Novel peptides and peptide combinations for immunotherapy of esophageal and other cancers

Abstract

The present invention relates to peptides, proteins, nucleic acids, and cells for use in immunotherapy methods. In particular, the present invention relates to cancer immunotherapy. The present invention also relates to tumor-associated T cell (CTL) peptide epitopes, used alone or in combination with other tumor-associated peptides (which can, for example, serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses or stimulate T cells in vitro and are then delivered to patients). Peptides that bind to major histocompatibility complex (MHC) molecules or peptides of similar ilk can also be targets for antibodies, soluble T cell receptors, and other binding molecules.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680038852.7 (filing date: July 5, 2016, invention name: Novel peptides and peptide compositions for immunotherapy of esophageal cancer and other cancers).

[0002] The present invention relates to peptides, proteins, nucleic acids, and cells for use in immunotherapy methods. In particular, the present invention relates to cancer immunotherapy. The present invention also relates to tumor-associated T cell (CTL) peptide epitopes, used alone or in combination with other tumor-associated peptides (active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses or stimulate T cells in vitro and are then delivered to patients). Peptides that bind to major histocompatibility complex (MHC) molecules or peptides of similar types can also be targets for antibodies, soluble T cell receptors, and other binding molecules.

[0003] The present invention relates to several novel peptide sequences and their variants, which are derived from HLA class I molecules of human tumor cells and can be used in vaccine compositions for eliciting anti-tumor immune responses or as targets for developing drugs / immunologically active compounds and cells. Background of the Invention

[0004] Esophageal cancer is the eighth most common cancer worldwide, with a five-year incidence of 464,063 patients in 2012. The mortality rate is very similar to the incidence rate (400,169 vs. 455,784 in 2012), suggesting a high mortality rate for esophageal cancer (World Cancer Report, 2014; Ferlay et al., 2013; Bray et al., 2013).

[0005] Squamous cell carcinoma and adenocarcinoma represent the two most common subtypes of esophageal cancer. Both subtypes are more common in men than in women, but they exhibit distinct geographic distributions. The greatest risk factors for developing esophageal squamous cell carcinoma include alcohol and smoking, while esophageal adenocarcinoma is primarily associated with obesity and gastroesophageal reflux disease. The incidence of esophageal adenocarcinoma has steadily increased in high-income countries, likely due to rising rates of obesity and gastroesophageal reflux disease, as well as changes in the classification of tumors at the gastroesophageal junction. Rare subtypes of esophageal cancer include neuroendocrine carcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, mucoepidermoid carcinoma, mixed adenoneuroendocrine carcinoma, various sarcomas, and melanoma (World Cancer Report, 2014).

[0006] The primary treatment strategy for esophageal cancer depends on tumor stage and location, histologic type, and patient condition. Surgery alone is insufficient except in a small subset of patients with squamous cell carcinoma. Generally, surgery should be combined with preoperative and postoperative chemotherapy or preoperative chemoradiotherapy, as preoperative or postoperative radiotherapy alone has been shown to provide no survival benefit. Chemotherapy regimens include oxaliplatin plus fluorouracil, carboplatin plus paclitaxel, cisplatin plus fluorouracil, FOLFOX, and cisplatin plus irinotecan. Patients whose tumors are HER2-positive should be treated with a combination of cisplatin, fluorouracil, and trastuzumab according to gastric cancer treatment guidelines, as randomized data on targeted therapies for esophageal cancer are very limited (Stahl et al., 2013; Leitlinie Magenkarzinom, 2012).

[0007] In general, most types of esophageal cancer are well treated if they are early-stage tumors, but treatment success is very limited in late-stage tumors. Therefore, developing new screening programs may be very effective in reducing esophageal cancer-related mortality (World Cancer Report, 2014).

[0008] Immunotherapy may be a promising new approach for treating advanced esophageal cancer. Several cancer-related genes and cancer-testis antigens have been shown to be overexpressed in esophageal cancer, including various MAGE genes, NY-ESO-1, and EpCAM (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 represent very interesting targets for immunotherapy, many of which are currently under investigation for the treatment of other malignancies (ClinicalTrials.gov, 2015). Furthermore, PD-L1 and PD-L2 have been described as upregulated in esophageal cancer and associated with a poor prognosis. Therefore, patients with esophageal cancer whose tumors are PD-L1 positive may benefit from anti-PD-L1 immunotherapy (Ohigashi et al., 2005).

[0009] Currently, clinical data on immunotherapy approaches for esophageal cancer remain relatively limited, as only a limited number of early-phase clinical trials have been completed (Toomey et al., 2013). In a phase I trial, patients with advanced esophageal cancer were given a vaccine containing proteins 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), with modest results (Kono et al., 2009). In a phase I / II study, in vitro stimulation with autologous tumor cells and interleukin 2 followed by intratumoral infusion of activated T cells induced complete or partial tumor responses in 4 of 11 patients (Toh et al., 2000; Toh et al., 2002). Further clinical trials are currently underway to evaluate the effects of different immunotherapies on esophageal cancer, including follow-up cell therapy (NCT01691625, NCT01691664, NCT01795976, NCT02096614, NCT02457650), vaccination strategies (NCT01143545, NCT01522820), and anti-PD-L1 therapy (NCT02340975) (ClinicalTrials.gov, 2015).

[0010] Given the severe side effects and costs associated with cancer treatment, there is a need to identify factors that can be used to treat 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, to better diagnose cancer, assess prognosis, and predict treatment success.

[0011] Cancer immunotherapy represents an option for specifically targeting cancer cells while minimizing side effects. Cancer immunotherapy exploits the presence of tumor-associated antigens.

[0012] The current classification of tumor-associated antigens (TAAs) mainly includes the following groups:

[0013] a) Cancer-testis antigens: The first TAAs recognized by T cells belong to this class of antigens. Because their members are expressed in histologically diverse human tumors, normal tissues, exclusively in testicular spermatocytes / spermatogonia, and occasionally in the placenta, they were initially referred to as cancer-testis (CT) antigens. Because testicular cells do not express HLA class I and II molecules, these antigens are not recognized by T cells in normal tissues and are therefore considered immunologically tumor-specific. Well-known examples of CT antigens include members of the MAGE family and NY-ESO-1.

[0014] b) Differentiation antigens: Both tumors and normal tissues (from which the tumor originates) contain TAAs. Most known differentiation antigens are found in melanoma and normal melanocytes. Many of these melanocyte lineage-associated proteins are involved in melanin biosynthesis, and therefore are not tumor-specific, but are nonetheless widely used in cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan-A / MART-1 in melanoma or PSA in prostate cancer.

[0015] c) Overexpressed TAAs: Genes encoding ubiquitously expressed TAAs have been detected in histologically diverse tumors as well as in many normal tissues, generally at low expression levels. It is possible that many epitopes processed and potentially presented by normal tissues are below the threshold for T cell recognition, and their overexpression in tumor cells can trigger anti-cancer responses by breaking previously established tolerance. Typical examples of such TAAs include Her-2 / neu, survivin, telomerase, and WT1.

[0016] d) Tumor-specific antigens: These unique TAAs arise from mutations in normal genes (e.g., β-catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and / or progression. Tumor-specific antigens can generally induce a strong immune response without the risk of autoimmune reactions in normal tissues. On the other hand, these TAAs are in most cases only associated with the exact tumor on which they are identified, and are not usually shared between many individual tumors. In the case of proteins containing tumor-specific (associated) isoforms, peptide tumor specificity (or association) may also occur if the peptide is derived from a tumor (associated) exon.

[0017] e) TAAs resulting from aberrant post-translational modifications: These TAAs may be produced from proteins that are neither specific nor overexpressed in tumors, but are nonetheless tumor-associated due to post-translational processing that is primarily active on tumors. These TAAs arise from altered glycosylation patterns that result in the production of novel epitopes on MUC1 by tumors or events such as protein splicing during degradation, which may or may not be tumor-specific.

[0018] f) Oncovirus proteins: These TTAs are viral proteins that play a key role in carcinogenesis and, because they are foreign (non-human), are able to stimulate T cell responses. Examples of such proteins are the human papillomavirus type 16 proteins, E6 and E7, which are expressed in cervical cancer.

[0019] T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by major histocompatibility complex (MHC) molecules. The antigens recognized by tumor-specific T lymphocytes, i.e., their epitopes, can be derived from all types of proteins, such as enzymes, receptors, and transcription factors. These are expressed in cells of the corresponding tumor and their expression is usually upregulated compared to unaltered cells of the same origin.

[0020] There are two classes of MHC molecules: MHC class I and MHC class II. MHC class I molecules consist of an α heavy chain and β-2-microglobulin, while MHC class II molecules consist of one α and one β chain. Their three-dimensional structure forms a binding groove for non-covalent interactions with peptides.

[0021] MHC class I molecules are found on most nucleated cells. They present peptides primarily from endogenous proteins, defective ribosomal products (DRIPs), and peptides generated by cleavage of larger peptides. However, peptides originating from endosomal structures or exogenous sources are also frequently found on MHC class I molecules. This non-classical presentation of class I molecules is referred to in the literature as cross-presentation (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules are primarily found on professional antigen-presenting cells (APCs) and primarily present peptides from exogenous or transmembrane proteins that, for example, are taken up by APCs during endocytosis and subsequently processed.

[0022] Complexes of peptides and MHC class I are recognized by CD8-positive T cells bearing the corresponding T cell receptor (TCR), while complexes of peptides and MHC class II molecules are recognized by CD4-positive helper T cells bearing the corresponding TCR. Therefore, it is generally accepted that TCR, peptide, and MHC are presented in a 1:1:1 stoichiometry.

[0023] CD4-positive helper T cells play an important role in inducing and maintaining effective CD8-positive cytotoxic T cell responses. The identification of CD4-positive T cell epitopes derived from tumor-associated antigens (TAAs) may be important for the development of pharmaceutical products that can elicit anti-tumor immune responses (Gnjatic et al., 2003). At the tumor site, T helper cells maintain a cytokine milieu that is friendly to cytotoxic T cells (CTLs) (Mortara et al., 2006) and attract effector cells such as CTLs, natural killer (NK) cells, macrophages, and granulocytes (Hwang et al., 2007).

[0024] In the absence of inflammation, the 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. MHC class II molecules have been found to be expressed in tumor cells from cancer patients (Dengjel et al., 2006).

[0025] The elongated (longer) peptides of the present invention can serve as MHC class II active epitopes. Helper T cells activated by MHC class II epitopes play an important role in orchestrating CTL effector functions in anti-tumor immunity. Helper T cell epitopes that trigger TH1 cell responses support the effector functions of CD8-positive killer T cells, including cytotoxic functions directed against tumor cells displaying tumor-associated peptide / MHC complexes on their surfaces. Thus, tumor-associated T helper cell epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compounds that stimulate anti-tumor immune responses.

[0026] Mammalian (e.g., mouse) models have shown that even in the absence of CD8-positive T lymphocytes, CD4-positive T cells can suppress tumor manifestation by secreting interferon-γ (IFNγ) to inhibit angiogenesis (Beatty and Paterson, 2001; Mumberg et al., 1999). However, there is no evidence that CD4 T cells act as direct anti-tumor effectors (Braumuller et al., 2013; Tran et al., 2014).

[0027] Because the constitutive expression of HLA class II molecules is usually limited to immune cells, it was previously considered impossible to isolate class II peptides directly from primary tumors. However, Dengjel et al. successfully identified multiple MHC class II epitopes directly in tumors (WO2007 / 028574, EP1760088B1).

[0028] Because both CD8-dependent and CD4-dependent responses contribute jointly and synergistically to antitumor effects, identifying and characterizing tumor-associated antigens recognized by CD8+ T cells (ligand: MHC class I molecules + peptide epitopes) or CD4-positive T helper cells (ligand: MHC class II molecules) is very important for the development of tumor vaccines.

[0029] For an MHC class I peptide to trigger (elicit) a cellular immune response, it must also bind to an MHC molecule. This process depends on the allele of the MHC molecule and the specific polymorphism of the peptide's amino acid sequence. MHC class I-binding peptides are typically 8-12 amino acid residues in length and typically contain two conserved residues ("anchors") within their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele has a "binding motif" that determines which peptides will specifically bind to the binding groove.

[0030] In MHC class I-dependent immune responses, peptides must not only bind to certain MHC class I molecules expressed by tumor cells, but they must then be recognized by specific T cell receptors (TCRs) carried by T cells.

[0031] For proteins to be recognized by T lymphocytes as tumor-specific or tumor-associated antigens and used therapeutically, specific conditions must be met. The antigen should be expressed primarily by tumor cells and not by normal healthy tissues, or expressed in relatively low amounts. In a preferred embodiment, the peptide should be over-presented in tumor cells compared to normal healthy tissues. More preferably, the corresponding antigen is not only present in a tumor but also at high concentrations (i.e., the number of copies of the corresponding peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins that are directly involved in the transformation of normal cells into tumor cells due to their functions in cell cycle control or apoptosis inhibition. Furthermore, downstream targets of these proteins directly responsible for the transformation event may be upregulated, thus indirectly associated with the tumor. These indirect tumor-associated antigens may also be targets for vaccination approaches (Singh-Jasuja et al, 2004). It is crucial that the epitope is present within the antigen's amino acid sequence to ensure that such a peptide derived from a tumor-associated antigen ("immunogenic peptide") can elicit a T cell response in vitro or in vivo.

[0032] Essentially, any peptide that can bind to an MHC molecule can potentially act as a T cell epitope. The induction of a T cell response in vitro or in vivo requires the presence of T cells with the corresponding TCR and the absence of immune tolerance to that specific epitope.

[0033] Therefore, TAAs are a starting point for the development of T cell-based therapies, including but not limited to tumor vaccines. Methods for identifying and characterizing TAAs are typically based on the use of T cells from patients or healthy subjects, or on the generation of differential transcriptional signatures or differential expression patterns between tumor and normal tissue peptides. However, the identification of genes that are overexpressed or selectively expressed in tumor tissue or human tumor cell lines does not provide accurate information for the use of the antigens transcribed by these genes in immunotherapy. This is because T cells with corresponding TCRs must be present and immune tolerance to this specific epitope must be absent or minimal. Therefore, only a subset of these antigenic epitopes are suitable for such applications. Therefore, in a highly preferred embodiment of the present invention, it is crucial to select only those peptides that are overexpressed or selectively presented in response to which functional and / or proliferating T cells can be found. Such functional T cells are defined as T cells that can clonally expand and perform effector functions ("effector T cells") after stimulation with a specific antigen.

[0034] In the case of peptide-MHC targeting by specific TCRs (e.g. soluble TCRs) and antibodies or other binding molecules (scaffolds) according to the invention, the immunogenicity of the underlying peptide is secondary. In these cases, presentation is the determining factor.

[0035] Introduction of the invention

[0036] In a first aspect, the present invention relates to a peptide or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 93, or a variant sequence 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 the variant binds to MHC and / or induces T cells to cross-react with the peptide, wherein the peptide is not the underlying full-length polypeptide.

[0037] The present invention further relates to a peptide of the present invention comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof having at least 77%, preferably at least 88% homology (preferably at least 77% or at least 88% identity) to SEQ ID NO: 1 to SEQ ID NO: 93, wherein the total length of the peptide or its variant is 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0038] The following table shows peptides according to the present invention, their respective SEQ ID NOs, and the possible source (potential) genes for these peptides. All peptides in Tables 1 and 2 bind to HLA-A*02. The peptides in Table 2 were previously 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 can be used in combination with other peptides of the present invention. The peptides in Table 4 can also be used to diagnose and / or treat various other malignant diseases that involve overexpression or overpresentation of the respective potential polypeptides.

[0039] Table 1: Peptides of the present invention

[0040]

[0041]

[0042]

[0043] Table 2: Other peptides of the present invention that have no known association with cancer

[0044] Serial ID number sequence Gene ID Official gene symbol 77 VLVPYEPPQV 8626 TP63 78 KVANIIAEV 5910 RAP1GDS1 79 GQDVGRYQV 6748 SSR4 80 ALQEALENA 9631 NUP155 81 AVLPHVDQV 23379 KIAA0947 82 HLLGHLEQA 63977 PRDM15 83 ALADGVVSQA 27238 GPKOW 84 SLAESLDQA 22894 DIS3 85 NIIELVHQV 6850 SYK 86 GLLTEIRAV 9263 STK17A 87 FLDNGPKTI 1982 eIF4G2 88 GLWEQENHL 79768 KATNBL1 89 SLADSLYNL 23271 CAMSAP2 90 SIYEYYHAL 3091 HIF1A 91 KLIDDVHRL 6734 SRPR 92 SILRHVAEV 1965 eIF2S1 93 VLINTSVTL 23036 ZNF292

[0045] Table 3: Peptides for use in, for example, personalized cancer therapy

[0046]

[0047] The present invention also relates to the use of the peptides of the present invention in the treatment of proliferative diseases, 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.

[0048] Particularly preferred are peptides of the present invention (alone or in combination) selected from SEQ ID NO: 1 to SEQ ID NO: 93. More preferred are peptides (alone or in combination) selected from SEQ ID NO: 1 to SEQ ID NO: 76 (see Table 1), and are used for 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.

[0049] Particularly preferred are peptides of 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 for use in 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. Further particularly preferred are peptides according to SEQ ID NO: 9.

[0050] As shown in Table 4A below, many of the peptides of the present invention are also found in other tumors and thus can also be used for immunotherapy of other indications. See also Figure 1 and Example 1.

[0051] Table 4A: Peptides of the present invention and their specific uses in other proliferative diseases, particularly other cancerous diseases. This table shows selected peptides for other tumor types that were found to be over-presented (specifically presented) in greater than 5% of the tumor samples tested, or presented in greater than 5% of the tumor samples tested with a geometric mean tumor to normal tissue ratio greater than 3. Over-presentation is defined as higher presentation in the tumor sample compared to the highest presenting normal sample. The normal tissues tested for over-presentation compared to the tissues tested were: adipose tissue, adrenal gland, artery, bone marrow, brain, central nervous system, colon, duodenum, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear leukocytes, pancreas, peripheral nerve, peritoneum, pituitary gland, pleura, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, ureter, bladder, and vein.

[0052]

[0053]

[0054] Table 4B: Peptides of the invention and their specific uses in other proliferative diseases, particularly other cancerous diseases (revised version of Table 4). This table (as in Table 4A) shows selected peptides for other tumor types that were found to be over-presented (specifically presented) in greater than 5% of the tumor samples measured, or presented in greater than 5% of the tumor samples measured with a geometric mean tumor to normal tissue ratio greater than 3. Over-presentation is defined as higher presentation in the tumor sample compared to the highest presenting normal sample. Normal tissues with over-presentation include: adipose tissue, adrenal gland, artery, bone marrow, brain, central nervous system, colon, duodenum, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear leukocytes, pancreas, peripheral nerve, peritoneum, pituitary gland, pleura, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, ureter, bladder, and vein.

[0055]

[0056]

[0057] NSCLC = non-small cell lung cancer, SCLC = small cell lung cancer, RCC = renal cell cancer, CRC = colon or rectal cancer, GC = gastric cancer, HCC = liver cancer, PC = pancreatic cancer, PrC = prostate cancer, BRCA = breast cancer, OC = ovarian cancer, NHL = non-Hodgkin lymphoma, AML = acute myeloid leukemia, CLL = chronic lymphocytic leukemia, HNSCC = head and neck squamous cell carcinoma

[0058] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to 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 in the treatment of non-small cell lung cancer. In a preferred embodiment, the above-mentioned peptides are used in the combined treatment of non-small cell lung cancer.

[0059] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to 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 treating lymphoma, and in a preferred embodiment, the use of the above peptide in a combination therapy for lymphoma. Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs: 22, 55, 58, 62, 57, 61, 76, 79, and 80 for treating small cell lung cancer, and in a preferred embodiment, the use of the above peptide in a combination therapy for small cell lung cancer.

[0060] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs. 17, 56, 76, 82 and 54 in the treatment of renal cell carcinoma, and in a preferred embodiment, in the combined treatment of renal cell carcinoma.

[0061] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs: 16, 22, 66, 67, 80, 81, 83, 86, 87 and 89 in the treatment of brain cancer. In a preferred embodiment, the use of the above-mentioned peptide in the combined treatment of brain cancer.

[0062] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ sequence numbers 31, 33, 35, 36, 37, 38, 39, 41, 42, 45, 46, 48, 50, 52, 53, 54, 55, 63, 68, 70, 75 and 71 in the treatment of gastric cancer, and in a preferred embodiment, the above-mentioned use in the combined treatment of gastric cancer.

[0063] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ sequence numbers 26, 34, 40, 74, 80, 88 and 92 in the treatment of colorectal cancer, and in a preferred embodiment, the above-mentioned use in the combined treatment of colorectal cancer.

[0064] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs: 15, 17, 22, 35, 49, 62, 67, 70, 72, 74, 75, 80, 82 and 92 in the treatment of hepatocellular carcinoma, and in a preferred embodiment, the use of the above-mentioned in the combined treatment of hepatocellular carcinoma.

[0065] Therefore, another aspect of the present invention relates to the use of at least one peptide of the invention according to any one of SEQ ID No. 37, 40, 68, 69, 71, 78, 79, 87 and 91 in the treatment of pancreatic cancer, in a preferred embodiment, in the combined treatment of pancreatic cancer.

[0066] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs. 79 and 91 in the treatment of prostate cancer, and in a preferred embodiment, in the combined treatment of prostate cancer.

[0067] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs. 4, 28, 58, 61, 72, 78, 79, 80, 82, 84, 86, 88, 92 and 85 in the treatment of leukemia, and in a preferred embodiment, the use of the above-mentioned in the combined treatment of leukemia.

[0068] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to 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 in the treatment of breast cancer, and in a preferred embodiment, the use of the above-mentioned in the combined treatment of breast cancer.

[0069] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to 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 in the treatment of melanoma, and in a preferred embodiment, the use of the above-mentioned in the combined treatment of melanoma.

[0070] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs. 8, 62, 70, 78, 79, 80 and 87 in the treatment of ovarian cancer, and in a preferred embodiment, in the combined treatment of ovarian cancer.

[0071] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to 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 in the treatment of bladder cancer, and in a preferred embodiment, the use of the above-mentioned in the combined treatment of bladder cancer.

[0072] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs: 13, 15, 29, 30, 40, 56, 57, 58, 80, 81, 83, 84 and 88 in the treatment of uterine cancer, and in a preferred embodiment, the use of the above-mentioned in the combined treatment of uterine cancer.

[0073] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to 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 in the treatment of gallbladder cancer and bile duct cancer, and in a preferred embodiment, the use of the above-mentioned in the combined treatment of gallbladder cancer and bile duct cancer.

[0074] Therefore, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any one of SEQ ID NOs: 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 in the treatment of HNSCC, and in a preferred embodiment, in the combination treatment of HNSCC.

[0075] Therefore, another aspect of the present invention relates to the use of the peptides of the present invention in the treatment - preferably in combination therapy - of a proliferative disease selected from the group consisting 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.

[0076] The present invention also relates to peptides according to the invention which have the ability to bind to major histocompatibility complex (MHC) class I or MHC class II molecules in an elongated form, for example of varying length.

[0077] The present invention further relates to the peptides of the present invention, wherein the peptide (each peptide) consists of or essentially consists of the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 93.

[0078] The present invention further relates to the peptides of the present invention, wherein the peptides are modified and / or comprise non-peptide bonds.

[0079] The present invention further relates to the peptides of the present invention, wherein the peptide is part of a fusion protein, in particular fused to the N-terminal amino acid of the invariant chain (Ii) associated with the HLA-DR antigen, or fused to an antibody (e.g., a dendritic cell-specific antibody), or fused to the sequence of an antibody.

[0080] The present invention further relates to a nucleic acid encoding the peptide of the present invention. The present invention further relates to a nucleic acid of the present invention, which is DNA, cDNA, PNA, RNA, or a combination thereof.

[0081] The present invention further relates to an expression vector capable of expressing and / or expressing the nucleic acid of the present invention.

[0082] The present invention further relates to the use of the peptide of the present invention, the nucleic acid of the present invention or the expression vector of the present invention in treating diseases and making pharmaceuticals, in particular in treating cancer.

[0083] The present invention further relates to antibodies specific for the peptides of the present invention or complexes of the peptides of the present invention and MHC, and methods for producing these antibodies.

[0084] The present invention further relates to the T cell receptors (TCRs) of the present invention, in particular soluble TCRs (sTCRs) and cloned TCRs processed into autologous or allogeneic T cells, as well as methods of making these TCRs, and NK cells carrying or cross-reacting with said TCRs.

[0085] Antibodies and TCRs are further embodiments of immunotherapeutic uses of the peptides according to the invention.

[0086] The present invention further relates to a host cell containing the nucleic acid of the present invention or the aforementioned expression vector. The present invention further relates to a host cell of the present invention, which is an antigen presenting cell, preferably a dendritic cell.

[0087] The present invention further relates to a method for preparing a peptide of the present invention, said method comprising culturing the host cell of the present invention, and isolating the peptide from said host cell or its culture medium.

[0088] The present invention further relates to the method of the present invention, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by contacting a sufficient amount of the antigen with the antigen-presenting cell.

[0089] The present invention further relates to the method of the present invention, wherein the antigen presenting cell comprises an expression vector capable of expressing a peptide comprising the amino acid sequence of SEQ ID NO. 1 to SEQ ID NO. 93, preferably comprising the amino acid sequence of SEQ ID No. 1 to SEQ ID No. 76, or a variant thereof.

[0090] The present invention further relates to activated T cells produced by the method of the present invention, wherein the T cells selectively recognize a cell expressing a polypeptide comprising the amino acid sequence of the present invention.

[0091] The present invention further relates to a method for killing target cells in a patient, wherein the target cells of the patient abnormally express a polypeptide comprising any amino acid sequence of the present invention, the method comprising administering to the patient an effective amount of T cells produced by the method of the present invention.

[0092] The present invention further relates to the use of any of the peptides, nucleic acids, expression vectors, cells, activated T lymphocytes, T cell receptors, antibodies, or other peptide- and / or peptide-MHC binding molecules as a medicament or in the preparation of a medicament. The medicament preferably has anticancer activity.

[0093] Preferably, the drug is a cell therapy drug, vaccine or protein based on soluble TCR or antibody.

[0094] The present invention also relates to the use of the present invention, wherein the cancer cells are 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 cells.

[0095] The present invention further relates to a biomarker based on a peptide of the present invention, referred to herein as a "target," which can be used to diagnose 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 likelihood of success of treatment, preferably immunotherapy, most preferably immunotherapy targeting the same target recognized by the biomarker. For example, antibodies or soluble TCRs can be used to stain tumor sections to detect the presence of the relevant peptide complexed with MHC.

[0096] Optionally, the antibody has further effector functions, such as an immunostimulatory domain or a toxin.

[0097] The present invention also relates to the use of these novel targets in cancer treatment.

