阅读说明：本技术 用于食管癌和其他癌症免疫治疗的新型肽和肽组合物 (Novel peptides and peptide compositions for immunotherapy of esophageal and other cancers ) 是由 安德烈·马尔 托尼·温斯切尼克 科莱特·宋 奥利弗·施尔 延斯·弗里切 哈普瑞特·辛格 于 2016-07-05 设计创作，主要内容包括：本发明涉及用于免疫治疗方法的肽、蛋白、核酸和细胞。特别是,本发明涉及癌症的免疫疗法。本发明还涉及单独使用或与其他肿瘤相关肽(能够例如作为刺激抗肿瘤免疫反应或体外刺激T细胞并转入患者的疫苗组合物的活性药物成分)联合使用的肿瘤相关T细胞(CTL)肽表位。与主要组织兼容性复合体(MHC)分子结合的肽或与此同类的肽也可以是抗体、可溶性T细胞受体和其他结合分子的靶标。(The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the invention relates to immunotherapy of cancer. The invention also relates to tumor-associated T Cell (CTL) peptide epitopes, used alone or in combination with other tumor-associated peptides, which can be used, for example, as active pharmaceutical ingredients of vaccine compositions for stimulating anti-tumor immune responses or stimulating T cells in vitro and into patients. Peptides that bind to or are cognate to Major Histocompatibility Complex (MHC) molecules can also be targets for antibodies, soluble T cell receptors, and other binding molecules.)

1. A peptide comprising an amino acid sequence selected from SEQ ID nos. 13, 1 to 8, 10 to 12 and 14 to 94, and variant sequences thereof which are at least 88% homologous to SEQ ID nos. 13, 1 to 8, 10 to 12 and 14 to 94, wherein the variant peptide binds to 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, and pharmaceutically acceptable salts thereof.

2. A nucleic acid encoding the peptide or variant thereof of claim 1, optionally linked to a heterologous activator sequence.

3. An expression vector expressing the nucleic acid of claim 2.

4. A recombinant host cell comprising the peptide of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, wherein the host cell is preferably an antigen presenting cell, such as a dendritic cell.

5. Use of the peptide or variant thereof of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, or the host cell of claim 4 in medicine.

6. A method of producing the peptide or variant thereof of claim 1, comprising culturing the host cell of claim 4 which presents the peptide of claim 1, or expresses the nucleic acid of claim 2, or carries the expression vector of claim 3, and isolating the peptide or variant thereof from the host cell or culture medium thereof.

7. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial mimetic of an antigen-presenting cell structure for a time sufficient to activate the T cells in an antigen specific manner, wherein the antigen is a peptide according to claim 1.

8. An activated T lymphocyte, produced by the method of claim 7, that selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence given in claim 1.

9. A method of killing a target cell in a patient, wherein the target cell presents a polypeptide comprising an amino acid sequence given in claim 1, the method comprising administering to the patient an effective amount of the activated T cell of claim 8.

10. An antibody, in particular a soluble or membrane-bound antibody, which specifically recognizes a peptide or variant thereof according to claim 1, preferably a peptide or variant thereof according to claim 1 when bound to an MHC molecule.

Background

Esophageal cancer is the eighth most common cancer worldwide, and 464063 patients have the 5-year-old onset number in 2012. Mortality rates were very similar to morbidity rates (400169 vs 455784 in 2012), suggesting high esophageal cancer mortality rates (World cancer report, 2014; Ferlay et al, 2013; Bray et al, 2013).

