant host cell comprising the peptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, the nucleic acid of claim 6, or the expression vector of claim 7, or the host cell is an antigen-presenting cell including a dendritic cell.

9. 9. A drug for use in medicine, comprising the peptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, the fusion protein of claim 3, the nucleic acid of claim 6, the expression vector of claim 7, or the recombinant host cell of claim 8.

10. 10. A method for producing the peptide or a pharmaceutically acceptable salt thereof of claim 1, the antibody or antigen-binding fragment thereof of claim 4, or the T cell receptor of claim 5, comprising the steps of culturing a recombinant host cell of claim 8 that presents the peptide of claim 1, expresses the nucleic acid of claim 6, or has the expression vector of claim 7, and isolating the peptide or a pharmaceutically acceptable salt thereof, the antibody or antigen-binding fragment thereof, or the T cell receptor from the recombinant host cell or its culture medium.

11. 10. An in vitro method for producing activated T lymphocytes, comprising ex vivo contacting T cells with antigen-loaded human MHC class I molecules expressed on the surface of suitable antigen-presenting cells or on the surface of an artificial construct that mimics an antigen-presenting cell, for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is the peptide of claim 1 or a pharmaceutically acceptable salt thereof.

12. Activated T lymphocytes produced by the in vitro method of claim 11, which selectively recognize cells presenting the peptide of claim 1.

13. A therapeutic agent for killing target cells in a patient, comprising the activated T lymphocytes of claim 12 and presenting the peptide of claim 1.

14. 13. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of the peptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, the fusion protein of claim 3, the antibody or antigen-binding fragment thereof of claim 4, the T cell receptor of claim 5, the nucleic acid of claim 6, the expression vector of claim 7, the recombinant host cell of claim 8, and the activated T lymphocyte of claim 12, and a pharmaceutically acceptable carrier, or the pharmaceutical composition further comprising an adjuvant.

15. Use of the peptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, the fusion protein of claim 3, the antibody or antigen-binding fragment thereof of claim 4, the T cell receptor of claim 5, the nucleic acid of claim 6, the expression vector of claim 7, the recombinant host cell of claim 8, the activated T lymphocyte of claim 12, or the pharmaceutical composition of claim 14 in the manufacture of a cancer therapeutic agent.

16. 16. The use according to claim 15, wherein the cancer is selected from the group consisting of esophageal cancer, non-small cell lung cancer, small cell lung cancer, renal cell, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, breast cancer, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder and bile duct cancer, and other tumors that show over-expression of the protein from which the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is derived.

17. 16. A drug for diagnosing and / or treating cancer, comprising the peptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, the fusion protein of claim 3, the antibody or antigen-binding fragment thereof of claim 4, the T cell receptor of claim 5, the nucleic acid of claim 6, the expression vector of claim 7, the recombinant host cell of claim 8, the activated T lymphocyte of claim 12, or the pharmaceutical composition of claim 14.

18. The drug of claim 17, wherein the cancer is selected from the group consisting of esophageal cancer, non-small cell lung cancer, small cell lung cancer, renal cell, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, breast cancer, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder and bile duct cancer, and other tumors that exhibit over-expression of proteins from which peptides consisting of an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 1 are derived.

19. (a) a container comprising the peptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, the fusion protein of claim 3, the antibody of claim 4 or an antigen-binding fragment of said antibody, the T cell receptor of claim 5, the nucleic acid of claim 6, the expression vector of claim 7, the recombinant host cell of claim 8, or the activated T lymphocyte of claim 12 in solution or lyophilized form; and (b) a second container containing a diluent or reconstitution solution for the lyophilisate; or In addition to (a) and (b), the kit further comprises one or more selected from the group consisting of (i) a buffer solution, (ii) a diluent, (iii) a filter, (iv) a needle, and (v) a syringe.

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