[0098] ABHD11 antisense RNA 1 (ABHD11-AS1) has been described as a long noncoding RNA that has been shown to be upregulated in gastric cancer and correlates with differentiation and Lauren histological classification. Therefore, ABHD11-AS1 may be a potential biomarker for gastric cancer diagnosis (Lin et al, 2014). ABHD11 activity has been shown to correlate with the development of distant metastases in lung adenocarcinoma and may therefore represent a potential new biomarker (Wiedl et al, 2011).

[0099] ADAMTS2 has been shown to be dysregulated in patients with mixed T / myeloid acute leukemia (Tota et al, 2014). ADAMTS2 has been described as being associated with the JNK signaling pathway following upregulation of 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 has been described as a potential marker for metastasis in tongue squamous cell carcinoma (Carinci et al, 2005). ADAMTS2 has been shown to be upregulated in renal cell carcinoma and associated with shorter patient survival (Roemer et al, 2004). ADAMTS2 has been shown to be regulated by the cell proliferation-associated transforming growth factor-β1 (Wang et al, 2003).

[0100] AHNAK2 encodes the scaffold protein AHNAK nuclear protein 2 (Marg et al, 2010). AHNAK2 is an important element of the non-canonical secretory pathway of fibroblast growth factor 1 (FGF1), which is involved in tumor growth and invasion (Kirov et al, 2015).

[0101] ANO1 encodes anoctamin 1, a calcium-activated chloride channel associated with small intestinal sarcomas and oral cancer (RefSeq, 2002). ANO1 is amplified in esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumors (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, and breast cancer (Qu et al, 2014).

[0102] 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 has been shown to be downregulated in the lung cancer cell line A549 after treatment with periplocin. Therefore, the growth inhibition of lung cancer cells by periplocin may be related to ARHGDIA (Lu et al, 2014). Knockdown of ARHGDIA has been shown to be associated with increased apoptosis in lung-derived normal and cultured tumor cells. 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 as being associated with the staging of ovarian clear cell and high-grade serous carcinoma (Canet et al, 2011). ARHGDIA has been described as an apoptosis pathway-associated gene, shown to be dysregulated in fibrosarcoma HT1080 cells following TRAIL-mediated apoptosis (Daigeler et al, 2008). ARHGDIA has been shown to be upregulated in the oxaliplatin-resistant colon cancer cell line THC8307 / L-OHP and has been described as a gene involved in anti-apoptosis. Therefore, ARHGDIA may be a potential marker associated with oxaliplatin sensitivity (Tang et al, 2007). ARHGDIA overexpression has been shown to be regulated by the putative tumor suppressor ACVR2 (a member of the cancer-associated TGFBR2 family) in an MSI-H colon cancer cell line transfected with wild-type ACVR2 carrying a frameshift mutation in ACVR2 (Deacu et al, 2004). ARHGDIA has been described as a key regulator of Rho GTPases. ARHGDIA depletion has been shown to induce constitutive activation of Rho GTPases 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 dysregulated in colorectal cancer (Sethi et al, 2015). ARHGDIA has been shown to target MEK1 / 2-ERK after SUMOylation, which is associated with inhibition of c-Jun / AP-1, cyclin D1 transcription, and cell cycle progression. Thus, ARHGDIA has been implicated in cancer cell growth inhibition (Cao et al, 2014). ARHGDIA has been described as a novel suppressor of prostate cancer and may play a key role in regulating androgen receptor signaling and prostate cancer growth and progression (Zhu et al, 2013b).

[0103] ATIC has been described as a potential fusion partner of the cancer-associated anaplastic lymphoma kinase (ALK) in anaplastic large cell lymphoma (Cheuk and Chan, 2001). ATIC has been shown to be expressed as a chimeric fusion gene with ALK in inflammatory myofibroblastic tumors of the bladder (Debiec-Rychter et al, 2003). Inhibition of ATIC aminoimidazolecarboxamide formyltransferase (AICAR) activity in breast cancer cell line models has been shown to result in a dose-dependent decrease in cell number and cell division rate. Therefore, ATIC may be a potential target for cancer therapy (Spurr et al, 2012).

[0104] CAPZB has been reported to be overexpressed in human papillomavirus 18-positive oral squamous cell carcinoma and has been identified as a prostate cancer susceptibility gene (Lo et al, 2007; Nwosu et al, 2001).

[0105] COL6A1 is upregulated in the reactive stroma of castration-resistant prostate cancer, promoting tumor growth (Zhu et al, 2015). COL6A1 is overexpressed in CD166- pancreatic cancer cells, which display enhanced invasive and migratory activity compared to 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).

[0106] 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 lung carcinoma, and head and neck squamous cell carcinoma, 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).

[0107] CYFIP1 has been shown to be downregulated during epithelial tumor invasion (Silva et al, 2009). CYFIP1 downregulation is associated with poor prognosis in epithelial tumors (Silva et al, 2009).

[0108] CYP2S1 has been shown to regulate colorectal cancer growth in the HCT116 cell line by linking it to PGE2-mediated activation of the β-catenin signaling pathway (Yang et al, 2015b). CYP2S1 has been described as upregulated in a variety of epithelial-derived cancers and in hypoxic tumor cells (Nishida et al, 2010; Madanayake et al, 2013). CYP2S1 depletion in bronchial epithelial cell lines 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 associated with drug sensitivity in both colorectal and breast cancers (Tan et al, 2011). CYP2S1 has been shown to be associated with breast cancer survival and 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,tert-8-dihydrodiol-tert-9,10-epoxide and, therefore, may 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 metastasis compared with primary ovarian cancer (Downie et al, 2005).

[0109] DES expression in the stroma of colorectal cancer is associated with advanced 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 colorectal cancer severity and differentiation, as well as decreased 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 members of a group of proteins that may aid in the staging of bladder cancer using immunohistochemistry (Council and Hameed, 2009).

[0110] 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 mutations in multiple myeloma have been associated with shorter overall survival. Compared with patients with major subclonal DIS3 mutations, minor subclonal mutations have been associated with a poorer response to treatment (Weissbach et al, 2015). DIS3 has been shown to be upregulated in colorectal cancer via 13q gain. DIS3 silencing has been shown to affect important tumorigenic features such as survival, migration, and invasiveness. Therefore, DIS3 may be a novel candidate gene that contributes to colorectal cancer progression (de Groen et al, 2014). DIS3 has been described as belonging to a panel of genes that could be used in combination with plasma protein-based biomarkers for earlier diagnosis of epithelial ovarian cancer (Pils et al, 2013). DIS3 may be a potential candidate gene for breast cancer susceptibility because breast cancer mutation screening has detected numerous polymorphisms (Rozenblumetal, 2002).

[0111] EEF1A1 has been shown to be upregulated in multiple cancer entities, including colorectal, ovarian, gastric, prostate, 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; Reiman et al, 2012). Mechanistically, EEF1A1 inhibits apoptosis by interacting with p53 and p73, promotes proliferation through transcriptional repression of the cell cycle inhibitor p21, and is involved in the regulation of epithelial-mesenchymal transition (Blanch et al, 2013; Choi et al, 2009; Hussey et al, 2011).

[0112] EEF1A2 has been described as upregulated in breast, ovarian, lung, pancreatic, gastric, and prostate cancers, as well as in TFE3 translocation 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 ductal, 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 involved in tumorigenesis because it stimulates phospholipid signaling and activates Akt-dependent cell migration and actin remodeling, which ultimately promotes tumorigenesis (Abbas et al, 2015). EEF1A2 has been described as suppressing p53 function in hepatocellular carcinoma 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 hepatocellular carcinoma and may therefore be a therapeutic target for some patients (Longerrich, 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 pancreatic cancer treatment (Zang et al., 2015). EEF1A2 has been shown to be associated with prostate cancer progression by promoting cell proliferation and inhibiting apoptosis, and therefore may serve as a potential therapeutic target for 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 cancer cell growth inhibition (Lee et al., 2013). EEF1A2 has been shown to be associated with lymph node metastasis and neural invasion in pancreatic ductal adenocarcinoma (Duanmin et al., 2013). EEF1A2 has been shown to be associated with breast cancer survival (Kulkarni et al, 2007). EEF1A2 has been described as a putative oncogene and tumor suppressor gene in lung adenocarcinoma cell lines and ovarian cancer (Lee, 2003; Zhu et al, 2007a).

[0113] EIF2S1 has been described as an activator of tumor progression and therapeutic resistance upon phosphorylation. However, EIF2S1 has also been implicated in tumorigenesis suppression (Zheng et al, 2014). EIF2S1 has been described as a downstream effector of mTOR, and hyperphosphorylation reduces cancer cell survival, making it a potential target for drug development (Tuval-Kochen et al, 2013).

[0114] EIF4G2 has been described as one of a core group of genes implicated in the abrogation of tumorigenesis in CD133+ cells of pediatric gliomas (Baxter et al, 2014). Downregulation of EIF4G2 by miR-520c-3p has been shown to be associated with the suppression of diffuse large B-cell lymphoma development (Mazan-Mamczarz et al, 2014). EIF4G2 has been shown to promote protein synthesis and cell proliferation by regulating cell cycle protein synthesis (Lee and McCormick, 2006). Downregulation of EIF4G2 in bladder tumors has been shown to correlate with tumor invasiveness (Buim et al, 2005). EIF4G2 has been described as involved in MycN / IFNγ-induced apoptosis and in the survival and death of neuroblastoma cells (Wittke et al, 2001).

[0115] F7, a member of the tissue factor complex, has been described as aberrantly expressed on the surface of cancer cells, including ovarian cancer. The 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, stimulating the expression of numerous malignant phenotypes in breast cancer cells. Therefore, the F7-tissue factor pathway is an attractive potential 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 regulate autophagy through mTOR signaling in liver cancer cell lines (Chen et al, 2014a). F7 has been shown to be associated with tumor invasion and metastasis in colorectal and ovarian cancers (Tang et al, 2010; Koizume et al, 2006). F7 has been shown to be ectopically upregulated in colorectal cancer (Tang et al, 2009). F7 in the tissue factor complex has been shown to be associated with chemotherapy resistance in neuroblastoma (Fang et al, 2008a).

[0116] FAM115C is upregulated in non-small cell lung cancer during hypoxia (Leithner et al, 2014).

[0117] FAM83A has been described as a potential biomarker for lung cancer (Li et al, 2005). FAM83A has been described as a marker gene that can be used, along with NPY1R and KRT19, to detect circulating tumor cells in breast cancer patients (Liu et al, 2014d). Ablation of FAM83A in breast cancer cells has been shown to reduce MAPK signaling, significantly inhibiting in vitro growth and in vivo tumorigenicity (Cipriano et al, 2014). Furthermore, the FAM83 protein family has been described as a novel oncogene family that regulates MAPK signaling in cancer and is therefore amenable to the development of cancer therapies aimed at inhibiting MAPK signaling (Cipriano et al, 2014). FAM83A has been shown to be associated with trastuzumab resistance in HER2-positive breast cancer cell lines (Boyer et al, 2013). In general, FAM83A has been shown to be a candidate gene associated with resistance to EGFR tyrosine kinase inhibitors in breast cancer (Lee et al, 2012). FAM83A has been described as being associated with poor prognosis in breast cancer (Lee et al, 2012). FAM83A has been shown to be upregulated in non-small cell lung cancer (Qu et al, 2010). FAM83A has been shown to be a potential specific and sensitive marker for detecting circulating tumor cells in the peripheral blood of patients with non-small cell lung cancer (Qu et al, 2010).

[0118] 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).

[0119] FAT1 has been described as being significantly mutated in head and neck squamous cell carcinoma, 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 being repressed in oral cancer and preferentially downregulated in invasive breast cancer (Katoh, 2012). FAT1 has been described as being upregulated in leukemias and associated with a poor prognosis in pre-B acute lymphoblastic leukemia (Katoh, 2012). FAT1 has been shown to be upregulated in pancreatic cancer and hepatocellular carcinoma (Valletta et al, 2014; Wojtalewicz et al, 2014). FAT1 has been described to inhibit tumor growth through activation of Hippo signaling and to promote tumor metastasis through induction of actin polymerization (Katoh, 2012). FAT1 has been shown to be a candidate cancer driver gene in cutaneous squamous cell carcinoma (Pickering et al, 2014). FAT1 has been described as a tumor suppressor associated with Wnt signaling and tumorigenesis (Morris et al, 2013).

[0120] Based on its subcellular localization, filamin A plays a dual role in cancer: in the cytoplasm, in addition to participating in cell migration and adhesion pathways, filamin A also plays a role in various growth signaling pathways. Therefore, its overexpression has a tumor-promoting effect. Compared with full-length filamin A, the C-terminal fragment (released after proteolysis of the protein) is localized to the cell nucleus, where it interacts with transcription factors, thereby inhibiting tumor growth and metastasis (Savoy and Ghosh, 2013).

[0121] Tumor-specific C-terminal truncations of GBP5 have been described as potentially responsible for GBP5 deregulation in lymphoma cells (Wehner and Herrmann, 2010). GBP5 has been described as potentially having cancer-related functions due to the restricted expression patterns of three GBP5 splice variants in cutaneous T-cell lymphoma tumor tissues and cell lines, as well as in melanoma cell lines (Fellenberg et al, 2004).

[0122] 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; Al Moustafa et al., 2002). GJB5 has been described as acting as a tumor suppressor in non-small cell lung cancer cell lines by inhibiting cell proliferation and metastasis (Zhang et al., 2012). GJB5 has been shown to be upregulated in sessile serrated adenomas / polyps, precancerous lesions that may account for 20-30% of colon cancers (Delker et al., 2014). GJB5 expression has been described as significantly altered during skin tumor promotion and progression in mouse models (Slaga et al., 1996).

[0123] GLS has been described as indirectly regulated by the MYC gene to increase glutamine metabolism in cancer cells (Dang et al, 2009). GLS has been shown to be inhibited by the tumor suppressor NDRG2 in colorectal cancer (Xu et al, 2015). GLS has been described as upregulated in pancreatic ductal adenocarcinoma, triple-negative breast cancer, hepatocellular carcinoma, oral squamous cell carcinoma, colorectal cancer, and malignant glial cell-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 correlate with survival in hepatocellular carcinoma and has been described as a sensitive and specific biomarker for pathological diagnosis and prognosis of hepatocellular carcinoma (Yu et al, 2015a). Loss of one copy of GLS has been shown to delay tumor progression in an immune-associated MYC-mediated mouse model of hepatocellular carcinoma (Xiang et al, 2015). GLS has been shown to be required for tumorigenesis and tumor-specific suppression, and has been described as a potential approach for cancer therapy (Xiang et al., 2015). GLS has been shown to be associated with paclitaxel 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 associated with deeper tumor invasion and the pathological type of tubular adenocarcinoma in colon cancer tumorigenesis. GLS may be a therapeutic target for colorectal cancer (Huang et al., 2014a). Silencing of the GLS isozyme KGA has been shown to result in decreased survival in the glioma cell lines SFxL and LN229 (Martin-Rufian et al., 2014). Silencing of GLS in the glioma cell lines SFxL and LN229 has also been shown to induce apoptosis by causing lower levels of c-myc and bcl-2 expression and higher expression of pro-apoptotic proteins (Martin-Rufian et al., 2014). ErbB2 activation has been shown to upregulate GLS expression through the NF-κB pathway, which promotes breast cancer cell proliferation (Qie et al, 2014). Knockdown or inhibition of GLS in breast cancer cells, as well as high levels of GLS, have been shown to significantly reduce proliferation (Qie et al, 2014).

[0124] GNA15 has been shown to be upregulated in primary and metastatic small intestinal neuroendocrine tumors (Zanini et al, 2015). Increased GNA15 expression has been shown to correlate with poor survival, suggesting a pathobiological role for GNA15 in small intestinal neuroendocrine tumorigenesis and a potential therapeutic target (Zanini et al, 2015). GNA15 has been described as downregulated in numerous non-small cell lung cancer cell lines (Avasarala et al, 2013). High GNA15 expression 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 cell proliferation and anchorage-independent cell growth in non-small cell lung cancer. Therefore, GNA15 is a potential therapeutic target for non-small cell lung cancer (Avasarala et al, 2013). GNA15 has been shown to be involved in tumor signaling in pancreatic cancer (Giovinazzo et al, 2013). GNA15 has been shown to stimulate STAT3 via c-Src / JAK- and ERK-dependent mechanisms after constitutive activation in human embryonic kidney 293 cells (Lo et al, 2003).

[0125] Underexpression of HAS3 has been shown to be associated with advanced tumor stage, lymph node metastasis, vascular invasion, and poor disease-specific and metastasis-free survival in upper urinary tract and bladder urothelial carcinoma (Chang et al, 2015). Therefore, HAS3 may serve as a potential prognostic biomarker and novel therapeutic target for urothelial carcinoma (Chang et al, 2015). HAS3 has been shown to promote pancreatic cancer growth through hyaluronan accumulation (Kultti et al, 2014). HAS3 inhibition has been shown to reduce the survival of 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 implicated in mediating colon cancer growth by inhibiting apoptosis (Teng et al, 2011). HAS3 has been shown to be upregulated in esophageal squamous cell carcinoma, adenocarcinoma, lung squamous cell carcinoma, and nodular basal cell carcinoma (Tzellos et al, 2011; Twarock et al, 2011; de Sa et al, 2013). HAS3 has been described as an independent prognostic factor in breast cancer, as HAS3 expression in stromal cells of breast cancer patients has been associated with a high recurrence rate and shorter overall survival (Auvinen et al, 2014). HAS3 has been associated with serous ovarian cancer, clear cell renal carcinoma, endometrial cancer, and osteosarcoma (Nykopp et al, 2010; Weiss et al, 2012; Cai et al, 2011; Tofuku et al, 2006).

[0126] HIF1A has been shown to be associated with tumor necrosis in invasive endometrial cancer. HIF1A has been further characterized as a potential therapeutic target for this disease (Bredholt et al, 2015). HIF1A has been implicated in hepatocarcinogenesis, sarcoma metastasis, and nasopharyngeal carcinoma (Chen et al, 2014c; El-Naggar et al, 2015; Li et al, 2015b). Single nucleotide polymorphisms in HIF1A have been shown to be significantly associated with clinical outcome in patients with invasive hepatocellular carcinoma after surgery (Guo et al, 2015). Aberrant HIF1A activity, along with aberrant STAT3 activity, has been shown to drive tumor progression in malignant schwannoma cell lines. Therefore, inhibition of the STAT3 / HIF1A / VEGF-A signaling axis has been described as a viable therapeutic strategy (Rad et al, 2015). HIF1A has been described as an important target for hypoxia-driven drug resistance in multiple myeloma (Maiso et al, 2015). HIF1A has been shown to be asymmetrically expressed in three different cell lines corresponding to different stages of multiple myeloma pathogenesis, suggesting that HIF1A is involved in multiple myeloma tumorigenesis and metastasis (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 by HIF1A has been described as crucial for the quiescence, maintenance, and survival of prostate cancer stem cells (Marhold et al, 2015). HIF1A has been described as one of the genes in a four-gene classifier that predicts stage I lung adenocarcinoma (Okayama et al, 2014). Polymorphisms in HIF1A have been shown to be associated with increased susceptibility to gastrointestinal cancer in Asian populations (Xu et al, 2014). HIF1A has been described as a prognostic marker for sporadic male breast cancer (Deb et al, 2014).

[0127] Intracellular HYOU1 protein activity has been shown to benefit cancer cell survival during tumor progression or metastasis. Extracellular HYOU1 protein plays a crucial role in generating antitumor immune responses by facilitating the delivery of tumor antigens for cross-presentation (Fu and Lee, 2006; Wang et al., 2014). HYOU1 protein has been incorporated into cancer immunotherapy and has demonstrated positive immunomodulatory effects (Yu et al., 2013; Chen et al., 2013; Yuan et al., 2012; Wang and Subjeck, 2013).

[0128] Studies have shown that IGHG1 is overexpressed in human pancreatic cancer tissue compared with adjacent noncancerous tissue. In contrast, IGHG1 protein is downregulated in invasive ductal carcinoma tissue (Kabbage et al, 2008; Li et al, 2011b). Targeted silencing of IGHG1 by siRNA inhibits cell viability and promotes apoptosis (Pan et al, 2013).

[0129] Researchers have found that IGHG3 is expressed in Saudi women affected by breast cancer. Similarly, increased copy number and elevated IGHG3 levels have been detected in African men with prostate cancer. Another report showed that IGHG3 expression was found in squamous non-small cell lung cancer, malignant mesothelioma, and tumor cells that occasionally appear in MALT lymphoma and show a propensity to differentiate into plasma cells (Remmelink et al, 2005; Bin Amer et al, 2008; Ledet et al, 2013; Zhang et al, 2013c; Sugimoto et al, 2014).

[0130] IGHG4 encodes immunoglobulin heavy chain constant gamma 4 (G4m marker) and is located on chromosome 14q32.33 (RefSeq, 2002). Recent work has detected rearrangements involving IGHG4 in primary testicular diffuse large B-cell lymphoma (Twa et al, 2015).

[0131] IGHM encodes the immunoglobulin heavy chain constant μ (RefSeq, 2002). Studies have found that IGHM is downregulated in Chinese patients affected by rhabdomyosarcoma. Others 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 the active IGH allele in the majority of IgM+ tumors. Furthermore, epithelial hamartoma samples showed no reactivity to transcription factors that bind to IGHM enhancer factor 3 or transcription factor EB (Kato et al, 2009; Blenk et al, 2007; Ruminy et al, 2011; Liu et al, 2014b).

[0132] IL36RN has been described as a marker that can significantly distinguish stage III from stage I and II lung adenocarcinoma (Liang et al, 2015).

[0133] Reduced INA expression is associated with metastasis, recurrence, and shorter overall survival in pancreatic neuroendocrine tumors. Therefore, INA may be a useful prognostic biomarker for the aggressiveness of pancreatic neuroendocrine tumors (Liu et al, 2014a). INA has been described as upregulated in gliomas with an oligodendroglial phenotype, 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 marker for neuroblastoma and can be used in the differential diagnosis of small round cell tumors in childhood (Willoughby et al, 2008).

[0134] ITGA6 expression is upregulated in various cancer entities (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 the Abeta4 variant of ITGA6 appear to be mediated through the Wnt / β-catenin pathway (Groulx et al, 2014). The Abeta4 variant of ITGA6 leads to VEGF-dependent activation of the PI3K / Akt / mTOR pathway. This pathway plays an important role in the survival of metastatic cancer cells (Chung et al, 2002).

[0135] KRT14 is highly expressed in various squamous cell carcinomas (e.g., esophageal, lung, laryngeal, and cervical cancers) as well as adenomatous odontogenic tumors. However, it is absent in small cell carcinoma of the bladder and weakly expressed in lung, gastric, colorectal, hepatocellular, pancreatic ductal, invasive breast, papillary thyroid, and endometrial adenocarcinomas (Xue et al, 2010; Terada, 2012; Vasca et al, 2014; Hammam et al, 2014; Shruthi et al, 2014). In bladder cancer, KRT14 expression is strongly associated with poor survival (Volkmer et al, 2012).

[0136] KRT16 overexpression has been found in basal-like breast cancer cell lines and carcinoma in situ. Others have not found significant differences in KRT16 immunohistochemical expression between non-recurrent and recurrent ameloblastoma (Joosse et al, 2012; Ida-Yonemochi et al, 2012; Safadi et al, 2016). Furthermore, in silico analysis has shown an association between KRT16 expression and shorter recurrence-free survival in metastatic breast cancer (Joosse et al, 2012).

[0137] KRT5 has been shown to be upregulated in breast cancers of young women (Johnson et al, 2015). KRT5 has been shown to be associated with poor disease-free survival in young women with breast cancer and clinical outcomes in premenopausal women 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 the breast cancer cell lines HCC1937 and T47D (Gorski et al, 2010). KRT5 has been shown to be dysregulated 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 be associated with the progression of endometrial cancer (Zhao et al, 2013). KRT5 has been shown to be downregulated in invasive tumor areas in patients with verrucous carcinoma (Schumann et al, 2012). KRT5 has been shown to be part of a family of four proteins that are differentially expressed in colorectal cancer biopsies compared with normal tissue samples (Yang et al, 2012). KRT5 and three other proteins from this family have been described as novel markers and potential targets for colorectal cancer therapy (Yang et al, 2012). KRT5 has been associated with basal cell carcinoma (Depianto et al, 2010). KRT5 has been described as a candidate gene for identifying urothelial cancer stem cells (Hatina and Schulz, 2012).

[0138] Activation of the kynurenine pathway, involving KYNU, has been shown to be significantly elevated in glioblastoma, suggesting that the kynurenine pathway is involved in the pathophysiology of glioma (Adams et al, 2014). KYNU has been described as a cancer-associated gene, and its expression is altered following aryl hydrocarbon receptor knockdown in the MDA-MB-231 breast cancer cell line (Goode et al, 2014). KNYNU has been shown to be differentially expressed in highly and non-invasive osteosarcoma cell lines, suggesting that it may play an important role in osteosarcoma tumorigenesis. Therefore, KYNU may also represent a candidate gene for future therapeutic targets (Lauvrak et al, 2013). KYNU has been shown to correlate with tumorigenic re-expression in a mixture of non-tumorigenic HeLa and human dermal fibroblast cells. Therefore, KYNU may provide a relevant candidate for regulation of tumorigenic expression (Tsujimoto et al, 1999).

[0139] Combined transcriptional analysis of LAMB3 with two other genes has been shown to be beneficial for the diagnosis of papillary thyroid carcinoma and the prediction of lymph node metastasis risk (Barros-Filho et al, 2015). LAMB3 has been shown to be associated with 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; Tanise et al, 2014). LAMB3 has been shown to be upregulated in cervical squamous cell carcinoma, lung cancer, gastric cancer, nasopharyngeal carcinoma, 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 is a protein known to affect cell differentiation, migration, adhesion, proliferation, and survival and to act as an oncogene in cervical squamous cell carcinoma (Yamamoto et al, 2013). LAMB3 knockdown has been shown to inhibit 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). LAMB3 silencing 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 correlates with the depth of invasion and venous invasion in esophageal squamous cell carcinoma (Kita et al, 2009). LAMB3 methylation has been shown to be associated with several poor prognostic parameters in bladder cancer (Sathyanarayana et al, 2004).

[0140] Inhibition of LAP3 has been shown to inhibit 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). Overexpression of LAMB3 has been shown to correlate with malignant tumor grade and poor prognosis in glioma patients (He et al, 2015). LAP3 has been shown to promote glioma progression by regulating cell growth, migration, and invasion, thus potentially representing a novel predictor (He et al, 2015). Frameshift mutations in genes involved in amino acid metabolism, including LAP3, have been detected in microsatellite instability-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 liver cancer cell proliferation by regulating the G1 / S checkpoint of the cell cycle and late cell migration (Tian et al, 2014). LAP3 expression has further been shown to correlate with the prognosis and malignant progression of hepatocellular carcinoma (Tian et al, 2014). LAP3 silencing in the esophageal squamous cell carcinoma line ECA109 has been shown to reduce cell proliferation and colony formation, while LAP3 knockdown leads to cell cycle arrest (Zhang et al, 2014). Overexpression of LAP3 in the esophageal squamous cell carcinoma line TE1 has been shown to promote cell proliferation and invasion (Zhang et al, 2014). Therefore, LAP3 has been shown to play a role in the malignant progression of esophageal squamous cell carcinoma (Zhang et al, 2014).