Squamous cell carcinoma and adenocarcinoma represent the two most common subtypes of esophageal cancer. These two subtypes are more common in men than women, but they exhibit distinctly different geographical distributions. Squamous cell carcinoma is more prevalent in resource-poor locations, particularly in the Islamic Lankansui, parts of China, and Zimbabwe, with high incidence. Adenocarcinoma is the most common type of esophageal cancer among caucasians and socioeconomic populations, with the united kingdom, australia, the netherlands, and the united states leading. The greatest risk factors for the development of 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 is steadily increasing in high-income countries, probably due to increased incidence of obesity and gastroesophageal reflux disease and changes in the classification of tumors at the gastroesophageal junction. Neuroendocrine cancers, adenoid cystic cancers, adenosquamous carcinomas, mucoepidermoid carcinomas, mixed adenoneuroendocrine cancers, various sarcomas, and melanomas represent a rare subtype of esophageal cancer (World cancer report, 2014).

The primary treatment strategy for esophageal cancer depends on the stage and location of the tumor, the histological type, and the patient's condition. Surgery alone is not sufficient unless used in a small fraction of patients with squamous cell carcinoma. Generally, surgery should be combined with preoperative and postoperative chemotherapy or preoperative chemoradiotherapy, and simple preoperative or postoperative radiotherapy has not been proven to provide survival benefits. Chemotherapeutic regimens include oxaliplatin + fluorouracil, carboplatin + paclitaxel, cisplatin + fluorouracil, FOLFOX and cisplatin + irinotecan. Patients positive for tumor HER2 should be treated with a combination of cisplatin, fluorouracil and trastuzumab according to the guidelines for gastric cancer therapy because the randomized data for esophageal cancer targeting therapy is very limited (Stahl et al, 2013; leitliie magenkrazinom, 2012).

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

Immunotherapy may be a promising new approach to the treatment of advanced esophageal cancer. Several cancer-related genes and cancer-testis antigens have been shown to be overexpressed in esophageal cancers, including different MAGE genes, NY-ESO-1 and EpCAM (Kimura et al, 2007; Liang et al, 2005 b; Inoue et al, 1995; Bujas et al, 2011; Tanaka et al, 1997; Quillien et al, 1997). These genes represent very interesting targets for immunotherapy, most of which are being investigated for the treatment of other malignancies (clinical trials. gov, 2015). In addition, PD-L1 and PD-L2 are described as being up-regulated in esophageal cancer, which correlates with a poor prognosis. Thus, esophageal cancer patients with tumors positive for PD-L1 would benefit from anti-PD-L1 immunotherapy (Ohigashi et al, 2005).

Currently, the clinical data for esophageal cancer immunotherapy approaches are still relatively poor, as only a limited number of early stage clinical trials have been completed (tomey et al, 2013). In a phase I trial, patients with advanced esophageal cancer were given a vaccine comprising peptides 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 the general results (Kono et al, 2009). In one phase I/II study, 4 of 11 patients had complete or partial tumor responses due to intratumoral injection of activated T cells following in vitro stimulation with autologous tumor cells and interleukin 2 (Toh et al., 2000; Toh et al., 2002). Further clinical trials are currently underway to assess the effects of different immunotherapies on esophageal cancer, including secondary cell therapy (NCT01691625, NCT01691664, NCT01795976, NCT02096614, NCT02457650) vaccination strategies (NCT01143545, NCT01522820) and anti-PD-L1 therapy (NCT02340975) (clinical trials.

Given the severe side effects and costs associated with treating cancer, it is often necessary to identify factors that can be used to treat cancer, particularly esophageal cancer. It is also often necessary to determine factors that represent biomarkers of cancer, particularly esophageal cancer, in order to better diagnose cancer, assess prognosis, and predict treatment success.

Cancer immunotherapy represents one option for cancer cell-specific targeting, while minimizing side effects. Cancer immunotherapy utilizes the presence of tumor-associated antigens.

The current classification of Tumor Associated Antigens (TAAs) mainly includes the following groups:

a) cancer-testis antigen: the first identified TAA that T cells can recognize belongs to this class of antigens, which were originally called cancer-testis (CT) antigens because their members are expressed in histologically distinct human tumors, in normal tissues, only in spermatocytes/spermatogonia of the testis, and occasionally in the placenta. Since testis cells do not express HLA class I and class II molecules, these antigens are not recognized by T cells in normal tissues and are therefore immunologically considered to be tumor-specific. Well known examples of CT antigens are members of the MAGE family and NY-ESO-1.

b) Differentiation antigen: both tumor 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 thus these proteins are not tumor specific, but are still widely used in cancer immunotherapy. Examples include, but are not limited to, tyrosinase for melanoma and PSA for Melan-A/MART-1 or prostate cancer.

c) Overexpressed TAA: genes encoding widely expressed TAAs have been detected in histologically distinct tumors as well as in many normal tissues, generally at low levels. It is likely that many epitopes processed and potentially presented by normal tissues are below the threshold level of T cell recognition, and their overexpression in tumor cells can trigger an anti-cancer response by breaking the previously established tolerance. Typical examples of such TAAs are Her-2/neu, survivin, telomerase or WT 1.

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 generally induce strong immune responses without risking autoimmune responses in normal tissues. On the other hand, these TAAs are in most cases only associated with the exact tumor on which they are confirmed, and are not usually shared between many individual tumors. Peptide tumor specificity (or relatedness) may also occur if the peptide is derived from a tumor (associated) exon, in the case of proteins containing tumor-specific (associated) isoforms.

e) TAA resulting from aberrant post-translational modifications: such TAAs may be produced by proteins that are neither specific nor overexpressed in tumors, but which still have tumor-related properties (the association results from post-translational processing that is primarily active against tumors). Such TAAs arise from alterations in the variant glycosylation pattern, resulting in the tumor developing a novel epitope for MUC1 or in degradation processes resulting in events such as protein splicing, which may or may not be tumor specific.

f) Oncoviral proteins: these TTAs are viral proteins that may play a key role in carcinogenesis and, since they are foreign proteins (non-human proteins), are capable of triggering T cell responses. Examples of such proteins are human papilloma virus type 16 proteins, E6 and E7, which are expressed in cervical cancer.

T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by Major Histocompatibility Complex (MHC) molecules. The antigen recognized by tumor-specific T lymphocytes, i.e. the epitope thereof, can be molecules derived from all protein types, such as enzymes, receptors, transcription factors, etc., which are expressed in the cells of the respective tumor and whose expression is usually upregulated compared to homologous unaltered cells.

MHC molecules are of two classes: MHC class I and MHC class II. MHC class I molecules consist of an alpha heavy chain and beta-2-microglobulin, and MHC class II molecules consist of an alpha chain and a beta chain. Its three-dimensional configuration forms a binding pocket for non-covalent interaction with the peptide.

MHC class I molecules are found on most nucleated cells. They presented peptides generated by cleavage of mainly endogenous proteins, defective ribosomal products (DRIP) and larger peptides. However, peptides derived from endosomal structures or from exogenous sources are also often found on MHC class I molecules. This non-classical way of presenting class I molecules is known in the literature as cross-presentation (Brossartand Bevan, 1997; Rock et al, 1990). MHC class II molecules are found predominantly on professional Antigen Presenting Cells (APCs) and present predominantly, for example, peptides of exogenous or transmembrane proteins that are occupied by APCs during endocytosis and subsequently processed.

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. Thus, it is well recognized that TCR, peptide and MHC are presented in a 1: 1 stoichiometric ratio.

CD 4-positive helper T cells play an important role in inducing and maintaining an effective response of CD 8-positive cytotoxic T cells. The recognition of a Tumor Associated Antigen (TAA) derived CD4 positive T cell epitope may be important for the development of pharmaceutical products capable of eliciting anti-tumor immune responses (Gnjatic et al, 2003). At the tumor site, T helper cells maintain a cytokine environment friendly to cytotoxic T Cells (CTL) (Mortara et al, 2006) and attract effector cells such as CTL, Natural Killer (NK) cells, macrophages and granulocytes (Hwang et al, 2007).