[0141] Researchers have reported that M6PR is expressed in colon cancer cell lines and chorionic villus cells (Braulke et al, 1992; O'Gorman et al, 2002). In breast cancer, low-level M6PR expression is associated with poor patient prognosis (Esseghir et al, 2006). Furthermore, overexpression of M6PR results in decreased cell growth in vitro and reduced tumor growth in nude mice (O'Gorman et al, 2002).

[0142] 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 has been described as part of the cancer-associated MAPK signaling pathway, associated with 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 invasion through phosphorylation of the oncogene SRC-3. Therefore, MAPK6 may be an attractive target for the treatment of aggressive lung cancer (Long et al, 2012). MAPK6 has also been described as a potential target for anticancer drug development in drug-resistant breast cancer cells (Yang et al, 2010). Overexpression of MAPK6 in gastric cancer has been shown to be associated with TNM stage, serosal invasion, and lymph node involvement (Liang et al, 2005a). MAPK6 has been shown to be a binding partner of cyclin D3, a core cell cycle machinery component, suggesting that MAPK6 has potential activity in cell proliferation (Sun et al, 2006).

[0143] MNAT1 has been shown to be associated with poor prognosis in estrogen receptor-positive / HER2-negative breast cancer (Santaripia et al., 2013). Loss of MNAT1-intrinsic fragments during granulopoiesis has been shown to promote the growth and metastasis of leukemic myeloblasts (Lou et al., 2013). MNAT1 has been shown to be dysregulated in the ovarian cancer cell line OAW42 following knockdown of the putative oncogene ADRM1 (Fejzo et al., 2013). siRNA-mediated silencing of MNAT1 has been shown to inhibit cell growth in the pancreatic cancer cell line BxPC3 in vitro and to exert antitumor effects against subcutaneously transplanted pancreatic tumors in vivo (Liu et al., 2007a). Genetic variants in MNAT1 have been described as being associated with susceptibility to lung cancer (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 increase in the proportion of cells in the G0 / G1 phase. Thus, it was shown that MNAT1 plays an important role in regulating the G1 to S phase transition of the cell cycle in the pancreatic cancer cell line BxPC3 (Zhang et al, 2005). MNAT1 regulation of cyclin-dependent kinase-activated kinase activity has been shown to cross-regulate G1 arrest in neuroblastoma cells and play a key role in the transition from proliferation to differentiation in neuroblastoma (Zhang et al, 2004).

[0144] DNA methylation-associated silencing of NEFH in breast cancer has been shown to be frequent, cancer-specific, and associated with clinical features of disease progression (Calmon et al, 2015). NEFH has further been described to be inactivated by DNA methylation in pancreatic, gastric, and colon cancers, potentially contributing to the progression of these malignancies (Calmon et al, 2015). NEFH CpG island methylation has been shown to correlate 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 vulvar myxoid chondrosarcoma (Dotlic et al, 2014). Overexpression of NEFH in hepatoma cell lines has been shown to reduce cell proliferation, while knockdown of NEFH promotes cell invasion and migration in vitro and increases tumor formation in mice. Thus, NEFH acts 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 associated with tumorigenesis (Alholle et al, 2013).

[0145] DNA methylation-mediated silencing of NEFL has been shown to be a frequent event in breast cancer and may contribute to the progression of breast cancer and 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 as potentially playing a role in cancer cell apoptosis and invasion in head and neck squamous cell carcinoma lines (Huang et al, 2014c). NEFL methylation has been described as a novel mechanism that confers cisplatin resistance in head and neck cancer cell lines by interacting with the mTOR signaling pathway (Chen et al, 2012). NEFL has been described as a candidate biomarker for predicting chemotherapy efficacy and survival in head and neck cancer (Chen et al, 2012). High NEFL expression has been associated with a better clinical outcome in supratentorial ependymomas (Hagel et al, 2013). NEFL has been shown to be ectopically expressed in breast cancer and to be downregulated in primary breast cancers with lymph node metastasis compared with node-negative cancers (Li et al., 2012). Low NEFL expression has been shown to correlate with poor 5-year disease-free survival in patients with early-stage breast cancer and may therefore be a potential prognostic factor for early-stage breast cancer (Li et al., 2012). NEFL has also been shown to be downregulated in glioblastoma multiforme (Khalil, 2007). Allelic loss at chromosome 8p21-23, where NEFL is located, has been described as an early and frequent event in carcinogenesis and lung cancer progression and has 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).

[0146] NEFM has been described as a gene involved in processes related to tumor progression and 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 associated with clinical features of disease progression (Calmon et al, 2015). NEFM has further been described as inactivated by DNA methylation in pancreatic, gastric, and colon cancers, potentially contributing to the progression of these malignancies (Calmon et al, 2015). NEFM has been associated with prostate cancer and astrocytoma (Wu et al, 2010; Penney et al, 2015). NEFM has been described as a novel candidate tumor suppressor gene that is methylated in renal cell carcinoma (Ricketts et al, 2013). Methylation of NEFM has been shown to correlate with prognosis in renal cell carcinoma (Ricketts et al, 2013). NEFM has been described as a potential diagnostic marker and has been shown to be differentially expressed in neuroendocrine tumor cell lines compared to non-neuroendocrine tumor cell lines (Hofsli et al, 2008).

[0147] NUP155 has been described as a potential epigenetic biomarker on white blood cell DNA associated with breast cancer susceptibility (Khakpour et al, 2015). NUP155 has been described as strictly required for the proliferation and survival of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia cells and thus constitutes a potential drug target for this disease (De et al, 2014).

[0148] OAS2 has been shown to be associated with impaired expression of the CD3-ζ chain, which is activated by caspase-3. CD3-ζ chain deficiency has been described as frequently observed in oral cancer (Dar et al, 2015). OAS2 has been described as a subpathway involved in innate immunity and inflammation pathways associated with risk of advanced prostate cancer (Kazma et al, 2012). Subpathway analysis revealed that OAS2 was nominally associated with risk of advanced prostate cancer (Kazma et al, 2012).

[0149] Low expression of PABPN1 in non-small cell lung cancer is associated with poor prognosis (Ichinose et al, 2014). Loss of PABPN1 has been described to release cancer cells from microRNA-mediated gene regulation in non-small cell lung cancer, potentially promoting tumor aggressiveness (Ichinose et al, 2014). An N-terminal polyalanine expansion variant of PABPN1 has been shown to be associated with apoptosis induction via the p53 pathway in HeLa and HEK-293 cell lines (Bhattacharjee et al, 2012).

[0150] PCBP1 has been described as being particularly important for cancer stem cell enrichment and function in prostate cancer cells (Chen et al., 2015a). PCBP1 has been described as a suppressor of gastric cancer pathogenesis, with its downregulation associated with a malignant phenotype in cultured and xenografted gastric cancer cells (Zhang et al., 2015e). Differential expression between benign and malignant serum and tissue samples from patients with ovarian serous adenocarcinoma suggests a role in the pathophysiology of ovarian cancer (Wegdam et al., 2014). PCBP1 has been shown to be an important mediator of TGF-β-induced epithelial-mesenchymal transition, a prerequisite for tumor metastasis in the gallbladder cancer cell line GBC-SD (Zhang and Dou, 2014). PCBP1 expression levels have been shown to regulate the in vitro migration and invasion capabilities of the gallbladder cancer cell line GBC-SD (Zhang and Dou, 2014). Therefore, PCBP1 may be a potential prognostic marker for gallbladder cancer metastasis (Zhang and Dou, 2014). Downregulation of PCBP1 has been described as potentially involved in the pathogenesis of cervical cancer (Pathak et al, 2014). PCBP1 has been described as a regulator of the tumor suppressor transcription factor p63 (Choet al, 2013). High PCBP1 expression in complete moles has been shown to be associated with a lower risk of developing gestational trophoblastic tumors, whereas PCBP1 expression is significantly reduced in malignantly transformed nevi (Shi et al, 2012). Thus, PCBP1 plays an important role in the pathogenesis of gestational trophoblastic tumors (Shi et al, 2012). Overexpression of PCBP1 has been shown to inhibit metastasis-associated PRL-3 protein translation and inactivate AKT, while knockdown of PCBP1 has been shown to activate AKT and promote tumorigenesis (Wang et al, 2010). PCBP1 has been described as playing a negative role in tumor invasion in the hepatocellular carcinoma cell line HepG2 (Zhang et al, 2010). Loss of PCBP1 in human liver tumors has been described to contribute to the development of a metastatic phenotype (Zhang et al, 2010).

[0151] 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 for oral cancer and epithelial mesothelioma (Swain et al., 2014; Ordonez, 2005). PDPN upregulation has been associated with lymph node metastasis and poor prognosis in upper aerodigestive tract squamous cell carcinoma (Chuang et al., 2013). PDPN has been described as expressed in vascular tumors, malignant mesothelioma, central nervous system tumors, germ cell tumors, squamous cell carcinoma, and aggressive tumors with increased invasiveness and metastatic potential (Raica et al., 2008). Therefore, PDPN may be considered an attractive therapeutic target for tumor cells (Raica et al., 2008).

[0152] PHTF2 was shown to be downregulated in tongue squamous cell carcinoma (Huang et al, 2007).

[0153] PKM2 has been shown to be critical for cancer cell proliferation and tumor growth (Chen et al., 2014b; Li et al., 2014; DeLaBarre et al., 2014). The N-myc gene acts as a transcriptional regulator of PKM2 in medulloblastoma (Tech et al., 2015). PKM2 appears to play a role in hepatocellular carcinogenesis, epithelial-mesenchymal transition, and angiogenesis (Nakao et al., 2014). PKM2 is one of two key players in the Warburg effect in oncology (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 has glycolytic functions, acting as a transcriptional coactivator and protein kinase. In the latter function, it translocates to the nucleus and phosphorylates histone 3, ultimately leading to cell cycle progression in glioblastoma (Semenza, 2011; Luo and Semenza, 2012; Tamada et al, 2012; Vennettian and Thompson, 2013; Yang and Lu, 2013; Gupta et al, 2014; Iqbal et al, 2014; Chen et al, 2014b; Warner et al, 2014). The low activity dimer PKM2, rather than the active tetramer form, may play a role in cancer (Mazurek, 2011; Wong et al, 2015; Iqbal et al, 2014; Mazurek, 2007).

[0154] 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-tumor, prostate BPH-1 cell line resulted in reduced apoptosis and differential expression of genes such as the prostate cancer-associated SPOCK1 gene (Yang et al, 2015a). Overall, altered PKP1 and SPOCK1 expression appear to be frequent and significant events in prostate cancer, suggesting a tumor suppressor function for PKP1 (Yang et al, 2015a). Decreased PKP1 expression has been associated with a significantly shorter time to distant metastasis in oral squamous cell carcinoma (Harris et al, 2015). Loss of PKP1 through activator methylation has been described as 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 distinguishing squamous cell carcinoma samples (Sanchez-Palencia et al, 2011). PKP1 has also been shown to be upregulated in the well-differentiated liposarcoma cell line GOT3 (Persson et al, 2008). Decreased PKP1 expression has been described as promoting increased activity in head and neck squamous cell carcinoma (Sobolik-Delmaire et al, 2007). PKP1 loss has been associated with cervical carcinogenesis (Schmitt-Graeff et al, 2007). PKP1 expression has been associated with local recurrence or metastasis and poor prognosis in patients with oropharyngeal squamous cell carcinoma (Papagerakis et al, 2003).

[0155] Increased PKP3 mRNA in the blood of patients with gastrointestinal tumors can serve as a biomarker and predictor of disease prognosis (Valladares-Ayerbes et al, 2010). PKP3 overexpression is associated with poor prognosis in breast, lung, and prostate cancers, while downregulation in bladder cancer is associated with aggressive behavior (Furukawa et al, 2005; Breuninger et al, 2010; Demirag et al, 2012; Takahashi et al, 2012). Loss of PKP3 leads to increased levels of MMP7 and PRL3 proteins, which are required for cell migration and tumor formation (Khapare et al, 2012; Basu et al, 2015).

[0156] Knockdown of PPP4R1 has been shown to inhibit cell proliferation in the breast cancer cell line ZR-75-30 (Qi et al, 2015). Therefore, PPP4R1 can promote breast cancer cell proliferation and may play a crucial role in breast cancer development (Qi et al, 2015). Knockdown of PPP4R1 in the hepatocellular carcinoma cell line HepG2 has been shown to induce cell proliferation, colony formation, and cell cycle arrest at the G2 / M phase (Wu et al, 2015). PPP4R1 knockdown has been further shown to inactivate p38 and c-Jun N-terminal kinase signaling cascades in HepG2 cells, suggesting that PPP4R1 promotes cell proliferation (Wu et al, 2015). Therefore, PPP4R1 plays a key role in promoting the growth of hepatocellular carcinoma cells (Wu et al, 2015). PPP4R1 has been described as a negative regulator of the inhibitor of NF-κB kinase activity in lymphocytes, whose downregulation promotes oncogenic NF-κB signaling in a subset of T-cell lymphomas (Brechmann et al, 2012).

[0157] PRC1 has been described as being associated with radiation resistance in cervical cancer, as cervical cancer tissues show high differential expression after irradiation (Fu et al, 2015b). A genetic locus within intron 14 of PRC1 has been described as being associated with breast cancer susceptibility (Cai et al, 2014). PRC1 is one of the genes in a five-gene signature that serves as a prognostic marker for 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 has been 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 of significant biological significance in tumorigenesis, and it can be used as a gene panel to predict prognosis in patients with resectable non-small cell lung cancer after adjuvant chemotherapy (Tang et al, 2013). PRC1 has been shown 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 has been 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, and the MPHOSPH1 / PRC1 complex has been shown to play a key role in bladder carcinogenesis and may represent a novel therapeutic target (Kanehira et al, 2007). PRC1 has also been shown to be regulated by p53 (Li et al, 2004).

[0158] Expressed sequence tag analysis identified PRDM15 as an upregulated gene in lymphoma (Giallourakis et al, 2013). PRDM15 has been described as a candidate tumor suppressor gene that may contribute to the development or progression of pancreatic cancer (Bashyam et al, 2005).

[0159] Different polymorphisms of PTHLH have been shown to be associated with lung cancer risk and prognosis (Manenti et al, 2000). PTHLH upregulation in a C57BL / 6 mouse-derived model of spontaneously metastatic breast cancer has been described as potentially involved in the metastatic spread of breast cancer (Johnstone et al, 2015). PTHLH has been shown to be upregulated in oral squamous cell carcinoma, enchondroma, adult T-cell leukemia / lymphoma, and clear cell renal 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 through p38 signaling, which promotes extravasation of colorectal cancer cells in the lung through caspase-independent cell death of endothelial cells in the lung microvasculature (Urosevic et al, 2014). PTHLH has been shown to be significantly differentially expressed in squamous cell carcinoma compared with normal skin (Prasad et al, 2014). PTHLH has been described as part of a four-gene signature associated with survival in patients with early-stage non-small cell lung cancer (Chang et al, 2012). Disruption of the antiproliferative effect of PTHLH through frameshift mutations has been described to promote 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 overall survival and disease-free survival in patients with clear cell renal cell carcinoma undergoing nephrectomy (Yao et al, 2014). PTHLH has been shown to positively regulate cell cycle progression and expression of proteins involved in cell cycle regulation through ERK1 / 2, p38, MAPK, and PI3K signaling pathways in the colorectal adenocarcinoma cell line Caco-2 (Calvo et al, 2014). RAP1GDS1 has been shown to promote proliferation in pancreatic cancer cells (Schuld et al, 2014). Simultaneous loss of both splice variants of RAP1GDS1 in xenografts of the mouse non-small cell lung cancer cell line NCI-H1703 has been shown to reduce tumorigenesis (Schuld et al, 2014). RAP1GDS1 has been shown to promote cell cycle progression in multiple cancer types, making it a valuable target for cancer therapy (Schuld et al, 2014). RAP1GDS1 has been shown to be upregulated in breast, prostate, and non-small cell lung cancers (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 activator 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 poor clinical outcome 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, thereby promoting the malignant phenotype of this disease. Therefore, RAP1GDS1 is an interesting therapeutic target for 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).

[0160] RNPEP activity has been shown to be upregulated in colorectal adenomas, papillary thyroid carcinomas, breast cancer, and clear cell renal 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 in rat C6 gliomas implanted subcutaneously (Mayas et al, 2012).

[0161] 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 OPCML, a potential tumor suppressor gene, 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 in 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 hepatocellular carcinoma cell line HepG2 has been shown to affect the expression of genes involved in glucose metabolism and hepatocarcinogenesis, suggesting that RORA is involved in carcinogenesis in liver-derived cells (Chauvet et al, 2011). RORA has been shown to be differentially methylated in gastric cancer compared with normal gastric mucosa (Watanabe et al, 2009). RORA has been described to be involved in the control of cell growth and differentiation as well as in the metastatic behavior of the androgen-independent prostate cancer cell line DU 145 (Moretti et al, 2002).

[0162] RPS17 has been shown to be differentially expressed in normal whole blood metastatic uveal melanoma and in tissues prone to uveal melanoma metastasis, suggesting that RPS17 may play a role in the metastatic tropism of uveal melanoma (Demirci et al, 2013). RPS17 has been shown to be upregulated in hepatocellular carcinoma (Liu et al, 2007b).

[0163] Knockdown of RPS26 has been shown to induce p53 stabilization and activation, leading to p53-dependent cell growth inhibition (Cui et al, 2014). RPS26 has further been shown to play a role in the DNA damage response by directly affecting the transcriptional activity of p53 (Cui et al, 2014).

[0164] S100A2 has been shown to be associated with non-small cell lung cancer and has been described as a predictive marker for poor overall survival in patients with squamous cell lung cancer (Hountis et al, 2014; Zhang et al, 2015d). S100A2 has been described as a downstream target of the oncogene KRAS and an activator of lung cancer tumor progression (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 S100A2 expression by the nitrosamine N-nitrosopyrrolidine has been described as a potential cause of tumor progression in esophageal squamous cell carcinoma in black South Africans (Pillay et al, 2015). S100A2 has been shown to be upregulated in the plasma of patients with early-stage non-small cell lung cancer and nasopharyngeal carcinoma, as well as in laryngeal, gastric, 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 be an important event in tumorigenesis in these diseases (Lee et al, 2015c). Cytoplasmic expression of S100A2 has been shown to be upregulated in oral squamous cell carcinoma, whereas 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 also 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 that plays a role in regulating mutant p53 stability by modulating its binding to HSP90 (Buckley et al, 2014). S100A2 has been described as a candidate tumor suppressor gene that is downregulated in recurrent nasopharyngeal carcinoma and may play an important role in the development of recurrent nasopharyngeal carcinoma (Huang et al, 2014b). S100A2 has been shown to be downregulated in gastric cancer, and downregulation has been associated with depth of invasion, lymph node metastasis, decreased recurrence-free probability, and decreased 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 further been shown to negatively regulate the MEK / ERK signaling pathway in MGC-803 cancer cells (Liu et al, 2014e).In immunodeficient mice, overexpression of S100A2 has been shown to induce epithelial-mesenchymal transition in A549 lung cancer cells, subsequently increasing invasiveness, enhancing Akt phosphorylation, and accelerating tumor growth (Naz et al, 2014). The proto-oncogenic behavior of S100A2 has been further characterized by its involvement in the regulation of PI3K / Akt signaling and its functional interaction with the TGFβ signaling pathway 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 serve as a prognostic marker for patients with cholangiocarcinoma (Sato et al, 2013).

[0165] S100A8 has been described as an important mediator of 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 marker, prognostic indicator, and therapeutic target for non-small cell lung cancer (Lim and Thomas, 2013). Overexpression of S100A8 has been shown to be associated with stage progression, 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 of anaplastic thyroid cancer cells in mice with S100A8 knockdown revealed reduced tumor growth and lung metastasis, and significantly prolonged animal survival (Reeb et al, 2015). S100A8 has been shown to promote the proliferation of anaplastic thyroid cancer cells by interacting with RAGE, which activates the p38, ERK1 / 2, and JNK signaling pathways in tumor cells (Reeb et al, 2015). Therefore, S100A8 may represent a relevant therapeutic target for 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 shown to be associated with renal cancer progression and has been described as a potential biomarker and therapeutic target for renal cancer (Mirza et al, 2014). S100A8 has been described as part of calprotectin, a heterodimer required for the progression of non-inflammatory-driven liver tumors, and may represent a therapeutic target for hepatocellular carcinoma (De et al, 2015). Si00A8 was shown to regulate colon cancer cell cycle and proliferation through induction of ID3 expression while inhibiting p21 (Zhang et al, 2015b).

[0166] SERPINH1 encodes serine protease inhibitor, clade H (heat shock protein 47) member 1, (collagen binding protein 1), a serine protease inhibitor. SERPINH1 functions as a collagen-specific molecular chaperone in the endoplasmic reticulum (RefSeq, 2002). SERPINH1 is overexpressed in many human cancers, including gastric cancer, lung cancer, pancreatic ductal adenocarcinoma, gliomas, and ulcerative colitis-associated cancers (Zhao et al, 2014a). SERPINH1 is upregulated in hepatocellular carcinoma, esophageal squamous cell carcinoma, cholangiocarcinoma, gastric cancer, lung cancer, pancreatic ductal adenocarcinoma, ulcerative colitis-associated cancers, and gliomas (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 SERPINH1 immunostaining levels and pathological stage have been shown to be significantly correlated with overall and recurrence-free survival (Lee et al, 2015b). Therefore, SERPINH1 may be a potential prognostic marker for esophageal squamous cell carcinoma (Lee et al, 2015b). SERPINH1 knockdown in glioma cells has been shown to inhibit glioma cell growth, 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 glioma (Zhao et al, 2014a). SERPINH1 has been shown to be downregulated in metastatic carcinomas compared with primary oral squamous cell carcinomas with multiple lymph node involvement, suggesting that SERPINH1 may be associated with the metastatic potential of these tumors (Nikitakis et al, 2003).

[0167] SLC7A11 has been shown to be downregulated in drug-resistant variants of the W1 ovarian cancer cell line, suggesting a role in cancer cell drug resistance (Januchowski et al., 2013). SLC7A11 has been described as modulating the tumor microenvironment, leading to a growth advantage for cancer (Savaskan and Eyupoglu, 2010). SLC7A11 has been described as participating in neurodegeneration in gliomas, making it a potential target for cancer therapy (Savaskan et al., 2015). SLC7A11 has been shown to be repressed by p53 in the context of ferroptosis, and the p53-SLC7A11 axis has been described as being conserved in p53(3KR) mutants and contributing to their ability to suppress tumorigenesis in the absence of classical tumor suppressor mechanisms (Jiang et al., 2015). SLC7A11 has been described as a functional subunit of system Xc, whose 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 poor clinical outcome, and SLC7A11 inhibition has been described as a promising therapeutic approach for this disease (Drayton et al, 2014). SLC7A11 was shown to be differentially expressed in the human promyelocytic leukemia cell line HL-60 that had been exposed to benzene and its metabolites, highlighting the potential relevance of SLC7A11 to leukemogenesis (Sarma et al, 2011). Disruption of SLC7A11 has been described to result in growth inhibition in a variety of cancers, including lymphomas, gliomas, prostate cancer, and breast cancer (Chen et al, 2009). SLC7A11 inhibition was shown to inhibit cell invasion of the esophageal cancer cell line KYSE150 in vitro and experimental metastasis in nude mice, establishing a role for SLC7A11 in tumor metastasis (Chen et al, 2009).

[0168] SRPR has been shown to be amplified in acute myeloid leukemias containing double minute chromosomes (Crossen et al, 1999).

[0169] The human ortholog of SSR4 was shown to be differentially expressed in the opossum melanoma cell lines TD6b and TD15L2 and upregulated in advanced tumors, suggesting that SSR4 is a candidate gene with potential functions in UV-induced melanoma and metastasis (Wang and VandeBerg, 2004). SSR4 mRNA levels were shown to be enriched in the osteosarcoma cell lines OHS, SAOS-2, and KPDXM compared to normal osteoblasts (Olstad et al, 2003).

[0170] Dysregulated STK17A expression is associated with diverse cancer types. Reduced expression in cervical and colorectal cancers correlates with the pro-apoptotic properties of STK17A, which are associated with tumor progression. STK17A is overexpressed in glioblastoma and head and neck cancer in a level-dependent manner, potentially through effects on other tumor-associated pathways such as TGF-β (Mao et al, 2013a; 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 regulator of reactive oxygen species (ROS) (Kerley-Hamilton et al, 2005; Mao et al, 2011).

[0171] SYK has been described as a regulator of tumorigenesis, acting as a tumor activator in some cells by providing survival functions and as a tumor suppressor in others by limiting epithelial-mesenchymal transition and inhibiting migration (Krisenko and Geahlen, 2015). SYK has been described as being involved in B cell receptor (BCR) activation in B cell lymphomas (Seda and Mraz, 2015). Inhibition of key kinases in the BCR pathway, such as SYK, has been found to reduce the viability of chronic lymphocytic leukemia cells 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 involved in the pathogenesis of chronic lymphocytic leukemia and may be valuable in assessing treatment efficacy and prognosis in this disease (Feng and Wang, 2014). SYK has been described as a potential tumor suppressor in breast cancer, and its absence in primary breast tumors is associated with poor outcome (Navara, 2004). SYK has been shown to play a key role in paclitaxel resistance in ovarian cancer (Yu et al, 2015b). SYK downregulation has been described as being associated with the development of various cancers, including colorectal cancer (Peng et al, 2015). Different genetic polymorphisms in the SYK activator have been shown to be independent risk factors for the development of colorectal cancer in a southern Chinese Han population (Peng et al, 2015). SYK has been shown to be frequently methylated in hepatocellular carcinoma, and SYK methylation has been shown to identify a subset of hepatocellular carcinoma with a poor prognosis (Shin et al, 2014).

[0172] TP63 translocations have been described as an event in a subset of anaplastic lymphoma kinase-positive anaplastic large cell lymphomas, where they are associated with the aggressive course of the disease (Hapgood and Savage, 2015). TP63 has been described as playing a complex role in cancer due to its involvement in epithelial cell differentiation, cell cycle arrest, and apoptosis (Lin et al, 2015). The TP63 isoform TAp63 has been described as overexpressed in hematological malignancies, while missense mutations of TP63 have been reported in squamous cell carcinomas and TP63 translocations in lymphomas and some lung adenocarcinomas (Orzol et al, 2015). Aberrant splicing resulting in overexpression of the TP63 isoform δNp63 is frequently found in human cancers (e.g., cutaneous squamous cell carcinoma) and likely contributes to tumorigenesis and progression (Missero and Antonini, 2014; Inoue and Fry, 2014).