In the absence of inflammation, MHC class II molecule expression is primarily restricted to immune system cells, especially professional Antigen Presenting Cells (APCs), e.g., monocytes, monocyte derived cells, macrophages, dendritic cells. Expression of MHC class II molecules is found in tumor cells of cancer patients (Dengjel et al, 2006).

The elongated (longer) peptides of the invention can serve as MHC class II active epitopes. MHC-II epitope-activated helper T cells play an important role in orchestrating CTL effector functions of anti-tumor immunity. Helper T cell epitopes that trigger TH1 cell responses support effector functions of CD8 positive killer T cells, including cytotoxic functions that act directly on tumor cells (tumor-associated peptide/MHC complexes are displayed on the surface of such tumor cells). Thus, the tumor-associated T helper cell epitopes, used alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compounds for stimulating anti-tumor immune responses.

Mammalian (e.g., mouse) models have shown that CD4 positive T cells can suppress angiogenesis sufficiently to suppress tumor expression by secreting interferon-gamma (IFN γ) even in the absence of CD8 positive T lymphocytes (Beatty and Paterson, 2001; Mumberg et al, 1999). There is no evidence for CD 4T cells as direct anti-tumor effectors (Braumuller et al, 2013; Tran et al, 2014).

Since constitutive expression of HLA class II molecules is usually restricted to immune cells, it was previously considered impossible to isolate class II peptides directly from primary tumors. However, Dengjel et al succeeded in directly recognizing multiple MHC class II epitopes in tumors (WO2007/028574, EP1760088B 1).

Since both CD 8-dependent and CD 4-dependent responses together and synergistically contribute to anti-tumor effects, the identification and characterization of tumor-associated antigens recognized by CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4 positive T helper cells (ligand: MHC class II molecule) is important for the development of tumor vaccines.

For a peptide to trigger (elicit) a cellular immune response by an MHC class I peptide, it must also bind to an MHC molecule. This process relies on alleles of MHC molecules and specific polymorphisms of peptide amino acid sequences. MHC class I-binding peptides are typically 8-12 amino acid residues in length and typically comprise two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele has a "binding motif" to determine which peptides are capable of specifically binding to the binding groove.

In an MHC class I dependent immune response, peptides not only bind to certain MHC class I molecules expressed by tumor cells, but they must then be recognized by a T cell-loaded specific T Cell Receptor (TCR).

Special conditions must be met for the proteins recognized by T lymphocytes as tumor-specific or related antigens and for therapy. The antigen should be expressed predominantly by tumor cells and not by normal healthy tissue, or in relatively small amounts. In a preferred embodiment, the peptide should be over-represented in tumor cells compared to normal healthy tissue. Preferably, the corresponding antigen is not only present in a tumor, but also in 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, which occurs due to their function in cell cycle control or apoptosis inhibition. In addition, downstream targets of these proteins that directly lead to a transformation event may be upregulated and thus may be indirectly associated with the tumor. These indirect tumor-associated antigens may also be targets for vaccination methods (Singh-Jasuja et al, 2004). It is essential that epitopes are present in the amino acid sequence of the antigen to ensure that such peptides from tumour associated antigens ("immunogenic peptides") can cause T cell responses in vitro or in vivo.

Basically, any peptide that binds to an MHC molecule may serve as a T cell epitope. The prerequisite for inducing an in vitro or in vivo T cell response is the presence of T cells with the corresponding TCR and the absence of immune tolerance to this particular epitope.

Thus, TAAs are the starting point for development based on T cell therapies, including but not limited to tumor vaccines. Methods for identifying and characterizing TAAs are generally based on the use of T cells in patients or healthy subjects, or on the generation of differential transcriptional profiles or differential expression patterns between tumor and normal tissue peptides. However, the identification of genes that are overexpressed or selectively expressed in tumor tissues or human tumor cell lines does not provide accurate information on the antigens transcribed using these genes in immunotherapy. This is because only a portion of the epitopes of these antigens are suitable for this application, since T cells with the corresponding TCR must be present and immune tolerance to this particular epitope must be absent or minimal. Therefore, in a very preferred embodiment of the invention, it is important to select only those peptides that are over-or selectively presented for situations where functional and/or proliferative T cells are found. Such functional T cells are defined as T cells that are clonally expanded upon stimulation with a specific antigen and are capable of performing effector functions ("effector T cells").