[0173] TPM1 has been shown to be downregulated in renal cell carcinoma, esophageal squamous cell carcinoma, 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 correlate with tumor size, Fuhrman grade, and prognosis in patients with renal cell carcinoma. TPM1 transfection of the renal cell carcinoma cell lines OSRC-2 and 786-0 was shown to reduce migration and invasion while enhancing apoptosis (Wang et al., 2015b). Thus, TPM1 has been described as displaying characteristics of a tumor suppressor gene and is overexpressed in renal cell carcinoma cells (Wang et al., 2015b). The RAS / PI3K / AKT and RAS / MEK signaling pathways have been described as being involved in TPM1 regulation and inhibition in the intrahepatic cholangiocarcinoma cell line HuCCT1 and in esophageal squamous cell carcinoma cell lines (Zare et al, 2012; Yang et al, 2013b). TPM1 has been described as a tumor suppressor, and its overexpression in the breast cancer cell line MCF-7 inhibits anchorage-independent cell growth (Zhu et al, 2007b). Epigenetic inhibition of TPM1 has been described as being associated with altered TGFβ tumor suppressor function and may contribute to the metastatic properties of tumor cells (Varga et al, 2005).

[0174] Tryptase has been shown to be upregulated in some patients with acute myeloid leukemia (Jin et al, 2014). Tryptase expression has been described as being regulated by SCF / c-KIT signaling via the ERK1 / 2 and p38 MAPK pathways (Jin et al, 2014). Mast cell tryptase has been described as being involved in colorectal cancer angiogenesis and has been shown to be more highly expressed in the serum of colorectal cancer patients before than after radical surgical resection (Ammendola et al, 2014).

[0175] TSHZ3 was shown to be downregulated in the oral squamous cell carcinoma cell line SCC-9 compared to the non-tumorigenic cell line OKF6-TERT1RTSHZ3 (Marcinkiewicz and Gudas, 2014). TSHZ3 was described as a transcriptional regulator gene that was found to be recurrently rearranged in some cases of high-grade serous ovarian carcinoma (McBride et al, 2012). TSHZ3 was described as a candidate tumor suppressor gene that is downregulated in breast and prostate cancers (Yamamoto et al, 2011).

[0176] TSPAN10 has been shown to be a gene differentially expressed between metastatic melanoma samples and normal skin samples and may be a potential biomarker for metastatic melanoma treatment (Liu et al, 2014c). Among other genes, TSPAN10 has been shown to be upregulated in uterine leiomyosarcoma metastases compared with primary leiomyosarcoma, thus helping to distinguish these diseases and potentially aiding in understanding tumor progression in this cancer (Davidson et al, 2014).

[0177] TTPAL has been described as a candidate oncogene showing mutations in microsatellite instability colorectal cancer (Tuupanen et al, 2014).

[0178] TUBGCP2 has been shown to be upregulated in paclitaxel-resistant ovarian cancer cell lines and has been described as being associated with paclitaxel sensitivity in the non-small cell lung cancer cell line NCI-H1155 (Huang and Chao, 2015). TUBGCP2 has also been 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).

[0179] VIM has been described as a downstream target of STAT3, implicated in breast tumor progression through STAT3 dysregulation (Banerjee and Resat, 2015). VIM has been described as a potential nasopharyngeal carcinoma-associated protein (Chen et al, 2015c). Negative methylation of vimentin has been shown to predict improved prognosis in patients with pancreatic cancer (Zhou et al, 2014). VIM has been shown to be upregulated in non-small cell lung cancer via C6orf106 and subsequently associated with enhanced cancer cell invasiveness (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 melanoma subgroups (Qendro et al, 2014). VIM has been shown to be upregulated in clear cell renal cell carcinoma (Shi et al, 2015). High VIM expression has been described as an independent predictor of clear cell renal cell carcinoma (Shi et al, 2015). VIM has been shown to act as a scaffold to recruit Slug to ERK and promote Slug phosphorylation, which has been described as required for activating the epithelial-mesenchymal transition, a developmental process employed during tumorigenesis that promotes metastatic capacity (Virtakoivu et al, 2015).

[0180] WDR1 has been shown to be upregulated in stromal fluid from ovarian cancer and in high-grade canine cutaneous mast cell tumors with a poor prognosis, compared with low-grade mast cell tumors with a good 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 be associated with poor overall survival (Kim et al., 2011). WDR1 has been shown to be upregulated in the region between the invasive front of breast cancer tumors and normal tissue (the interface zone) and, therefore, may be involved in breast cancer progression and metastasis (Kang et al., 2010).

[0181] Fusion of YWHAE with NUTM2B / NUTM2E has been described as an event observed in a small number of renal clear cell sarcomas (Karlsson et al, 2015). YWHAE-NUTM2 fusion has been described as a common event in high-grade endometrial stromal sarcomas (Ali et al, 2014). High-grade endometrial stromal sarcomas harboring YWHAE-NUTM2 fusions have been described as a subset of endometrial stromal sarcomas with aggressive clinical behavior and a poor prognosis (Kruse et al, 2014). Disruption of three loci, including YWHAE, has been described as a potential factor in the development of uterine sarcomas (Suzuki et al, 2014). YWHAE has been shown to be downregulated in gastric cancer, with decreased YWHAE levels associated with diffuse gastric cancer and 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 breast cancer tissues from patients with and without recurrence, and has been shown to be associated with both disease-free and overall survival (Cimino et al, 2008). Therefore, YWHAE can be used as an independent prognostic marker and potential drug target for breast cancer (Cimino et al, 2008).

[0182] Alterations in ZNF292 have been described as driver alterations in 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 a clinically relevant immunogenic antigen in head and neck squamous cell carcinoma (Heubeck et al, 2013).

[0183] Detailed Description of the Invention

[0184] Whether an immune response can be stimulated depends on the presence of antigens that are recognized as foreign by the host immune system. The discovery of tumor-associated antigens has raised the possibility of harnessing the host immune system to intervene in tumor growth. Currently, various mechanisms of immune response, both humoral and cellular, are being explored for cancer immunotherapy.

[0185] Specific elements of the cellular immune response can specifically recognize and destroy tumor cells. T cells isolated from tumor-infiltrating cell populations or peripheral blood have demonstrated that these cells play a crucial role in the innate immune defense against cancer. CD8+ T cells, in particular, play a crucial role in this response. TCD8+ T cells recognize class I molecules contained in peptides carried by the major histocompatibility complex (MHC) and typically consist of 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRIPs) located in the cytoplasm. Human MHC molecules are also known as human leukocyte antigens (HLA).

[0186] Unless otherwise stated, all terms used herein are defined below.

[0187] 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 naturally peptide-presenting target cells, secretion of cytokines, preferably peptide-induced interferon-γ, TNF-α, or IL-2, secretion of effector molecules, preferably peptide-induced granzymes or perforins, or degranulation.

[0188] As used herein, the term "peptide" refers to a series of amino acid residues, typically linked by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. These peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids in length and as long as 10, 11, 12, 13, or 14 amino acids or longer. In the case of MHC class II peptides (elongated variants of the peptides of the present invention), they can be as long as 14, 15, 16, 17, 18, 19, or 20 amino acids or longer.

[0189] Thus, the term "peptide" shall include salts of a series of amino acid residues, typically linked 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 or acetate (trifluoroacetic acid) salt. It should be noted that the salts of the peptides of the present invention are substantially different from the peptides in their in vivo state, as they are not salts in vivo.

[0190] The term "peptide" shall also include "oligopeptide". As used herein, the term "oligopeptide" refers to a series of amino acid residues, typically linked 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, as long as the correct epitope is maintained in the oligopeptide. Typically, the oligopeptide is less than about 30 amino acid residues in length and greater than about 15 amino acids in length.

[0191] The term "polypeptide" refers to a series of amino acid residues, typically linked 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, as long as the correct epitope is maintained. In contrast to the terms peptide or oligopeptide, the term "polypeptide" refers to molecules containing more than about 30 amino acid residues.

[0192] A peptide, oligopeptide, protein, or nucleic acid encoding such a molecule is "immunogenic" (and therefore an "immunogen" for purposes of the present invention) if it is capable of inducing an immune response. In the context of the present invention, immunogenicity is more specifically defined as the ability to induce a T cell response. Thus, an "immunogen" is 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 aspect, the immunogen can be a peptide, a complex of a peptide with MHC, and / or a protein for enhancing specific antibody or TCR resistance.

[0193] A class I T cell "epitope" requires a short peptide that binds to an MHC class I receptor, forming a ternary complex (MHC class I alpha chain, beta-2-microglobulin, and peptide) that can be recognized by a T cell bearing a matching T cell receptor bound to the MHC / peptide complex with appropriate affinity. Peptides that bind to MHC class I molecules are typically 8-14 amino acids in length, with 9 amino acids being the most typical length.

[0194] In humans, there are three different gene loci that encode MHC class I molecules (human MHC molecules are also designated 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.

[0195] Table 5: Frequency of expression of HLA-A*02 and HLA-A*24 and the most common HLA-DR serotypes, F. Frequencies are derived from haplotype frequencies within the US population, adapted from the Hardy-Weinberg formula F = 1-(1-Gf)² used by Mori et al. (Mori et al, 1997). Due to linkage disequilibrium, combinations of A*02 or A*24 within certain HLA-DR alleles may be enriched or have lower frequencies compared to their expected individual frequencies. For more information, see Chanock et al. (Chanock et al, 2004).

[0196]

[0197]

[0198]

[0199] The peptides of the present invention, when incorporated into the vaccines of the present invention as described herein, preferably bind to A*02. Vaccines may also include pan-binding MHC class II peptides. Thus, the vaccines of the present invention can be used to treat cancer in A*02-positive patients, but do not necessarily require selection for MHC class II allotypes due to the broad binding properties of these peptides.

[0200] If the A*02 peptide of the present invention is combined with a peptide that binds to another allele, such as A*24, a higher proportion of the patient population can be treated compared to each MHC class I allele alone. While in most populations, less than 50% of patients can be treated with either allele alone, a vaccine comprising both HLA-A*24 and HLA-A*02 epitopes can treat at least 60% of patients in any relevant population. Specifically, the following proportions of patients in each region have a positive response to 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 according to www.allelefrequencies.net).

[0201] In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxynucleotides.

[0202] The nucleotide sequence encoding a specific peptide, oligopeptide, or polypeptide can be a natural nucleotide sequence or a synthetic nucleotide sequence. Generally, the DNA fragment encoding the peptide, polypeptide, and protein of the present invention is composed of an eDNA fragment and a short oligonucleotide linker, or a series of oligonucleotides, to provide a synthetic gene that can be expressed in a recombinant transcription unit containing regulatory elements derived from a microbial or viral operon.

[0203] As used herein, the term "nucleotide encoding a peptide" refers to a nucleotide sequence encoding a peptide, wherein the peptide includes artificial (man-made) activation and stop codons that are compatible with the biological system in which the sequence is to be expressed by dendritic cells or another cell system used to generate TCRs.

[0204] The nucleic acid sequences mentioned herein include both single-stranded nucleic acids and double-stranded nucleic acids. Therefore, unless otherwise indicated herein, for example, for DNA, a specific sequence is the single-stranded DNA of the sequence, the duplex (double-stranded DNA) of the sequence and its complementary sequence, and the complementary sequence of the sequence.

[0205] The term "coding region" refers to that portion of a gene that naturally or normally encodes the expression product of the gene in its natural genomic environment, ie, the region that encodes the native expression product of the gene in vivo.

[0206] The coding region can be derived from a non-mutated ("normal") gene, a mutant gene, or an abnormal gene, or can even be derived from a DNA sequence, which can be synthesized entirely in the laboratory using DNA synthesis methods well known in the art.

[0207] The term "expression product" refers to a polypeptide or protein that is the translation product of a gene and any nucleic acid sequence encoding the equivalent resulting from degeneration of the genetic code and thereby encoding the same amino acids.

[0208] The term "fragment", when referring to a coding sequence, means a portion of DNA comprising a non-complete coding region, the expression product of which has substantially the same biological function or activity as the expression product of the complete coding region.

[0209] The term "DNA fragment" refers to a DNA polymer, either as an individual fragment or as a component of a larger DNA structure, obtained from DNA that has been isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials, and in quantities or concentrations that permit identification, manipulation, and recovery of the fragment and its component nucleotide sequences using standard biochemical methods, such as cloning vectors. Such fragments are present in the form of open reading frames (uninterrupted by internal untranslated sequences) or introns (commonly present in eukaryotic genes). Untranslated DNA sequences may be present downstream of the open reading frame, where they do not interfere with manipulation or expression of the coding region.

[0210] The term "primer" refers to a short nucleic acid sequence that pairs with a DNA strand and provides a free 3'-OH end at which DNA polymerase begins to synthesize a deoxyribonucleic acid chain.

[0211] The term "activator" refers to a region of DNA that is involved in the binding of RNA polymerase, thereby activating transcription.

[0212] The term "isolated" means that a material is removed from its original environment (e.g., its natural environment if naturally occurring). For example, a naturally occurring nucleotide or polypeptide in a living animal is not isolated, but a nucleotide or polypeptide separated from some or all coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector and / or such polynucleotides and polypeptides may be part of a composition and still be isolated because the vector or composition is not part of its natural environment.

[0213] The polynucleotides and recombinant or immunogenic polypeptides disclosed herein may also be in a "purified" form. The term "purified" does not require absolute purity; it is a relative definition and can include highly purified or partially purified preparations, as those skilled in the relevant art will understand. For example, individual clones isolated from a cDNA library that has been purified to an electrophoretic isoform using conventional methods. Purification of the starting material or natural substance by 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, it is expressly contemplated that the polypeptides are preferably 99.999% pure, or at least 99.99% or 99.9% pure; and even suitably 99% or more by weight.

[0214] The nucleic acid and polypeptide expression products disclosed herein, as well as expression vectors comprising such nucleic acids and / or polypeptides, may be present in a "concentrated form." As used herein, the term "concentrated" refers to a concentration of a material that is at least about 2, 5, 10, 100, or 1000 times its natural concentration, advantageously 0.01% by weight, preferably at least 0.1%. Concentrated preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also expressly contemplated. The sequences, constructs, vectors, clones, and other materials comprising the invention may advantageously be present in a concentrated or isolated form. The term "active fragment" refers to a fragment that produces an immune response (i.e., possesses immunogenic activity), typically a fragment of a peptide, polypeptide, or nucleic acid sequence, administered alone or optionally with a suitable adjuvant or in a carrier to an animal, such as a mammal, e.g., a rabbit or mouse, including a human; such immune response takes the form of stimulating a T cell response in the recipient animal (e.g., a human). Alternatively, "active fragments" may also be used to induce T cell responses in vitro.

[0215] As used herein, the terms "portion," "segment," and "fragment," when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which form a subset of a larger sequence. For example, if a polypeptide is treated with any endopeptidase (such as trypsin or chymotrypsin), the oligopeptides obtained from such treatment will represent a portion, segment, or fragment of the starting polypeptide. When used in relation to polynucleotides, these terms refer to the products produced by treating the polynucleotide with any endonuclease.

[0216] According to the present invention, the term "percent identity" or "percent equivalence", when referring to sequences, means that the sequence to be compared ("compared sequence") is compared to the sequence or sequence of the claim ("reference sequence") after the sequence to be compared ("compared sequence") is aligned with the sequence or sequence of the claim. The percent identity is then calculated according to the following formula:

[0217] Percentage of identity = 100[1-(C / R)]

[0218] Where C is the number of differences between the reference sequence and the compared sequence over the length of the alignment between the reference sequence and the compared sequence,

[0219] (i) Each base or amino acid sequence in the reference sequence has no corresponding aligned base or amino acid in the compared sequence;

[0220] (ii) each gap in the reference sequence, and

[0221] (iii) each aligned base or amino acid in the reference sequence that differs from the aligned base or amino acid in the compared sequence constitutes a difference and

[0222] (iiii) Alignment must start at position 1 of the alignment sequence;

[0223] And R is the number of bases or amino acids in the reference sequence over the length of the alignment between the reference sequence and the compared sequence, with any gaps in the reference sequence also being counted as one base or amino acid.

[0224] If there is an alignment between the "compared sequence" and the "reference sequence" with a percent identity, as calculated above, that is approximately equal to or greater than the specified minimum percent identity, then the compared sequence has the specified minimum percent identity to the reference sequence, although there may be alignments with percent identities, as calculated herein above, that are lower than the specified percent identity.

[0225] Therefore, as described 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 having 88% homology to SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that induces T cell cross-reactivity with the peptide. The peptide of the present invention has the ability to bind to a major histocompatibility complex (MHC) class I or an elongated version of the peptide class II molecule.

[0226] In the present invention, the term "homology" refers to the degree of identity between two amino acid sequences (see the percentage of identity above, such as peptide or polypeptide sequences. The "homology" mentioned above is determined by aligning the two sequences adjusted under ideal conditions with the sequences to be compared. Such sequence homology can be calculated by creating an alignment using an algorithm such as ClustalW. General sequence analysis software, more specifically Vector NTI, GENETYX or other tools provided by public databases can also be used.

[0227] One skilled in the art can assess whether T cells induced by a particular peptide variant can cross-react with the peptide itself (Appay et al, 2006; Colombetti et al, 2006; Fong et al, 2001; Zaremba et al, 1997).

[0228] The inventors use the term "variant" of a given amino acid sequence to mean that the side chains of one or two amino acid residues, for example, are altered by substitution with the side chains of another naturally occurring amino acid residue or with other side chains, such that the peptide is still capable of binding to an HLA molecule in substantially the same manner as a peptide comprising the given amino acid sequence (consisting of SEQ ID NO: 1 to SEQ ID NO: 93). For example, a peptide may be modified so as 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 at least maintain (if not improve) its ability to bind to the TCR of activated T cells.

[0229] These T cells can then cross-react with and kill cells expressing polypeptides comprising the natural amino acid sequence of a cognate peptide as defined herein. As described in scientific literature and databases (Rammensee et al., 1999; Godkin et al., 1997), certain positions in HLA-A binding peptides are typically anchor residues, forming a core sequence that aligns with the binding motif of the HLA binding groove, defined by the polarity, electrophysical, hydrophobicity, and steric properties of the polypeptide chains comprising the binding groove. Therefore, one skilled in the art can modify the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 93 by retaining known anchor residues and determine whether these variants retain the ability to bind to MHC class I or II molecules. The variants of the present invention retain the ability to bind to the TCR of activated T cells, and these T cells can then cross-react with and kill cells expressing a polypeptide comprising the natural amino acid sequence of a cognate peptide as defined herein.

[0230] If not otherwise stated, the original (unmodified) peptides disclosed herein can be modified by replacing one or more residues at different (possibly selective) sites within the peptide chain. Preferably, these substitutions are located at the termini of the amino acid chain. This substitution may be conservative, for example, where one amino acid is replaced by another amino acid having similar structure and characteristics, such as where one hydrophobic amino acid is replaced by another hydrophobic amino acid. A more conservative substitution is a substitution between amino acids of the same or similar size and chemical properties, for example, leucine is replaced by isoleucine. In studies of sequence variations in natural homologous protein families, substitutions of certain amino acids are often more tolerated than others, and these amino acids often show similarities with the size, charge, polarity and hydrophobicity of the original amino acids, which is the basis for determining "conservative substitutions".

[0231] In this article, conservative substitutions are defined as exchanges within one of the following five groups: Group 1 - small aliphatic, nonpolar 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 nonpolar residues (Met, Leu, Ile, Val, Cys) and Group 5 - large aromatic residues (Phe, Tyr, Trp).

[0232] Less conservative substitutions might involve replacing one amino acid with another with similar characteristics but a different size, such as replacing an alanine residue with an isoleucine residue. Highly nonconservative substitutions might involve replacing an acidic amino acid with one with polar or even basic properties. However, such "radical" substitutions should not be dismissed as ineffective, as chemical reactions are not entirely predictable, and radical substitutions may have unforeseen effects not foreseen by simple chemical principles.

[0233] Of course, such substitutions may involve structures other than common L-amino acids. Thus, D-amino acids may be substituted with common L-amino acids in the antigenic peptides of the present invention and remain within the scope of this disclosure. Furthermore, non-standard amino acids (i.e., other than common naturally occurring proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

[0234] If substitutions at more than one position are found to result in peptide antigenic activity substantially equal to or greater than the values ​​defined below, combinations of these substitutions are tested to determine whether the combined substitutions produce additive or synergistic effects on the antigenicity of the peptide. No more than four positions within the peptide may be substituted simultaneously.

[0235] A peptide consisting essentially of an amino acid sequence as described herein may have one or two non-anchor amino acids (see below regarding anchor motifs) exchanged, without substantially altering or adversely affecting the ability of the peptide to bind to human major histocompatibility complex (MHC) class I or II molecules compared to the unmodified peptide. In another embodiment, in a peptide consisting essentially of an amino acid sequence as described herein, one or two amino acids may be exchanged with their conserved exchange partners (see below), without substantially altering or adversely affecting the ability of the peptide to bind to human major histocompatibility complex (MHC) class I or II molecules compared to the unmodified peptide.

[0236] These amino acid residues that do not substantially interact with T cell receptors can be modified by substituting other amino acids that have little effect on T cell responses and do not interfere with binding to the relevant MHC. Therefore, except for specific limiting conditions, the peptides of the present invention may be any peptide (the term used by the inventors includes oligopeptides or polypeptides) that includes a given amino acid sequence or a portion or variant thereof.

[0237] Table 6: Variants and motifs of peptides according to SEQ ID NO: 4, 9 and 18

[0238]

[0239]

[0240]

[0241] Longer (elongated) peptides may also be suitable. MHC class I epitopes (typically 8 to 11 amino acids in length) may be produced by processing of the peptide from a longer peptide or protein containing the actual epitope. The residues flanking the actual epitope are preferably residues that do not significantly interfere with the protein cleavage required to expose the actual epitope during processing.

[0242] The peptides of the present invention can be elongated by up to four amino acids, i.e., 1, 2, 3, or 4 amino acids, and can be added to either end in any combination between 4:0 and 0:4. The elongation combinations of the present invention can be found in Table 7.

[0243] Table 7: Elongated combinations of peptides of the present invention

[0244]

[0245]

[0246] The stretched / elongated amino acids can be the original sequence peptide of the protein or any other amino acid. Elongation can be used to enhance the stability or solubility of the peptide.

[0247] Therefore, the epitopes of the present invention may be identical to naturally occurring tumor-associated epitopes or tumor-specific epitopes, or may include peptides that differ by no more than four residues from a reference peptide, as long as they have substantially the same antigenic activity.

[0248] In an alternative embodiment, one or both sides of the peptide are extended by more than 4 amino acids, preferably to a total length of up to 30 amino acids. This can form an MHC class II binding peptide. Binding to MHC class II peptides can be tested by methods known in the art.

[0249] Thus, the present invention provides peptides and variants of MHC class I epitopes, wherein the total length of the peptide or antibody is 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids in length (i.e., 10, 11, 12, 13, 14 amino acids, and in the case of elongated class II binding peptides, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids).

[0250] Of course, the peptides or variants of the present invention can bind to human major histocompatibility complex (MHC) class I or II molecules. The binding of the peptides or variants to the MHC complex can be tested using methods known in the art.

[0251] Preferably, when peptide-specific T cells of the present invention are tested in comparison to the substituted peptide, if the substituted peptide achieves a half-maximal increase in peptide solubility relative to background, the peptide concentration is no more than about 1 mM, preferably no more than about 1 μM, more preferably no more than about 1 nM, even more preferably no more than about 100 pM, and most preferably no more than about 10 pM. It is also preferred that the substituted peptide is recognized by more than one T cell, at least two, and more preferably three.

[0252] In a particularly preferred embodiment of the present invention, the peptide consists or essentially consists of an amino acid sequence selected according to SEQ ID NO: 1 to SEQ ID NO: 93.

[0253] Essentially consisting of "..." means that the peptide of the present invention, in addition to consisting of any one of SEQ ID NO: 1 to SEQ ID NO: 93 or a variant thereof, further contains amino acids located at other N- and / or C-terminal extensions, which are not necessarily capable of forming a peptide that serves as an epitope on an MHC molecule.

[0254] However, these extended regions are important for effectively introducing the peptides of the present invention into cells. In one embodiment of the present invention, the peptide is part of a fusion protein comprising, for example, the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33, hereinafter referred to as "Ii") from NCBI GenBank Accession No. X00497. In other fusions, the peptides of the present invention can be fused to antibodies described herein, or functional portions thereof, particularly into antibody sequences to enable specific targeting of the antibodies, or, for example, into dendritic cell-specific antibodies described herein.

[0255] In addition, the peptide or variant can be further modified to improve stability and / or binding to MHC molecules, thereby eliciting a stronger immune response. Such optimization methods for peptide sequences are well known in the art and include, for example, the introduction of trans-peptide bonds and non-peptide bonds.

[0256] In a trans-peptide bond, the amino acid residues are not linked by a peptide bond (-CO-NH-), but the peptide bond is reversed. Such retro-inverso peptidomimetics can be prepared by methods known in the art, such as those described by Meziere et al. (Meziere et al., 1997), which is incorporated herein by reference. This method involves preparing peptidomimetics that contain backbone (rather than side chain) alterations. Studies by Meziere et al. (Meziere et al., 1997) have shown that these peptidomimetics facilitate MHC binding and helper T cell responses. Retro-inverso peptidomimetics, which replace the CO-NH peptide bond with an NH-CO bond, significantly improve resistance to hydrolysis.

[0257] Non-peptide bonds include -CH2-NH, -CH2S-, -CH2CH2-, -CH=CH-, -COCH2-, -CH(OH)CH2-, and -CH2SO-, etc. U.S. Patent No. 4,897,445 proposes a non-solid phase synthesis method for non-peptide bonds (-CH2-NH) in polypeptide chains, which involves a polypeptide synthesized according to a standard procedure and a non-peptide bond synthesized by the interaction of an aminoaldehyde and an amino acid containing NaCNBH3.

[0258] Peptides containing the above sequences may be synthesized with additional chemical groups at their amino and / or carboxyl termini to enhance their stability, bioavailability, and / or affinity. For example, a hydrophobic group such as a benzyloxycarbonyl group, a dansyl group, or a tert-butyloxycarbonyl group may be added to the amino terminus of the peptide. Similarly, an acetyl group or a 9-fluorenylmethyloxycarbonyl group may be located at the amino terminus of the peptide. Furthermore, a hydrophobic group, a tert-butyloxycarbonyl group, or an amino group may be added to the carboxyl terminus of the peptide.

[0259] In addition, all peptides of the present invention may be synthesized to alter their spatial configuration. For example, the dextrorotatory form of one or more amino acid residues of these peptides may be used, rather than the levorotatory form. Furthermore, at least one amino acid residue of a peptide of the present invention may be substituted with a well-known non-natural amino acid residue. Such alterations may help to increase the stability, bioavailability, and / or binding activity of the peptides of the present invention.

[0260] Similarly, the peptides or variants of the present invention can be chemically modified by reacting specific amino acids 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. Although the methods for chemical modification of amino acids are not limited, they include (but are not limited to) modification by the following methods: acylation, amidination, pyridoylation of lysine, reductive alkylation, trinitrophenylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amino modification of carboxyl groups and sulfhydryl groups by performic acid oxidation of cysteine ​​to cysteic acid, formation of labile derivatives, formation of mixed disulfide compounds with other sulfhydryl compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanate at alkaline pH. In this regard, the skilled person is 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 extensive methods related to chemical modification of proteins.

[0261] In short, modification of arginyl residues in proteins is often based on the reaction of ortho-dicarbonyl compounds (such as phenylglyoxal, 2,3-butanedione, and 1,2-hexanedione) to form adducts. Another example is the reaction of methylglyoxal with arginine residues. Cysteine ​​can be modified without concurrent modification of nucleophilic sites such as lysine and histidine. Therefore, a large number of reagents are available for cysteine ​​modification. The websites of companies such as Sigma-Aldrich (http: / / www.sigma-aldrich.com) contain information on specific reagents.

[0262] Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during heat treatment of biopharmaceuticals. Woodward's reagent K can be used to modify specific glutamic acid residues. N-(3-Dimethylaminopropyl)-N′-ethyl-carbodiimide can be used to form intramolecular crosslinks between lysine residues and glutamic acid residues. For example, diethyl pyrocarbonate is a reagent for modifying histidine residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. Reaction of lysine residues with other α-amino groups, for example, facilitates peptide binding to protein / peptide surfaces or crosslinking sites. Lysine is the attachment point for poly(ethylene) glycol and is also the main modification site for protein glycosylation. Methionine residues in proteins can be modified by iodoacetamide, bromoethylamine, chloramine T, etc.

[0263] Tetranitromethane and N-acetylimidazole can be used to modify tyrosine residues. Cross-linking via dityrosine can be accomplished with hydrogen peroxide / copper ions.

[0264] Recent studies on tryptophan modification have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

[0265] Successful modification of therapeutic proteins and peptides containing polyethylene glycol often results in increased circulation half-life when cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used to formulate hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

[0266] A peptide or variant, wherein the peptide is modified or contains non-peptide bonds, is a preferred embodiment of the present invention. Generally, peptides and variants (at least containing peptide linkages between amino acid residues) can be synthesized using the Fmoc-polyamide format for solid-phase peptide synthesis as described by Lukas et al. (Lukas et al., 1981) and references cited therein. The fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection for the N-amino group. Repeated cleavage of this highly base-labile protecting group is performed using 20% ​​dimethylpiperidine in N,N-dimethylformamide. Side chain functionalities may be protected as butyl ethers (in the case of serine, threonine, and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), tert-butyloxycarbonyl derivatives (in the case of lysine and histidine), trityl derivatives (in the case of cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (in the case of arginine). For glutamine and asparagine, the side-chain amino functionalities were protected with 4,4'-dimethoxydiphenyl groups, provided they were C-terminal residues. The solid support was based on a polydimethylacrylamide polymer, composed of the three monomers dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and N-acryloylsarcosine methyl ester (functionalizing agent). The peptide-resin linker used was an acid-labile 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives were added as preformed symmetrical anhydride derivatives, with the exception of asparagine and glutamine, which were added using a reversed N,N-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions were monitored using ninhydrin, nitrobenzenesulfonic acid, or isotin assays. After synthesis, the peptides were cleaved from the resin support with concomitant removal of the side-chain protecting groups using 95% trifluoroacetic acid containing a 50% scavenger mixture. Commonly used scavenger mixtures include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the amino acid composition of the peptide being synthesized. In addition, it is possible to synthesize peptides using a combination of solid-phase and liquid-phase methods (e.g., see (Bruckdorfer et al, 2004) and references cited therein).

[0267] Trifluoroacetic acid was removed by evaporation in vacuo followed by titration with diethyl ether loaded with the crude peptide. Any scavenger compounds present were removed by a simple extraction procedure (which yielded scavenger compound-free peptides after lyophilization of the aqueous phase). Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nottingham, UK).

[0268] Purification may be performed by any one or a combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and (usually) reversed-phase high performance liquid chromatography (e.g. using an acetonitrile / water gradient).

[0269] Peptide analysis can be performed using thin layer chromatography, electrophoresis, especially capillary electrophoresis, solid phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.

[0270] To select over-presented peptides, a presentation plot was calculated that shows the median presentation level of each sample and the variation in replicates. This feature juxtaposes samples of relevant tumor entities with baseline values ​​of normal tissue samples. Each of the above features can be incorporated into the over-presentation score by calculating a p-value from an adjusted linear mixed-effects model (Pinheiro et al, 2015), thereby adjusting for multiple testing using the false discovery rate (Benjamini and Hochberg, 1995) (see Example 1).

[0271] For the identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock-frozen tissue samples were purified and HLA-associated peptides were isolated. The isolated peptides were separated and identified by online nano-electrospray ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequences were validated by comparing the fragmentation patterns of natural tumor-associated peptides (TUMAPs) recorded from esophageal cancer samples (N=16 A*02-positive samples) with the fragmentation patterns of corresponding synthetic reference peptides of the same sequence. Because these peptides were directly identified as ligands for HLA molecules of the primary tumor, these results provide direct evidence for the natural processing and presentation of the identified peptides on primary cancer tissue from 16 esophageal cancer patients.

[0272] Discovery Pipeline v2.1 (e.g., see US 2013-0096016, which is hereby incorporated by reference in its entirety) allows for the identification and selection of relevant over-presented peptide vaccine candidates based on direct relative quantification of HLA-restricted peptide levels in cancerous or other infected tissues compared to several different non-cancerous tissues and organs. This is achieved by developing a label-free differential quantification method using LC-MS acquired data processed using a proprietary data analysis pipeline, incorporating sequence identification algorithms, spectral clustering, ion counting, retention time adjustment, charge state convolution, and normalization.

[0273] Presentation levels, including error estimates, were established for each peptide and sample. Peptides abundantly presented by tumor tissues, as well as peptides over-presented in tumor versus non-tumor tissues and organs, were identified.

[0274] HLA-peptide complexes from esophageal cancer tissue samples were purified, and HLA-associated peptides were separated and analyzed using LC-MS (see Examples). All TUMAPs included in this application were identified using methods from primary esophageal cancer samples to confirm their presentation on primary esophageal cancer.

[0275] TUMAPs identified in multiple esophageal cancer and normal tissues were quantified using an ion counting method based on label-free LC-MS data. This method assumes that the LC-MS signal area of ​​a peptide correlates with its abundance in the sample. All quantified peptide signals from various LC-MS experiments were normalized based on central tendency, averaged per sample, and combined into a histogram (referred to as a presentation plot). This presentation plot integrates various analytical methods, such as protein database searching, spectral clustering, charge state deconvolution (deconvolution), and retention time alignment and normalization.

[0276] In addition to overexpressing peptides, mRNA expression of potential genes was also tested. mRNA data was obtained by RNA sequencing analysis of normal and cancer tissues (see Example 2). An additional source of normal tissue data was a database of publicly available RNA expression data from 3,000 normal tissue samples (Lonsdale, 2013). Peptides derived from protein-coding mRNAs that are highly expressed in cancer tissues but very low or absent in important normal tissues are preferably included in the present invention.

[0277] In addition, pipeline v2.x allows for direct absolute quantification of MHC-peptides (preferably HLA-restricted peptides) on cancer or other infected tissues. Briefly, the total cell count is calculated based on the total DNA content of the tissue sample being analyzed. The total peptide amount of TUMAPs in the tissue sample is determined by nanoLC-MS / MS as the ratio of the native TUMAP and a known amount of an isotope-labeled version of the TUMAP, referred to as the internal standard. TUMAP isolation efficiency is determined by adding the peptide:MHC of all selected TUMAPs to the tissue lysate at the earliest possible time point in the TUMAP isolation procedure and detecting them by nanoLC-MS / MS after peptide separation is complete. Total cell counts and total peptide amounts are calculated based on three measurements per tissue sample. The peptide-specific isolation efficiency is calculated as the average of 10 spike-in experiments with three measurements (see Example 6 and Table 12).

[0278] The present invention provides methods for treating cancer / tumors, preferably esophageal cancer, that overexpress or exclusively express the peptides of the present invention. These peptides were directly shown by mass spectrometry to be naturally presented by HLA molecules in primary human esophageal cancer samples.

[0279] Compared to normal tissues, many of the source genes / proteins (also designated as "full-length proteins" or "potential proteins") from which the peptides are derived are highly overexpressed in cancer - the "normal tissue" associated with the present invention is healthy esophageal cells or other normal cells, indicating a high degree of association between tumors and these source genes (see Example 2). In addition, these peptides themselves are also overexpressed in tumor tissues (the "tumor tissue" associated with the present invention refers to samples from esophageal cancer patients), but not in normal tissues (see Example 1).

[0280] HLA-bound peptides are recognized by the immune system, specifically T lymphocytes. These T cells then destroy cells presenting the recognized HLA / peptide complex (e.g., esophageal cancer cells presenting the derived peptide).

[0281] All peptides of the present invention have been shown to have the ability to stimulate T cell responses and are over-presented, and can therefore be used to prepare antibodies and / or TCRs of the present invention, such as soluble TCRs (see Examples 3 and 4). In addition, when the peptides are combined with the corresponding MHC, they can also be used to prepare antibodies and / or TCRs of the present invention, in particular sTCRs. Each method is well known to technicians and can be found in various literature. Therefore, the peptides of the present invention can be used to generate an immune response in patients, thereby destroying tumor cells. The patient's immune response can be induced by directly administering the peptide or precursor substance (e.g., extended peptides, proteins or nucleic acids encoding these peptides) to the patient, preferably in combination with an immunogenicity-enhancing agent. The immune response derived from this therapeutic vaccine is expected to be highly specific against tumor cells because the target peptide of the present invention is presented in a relatively small number of copies on normal tissues, preventing the risk of adverse autoimmune reactions against normal cells in patients.

[0282] The present specification also relates to a T cell receptor (TCR) comprising an alpha chain and a beta chain ("α / βTCR"). Also provided is an HAVCR1-001 peptide that can bind to a TCR and an antibody when presented by an MHC molecule. The present specification also relates to nucleic acids, vectors, and host cells for expressing the TCR and peptides of the present specification; and methods of using them. The term "T cell receptor" (abbreviated TCR) refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), wherein the heterodimeric receptor is capable of binding to a peptide antigen presented by an HLA molecule. The term also includes so-called gamma / delta TCRs.

[0283] In one embodiment, the disclosure provides a method of producing a TCR as described herein, comprising culturing a host cell capable of expressing the TCR under conditions suitable for promoting expression of the TCR.

[0284] In another aspect, the present description relates to a method according to the present description, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of suitable antigen-presenting cells or artificial antigen-presenting cells by binding to a sufficient amount of antigen-containing antigen-presenting cells, or the antigen is loaded onto class I or II MHC tetramers / class I or II MHC complex monomers by tetramerization.

[0285] The α and β chains of α / βTCR and the γ and δ chains of γ / δTCR are generally considered to have two "domains" each, that is, variable and constant domains. The variable domain is composed of a combination of a variable region (V) and a connecting region (J). The variable domain may also include a leader region (L). The β and δ chains may also include a diversity region (D). The α and β constant domains may also include a C-terminal transmembrane (TM) domain that anchors the α and β chains to the cell membrane. Relative to the TCR of γ / δ, as used herein, the term "TCRγ variable domain" refers to a combination of the TCRγV (TRGV) region without a leader region (L) and the TCRγ (TRGJ) region, and the term TCRγ constant domain refers to the extracellular TRGC region, or the C-terminal truncated TRGC sequence. Likewise, the term "TCRδ variable domain" refers to the combination of the TCRδV (TRDV) region without the leader (L) and the TCRδD / J (TRDD / TRDJ) region, and the term "TCR 6 constant domain" refers to the extracellular TRDC region, or a C-terminal truncated TRDC sequence.

[0286] The TCR of the present specification preferably binds to the HAVCR1-001 peptide HLA molecule complex with a binding affinity (KD) of about 100 μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM or less. More preferably, a high-affinity TCR with a binding affinity of about 1 μM or less, about 100 nM or less, about 50 nM or less, or about 25 nM or less is used. Non-limiting examples of preferred binding affinity ranges for 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.

[0287] As used herein with respect to the TCRs of this specification, "specifically binds" and its grammatical variations are used to indicate a TCR that has a binding affinity (KD) for the HAVCR1-001 peptide-HLA molecule complex of 100 μM or less.

[0288] The α / β heterodimeric TCRs of the present disclosure may have introduced disulfide bonds between their constant domains. Preferred TCRs of this type include those having a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, except that threonine 48 of TRAC and serine 57 of TRBC1 or TRBC2 are substituted with cysteine ​​residues that form disulfide bonds between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

[0289] With or without the above-mentioned introduced interchain bond, the α / β heterodimeric TCR of the present specification may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by a native disulfide bond between Cys4 of TRAC exon 2 and Cys4 of TRBC1 or TRBC2 exon 2.

[0290] The TCR of the present disclosure may include a detectable label selected from the group consisting of a radionuclide, a fluorophore, and biotin. The TCR of the present disclosure may be conjugated to a therapeutically active agent, such as a radionuclide, a chemotherapeutic agent, or a toxin.

[0291] In one embodiment, the TCR having at least one mutation in the alpha chain and / or having at least one mutation in the beta chain has modified glycosylation compared to the non-mutated TCR.

[0292] In one embodiment, a TCR comprising at least one mutation in the TCR α chain and / or TCR β chain has a binding affinity and / or binding half-life for a complex of an HAVCR1-001 peptide HLA molecule that is at least twice the binding affinity of a TCR comprising a non-mutated TCR α chain and / or non-mutated TCR β chain. Tumor-specific TCR affinity enhancement and its development rely on the existence of a window of optimal TCR affinity. The existence of such a window is based on the observation that HLA-A2-restricted pathogen-specific TCRs typically have KD values ​​approximately 10-fold lower than those specific for HLA-A2-restricted tumor-associated self-antigens. It is known that although tumor antigens may be immunogenic, because the tumor is derived from an individual's own cells, only mutated proteins or proteins with altered translational processing will be recognized 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 TCRs highly reactive to these antigens are negatively selected within the thymus in a process known as central tolerance, meaning that only cells with low-affinity TCRs for self-antigens remain. Therefore, the affinity of the TCRs or variants of the present disclosure for HAVCR1-001 can be enhanced by methods well known in the art.

[0293] The present specification also relates to a method for identifying and isolating the TCR of the present invention, comprising: incubating PBMC from an HLA-A*02 negative healthy donor with A2 / HAVCR1-001 peptide monomers, incubating PBMC with tetramer-phycoerythrin (PE) and isolating high-affinity T cells by fluorescence-activated cell sorting (FAGS)-Calibur analysis.

[0294] The present specification also relates to a method for identifying and isolating the TCR of the present invention, which comprises: obtaining transgenic mice containing the entire human TCRαβ gene locus (1.1 and 0.7 Mb) (whose T cells express diversified human TCRs to compensate for the TCR deficiency of mice), immunizing the mice with HAVCR1-001, incubating PBMCs obtained from the transgenic mice with tetramer-phycoerythrin (PE), and analyzing and isolating high-affinity T cells by fluorescence-activated cell sorting (FAGS)-Calibur method.

[0295] On the one hand, in order to obtain the T cell expressing this specification sheets TCR, the nucleic acid encoding this specification sheets TCR-α and / or TCR-β chain is cloned into an expression vector, such as a gamma retrovirus or a slow virus. Recombinant virus is produced, and then the function is tested, such as antigen specificity and functional affinity. Then, an aliquot of the final product is used to transduce the target T cell colony (generally purified from the patient's PBMC), which is launched before the patient is input. On the other hand, in order to obtain the T cell expressing this specification sheets TCR, TCR RNA is synthesized by technology known in the art (for example, in vitro transcription system). Then, the TCR RNA synthesized in vitro is introduced into the primary CD8+T cells obtained from healthy donors by electroporation to express tumor-specific TCR-α and / or TCR-β chains again.

[0296] To increase expression, the nucleic acid encoding the TCR of the present disclosure can be operably linked to a potent activator, such as the retroviral long terminal repeat (LTR), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin, ubiquitin, the simian virus 40 (SV40) / CD43 composite activator, elongation factor (EF)-1a, and spleen focus forming virus (SFFV) activator. In a preferred embodiment, the activator is heterologous to the expressed nucleic acid. In addition to the potent activator, the TCR expression cassette of the present disclosure may contain additional elements that enhance transgene expression, including the central polypurine tract (CPPT), which promotes nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), which increases transgene expression levels by improving RNA stability (Zufferey et al., 1999).

[0297] The α and β chains of the TCR of the present invention can be encoded by nucleic acids located on separate vectors, or can be encoded by polynucleotides located on the same vector.

[0298] Achieving high levels of TCR surface expression requires the introduction of high-level transcription of the TCR-α and TCR-β chains of the TCR. To achieve this, the TCR-α and TCR-β chains of the present disclosure can be cloned into a bicistronic construct in a single vector, which has been shown to overcome this obstacle. The use of a viral interribosomal entry site (IRES) between the TCR-α and TCR-β chains results in the coordinated expression of the two chains, because both the TCR-α and TCR-β chains are produced by a single transcript that is divided into two proteins during translation, thereby ensuring an equal molar ratio of the TCR-α and TCR-β chains. (Schmitt et al. 2009).

[0299] The nucleic acids encoding the TCRs of the present disclosure may be codon-optimized to increase expression from host cells. Redundancy in the genetic code allows some amino acids to be encoded by more than one codon, but some codons are less "optimal" than others due to the relative availability of matching tRNAs and other factors (Gustafsson et al, 2004). Modifying the TCR-α and TCR-β gene sequences so that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as eliminating mRNA instability motifs or cryptic splice sites, has been shown to significantly increase TCR-α and TCR-β gene expression (Scholten et al, 2006).

[0300] Furthermore, mispairing between introduced and endogenous TCR chains may result in acquired specificities that pose a significant risk for autoimmunity. For example, the formation of mixed TCR dimers may reduce the number of CD3 molecules available to form correctly paired TCR complexes and, therefore, can significantly reduce the functional avidity of cells expressing the introduced TCR (Kuball et al, 2007).

[0301] To reduce mispairing, the C-terminal domains of the TCR chains introduced herein can be modified to promote interchain affinity while reducing the ability of the introduced chains to pair with endogenous TCRs. These strategies may include replacing the human TCR-α and TCR-β C-terminal domains with mouse counterparts (murinized C-terminal domains); generating a second interchain disulfide bond in the C-terminal domain by introducing a second cysteine ​​residue into the TCR-α and TCR-β chains of the introduced TCR (cysteine ​​modification); exchanging the interacting residues of the C-terminal domains of the TCR-α and TCR-β chains ("knob-in-hole structure"); directly fusing the variable domains of the TCR-α and TCR-β chains to CD3ζ (CD3ζ fusion) (Schmitt et al. 2009).

[0302] In one embodiment, the host cell is modified to express the TCR of the present specification. 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 of the present specification can be allogeneic or autologous relative to the patient to be treated. In one embodiment, the host is a γ / δ T cell transformed to express an α / β TCR.

[0303] A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. Preferably, the pharmaceutical composition is sterile and manufactured according to GMP guidelines.

[0304] Pharmaceutical compositions include the peptides in free form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed peptides wherein the peptide is modified by making an acid or base salt of a pharmaceutical agent. For example, acid salts are prepared by reacting a free base (generally where the neutral drug has a neutral -NH2 group) with a suitable acid. Suitable acids for preparing acid salts include organic acids such as acetic acid, propionic acid, hydroxy acids, 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, methanesulfonic acid, benzenesulfonic acid, salicylic acid, and the like, as well as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Conversely, base salts of acidic groups present on a peptide are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

[0305] In particularly preferred embodiments, the pharmaceutical composition comprises the peptide in the form of acetic acid (acetate), trifluoroacetate, or hydrochloric acid (chloride).

[0306] The agent described herein is preferably an immunotherapeutic agent, such as a vaccine. The vaccine can be administered directly to the affected organ of the patient, systemically by intravenous, intravenous, intravenous, intravenous, or intravenous injection, or applied ex vivo to cells derived from the patient or their cell line (which are then reinfused into the patient), or ex vivo to a subset of immune cells derived from the patient (which are then reinfused into the patient). If the nucleic acid is administered ex vivo to cells, it may be beneficial to transfect the cells to co-express an immunostimulatory cytokine (e.g., interleukin-2). The peptide can be administered alone, in combination with an immunostimulatory adjuvant (see below), in combination with an immunostimulatory cytokine, or via an appropriate delivery system (e.g., liposomes). The peptide can also be conjugated to a suitable carrier (e.g., keyhole limpet hemocyanin (KLH) or mannan) (see WO 95 / 18145 and (Longenecker et al., 1993)). The peptide can also be labeled, as a fusion protein, or as a hybrid molecule. The peptides whose sequences are given herein are expected to stimulate CD4 or CD8 T cells. However, CD8 T cell stimulation is more effective with the help of CD4 T-helper cells. Therefore, for MHC class I epitopes that stimulate CD8 T cells, a fusion partner or fragment of the hybrid molecule provides an appropriate epitope for stimulating CD4-positive T cells. CD4- and CD8-stimulatory epitopes are well known in the art and include those identified in the present invention.

[0307] In one aspect, the vaccine comprises at least one peptide 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.

[0308] In another aspect, the present invention provides a nucleic acid (e.g., a polynucleotide) encoding a peptide or peptide variant of the present invention. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA, or combinations thereof, and may be single-stranded and / or double-stranded, or a native or stabilized form of a polynucleotide (e.g., a polynucleotide having a phosphorothioate backbone), and may or may not contain introns, as long as it encodes a peptide. Of course, a polynucleotide can only encode a peptide that incorporates naturally occurring peptide bonds and contains naturally occurring amino acid residues. In another aspect, the present invention provides an expression vector that can be used to express a polypeptide according to the present invention.

[0309] Various methods have been developed for ligating polynucleotides, particularly DNA, by adding ligatable ends to vectors. For example, complementary homopolymer tails can be added to DNA segments, which are then inserted into vector DNA. Hydrogen bonding between the complementary homopolymer tails then binds the vector and DNA segments, forming a recombinant DNA molecule.

[0310] Synthetic linkers containing one or more restriction endonucleases sites provide another method for connecting DNA fragments to vectors. Synthetic linkers containing various restriction endonucleases can be purchased from a variety of channels, including International Biotechnologies Inc, New Haven, CN, USA.

[0311] The DNA encoding the polypeptides of the present invention is ideally modified using the polymerase chain reaction method employed by Saiki et al. (Saiki et al., 1988). This method can be used to introduce DNA into a suitable vector (e.g., by designing suitable restriction sites), or it can be used to modify DNA using other methods known in the art. If a viral vector is used, poxvirus or adenovirus vectors are preferred.

[0312] The DNA (or RNA in the case of a retroviral vector) 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 can be used according to known techniques, appropriately modified as described herein, to construct an expression vector, which can then be used to transform a suitable host cell to express and produce the polypeptide of the invention. Such techniques include those disclosed in, for example, U.S. Patents 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.

[0313] The DNA (or in the case of retroviral vectors, RNA) encoding a polypeptide containing a compound of the invention may be joined to a variety of other DNA sequences for introduction into a suitable host. The companion DNA will depend on the nature of the host, the manner in which the DNA is introduced into the host, and whether it is desired to be maintained episomal or to be bound to one another.

[0314] In general, DNA can be attached to an expression vector (such as a plasmid) in the appropriate direction and correct reading frame for expression. If necessary, the DNA may be connected to the corresponding transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control functions are generally present in the expression vector. The vector is then introduced into the host by standard methods. Generally, not all hosts will be transformed by the vector. Therefore, it is necessary to select transformed host cells. The selection method includes inserting a DNA sequence into the expression vector with any necessary control elements, which encodes a selectable attribute (such as antibiotic resistance) in the transformed cell.

[0315] Alternatively, the gene conferring such a selectable trait may be on a separate vector which is used to co-transform the desired host cell.

[0316] The host cells transformed with the recombinant DNA of the present invention are then cultured under appropriate conditions familiar to those skilled in the art as described herein and for a sufficient period of time to express the peptide which can then be recovered.

[0317] There are many known expression systems, including bacteria (such as Escherichia coli and Bacillus subtilis), yeast (such as Saccharomyces cerevisiae), filamentous fungi (such as Aspergillus), plant cells, animal cells and insect cells. The system can preferably be mammalian cells, such as CHO cells from the ATCC Cell Biology Collection.

[0318] Typical mammalian cell constitutive expression vector plasmids include CMV or SV40 activators containing a suitable poly A tail and resistance markers (such as neomycin). An example is pSVL obtained from Pharmacia (Piscataway, New Jersey, USA). An example of an inducible mammalian expression vector is pMSG, which can also be obtained from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, which are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids pRS403, pRS404, pRS405, and pRS406 are yeast integrative plasmids (YIPs) and have yeast selectable markers HIS3, TRP1, LEU2, and URA3 inserted into them. The pRS413-416 plasmids are yeast centromeric plasmids (Ycp). Vectors based on the CMV activator (e.g., from Sigma-Aldrich) offer transient or stable expression, cytoplasmic expression or secretion, and N-terminal or C-terminal tagging in various combinations of FLAG, 3xFLAG, c-myc, or MATN. These fusion proteins can be used to detect, purify, and analyze recombinant proteins. Dual-labeled fusions provide flexibility for detection.

[0319] The robust human cytomegalovirus (CMV) activator regulatory region allows for constitutive protein expression levels as high as 1 mg / L in COS cells. For weaker cell lines, protein levels are generally below 0.1 mg / L. The presence of the SV40 origin of replication results in high levels of DNA replication in SV40-permissive COS cells. For example, CMV vectors can contain the pMB1 (a derivative of pBR322) origin of replication in bacterial cells, the calcium-lactamase gene for ampicillin resistance selection in bacteria, the hGH polyA, and the f1 origin. Vectors containing the preproinsulin leader (PPT) sequence can direct the secretion of FLAG fusion proteins into the culture medium for purification using anti-FLAG antibodies, resins, and plates. Other vectors and expression systems for use with various host cells are well known in the art.

[0320] In another embodiment, two or more peptides or peptide variants of the present invention are encoded and thus expressed in a continuous order (similar to a "beads on a string" construct). Upon reaching the target, the peptides or peptide variants may be linked or fused together by a stretch of linker amino acids (e.g., LLLLLL), or they may be linked without any additional peptides between them. These constructs can also be used for cancer treatment and can induce immune responses involving MHC class I and MHC class II molecules.

[0321] The present invention also relates to a host cell transformed with the polynucleotide vector construct of the present invention. The host cell can be a prokaryotic cell or a eukaryotic cell. In some cases, bacterial cells are preferred prokaryotic host cells, typically E. coli strains, for example, E. coli strain DH5 (available from Bethesda Research Laboratories, Bethesda, MD, USA) and RR1 (available from the American Type Culture Collection (ATCC, Rockville, MD, USA), ATCC No. 31343). Preferred eukaryotic host cells include yeast, insect, and mammalian cells, preferably vertebrate cells, such as cells from mouse, rat, monkey, or human fibroblast and colon cancer cell lines. Yeast host cells include YPH499, YPH500, and YPH501, generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells (available as CCL61 cells in ATCC), NIH Swiss mouse embryonic cells (NIH / 3T3 cells available as CRL1658 cells in ATCC), monkey kidney-derived COS-1 cells (available as CRL1650 cells in ATCC), and human embryonic kidney cells (293 cells). Preferred insect cells are Sf9 cells, which can be transfected with baculovirus expression vectors. A general guide to selecting suitable host cells for expression can be found in the textbook "Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols" Part 1, Second Edition, ISBN 978-1-58829-262-9 by Paulina Balbás and Argelia Lorence, and other references known to the skilled artisan.

[0322] The transformation of appropriate host cells containing the DNA structure of the present invention can be completed using well-known methods, generally depending on the type of vector used. Regarding the transformation of prokaryotic host cells, see, for example, the literature of Cohen et al. (Cohen et al., 1972) and (Green and Sambrook, 2012). The transformation of yeast cells is described in the article of Sherman et al. (Sherman et al., 1986). The method described in Beggs (Beggs, 1978) is also very useful. For vertebrate cells, reagents for transfecting these cells, such as calcium phosphate and DEAE-dextran or liposome formulations, can be obtained from Stratagene Cloning Systems or Life Technologies (Gaithersburg, MD20877, USA). Electroporation can also be used to transform and / or transfect cells, which is a method well known in the art for transforming yeast cells, bacterial cells, insect cells and vertebrate cells.

[0323] Successfully transformed cells (i.e., cells containing the DNA construct of the present invention) can be identified using well-known methods (e.g., PCR). Alternatively, proteins present in the supernatant can be detected using antibodies.

[0324] It will be appreciated that certain host cells of the present invention are useful for producing the peptides of the present invention, such as bacterial cells, yeast cells, and insect cells. However, other host cells may be useful for certain therapeutic methods. For example, antigen-presenting cells (e.g., dendritic cells) can be used to express the peptides of the present invention so that they can be loaded with corresponding MHC molecules. Thus, the present invention provides a host cell containing a nucleic acid or expression vector of the present invention.

[0325] In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell or an antigen-presenting cell. On April 29, 2010, the U.S. Food and Drug Administration (FDA) approved a recombinant fusion protein containing prostatic acid phosphatase (PAP) for the treatment of asymptomatic or minimally symptomatic metastatic HRPC (Rini et al, 2006; Small et al, 2006).

[0326] In another aspect, the present invention provides a method for preparing a peptide and variants thereof, the method comprising culturing host cells and isolating the peptide from the host cells or their culture medium.

[0327] In another embodiment, the peptide, nucleic acid, or expression vector of the present invention is used in a pharmaceutical. For example, the peptide or variant thereof can be prepared as an intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. Preferred methods for peptide injection include sc, id, ip, im, and iv injection. Preferred methods for DNA injection are id, im, sc, ip, and iv injection. For example, 50 μg to 1.5 mg, preferably 125 μg to 500 μg of peptide or DNA is administered, depending on the specific peptide or DNA. The above dosage range has been successfully used in previous experiments (Walter et al, 2012).

[0328] Polynucleotides used for active immunization can be in substantially purified form or encapsulated in a 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. For example, a general overview is provided in the literature (Teufel et al, 2005). Polynucleotide vaccines are readily prepared, but the mode 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 those based on adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus, or hybrid viruses containing elements of more than one virus. Non-viral delivery systems include cationic liposomes and cationic polymers, which are well known in the art of DNA delivery. Physical delivery systems, such as via a "gene gun," may also be used. The peptide or nucleic acid-encoded peptide may be a fusion protein, for example, containing an epitope that stimulates T cells to carry out the aforementioned CDRs.

[0329] The medicaments of the present invention may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or strengthen the immune response (e.g., the immune response to an antigen mediated by CD8-positive T cells and helper T (TH) cells) and are therefore considered useful for the medicaments of the present invention. Suitable adjuvants include (but are not limited to) 1018ISS, aluminum salts, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod resiquimod, ImuFactIMP321, interleukin IL-2, IL-13, IL-21, interferon alpha or beta, or their pegylated derivatives, IS Patch, ISS, ISCOMATRIX, ISCOMs, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS1312, MontanideISA 206, Montanide ISA 50V, Montanide ISA-51, oil-in-water and water-in-oil emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, Delivery systems, poly(lactide-co-glycolide) (PLG)- and dextran-based microparticles, recombinant human lactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGFtrap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulator, derived from saponins, mycobacterial extracts, and synthetic bacterial cell wall mimics, and proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Preferred adjuvants include Freund's adjuvant or GM-CSF. Several dendritic cell-specific adjuvants (e.g., MF59) and their preparation methods have been described previously (Allison and Krummel, 1995). Cytokines may also be used. Some cytokines directly affect the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerate the maturation of dendritic cells into efficient antigen-presenting cells of T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, specifically incorporated herein by reference in its entirety), and act as immune adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al, 1996).

[0330] CpG immunostimulatory oligonucleotides have been reported to enhance the effects of adjuvants in vaccines. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in prophylactic and therapeutic vaccines. Importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced activation of Th1 cells and enhanced production of cytotoxic T lymphocytes (CTLs), even with the loss of CD4 T cell expression. The Th1 shift induced by TLR9 activation is maintained even in the presence of vaccine adjuvants, such as alum or incomplete Freund's adjuvant (IFA), which normally promote a Th2 shift. CpG oligonucleotides exhibit enhanced adjuvant activity when formulated or co-administered with other adjuvants or formulations, such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly necessary to induce a strong response when the antigen is relatively weak. They can also accelerate the immune response, reducing the antigen dose by approximately two orders of magnitude, and in some experiments, have produced similar antibody responses to full-dose vaccines without CpG (Krieg, 2006). U.S. Patent No. 6,406,705 B1 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens in combination to promote antigen-specific immune responses. One CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical compositions of the present invention. Other TLR binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, can also be used.

[0331] Other useful adjuvant examples include (but are not limited to) chemically modified CpG (e.g., CpR, Idera), dsRNA mimics, such as Poly(I:C) and its derivatives (e.g., AmpliGen, Hiltonol, poly-(ICLC), poly(IC-R), poly(I:C12U)), non-CpG bacterial DNA or RNA, and immunoreactive small molecules and antibodies, such as cyclophosphamide, sunitinib, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting major structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptors), and SC58175, all of which may have therapeutic effects and / or act as adjuvants. The skilled artisan can readily determine the amounts and concentrations of adjuvants and additives useful in the present invention without undue experimentation.

[0332] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon α, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, and microparticle preparations of PLG or virus particles.

[0333] In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is selected from a colony stimulating factor-containing preparation, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod and interferon-α.

[0334] In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is selected from a colony stimulating factor preparation, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resiquimod. In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. More preferred adjuvants are Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, poly-ICLC and anti-CD40 mAB or a combination thereof.

[0335] This combination drug is used for parenteral injection, such as subcutaneous, intradermal, intramuscular injection, and can also be taken orally. For this reason, the peptide and other selective molecules are decomposed or suspended in a pharmaceutical carrier, preferably an aqueous carrier. In addition, the composition may contain excipients, such as buffers, binding agents, shock agents, diluents, spices, lubricants, etc. These peptides can also be used in combination with immunostimulants, such as cytokines. More excipients that can be used for such compositions can be found in books such as Handbook of Pharmaceutical Excipients (Kibbe, 2000) written by A.Kibbe. This combination drug can be used to prevent, prevent and / or treat adenomas or cancerous diseases. For example, example preparations are provided in EP2112253.

[0336] It is important to recognize that the immune response elicited by the vaccine of the present invention attacks cancer at different cellular stages and at different stages of development. Furthermore, different cancer-related signaling pathways are attacked. This offers an advantage over other vaccines that target only one or a few targets, which can lead to tumors easily adapting to attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Therefore, the combination of several tumor-associated peptides ensures that each tumor targets at least some of the targets. The composition is designed in such a way that it is expected that each tumor will express several antigens and cover several independent pathways required for tumor growth and maintenance. Therefore, the vaccine can be easily used "off-the-shelf" for larger patient populations. This means that preselection of patients for vaccine treatment can be limited to HLA typing, without the need for any additional biomarker assessment of antigen expression, while still ensuring that multiple targets are simultaneously attacked by the induced immune response, which is important for efficacy (Banchereau et al, 2001; Walter et al, 2012).

[0337] As used herein, the term "scaffold" refers to a molecule that specifically binds to a (e.g., antigen) determinant. In one embodiment, the scaffold is capable of guiding the entity to which it is attached (e.g., a (second) antigen binding portion) to a target of interest, for example, to a specific type of tumor cell or a tumor stroma bearing an antigenic determinant (e.g., a complex of a peptide and MHC according to the current application). In another embodiment, the scaffold is capable of activating a signaling pathway through its target antigen (e.g., a T cell receptor complex antigen). Scaffolds include, but are not limited to, antibodies and fragments thereof, antigen binding regions of antibodies comprising an antibody heavy chain variable region and an antibody light chain variable region, bound proteins comprising at least one ankyrin repeat motif and a single domain antigen binding (SDAB) molecule, an aptamer, a (soluble) TCR, and (modified) cells, such as allogeneic or autologous T cells. In order to assess whether a molecule is a scaffold that binds to a target, a binding assay can be performed.

[0338] "Specific" binding means that the scaffold binds better to the peptide-MHC complex of interest than to other naturally occurring peptide-MHC complexes, to the extent that a scaffold with active molecules capable of killing cells bearing the specific target is unable to kill another cell without the specific target but presenting one or more other peptide-MHC complexes. If the peptide of the cross-reactive peptide-MHC is not naturally occurring, i.e., not from the human HLA-peptide group, binding to other peptide-MHC complexes is irrelevant. Assays for assessing target cell killing are well known in the art. They should be performed with target cells (primary cells or cell lines) presenting unaltered peptide-MHC or with cells loaded with the peptide so that the levels of naturally occurring peptide-MHC are achieved.

[0339] Each scaffold can include a label that allows detection of bound aptamers by determining the presence or absence of a signal provided by the label. For example, the scaffold can be labeled with a fluorescent dye or any other suitable cell marker molecule. Such marker molecules are well known in the art. For example, fluorescent labeling with a fluorescent dye can provide visualization of bound aptamers by fluorescence or laser scanning microscopy or flow cytometry.

[0340] Each scaffold can be conjugated to a second active molecule (eg, IL-21, anti-CD3, anti-CD28).

[0341] For further information on polypeptide scaffolds, see, for example, the background section of WO 2014 / 071978 A1, which is incorporated herein by reference.

[0342] The present invention also relates to aptamers. Aptamers (e.g., see WO2014 / 191359 and references cited therein) are short, single-stranded nucleic acid molecules that can fold into a defined three-dimensional structure and recognize a specific target structure. They appear to be a suitable alternative approach for developing targeted therapies. Aptamers have been shown to selectively bind to complex targets with high affinity and specificity.

[0343] Aptamers that recognize cell surface molecules have been identified over the past decade and offer a promising approach for developing diagnostic and therapeutic approaches. Aptamers have been shown to be virtually non-toxic and immunogenic, making them promising candidates for biomedical applications. Aptamers, such as those recognizing prostate-specific membrane antigen (PSMA), have been successfully used for targeted therapy and demonstrated functionality in xenografts in in vivo models. Furthermore, aptamers that recognize specific tumor cell lines have been identified.

[0344] DNA aptamers can be selected to reveal broad-spectrum signatures of various cancer cells, particularly those originating from solid tumors, while non-tumorigenic and predominantly healthy cells remain unrecognized. If the identified aptamers not only recognize tumor-specific subtypes but also interact with a range of tumors, this makes aptamers suitable for so-called broad-spectrum diagnostics and therapeutics.

[0345] Furthermore, studies of the cell binding behavior using flow cytometry revealed that the aptamer exhibited good affinity in the nanomolar range.

[0346] Aptamers are used for diagnostic and therapeutic purposes. Furthermore, it has also been possible to show that some aptamers are taken up by tumor cells and can therefore serve as molecular vehicles for the targeted delivery of anticancer agents, such as siRNA, into tumor cells.

[0347] Aptamers can be selected against complex targets such as cells and tissues and complexes comprising, preferably including, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 93, a peptide according to the current invention with an MHC molecule using the cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique.

[0348] The peptides of the present invention can be used to generate and develop specific antibodies against MHC / peptide complexes. These antibodies can be used therapeutically to target toxins or radioactive substances to diseased tissue. Another use of these antibodies is to target radionuclides to diseased tissue for imaging purposes (such as PET). This can help detect small metastases or determine the size and exact location of diseased tissue.

[0349] Therefore, another aspect of the present invention is a method for producing a recombinant antibody that specifically binds to class I or II human major histocompatibility complex (MHC) in complex with an HLA-restricted antigen, the method comprising: immunizing a genetically engineered non-human mammal containing cells expressing the major histocompatibility complex (MHC) class I or II with a soluble form of the (MHC) class I or II molecule in complex with the HLA-restricted antigen; isolating mRNA molecules from antibody-producing cells of the non-human mammal; producing a phage display library that displays protein molecules encoded by the mRNA molecules; and isolating at least one phage from the phage display library, the at least one phage displaying that the antibody specifically binds to the human major histocompatibility complex (MHC) class I or II in complex with the HLA-restricted antigen.

[0350] Another aspect of the present invention provides an antibody that specifically binds to a human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and / or a chimeric antibody.

[0351] Corresponding 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 publications (Cohen et al, 2003a; Cohen et al, 2003b; Denkberg et al, 2003), all of which are incorporated herein by reference in their entirety for the purposes of the present invention.

[0352] Preferably, the antibody binds to the complex with an affinity of less than 20 nanomolar, preferably less than 10 nanomolar, which is also considered "specific" in the context of the present invention.

[0353] 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 having 88% homology (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof that induces T cells to cross-react with the variant peptide, wherein the peptide is not a full-length polypeptide.

[0354] The present invention further relates to a peptide comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof having at least 88% homology (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 93, wherein the total length of the peptide or variant is 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0355] The present invention further relates to the peptides of the present invention, which have the ability to bind to major histocompatibility complex (MHC) class I or II molecules.

[0356] The present invention further relates to the peptide of the present invention, wherein the peptide consists of or essentially consists of the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 93.

[0357] The present invention further relates to the peptides of the present invention, wherein the peptide is (chemically) modified and / or comprises non-peptide bonds.

[0358] The present invention further relates to the peptides of the present 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 wherein the peptide is fused to an antibody (e.g., a dendritic cell-specific antibody).

[0359] Another embodiment of the present invention relates to a non-natural peptide, wherein the peptide consists of or consists essentially of an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 48 and is synthetically produced (i.e., synthesized) as a pharmaceutically acceptable salt. Methods for synthetically producing peptides are well known in the art. Salts of the peptides of the present invention are substantially different from the peptides in their in vivo state, because these peptides produced in vivo are not salts. The non-natural salt form of the peptide mediates the solubility of the peptide, particularly in the case of a pharmaceutical composition comprising the peptide, for example, a peptide vaccine disclosed herein. In order to effectively provide the peptide to a subject in need of treatment, it is necessary for the peptide to have sufficient, at least basic, solubility. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. These salts of the present invention include alkali and alkaline earth salts, such as salts of the Hofmeister series, containing an anion PO4 3- 、SO4 2- 、CH3COO - 、Cl - Br - 、NO3- 、ClO4 - , I - 、SCN - and cation NH4 + , Rb + , K + 、Na + 、Cs + 、Li + 、Zn 2+ Mg 2+ , Ca 2+ 、Mn 2+ 、Cu 2+ and Ba 2+ In particular, the salt is selected from (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CH3COO, NH4Cl, NH4Br, NH4NO3, NH4CIO4, NH4I, NH4SCN, Rb3PO4, Rb2HPO4, RbH2PO4, Rb2SO4, Rb4CH3COO, Rb4Cl, Rb4Br, Rb4NO3, Rb 4CIO4, Rb4I, Rb4SCN, K3PO4, K2HPO4, KH2PO4, K2SO4, KCH3COO, KCl, KBr, KNO3, KClO4, KI, KSCN, Na3PO4, Na2HPO4, NaH2PO4, Na2SO4, NaCH3COO, NaCl, NaBr, NaNO3, NaCIO4, NaI, NaSCN, ZnCI2, Cs aPO4, 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)2, CaSO4, Ca(CH3COO)2, CaCl2, CaBr2, Ca(NO3)2, Ca(ClO4)2, CaI2, Ca(SCN)2, Ba3(PO4)2, Ba2HPO4, Ba(H2PO4)2, BaSO4, Ba(CH3COO)2, BaCl2, BaBr2, Ba(NO3)2, Ba(ClO4)2, BaI2 and Ba(SCN)2. Particularly preferred are NH3-acetic acid, MgCl2, KH2PO4, Na2SO4, KCl, NaCl and CaCl2, for example chlorides or acetates (trifluoroacetic acid).

[0360] In general, peptides and variants (at least containing peptide linkages between amino acid residues) can be synthesized using the Fmoc-polyamide format for solid-phase peptide synthesis as described by Lukas et al. (Lukas et al., 1981) and references cited therein. A fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection of the N-amino group. This highly base-labile protecting group is cleaved repeatedly using 20% ​​dimethylpiperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as butyl ethers (in the case of serine, threonine, and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), tert-butyloxycarbonyl derivatives (in the case of lysine and histidine), trityl derivatives (in the case of cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (in the case of arginine). Where glutamine and asparagine are C-terminal residues, side-chain amino functionalities are protected using a 4,4'-dimethoxydiphenyl group. The solid support is based on a polydimethylacrylamide polymer, composed of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and N-acryloylsarcosine methyl ester (functionalizing agent). The peptide-resin linker used is an acid-labile 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives were added as preformed symmetrical anhydride derivatives, with the exception of asparagine and glutamine, which were added using a reversed N,N-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions were monitored using ninhydrin, nitrobenzenesulfonic acid, or isotin assays. After synthesis, the peptide was cleaved from the resin support using a 95% trifluoroacetic acid solution containing a 50% scavenger mixture, accompanied by removal of side-chain protecting groups. Common scavenger mixtures include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the amino acid composition of the synthesized peptide. Furthermore, it is possible to synthesize peptides using a combination of solid and solution phase methods (see, for example, (Bruckdorfer et al, 2004) and references cited therein).

[0361] Trifluoroacetic acid was removed by evaporation in vacuo followed by titration with diethyl ether loaded with the crude peptide. Any scavenger compounds present were removed by a simple extraction procedure (which yielded scavenger compound-free peptides after lyophilization of the aqueous phase). Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nottingham, UK).

[0362] Purification may be performed by any one or a combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and (usually) reversed-phase high performance liquid chromatography (e.g. using an acetonitrile / water gradient).

[0363] The present invention further relates to a nucleic acid encoding a peptide according to the present invention, with the proviso that the peptide is not a complete (full) human protein.

[0364] The present invention further relates to a nucleic acid of the present invention, which is DNA, cDNA, PNA, RNA or a combination thereof.

[0365] The present invention further relates to an expression vector capable of expressing the nucleic acid of the present invention.

[0366] The present invention further relates to the use of the peptide of the present invention, the nucleic acid of the present invention or the expression vector of the present invention in medicine, in particular for treating esophageal cancer.

[0367] The present invention further relates to host cells comprising a nucleic acid according to the invention or an expression vector according to the invention.

[0368] The present invention further relates to the host cell of the present invention, which is an antigen presenting cell, preferably a dendritic cell.

[0369] The present invention further relates to a method for preparing the peptide of the present invention, said method comprising culturing the host cell of the present invention, and isolating the peptide from said host cell or its culture medium.

[0370] The present invention further relates to the method of the present invention, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell by contacting a sufficient amount of the antigen with the antigen-presenting cell.

[0371] The present invention further relates to the method of the present invention, wherein the antigen presenting cell comprises an expression vector capable of expressing a peptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 93 or a variant thereof.

[0372] The present invention further relates to activated T cells produced by the method of the present invention, wherein the T cells selectively recognize a cell that abnormally expresses a polypeptide comprising the amino acid sequence of the present invention.

[0373] The present invention further relates to a method for killing target cells of a patient, wherein the target cells of the patient abnormally express a polypeptide comprising any amino acid sequence of the present invention, the method comprising administering to the patient an effective amount of T cells of the present invention.

[0374] The present invention further relates to the use of any of the peptides, nucleic acids, expression vectors, cells, or activated cytotoxic T lymphocytes as a medicament or in the manufacture of a medicament. The present invention further relates to the use of the present invention, wherein the medicament is effective against cancer.

[0375] 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.

[0376] The present invention also generally relates to the use of the present invention, wherein the cancer cells are esophageal cancer cells or other solid or blood 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.

[0377] The present invention further relates to specific marker proteins and biomarkers based on the peptides of the present invention, referred to herein as "targets," which can be used to diagnose and / or determine the prognosis of esophageal cancer. The present invention also relates to these novel targets for use in cancer treatment.

[0378] The term "antibody" herein is defined in a broad sense, including both polyclonal and monoclonal antibodies. In addition to intact or "whole" immunoglobulin molecules, the term "antibody" also includes fragments (e.g., CDRs, Fv, Fab, and Fc fragments) or polymers of these immunoglobulin molecules and humanized immunoglobulin molecules, as long as they exhibit any of the desired properties of the present invention (e.g., specific binding to esophageal cancer marker (poly)peptides, delivery of toxins to esophageal cancer cells when cancer marker gene expression levels are increased, and / or inhibition of the activity of esophageal cancer marker polypeptides).

[0379] Whenever possible, the antibodies of the present invention can be purchased from commercial sources. The antibodies of the present invention may also be produced using known methods. A skilled artisan will appreciate that full-length esophageal cancer marker polypeptides or fragments thereof can be used to prepare the antibodies of the present invention. The polypeptides used to generate the antibodies of the present invention can be partially or fully purified from natural sources or produced using recombinant DNA technology.

[0380] For example, a cDNA encoding a peptide of the present invention, e.g., a peptide according to SEQ ID NO: 1 to SEQ ID NO: 93, 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). The recombinant protein can then be purified and used to produce a monoclonal or polyclonal antibody preparation that specifically binds to the esophageal cancer marker polypeptide used to generate the antibody of the present invention.

[0381] Those skilled in the art will recognize that two or more different sets of monoclonal or polyclonal antibodies can maximize the likelihood of obtaining an antibody with the specificity and affinity required for the intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). Depending on the intended use of the antibody, its desired activity is tested using known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on generating and testing antibodies, see, for example, Greenfield, 2014). For example, the antibody can be tested using ELISA or immunoblotting, immunohistochemical staining of formalin-fixed cancer tissue or frozen tissue sections. After initial in vitro characterization, antibodies for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.

[0382] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of homogeneous antibodies, i.e., a population of antibodies consisting of identical antibodies except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical (homogeneous) to corresponding sequences in antibodies obtained from a particular species or belonging to a particular antibody class and subclass, while the remaining chains are identical (homogeneous) to corresponding sequences in antibodies obtained from other species or belonging to a particular antibody class and subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. Patent No. 4,816,567, which is incorporated herein in its entirety).

[0383] The monoclonal antibodies of the present invention may be produced using a hybridoma method. In the hybridoma method, mice or other appropriate host animals are typically immunized with an immunizing agent to elicit or produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0384] Monoclonal antibodies can also be made by recombinant DNA methods, such as 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 that specifically bind to genes encoding the heavy and light chains of murine antibodies).

[0385] In vitro methods are also applicable to the preparation of monovalent antibodies. Antibody digestion can be performed to produce fragments of antibodies, particularly Fab fragments, 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. Patent No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, referred to as Fab fragments (each fragment having an antigen binding site) and residual Fc fragments. Pepsin treatment produces a F(ab')2 fragment and a pFc' fragment.

[0386] Antibody fragments, whether or not attached to other sequences, may include insertions, deletions, substitutions, or other selective 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 deleting / adding amino acids that can bind to disulfide bonds, increasing their biological lifespan, altering their secretion characteristics, etc. In any case, the antibody fragment must possess biologically active properties, such as binding activity, regulating the binding force of the binding domain, etc. The functional or active regions of the antibody can be determined by genetic mutation of specific regions of the protein, followed by expression and testing of the expressed polypeptides. These methods are well known to those skilled in the art and may include site-specific genetic mutations of the nucleic acid encoding the antibody fragment.

[0387] The antibodies of the present invention may further include humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', or other antigen-binding sequences of antibodies) that contain minimal sequence derived from a 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, with the same 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 include residues that are not found in either the recipient antibody or the imported CDR or framework sequences. Generally, a humanized antibody will include substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin and all or substantially all of the FR regions are regions of human immunoglobulin sequence identity. The humanized antibody ideally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0388] Methods for humanizing non-human antibodies are well known in the industry. Generally speaking, humanized antibodies have one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are often referred to as "input" residues and are typically obtained from "input" variable domains. Humanization can essentially be accomplished by replacing rodent CDRs or CDR sequences with corresponding human antibody sequences. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which much less than the complete human variable domain is replaced 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 replaced by residues from similar sites in rodent antibodies.

[0389] Transgenic animals (e.g., mice) that are capable of producing fully human antibodies in the absence of endogenous immunoglobulin production following immunization can be used. For example, it has been described that homozygous deletion of the antibody heavy chain joining region gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies following antigen challenge. Human antibodies can also be produced using phage display libraries.

[0390] The antibodies of the present invention are preferably administered to a subject in the form of a pharmaceutical carrier. Typically, an appropriate amount of a pharmaceutical salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutical carriers include physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to 8, more preferably about 7 to 7.5. In addition, carriers also include sustained-release formulations, such as a solid hydrophobic polymer semipermeable matrix containing the antibody, wherein the matrix is ​​in the form of a tangible article, such as a film, liposome, or microparticle. It is well known to those skilled in the art that certain carriers may be more preferred, depending on, for example, the route of administration and concentration of the antibody.

[0391] The antibodies can be administered to the subject, patient, or cells by injection (e.g., intravenously, intraperitoneally, subcutaneously, intramuscularly) or by other methods such as infusion to ensure that they are delivered to the bloodstream in an effective form. These antibodies can also be administered intratumorally or peritumorally to exert local and systemic therapeutic effects. Local or intravenous injection is preferred.

[0392] The effective dosage and schedule for administration of the antibody can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will appreciate that the dosage of the antibody that must be administered will vary depending on factors such as the subject receiving the antibody, the route of administration, the specific type of antibody being used, and other drugs being used. A typical daily dosage of the antibody used alone may be from about 1 μg / kg to a maximum of 100 mg / kg of body weight or more, depending on the factors mentioned above. Following administration of the antibody, preferably to treat esophageal cancer, the efficacy of the therapeutic antibody can be assessed by various methods well known to those skilled in the art. For example, the size, number, and / or distribution of cancer in a subject receiving treatment can be monitored using standard tumor imaging techniques. An antibody administered for treatment that prevents tumor growth, causes tumor shrinkage, and / or prevents the development of new tumors compared to the course of the disease without the administration of the antibody is an antibody that is effective in treating cancer.

[0393] Another aspect of the present invention proposes a method for preparing a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex. Such soluble T cell receptors can be produced from specific T cell clones, and their affinity can be increased by targeted mutagenesis of the complementary determining region. For the purpose of T cell receptor selection, phage display can be used (U.S. 2010 / 0113300, (Liddy et al, 2012)). For the purpose of stabilizing T cell receptors during phage display and when actually used as drugs, α and β chains can be connected by non-natural disulfide bonds, other covalent bonds (single-chain T cell receptors) or by dimerization domains (Boulter et al, 2003; Card et al, 2004; Willcox et al, 1999). T cell receptors can be connected to toxins, drugs, cytokines (see US 2013 / 0115191), domains recruiting effector cells, such as anti-CD3 domains, etc., in order to perform specific functions on target cells. In addition, it may be expressed in T cells for adoptive transfer. Further information can be found in WO 2004 / 033685 A1 and WO 2004 / 074322 A1. Combinations of sTCRs are described in WO 2012 / 056407 A1. Further methods of preparation are disclosed in WO 2013 / 057586 A1.

[0394] Furthermore, the peptides and / or TCRs or antibodies or other binding molecules of the invention can be used to confirm a pathologist's diagnosis of cancer based on a biopsy sample.

[0395] The antibodies or TCRs can also be used in in vivo diagnostic assays. Generally, the antibodies are labeled with a radionuclide (e.g., 111In, 99Tc, 14C, 131I, 3H, 32P, or 35S) to allow tumor localization by immunoscintigraphy. In one embodiment, the antibodies or fragments bind to two or more extracellular domains of target proteins selected from the group consisting of the aforementioned proteins with an affinity (Kd) of less than 1 x 10 μM.

[0396] Diagnostic antibodies can be labeled with probes suitable for detection using 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 rare earth elements, paramagnetic iron, fluorine-18, and other positron-emitting radionuclides. Furthermore, probes may be bifunctional or multifunctional and detectable using more than one of the aforementioned methods. These antibodies may be labeled directly or indirectly with the aforementioned probes. Antibody-probe attachment includes covalent attachment of the probe, fusion of the probe into the antibody, covalent attachment of a chelating compound to bind the probe, and other methods well known in the art. For immunohistochemistry, the diseased tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin. The fixed or embedded sections contain the sample, which is contacted with a labeled primary antibody and a secondary antibody for detecting protein expression in situ.

[0397] Another aspect of the present invention includes an in vitro method for producing activated T cells, comprising contacting T cells with antigen-loaded human MHC molecules expressed on the surface of suitable antigen-presenting cells for a sufficient period of time to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide according to the present invention. Preferably, a sufficient amount of antigen is used together with the antigen-presenting cells.

[0398] Preferably, the mammalian cells lack or have reduced levels or reduced function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter associated with antigen processing.

[0399] The human peptide loading-deficient cell line T2 is from the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852, USA) catalog number CRL1992; the Drosophila cell line Schneider strain 2 is from ATCC catalog number CRL 19863; and the mouse RMA-S cell line was described by Ljunggren et al. (Ljunggren and Karre, 1985).

[0400] Preferably, the host cells do not express substantially any MHC class I molecules prior to transfection. Stimulator cells also preferably express molecules that play an important role in T cell co-stimulatory signals, such as any of B7.1, B7.2, ICAM-1, and LFA3. Nucleic acid sequences for a large number of MHC class I molecules and co-stimulatory molecules are publicly available from the GenBank and EMBL databases.

[0401] When an MHC class I epitope is used as an antigen, the T cells are CD8-positive T cells.

[0402] If antigen presenting cells are transfected to express such an epitope, preferably the cells comprise an expression vector capable of expressing a peptide comprising SEQ ID NO: 1 to SEQ ID NO: 93 or a variant amino acid sequence.

[0403] Several other methods can be used to generate T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTLs. Plebanski et al. (Plebanski et al., 1995) used autologous peripheral blood lymphocytes (PLBs) to generate T cells. Alternatively, autologous T cells can be generated by pulsing dendritic cells with peptides or polypeptides or by infecting them with recombinant viruses. Furthermore, B cells can be used to generate autologous T cells. Furthermore, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses can be used to generate autologous T cells. S. Walter et al. (Walter et al., 2003) described in vitro activation of T cells using artificial antigen-presenting cells (aAPCs), which is also a suitable method for generating T cells specific for selected peptides. In the present invention, aAPCs are generated by coupling preformed MHC:peptide complexes to polystyrene particles (microspheres) using a biotin:streptavidin biochemical method. This system allows for precise control of the MHC density on the aAPCs, enabling the selective induction of high- or low-avidity, highly potent, antigen-specific T cell responses in blood samples. In addition to the MHC:peptide complex, aAPCs should also carry other proteins with co-stimulatory activity, such as anti-CD28 antibodies coupled to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, for example, cytokines such as interleukin-12.

[0404] T cells can also be generated using allogeneic cells, a method described in detail in WO 97 / 26328, which is incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells can be used to present peptides, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and vaccinia-infected target cells. In addition, plant viruses can also be used (for example, see Porta et al. (Porta et al., 1994) which describes the development of cowpea mosaic virus as a high-yield system for presenting foreign peptides).

[0405] The activated T cells directly target the peptides of the present invention, which is helpful for treatment. Therefore, another aspect of the present invention provides activated T cells prepared by the above method of the present invention.

[0406] The activated T cells prepared according to the above method will selectively recognize the abnormally expressed polypeptide containing the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 93.

[0407] Preferably, the T cell recognizes the cell by interacting (e.g., binding) with its TCR containing the HLA / peptide complex. T cells are useful cells in a method of killing a patient's target cells, wherein the target cells abnormally express a polypeptide containing the amino acid sequence of the present invention. Such patients are given an effective amount of activated T cells. 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 not derived from the patient, but from another person. Of course, it is preferred that the donor is a healthy person. The inventors use "healthy individual" to refer to a person who is generally in good condition, preferably has a competent immune system, and more preferably does not have any disease that can be easily tested or detected.

[0408] According to the present invention, the in vivo target cells of CD8-positive T cells can be tumor cells (sometimes expressing MHC class II antigens) and / or stromal cells (tumor cells) surrounding the tumor (sometimes also expressing MHC class II antigens; (Dengjel et al, 2006)).

[0409] The T cells of the present invention can be used as an active ingredient in a therapeutic composition. Therefore, the present invention also provides a method for killing target cells in a patient, wherein the target cells in the patient abnormally express a polypeptide comprising the amino acid sequence of the present invention, the method comprising administering to the patient an effective amount of the T cells described above.

[0410] As used by the inventors, "abnormal expression" also includes overexpression of a polypeptide compared to expression levels in normal (healthy) tissue, or expression of the gene in the tumor despite not being expressed in tumor-derived tissue. "Overexpression" means that the polypeptide level is at least 1.2 times that in normal tissue, preferably at least 2 times, and more preferably at least 5 or 10 times that in normal tissue.

[0411] T cells can be prepared by methods known in the art (eg, as described above).

[0412] T cell adoptive transfer protocols are well known in the art. Reviews can be found in: Gattioni et al. and Morgan et al. (Gattinoni et al, 2006; Morgan et al, 2006).

[0413] Another aspect of the present invention involves the use of peptides complexed with MHC to generate T cell receptors, the nucleic acid of which is cloned and introduced into a host cell, preferably a T cell. The genetically engineered T cell can then be transferred to a patient for cancer treatment.

[0414] Any molecule of the present invention (i.e., a peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor, or encoding nucleic acid) is useful for treating diseases characterized by cellular evasion of an immune response. Thus, any molecule of the present invention can be used as a medicament or in the manufacture of a medicament. Such a molecule can be used alone or in combination with other molecules of the present invention or known molecules.

[0415] The present invention also relates to a kit comprising:

[0416] (a) a container comprising the pharmaceutical composition in the form of a solution or lyophilized powder;

[0417] (b) an optional second container containing a diluent or reconstitution fluid for the lyophilized powder dosage form; and

[0418] (c) optionally, instructions for (i) use of the solution or (ii) reconstitution and / or use of the lyophilized formulation.

[0419] 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, and may be a multi-purpose container. The pharmaceutical composition is preferably lyophilized.

[0420] The kit of the present invention preferably comprises a lyophilized formulation placed in a suitable container and instructions for reconstitution and / or use. Suitable containers include, for example, bottles, vials (such as dual-chamber bottles), syringes (such as dual-chamber syringes), and test tubes. The container may be made of a variety of materials, such as glass or plastic. The kit and / or container preferably has instructions on or about the container indicating directions for reconstitution and / or use. For example, the label may indicate that the lyophilized formulation will be reconstituted to the above-mentioned peptide concentration. The label may further indicate that the formulation is for subcutaneous injection.

[0421] The container for storing the preparation can be a multi-use vial, so that the reconstituted dosage form can be administered repeatedly (for example, 2-6 times). The kit may further include a second container containing a suitable diluent (such as sodium bicarbonate solution).

[0422] After mixing 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 not more than 3 mg / mL / peptide (=1500 μg). The kit may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0423] The kit of the present invention may have a single container containing the pharmaceutical composition preparation of the present invention, which may have other ingredients (e.g., other compounds or pharmaceutical compositions thereof), or no other ingredients, or each ingredient may have its own different container.

[0424] Preferably, the kit of the present invention includes a formulation of the present invention packaged for use in combination with a second compound, such as an adjuvant (e.g., GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic agent or inhibitor, an apoptosis inducer, or a chelating agent, or a pharmaceutical combination thereof. The components of the kit can be pre-complexed or each component can be placed in a separate container prior to administration to the patient. The components of the kit can be in one or more solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit can also be in solid form, converted to liquid form by addition of a suitable solvent, preferably placed in a separate container.

[0425] The container of the treatment kit may be a vial, test tube, flask, bottle, syringe, or any other means for holding solids or liquids. Typically, when there is more than one component, the kit will contain a second vial or other container so that it can be quantified separately. The kit may also contain another container for loading a medicinal liquid. Preferably, the treatment kit will contain a device (e.g., one or more needles, syringes, eye droppers, pipettes, etc.) that allows the injection of the drug of the present invention (the composition of the kit).

[0426] The pharmaceutical formulations of the present invention are suitable for administration of the peptides by any acceptable route, such as oral (enteral), intranasal, intraocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal administration. Subcutaneous administration is preferred, intradermal administration is most preferred, and administration by infusion pump is also possible.

[0427] Since the peptide of the present invention is isolated from esophageal cancer, the agent of the present invention is preferably used for treating esophageal cancer.

[0428] The present invention further relates to a method for preparing a personalized medicine for an individual patient, comprising: preparing a pharmaceutical composition comprising at least one peptide selected from a prescreened TUMAP library, wherein the at least one peptide used in the pharmaceutical composition is selected to be appropriate for the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be adapted to generate T cell clones for downstream applications, such as TCR isolates or soluble antibodies and other therapeutic options.

[0429] “Personalized medicine” refers to treatments that are tailored to an individual patient and are intended for use only with that individual patient, including personalized active cancer vaccines and adoptive cell therapies using autologous tissue.

[0430] As used herein, a "repository" shall refer to a group or collection of peptides that have been pre-screened for immunogenicity and / or are overexpressed in specific tumor types. The term "repository" does not imply that the specific peptides included in the vaccine have been pre-manufactured and stored in a physical facility, although this possibility is contemplated. It is expressly contemplated that the peptides described may be used to create each personalized vaccine de novo, or may be pre-manufactured and stored. The repository (e.g., in the form of a database) consists of tumor-associated peptides that are highly overexpressed in tumor tissue from patients with esophageal cancer across a range of HLA-A, HLA-B, and HLA-C alleles. It may contain peptides that include MHC class I and MHC class II peptides or elongated MHC class I peptides. In addition to tumor-associated peptides collected from several esophageal cancer tissues, the repository may also contain HLA-A*02 and HLA-A*24-tagged peptides. These peptides allow for the quantitative comparison of T cell immunity induced by TUMAPs, which can lead to important conclusions about the anti-tumor response potential of the vaccine. Second, they serve as important positive control peptides derived from “non-self” antigens when no vaccine-induced T cell responses are observed against TUMAPs derived from the patient’s “self” antigens. Third, it allows conclusions to be drawn about the patient’s immune function status.

[0431] The repository's TUMAPs were identified using a functional genomics approach that combines gene expression analysis, mass spectrometry, and T cell immunology. This approach ensures that only TUMAPs that are truly present in a high percentage of tumors but not expressed or expressed only at very low levels in normal tissues are selected for further analysis. For the initial peptide selection, patient esophageal cancer samples and blood from healthy donors were analyzed in a step-by-step approach:

[0432] 1. HLA ligands of malignant material are determined by mass spectrometry;

[0433] 2. Use genome-wide messenger RNA (mRNA) expression analysis to identify genes that are overexpressed in malignant tumor tissue (esophageal cancer) compared with a range of normal organs and tissues;

[0434] 3. Compare the identified HLA ligands with gene expression data. Peptides that are over- or selectively presented on tumor tissue, preferably peptides encoded by the selectively expressed or over-expressed genes detected in step 2, are considered as suitable candidate TUMAPs for peptide vaccines.

[0435] 4. Literature search to identify additional evidence to support the relevance of the peptide identified as TUMP;

[0436] 5. The relevance of mRNA level overexpression was determined by re-detection of the TUMAPs selected in step 3 on tumor tissues and their absence (or low frequency) on healthy tissues;

[0437] 6. To evaluate whether the selected peptides induce T cell responses in vivo, in vitro immunogenicity assays were performed using human T cells from healthy donors as well as from esophageal cancer patients.

[0438] In one aspect, the peptides are screened for immunogenicity before being added to the library. For example, but not limited to, the immunogenicity of the peptides included in the library can be determined by in vitro T cell activation, specifically by repeatedly stimulating CD8+ T cells from healthy donors with artificial antigen-presenting cells loaded with peptide / MHC complexes and anti-CD28 antibodies.

[0439] This approach is preferred for rare cancers and patients with unusual expression profiles. In contrast to peptide cocktails currently developed with fixed components, a library allows for a higher degree of matching of the actual expression of antigens in tumors to the vaccine. In a multi-target approach, each patient would receive a single peptide or a combination of several "off-the-shelf" peptides. Theoretically, an approach based on selecting, for example, five different antigenic peptides from a library of 50 antigenic peptides could provide approximately 1.7 million possible drug product (DP) components.

[0440] In one aspect, the peptides are selected for inclusion in a vaccine based on suitability for an individual patient and using the methods of the invention described herein or hereinafter.

[0441] HLA phenotypic, transcriptomic, and peptidomic profiles are collected from patient tumor material and blood samples to identify peptides that are most appropriate for each patient and contain both the "reservoir" and the patient's unique (i.e., mutation) TUMAP. Peptides are selected that are selectively or overexpressed in the patient's tumor and, if possible, exhibit strong in vitro immunogenicity when tested in the patient's individual PBMCs.

[0442] Preferably, a method for identifying peptides included in the vaccine comprises: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from an individual patient; (b) comparing the peptides identified in (a) to a repository (database) of such peptides; and (c) selecting at least one peptide from the repository (database) that is related to the tumor-associated peptide identified in the patient. For example, the method for identifying TUMAPs presented by a tumor sample comprises: (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 tissue; 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. Preferably, the sequences of the MHC ligands are determined by eluting peptides bound to 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.

[0443] In addition to using a repository (database) model to select peptides, as an alternative, TUMAPs can be identified in vitro in patients and then included in vaccines. As an example, candidate TUMAPs in patients can be identified by: (a1) comparing expression data from a tumor sample with expression data from a normal tissue sample corresponding to the tumor sample tissue type to identify proteins that are overexpressed or abnormally expressed in the tumor tissue; and (a2) correlating the expression data with the sequences of MHC ligands that bind to class I MHC and / or class II molecules in the tumor sample to determine the MHC ligands derived from proteins that are overexpressed or abnormally expressed in the tumor. As another example, a protein can be identified as containing a mutation that is unique to the tumor sample relative to the corresponding normal tissue of an individual patient, and a TUMAP can be identified as specifically targeting the mutation. For example, the genomes of the tumor and the corresponding normal tissue can be sequenced using whole genome sequencing methods: to discover non-synonymous mutations in the protein-coding regions of genes, genomic DNA and RNA are extracted from the tumor tissue, and normal, non-mutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMCs). The NGS method used is limited to the resequencing of protein coding regions (exome resequencing). For this purpose, a target sequence enrichment kit provided by the supplier is used to capture exon DNA from human samples, followed by sequencing using HiSeq2000 (Illumina). In addition, the mRNA of the tumor is sequenced to directly quantify gene expression and confirm that the mutant gene is expressed in the patient's tumor. The millions of sequence reads obtained are processed by software algorithms. The output list contains mutations and gene expression. Tumor-specific somatic mutations are determined by comparison with PBMC-derived germline changes and optimized. Then, the newly determined peptides may be tested for immunogenicity as described above for the library, and candidate TUMAPs with suitable immunogenicity are selected for use in vaccines.

[0444] In one exemplary embodiment, the peptides included in the vaccine are identified by the following method: (a) identifying tumor-associated peptides (TUMAPs) presented by an individual patient's tumor sample using the above-described method; (b) comparing the peptides identified in (a) with a peptide library that has been pre-screened for immunogenicity and over-presentation in tumors (compared to corresponding normal tissues); (c) selecting at least one peptide from the library that is related to the tumor-associated peptide identified in the patient; and (d) optionally selecting at least one peptide newly identified in (a) and confirming its immunogenicity.

[0445] In one exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by an individual patient's tumor sample; and (b) selecting at least one newly identified peptide in (a) and confirming its immunogenicity.

[0446] Once the peptides for the personalized peptide vaccine are selected, the vaccine is produced. The vaccine is preferably a liquid formulation comprising the individual peptides dissolved in between 20-40% DMSO, preferably about 30-35% DMSO, for example, about 33% DMSO.

[0447] Each peptide included in the product is dissolved in DMSO. The concentration of the individual peptide solutions is determined by the number of peptides to be included in the product. The individual peptide-DMSO solutions are mixed equally to achieve a single solution containing all peptides at a concentration of approximately 2.5 mg / ml per peptide. This mixed solution is then diluted 1:3 with water for injection to a concentration of 0.826 mg / ml per peptide in 33% DMSO. The diluted solution is then sterile filtered through a 0.22 μm filter to obtain the final bulk solution.

[0448] The final bulk solution was filled into vials and stored at -20°C before use. One vial contained 700 μL of solution, containing 0.578 mg of each peptide, of which 500 μL (approximately 400 μg of each peptide) was used for intradermal injection.

[0449] In addition to being used to treat cancer, the peptides of the present invention can also be used for diagnosis. Since the peptides are produced by esophageal cancer cells and have been determined to be absent or present at low levels in normal tissues, these peptides can be used to diagnose the presence of cancer.

[0450] Blood samples and tissue biopsies containing the claimed peptides can aid pathologists in diagnosing cancer. Detection of certain peptides using antibodies, mass spectrometry, or other methods known in the art can enable pathologists to determine whether the tissue sample is malignant, inflammatory, or generally pathological. These peptides can also serve as biomarkers for esophageal cancer. Presentation of peptide moieties allows for the classification or further subdivision of diseased tissue.

[0451] Detection of peptides in lesion specimens allows for the assessment of the benefits of immune system-directed therapies, particularly if T-lymphocytes are known or suspected to be involved in the mechanism of action. Loss of MHC expression is a well-characterized mechanism by which infected malignant cells evade immune surveillance. Therefore, peptide presentation indicates that the analyzed cells do not utilize this mechanism.

[0452] The peptides of the present invention can be used to analyze the response of lymphocytes to the peptides (such as T cell responses), or the response of antibodies to the peptides or peptides complexed with MHC molecules. These lymphocyte responses can be used as prognostic indicators to determine whether further treatment should be taken. These responses can also be used as alternative response indicators in immunotherapy, which aims to induce lymphocyte responses in different ways, such as protein vaccines, nucleic acids, autologous materials, and adoptive transfer of lymphocytes. In gene therapy, the response of lymphocytes to peptides can be considered in the assessment of side effects. Lymphocyte response monitoring may also become a valuable tool in follow-up examinations of transplantation therapy, such as for detecting graft-versus-host and host-versus-graft diseases.

[0453] The present invention will be illustrated by the following examples which describe preferred embodiments thereof, with reference to (but not limitation to) the accompanying drawings. For the purposes of the present invention, all references cited herein are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0454] Figure 1A to 1V Overexpression 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 glands, 8 arteries, 5 bone marrow, 7 brains, 5 breasts, 2 cartilages, 1 central nervous system, 13 colons, 1 duodenum, 2 gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4 lymph nodes, 4 white blood cell samples, 3 ovaries, 8 pancreases, 5 peripheral nerves, 1 peritoneum, 3 pituitary glands, 4 placentas, 3 pleuras, 3 prostates, 6 rectus muscles, 7 salivary glands, 4 skeletal muscles, 6 skins, 2 small intestines, 4 spleens, 5 stomachs, 6 testes, 3 thymuses, 3 thyroid glands, 7 tracheas, 2 ureters, 6 bladders, 2 uteri, 2 veins, 6 esophagi, 16 esophageal cancer specimens. This peptide was also detected in 4 of 91 lung cancer specimens. Figure 1B Gene symbol: GJB5, peptide: SIFEGLLSGV (SEQ ID NO: 7). Tissues from left to right: 1 adipose tissue, 3 adrenal glands, 8 arteries, 5 bone marrow, 7 brains, 5 breasts, 2 cartilages, 1 central nervous system, 13 colons, 1 duodenum, 2 gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4 lymph nodes, 4 white blood cell samples, 3 ovaries, 8 pancreases, 5 peripheral nerves, 1 peritoneum, 3 pituitary glands, 4 placentas, 3 pleuras, 3 prostates, 6 rectus muscles, 7 salivary glands, 4 skeletal muscles, 6 skins, 2 small intestines, 4 spleens, 5 stomachs, 6 testes, 3 thymuses, 3 thyroid glands, 7 tracheas, 2 ureters, 6 bladders, 2 uteri, 2 veins, 6 esophaguses, and 16 esophageal cancer specimens. This peptide was also detected in 1 / 43 prostate cancer specimens, 1 / 3 gallbladder cancer specimens, 1 / 20 ovarian cancer specimens, 5 / 91 lung cancer specimens, and 1 / 4 bladder cancer specimens. Figure 2CGene symbol: PKP3, peptide: SLVSEQLEPA (SEQ ID NO: 34). Tissues from left to right: 1 adipose tissue, 3 adrenal glands, 8 arteries, 5 bone marrow, 7 brains, 5 breasts, 2 cartilages, 1 central nervous system, 13 colons, 1 duodenum, 2 gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4 lymph nodes, 4 white blood cell samples, 3 ovaries, 8 pancreases, 5 peripheral nerves, 1 peritoneum, 3 pituitary glands, 4 placentas, 3 pleurae, 3 prostates, 6 rectus muscles, 7 salivary glands, 4 skeletal muscles, 6 skins, 2 small intestines, 4 spleens, 5 stomachs, 6 testes, 3 thymuses, 3 thyroid glands, 7 tracheas, 2 ureters, 6 bladders, 2 uteri, 2 veins, 6 esophaguses, and 16 esophageal cancer specimens. This peptide was also detected in 8 / 24 colorectal cancers, 1 / 20 ovarian cancers, 1 / 46 gastric cancers, 5 / 91 lung cancers, and 2 / 4 bladder cancers. Figure 2DGene symbol: RNPEP, peptide: YTQPFSHYGQAL (SEQ ID NO: 37). Tissues from left to right: 1 adipose tissue, 3 adrenal glands, 8 arteries, 5 bone marrow, 7 brains, 5 breasts, 2 cartilages, 1 central nervous system, 13 colons, 1 duodenum, 2 gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4 lymph nodes, 4 white blood cell samples, 3 ovaries, 8 pancreases, 5 peripheral nerves, 1 peritoneum, 3 pituitary glands, 4 placentas, 3 pleurae, 3 prostates, 6 rectus muscles, 7 salivary glands, 4 skeletal muscles, 6 skins, 2 small intestines, 4 spleens, 5 stomachs, 6 testes, 3 thymuses, 3 thyroid glands, 7 tracheas, 2 ureters, 6 urinary bladders, 2 uteri, 2 veins, 6 esophaguses, and 16 esophageal cancer specimens. This peptide was also detected in 1 / 19 pancreatic cancers, 7 / 46 gastric cancers, and 1 / 91 lung cancers. 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 myeloid 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, 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, 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, 1 uterine cancer). Figure 10K) Gene symbol: AN01, 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, 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, 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 cancers, 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 cancers, 4 bladder cancers). 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 cecal 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 placentas), 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 cancers, 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 cancer, 12 brain cancer, 4 breast cancer, 3 esophageal cancer, 1 gallbladder cancer, 5 kidney cancer, 21 lung cancer, 1 lymph node cancer, 7 ovarian cancer, 1 pancreatic cancer, 1 skin cancer, 2 uterine cancer). Figure 17R) Gene symbol: PRC1, 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 cancer, 1 colon cancer, 2 esophageal cancers, 1 leukemia cancer, 8 lung cancer, 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 12U) 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 cancers, 4 esophageal cancers, 1 gallbladder cancer, 14 lung cancers, 7 lymph node cancers, 2 prostate cancers, 1 skin cancer, 8 bladder cancers). Figure 21V) Gene symbol: KIAA0947, peptide: ALVPHVDQV (SEQ ID NO: 81). Tissues from left to right: 3 cell lines (1 blood cell, 1 pancreas), 12 cancer tissues (5 brain cancers, 2 esophageal cancers, 1 lung cancer, 3 lymph node cancers, 1 uterine cancer).

[0455] Figures 2A to 2D Representative expression profiles of the source genes of the present invention are shown, which are highly overexpressed or exclusively expressed in esophageal cancer across a range 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 esophaguses, 2 gallbladders, 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 gland, 5 tracheas, 1 bladder, 1 breast, 3 ovaries, 3 placentas, 1 prostate, 1 testis, 1 uterus, and 11 esophageal cancer samples. Figure 2A )CT45A1, CT45A3, CT45A5, CT45A6, CT45A2, RP11-342L5.1, gene symbol: PTHLH; Figure 2B )CLDN16, gene symbol: KRT14; Figure 2C )ESR1, gene symbol: FAM83A; Figure 2D )IDO1, gene symbol: PDPN.

[0456] Figure 3 A to E show exemplary results of peptide-specific CD8+ T cell responses in vitro from a healthy HLA-A*02+ donor, i.e., exemplary immunogenicity data: flow cytometry results after peptide-specific multimer staining. Figure 3 A) Gene symbol: SF3B3, peptide: ELDRTPPEV (SEQ ID NO: 97); Figure 3B) Gene symbol: TNC, peptide: AMTQLLAGV (SEQ ID NO: 101). Additionally, CD8+ T cells were generated using artificial APCs coated with anti-CD28 mAb and HLA-A*02 and synthesized with peptides SEQ ID NO: 5 (C, left panel), SEQ ID NO: 2 (D, left panel), and SEQ ID NO: 77 (E, left panel). After three cycles of stimulation, peptide-reactive cells were detected using 2D multimer staining with A*02 / SeqID No 5 (C), A*02 / SeqID No 2 (D), and A*02 / SeqID No 77 (E). The right panels (C, D, and E) show control staining of cells stimulated with irrelevant A*02 / peptide complexes. Live single cells were gated on CD8+ lymphocytes. Boolean gating helps exclude false positive events detected with multimers specific for different peptides. The frequencies of specific multimer+ cells and CD8+ lymphocytes are indicated. Example

[0457] Example 1

[0458] Identification and quantification of tumor-associated peptides presented on the cell surface

[0459] tissue samples

[0460] Tumor tissue was obtained from Asterand (Detroit, USA and Royston, Herts, UK), ProteoGenex Inc. (Culver City, CA, USA), Tissue Solutions Ltd. (Glasgow, UK), and University Hospital Tübingen. Normal tissue was 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), Geneva University Hospital, Heidelberg University Hospital, Kyoto Prefectural University of Medicine (KPUM), University Hospital of Munich, ProteoGenex Inc. (Culver City, CA, USA), University Hospital Tübingen, Germany, and Tissue Solutions Ltd. (Glasgow, UK). Written informed consent was obtained from all patients before surgery or autopsy. Tissues were immediately frozen after resection and stored at -70°C or below before TUMAP isolation.

[0461] Isolation of HLA peptides from tissue samples

[0462] HLA peptide libraries were obtained from frozen tissue samples by immunoprecipitation according to the protocol (Falk et al, 1991; Seeger et al, 1999) with slight modifications, using the HLA-A*02-specific antibody BB7.2 and the HLA-A, HLA-B, and HLAC-specific antibody W6 / 32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

[0463] Mass spectrometry analysis

[0464] The resulting HLA peptide library was separated according to their hydrophobicity using reversed-phase chromatography (nanoAcquity UPLC system, Waters), and the eluted peptides were analyzed using an LTQ-Velos Fusion Hybrid Mass Spectrometer (ThermoElectron) equipped with an electrospray source. The peptide library was directly loaded onto an analytical fused silica microcapillary column (75 μm ID x 250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) at a flow rate of 400 nL / min. Subsequently, the peptides were separated using a two-step, 180-minute binary gradient from 10% to 33% solvent B at a flow rate of 300 nL / min. The gradient consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). Gold-coated glass capillaries (PicoTip, New Objective) were used for introduction into the nanoelectrospray source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using a top-5 strategy. Briefly, a scan cycle was initiated with a full orbitrap scan using high-accuracy mass (R = 30,000), followed by an orbitrap MS / MS scan of the five most abundant precursor ions (R = 7500) using dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted using SEQUEST and another manual controller. The fragmentation pattern of the resulting natural peptide was compared with that of a synthetic reference peptide of the same sequence to ensure the identification of the peptide sequence.

[0465] Label-free relative LC-MS quantification was performed by ion counting (i.e., extraction and analysis of LC-MS features) (Mueller et al, 2007). This 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; Sturmet et al, 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to combine quantitative data from different samples and tissues with peptide presentation profiles. Quantitative data were normalized in a two-tiered manner based on pooled data to account for technical and biological replicate variation. Thus, each identified peptide could be associated with quantitative data, allowing for relative quantification across samples and tissues. Furthermore, all quantitative data obtained for candidate peptides were manually reviewed to ensure data consistency and validate the accuracy of the automated analysis. For each peptide, a presentation plot was calculated, showing the sample mean presentation and replicate variation. These profiles juxtapose baseline values ​​for esophageal cancer samples with those for normal tissue samples.

[0466] The presentation profiles of exemplary over-presented peptides are shown in Figure 1. The presentation scores of exemplary peptides are shown in Table 8.

[0467] Table 8: Presentation Scores. This table lists peptides that are very highly over-presented (+++), highly over-presented (++), or over-presented (+) on tumors compared to a panel of normal tissues. The panel of normal tissues includes: adipose tissue, adrenal glands, arteries, veins, bone marrow, brain, central and peripheral nerves, colon, rectum, small intestine (including duodenum), esophagus, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear leukocytes, pancreas, peritoneum, pituitary gland, pleura, salivary glands, skeletal muscle, skin, spleen, stomach, thymus, thyroid gland, trachea, ureters, and bladder.

[0468]

[0469]

[0470]

[0471] Example 2

[0472] Expression profile of the gene encoding the peptide of the present invention

[0473] Overexpression or specific expression of a peptide on tumor cells compared to normal cells is sufficient for its effective use in immunotherapy. Some peptides are tumor-specific, even though their source protein is also present in normal tissues. However, mRNA expression profiling adds an additional level of safety to the selection of immunotherapy target peptides. Especially for therapeutic options with high safety risks, such as affinity-matured TCRs, the ideal target peptide will be derived from a protein that is unique to the tumor and not present in normal tissues.

[0474] RNA source and preparation

[0475] Surgical tissue specimens were obtained as described above (see Example 1) after written informed consent was obtained from each patient. Tumor tissue specimens were snap-frozen immediately after surgery and then homogenized in liquid nitrogen using a mortar and pestle. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, Germany) followed by RNeasy (QIAGEN, Hilden, Germany) cleanup; both methods were performed according to the manufacturer's protocols.

[0476] Total RNA from tumor tissues used for RNASeq experiments was obtained from: ProteoGenex Inc. (Culver City, CA, USA), Tissue Solutions Ltd. (Glasgow, UK).

[0477] Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, USA and Royston, Herts, UK), ProteoGenex 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 Heidelberg, Germany, BioCat GmbH (Heidelberg, Germany).

[0478] 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).

[0479] RNAseq experiments

[0480] Gene expression analysis was performed on RNA samples from tumors and normal tissues by CeGaT (Tübingen, Germany) using next-generation sequencing (RNAseq). Briefly, sequencing libraries were prepared using the Illumina HiSeq v4 kit according to the supplier's protocol (Illumina Inc, San Diego, CA, USA), which includes RNA fragmentation, cDNA conversion, and the addition of sequencing adapters. Libraries obtained from multiple samples were mixed in equal moles according to the manufacturer's instructions and sequenced on an Illumina HiSeq 2500 sequencer, generating 50 bp single-end reads. The processed reads were mapped to the human genome (GRCh38) using STAR software. According to the instructions of the ENSEMBL sequence database (Ensembl77), expression data were set at the transcript level as RPKM (reads per kilobase per million mapped reads, generated by Cufflinks software) and at the exon level (total reads, generated by Bedtools software). Exon reads were normalized to exon length and calibrated to obtain RPKM values.

[0481] The expression profiles of representative source genes of the present invention that are highly overexpressed in esophageal cancer are shown in Figure 2. The expression scores of further representative genes are shown in Table 9.

[0482] Table 9: Expression scores. This table lists peptides for genes that are very highly overexpressed (+++) on tumors compared to a range of normal tissues, highly overexpressed (++) on tumors compared to a range of normal tissues, or overexpressed (+) on tumors compared to a range of normal tissues. This score baseline was calculated based on measurements of the following normal tissues: adipose tissue, adrenal gland, artery, bone marrow, brain, colon, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, pancreas, pituitary, rectum, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid, trachea, bladder, vein.

[0483]

[0484]

[0485] Example 3

[0486] In vitro immunogenicity of MHC class I presented peptides

[0487] To obtain information about the immunogenicity of the TUMAPs of the invention, the inventors conducted studies using an in vitro T cell expansion assay based on repeated stimulation with artificial antigen-presenting cells (aAPCs) loaded with peptide / MHC complexes and anti-CD28 antibodies. Using this method, the inventors were able to show that the HLA-A*0201-restricted TUMAPs of the invention are immunogenic, indicating that these peptides are T cell epitopes against human CD8+ precursor T cells (Table 10).

[0488] In vitro activation of CD8+ T cells

[0489] For in vitro stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD28 antibodies, the inventors first isolated CD8+ T cells from healthy donors obtained from the University clinics Mannheim, Germany, using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) by positive selection of fresh HLA-A*02 products after leukapheresis.

[0490] PBMC and isolated CD8+ lymphocytes were cultured in T cell culture medium (TCM) before use. The culture medium consists 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). At this step, 2.5 ng / ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U / ml IL-2 (Novartis Pharma, Nürnberg, Germany) were also added to TCM.

[0491] For the generation of pMHC / anti-CD28-coated beads, T cell stimulation, and readout, four different pMHC molecules per stimulation condition and eight different pMHC molecules per readout condition were used in a highly defined in vitro system. Purified co-stimulatory mouse IgG2a anti-human CD28 antibody 9.3 (Jung et al, 1987) was chemically biotinylated using N-hydroxysuccinimide-biotin as recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were 5.6 μm streptavidin-coated polystyrene particles (Bangs Laboratories, IL, USA).

[0492] The pMHC used for positive and negative control stimulators were A*0201 / MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 102) modified from Melan-A / MART-1) and A*0201 / DDX5-001 (YLLPAIVHI (SEQ ID NO. 103) obtained from DDX5), respectively.

[0493] 800,000 beads / 200 μl were coated in a 96-well plate containing 4 x 12.5 ng of different biotin-pMHCs, washed, and then 600 ng of biotin anti-CD28 was added in a volume of 200 μl. Stimulation was activated by co-culturing 1 x 10 CD8+ T cells with 2 x 105 washed, coated beads in 200 μl of TCM containing 5 ng / ml IL-12 (PromoCell) at 37°C for 3 days. Half of the culture medium was then exchanged with fresh TCM supplemented with 80 U / ml IL-2, and incubation continued at 37°C for 4 days. This stimulation cycle was repeated three times in total. For pMHC multimer readout using eight different pMHC molecules per condition, a two-dimensional combinatorial encoding method was used as previously described (Andersen et al, 2012), with minor modifications to include coupling to five different fluorescent dyes. Finally, multimer analysis was performed using Live / dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD Biosciences, Heidelberg, Germany), and fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer equipped with appropriate lasers and screening programs was used. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis results were evaluated using FlowJo software (Tree Star, Oregon, USA). In vitro loading of specific multimer+ CD8+ lymphocytes was determined by comparison with negative control stimulation groups. The immunogenicity of a given antigen was determined if at least one evaluable in vitro stimulated well from a healthy donor was found to contain a specific CD8+ T cell line after in vitro stimulation (i.e., the well contained at least 1% specific multimer+ CD8+ T cells and the percentage of specific multimer+ was at least 10 times the median of the negative control stimulation).

[0494] In vitro immunogenicity of esophageal cancer peptides

[0495] For the tested HLA class I peptides, their in vitro immunogenicity can be demonstrated by the generation of peptide-specific T cell lines. Typical results of flow cytometry detection after staining of two peptides of the present invention (SEQ ID No 97 and SEQ ID No 101) with TUMAP-specific multimers are shown in Figure 2. Figure 3 The results for the five peptides of the present invention are summarized in Table 10A.

[0496] Table 10A: In vitro immunogenicity of HLA class I peptides of the present invention. Exemplary results of in vitro immunogenicity experiments conducted by applicants on peptides of the present invention. <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++

[0497] SEQ ID NO: sequence hole donor 94 TLLQEQGTKTV + ++ 95 LIQDRVAEV + ++ 97 ELDRTPPEV ++ ++++ 98 VLFPNLKTV + ++++ 101 AMTQLLAGV ++ +++

[0498] Table 10B: In vitro immunogenicity of HLA class I peptides of the invention.

[0499] Exemplary results of in vitro immunogenicity experiments conducted by the applicant on the peptides of the present invention. The results of the in vitro immunogenicity experiments are shown. The percentage of positive wells and donors (others can be evaluated) is summarized as <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++

[0500]

[0501] Example 4

[0502] Peptide synthesis

[0503] All peptides were synthesized using the standard, well-established solid-phase peptide synthesis method using the Fmoc strategy. The identity and purity of each peptide were confirmed using mass spectrometry and RP-HPLC analysis. Lyophilization (trifluoroacetate) yielded white to off-white peptides with a purity of >50%. All TUMAPs are preferably administered as trifluoroacetate or acetate salts, although other pharmaceutically acceptable salt forms are also possible.

[0504] Example 5

[0505] MHC binding assay

[0506] The present invention is based on candidate peptides for T cell therapy that are further tested for their MHC binding ability (affinity). Single peptide-MHC complexes are generated by UV-ligand exchange, wherein the UV-sensitive peptide is cleaved after ultraviolet irradiation and exchanged with the relevant peptide to be analyzed. Only candidate peptides that can effectively bind to and stabilize the peptide-receiving MHC molecule can prevent the dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA assay is performed based on the detection results of the light chain (β2m) that stabilizes the MHC complex. The test is generally performed according to the method described by Rodenko et al. (Rodenko et al, 2006).

[0507] 96-well Maxisorp plates (NUNC) were coated with 2 μg / ml streptavidin in PBS at room temperature overnight, washed four times, and blocked in 2% BSA in blocking buffer at 37°C for 1 hour. Folded HLA-A*02:01 / MLA-001 monomers were used as standards, covering a range of 15,500 ng / ml. Peptide-MHC monomers for UV exchange reactions were diluted 100-fold in blocking buffer. Samples were incubated at 37°C for 1 hour, washed four times, incubated with 2 μg / ml HRP-conjugated anti-β2m at 37°C for 1 hour, washed again, and detected with TMB solution blocked with NH2SO4. Absorbance was measured at 450 nm. When generating and producing 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 because they exhibit sufficient affinity for MHC molecules and prevent dissociation of the MHC complex.

[0508] Table 11: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-A*02:01 is categorized according to peptide exchange yield: ≥10% = +; ≥20% = ++; ≥50% = +++; ≥75% = ++++

[0509]

[0510]

[0511]

[0512] Example 6

[0513] Absolute quantification of tumor-associated peptides presented on the cell surface

[0514] The generation of binders such as antibodies and / or TCRs is a laborious process that can only be performed against a few selected targets. In the case of tumor-associated and specific peptides, selection criteria include, but are not limited to, exclusion of peptide presentation and concentration on the cell surface. Quantification of TUMAP copy number per cell in solid tumor samples requires absolute quantification of isolated TUMAPs, TUMAP isolation efficiency, and cell count of the analyzed tissue sample.

[0515] nanoLC-MS / MS peptide quantification

[0516] For accurate quantification of peptides by mass spectrometry, a calibration curve for each peptide is generated using an internal standard. The internal standard is a doubly isotopically labeled variant of each peptide; that is, two isotopically labeled amino acids are incorporated into the TUMAP synthesis. It differs from the tumor-associated peptide only in mass, but does not differ in other physicochemical properties (Anderson et al, 2012). The internal standard is spiked into each MS sample, and all MS signals are normalized to the internal standard MS signal to balance potential technical variations between MS experiments.

[0517] Calibration curves were prepared using at least three different matrices, i.e., HLA peptide eluates from natural samples similar to conventional MS samples, and each preparation was measured in duplicate MS runs. For evaluation, MS signals were normalized to the signal of an internal standard and calibration curves were calculated by logistic regression.

[0518] For quantification of tumor-associated peptides from tissue samples, each sample was also spiked with an internal standard; the MS signal was normalized to the internal standard and quantified using this peptide calibration curve.

[0519] Efficiency of peptide / MHC separation

[0520] As with any protein purification process, the isolation of proteins from tissue samples is associated with a certain loss of the associated proteins. To determine the efficiency of TUMAP isolation, peptide / MHC complexes were generated for all TUMAPs selected for absolute quantification. To enable the identification of native peptide / MHC complexes with the spiked material, monoisotopically labeled versions of the TUMAPs were used, i.e., one isotopically labeled amino acid was incorporated during TUMAP synthesis. These complexes were spiked into freshly prepared tissue lysates, i.e., at the earliest possible time point during TUMAP isolation, and subsequently recovered as native peptide / MHC complexes during affinity purification. Therefore, measuring the recovery of monolabeled TUMAPs allows conclusions to be drawn regarding the efficiency of individual TUMAP isolation.

[0521] Separation efficiency was analyzed using a small number of samples, and these tissue samples were comparable. In contrast, separation efficiency varied between individual peptides. This suggests that separation efficiency, although measured in a limited number of samples, can be extrapolated to any other tissue preparation. However, because separation efficiency cannot be extrapolated from one peptide to another, it is necessary to analyze each TUMAP individually.

[0522] Determination of cell counts in solid and frozen tissues

[0523] To determine the cell number of tissue samples undergoing absolute peptide quantification, the inventors employed DNA content analysis. This method is applicable to a wide range of samples from different sources, most notably frozen samples (Alcoser et al, 2011; Forsey and Chaudhuri, 2009; Silva et al, 2013). During the peptide isolation protocol, the tissue sample is processed into a homogenous lysate, from which a small aliquot of the lysate is taken. The sample is aliquoted into triplicates, from which DNA is isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content of each DNA isolation is quantified in at least two replicates using a fluorescence-based DNA quantification assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany).

[0524] To calculate cell number, a DNA standard curve was generated from a single aliquot of healthy blood cells, using a range of specified cell numbers. The standard curve was used to calculate the total cell content for each DNA isolation. The average total cell count for the tissue samples used for peptide isolation was extrapolated, taking into account the known volume of the lysate aliquot and the total lysate volume.

[0525] Peptide copies per cell

[0526] Using the data from the previous experiments, the inventors calculated the TUMAP copy number per cell by dividing the total peptide amount by the total cell count of the sample, and then divided by the isolation efficiency. The cell copy number of the selected peptides is shown in Table 12.

[0527] Table 12: Absolute copy number. This table lists the results of absolute peptide quantification in NSCLC tumor samples. For each peptide, the median copy number per cell is indicated as follows: <100 = +; >=100 = ++; >=1,000 +++; >=10,000 = ++++. The number of samples for which high-quality MS data were available for evaluation is indicated.

[0528]

[0529] In summary, this application includes but is not limited to the following:

[0530] 1. A peptide and a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid sequence selected from SEQ ID No. 1 to SEQ ID No. 93, and a variant sequence thereof having at least 88% homology with SEQ ID No. 1 to SEQ ID No. 93, wherein the variant peptide binds to the major histocompatibility complex (MHC) and / or induces T cells to cross-react with the variant peptide, wherein the peptide is not a full-length polypeptide.

[0531] 2. The peptide according to item 1, wherein the peptide is capable of binding to MHC class I or -II molecules, wherein the peptide can be recognized by CD4 and / or CD8 T cells when bound to MHC.

[0532] 3. The peptide or variant thereof according to item 1 or 2, wherein the amino acid sequence comprises a continuous stretch of amino acids of any one of SEQ ID No. 1 to SEQ ID No. 93.

[0533] 4. The peptide or variant thereof according to any one of items 1 to 3, wherein the total length of the peptide or variant thereof is 8 to 100 amino acids, preferably 8 to 30 amino acids, more preferably 8 to 16 amino acids, and most preferably the peptide consists of or essentially consists of the amino acid sequence of any one of SEQ ID No. 1 to SEQ ID No. 93.

[0534] 5. The peptide or variant thereof according to any one of items 1 to 4, wherein the peptide is modified and / or comprises non-peptide bonds.

[0535] 6. The peptide or variant thereof according to any one of items 1 to 5, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii).

[0536] 7. A nucleic acid encoding the peptide or variant thereof according to any one of items 1 to 6, optionally linked to a heterologous activator sequence.

[0537] 8. An expression vector expressing the nucleic acid described in item 7.

[0538] 9. A recombinant host cell comprising the peptide of Items 1 to 6, the nucleic acid of Item 7, and the expression vector of Item 8, wherein the host cell is preferably an antigen-presenting cell, such as a dendritic cell.

[0539] 10. Use of the peptide or variant thereof according to any one of items 1 to 6, the nucleic acid according to item 7, the expression vector according to item 8, or the host cell according to item 9 in medicine.

[0540] 11. A method for preparing the peptide or variant thereof described in any one of items 1 to 6, the method comprising culturing the host cell described in item 9, wherein the host cell presents the peptide described in items 1 to 6, or expresses the nucleic acid described in item 7, or carries the expression vector described in item 8, and isolating the peptide or variant thereof from the host cell or its culture medium.

[0541] 12. A method for preparing activated T lymphocytes in vitro, the method comprising contacting T cells with antigen-loaded human class I or II MHC molecules in vitro for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the human class I or II MHC molecules are expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificially simulated antigen-presenting cell structure, wherein the antigen is a peptide described in any one of items 1 to 4.

[0542] 13. An activated T lymphocyte produced by the method of item 12, which selectively recognizes a cell presenting a polypeptide comprising the amino acid sequence given in any one of items 1 to 4.

[0543] 14. A method for killing target cells in a patient, wherein the target cells present a polypeptide comprising the amino acid sequence given in any one of items 1 to 4, the method comprising administering to the patient an effective amount of the activated T cells of item 13.

[0544] 15. An antibody, particularly a soluble or membrane-bound antibody, which specifically recognizes a peptide or variant thereof according to any one of items 1 to 5, preferably a peptide or variant according to any one of items 1 to 5 when bound to an MHC molecule.

[0545] 16. Use of the peptide described in any one of items 1 to 6, the nucleic acid described in item 7, the expression vector described in item 8, the cell described in item 9, or the activated toxic T lymphocyte described in item 13, or the antibody described in item 15 in the diagnosis and / or treatment of cancer or the manufacture of an anticancer agent.

[0546] 17. The use according to claim 16, wherein the cancer is selected from esophageal cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, breast cancer, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder cancer, bile duct cancer and other tumors, which overexpress proteins from which peptides of SEQ ID No. 1 to SEQ ID No. 93 can be derived.

[0547] 18. A kit comprising:

[0548] (a) a container comprising a pharmaceutical composition comprising the peptide or variant according to any one of Items 1 to 6, the nucleic acid according to Item 7, the expression vector according to Item 8, the cell according to Item 10, the activated T lymphocyte according to Item 13, or the antibody according to Item 15, in the form of a solution or lyophilized product;

[0549] (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized dosage form;

[0550] (c) optionally, at least one peptide selected from SEQ ID No. 1 to SEQ ID No. 101, and

[0551] (d) Optionally, instructions for (i) using the solution or (ii) reconstitution and / or use of the lyophilized dosage form.

[0552] 19. The kit of claim 18, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

[0553] 20. The kit according to item 18 or 19, wherein the peptide is selected from SEQ ID No. 1 to SEQ ID No. 93.

[0554] 21. A method for producing a personalized anti-cancer vaccine for use as a compound-based and / or cell therapy for an i...

Claims

A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence STYGGGLSV (SEQ ID NO: 1).

2. The peptide according to claim 1, wherein the peptide has the ability to bind to major histocompatibility complex (MHC) class I molecules, and wherein when bound to the MHC, the peptide can be recognized by CD4 and / or CD8 T cells.

3. The peptide according to claim 1 or 2, wherein the pharmaceutically acceptable salt is a chloride salt or an acetate salt.

4. A nucleic acid encoding the peptide according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, linked to a heterologous promoter sequence. The nucleic acid according to claim 4 , which is contained in an expression vector.

7. A recombinant host cell comprising the peptide according to any one of claims 1 to 3 and the nucleic acid according to any one of claims 4 to 6. The recombinant host cell according to claim 7 , wherein the host cell is an antigen presenting cell. The recombinant host cell according to claim 8 , wherein the antigen-presenting cell is a dendritic cell.

10. A pharmaceutically acceptable salt of the peptide according to any one of claims 1 to 3.

11. The pharmaceutically acceptable salt according to claim 10, wherein the salt is chloride, acetate or trifluoroacetate.

12. A method for preparing activated T lymphocytes in vitro, the method comprising contacting T cells with antigen-loaded human class I MHC molecules for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the human class I MHC molecules are expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificial construct that mimics an antigen-presenting cell, wherein the antigen is a peptide according to any one of claims 1 to 3.

13. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of a peptide according to any one of claims 1 to 3, a nucleic acid according to any one of claims 4 to 6, a recombinant host cell according to any one of claims 7 to 9, or a pharmaceutically acceptable salt according to claim 10 or 11, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13, wherein (i) the pharmaceutical composition further comprises an adjuvant, and / or (ii) the pharmaceutical composition is a vaccine or a cell therapy agent.

15. The pharmaceutical composition according to claim 14, wherein the adjuvant is an interleukin. The pharmaceutical composition according to claim 15 , wherein the interleukin is IL-2 and / or IL-15.

17. A method for preparing the peptide according to any one of claims 1 to 3, comprising culturing the recombinant host cell according to any one of claims 7 to 9, and isolating the peptide from the host cell and / or its culture medium.

18. Use of the peptide according to any one of claims 1 to 3, the nucleic acid according to any one of claims 4 to 6, the recombinant host cell according to any one of claims 7 to 9, or the pharmaceutical composition according to any one of claims 13 to 16 in the preparation of a medicament for preventing and / or treating cancer.

19. The use according to any one of claims 18, wherein the cancer is selected from esophageal cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, breast cancer, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder cancer, bile duct cancer and other tumors, which show over-presentation of the peptide comprising SEQ ID No.

1.

20. A kit comprising: (a) a container comprising a pharmaceutical composition comprising the peptide according to any one of claims 1 to 3, the nucleic acid according to any one of claims 4 to 6, the recombinant host cell according to any one of claims 7 to 9, or the pharmaceutically acceptable salt according to claim 10 or 11, wherein the pharmaceutical composition is in the form of a solution or a lyophilized form; and (b) A second container containing a diluent or reconstitution fluid for the lyophilized dosage form.

21. The kit of claim 20, further comprising one or more of: (i) a buffer, (ii) a diluent, (iii) a filter, (iv) a needle, (v) a syringe, or (vi) an adjuvant.

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