Where targeting is to a peptide-MHC by a particular TCR (e.g. a soluble TCR) and an antibody or other binding molecule (scaffold) according to the invention, immunogenicity of the potential peptide is of minor importance. In these cases, presentation is the determining factor.

Brief introduction to the invention

In a first aspect of the invention, the invention relates to a peptide, or a pharmaceutically acceptable salt thereof, comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 93, or an amino acid sequence of this sequence that is 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 said peptide, and wherein said peptide is not a potential full-length polypeptide.

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

The following table shows the peptides according to the invention, their respective SEQ ID NOs, and possible source (potential) genes for these peptides. All peptides in tables 1 and 2 bound to HLA-a 02. The peptides in table 2 were previously disclosed in a large list as high-throughput screening results with high error rates or calculated using algorithms, but had no previous association with cancer. The peptides in table 3 are other peptides that can be used in combination with other peptides of the present invention. The peptides in table 4 may also be used in the diagnosis and/or treatment of various other malignant diseases involving the overexpression or over-presentation of each potential polypeptide.

Table 1: peptides of the invention

Table 2: other peptides of the invention, previously not known to be associated with cancer

Sequence ID number	Sequence of	Gene ID	Formal 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

Table 3: peptides for use in, e.g., personalized cancer therapy

The invention also relates to the use of the peptides of the invention in the treatment of proliferative diseases, such as lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular cancer, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, prostate cancer and leukemia.

Particularly preferred are peptides of the invention (alone or in combination) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93. more preferably the peptides (alone or in combination) are selected from SEQ ID NOs: 1 to SEQ ID NO: 76 (see table 1) and for the immunotherapy of esophageal cancer, lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular cancer, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gall bladder cancer, bile duct cancer, prostate cancer and leukemia, preferably esophageal cancer.

Particularly preferred are peptides of the invention (alone or in combination) selected from 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 which are useful in the immunotherapy of oesophageal 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 oesophageal cancer. Further particularly preferred is a polypeptide according to SEQ ID NO: 9.

As shown in table 4A below, many of the peptides of the invention are also found in other tumors and therefore can be used for immunotherapy for other indications. Please refer to fig. 1 and example 1.

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

Table 4B: the peptides of the invention and their particular use in other proliferative diseases, in particular other cancerous diseases (table 4 revision). The table (e.g., table 4A) shows that for selected peptides of other tumor types, they were found to be over-presented (specifically presented) in tumor samples assayed at greater than 5%, or in tumor samples assayed at greater than 5% and the geometric mean tumor to normal tissue ratio was greater than 3. Over-presentation is defined as higher presentation in tumor samples compared to the highest presented normal samples. The normal tissues presented by the degree of history are: adipose tissue, adrenal gland, artery, bone marrow, brain, central nerve, colon, duodenum, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear leukocyte, pancreas, peripheral nerve, peritoneum, pituitary, pleura, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid, trachea, ureter, bladder, vein.

NSCLC ═ non-small cell lung cancer, SCLC ═ small cell lung cancer, RCC ═ kidney cancer, CRC ═ colon or rectal cancer, GC ═ stomach cancer, HCC ═ liver cancer, PC ═ pancreatic cancer, PrC ═ prostate cancer, BRCA ═ breast cancer, OC ═ ovarian cancer, NHL ═ non-hodgkin lymphoma, AML ═ acute myelogenous leukemia, CLL ═ chronic lymphocytic leukemia, HNSCC ═ head and neck squamous cell carcinoma

Thus, another aspect of the invention relates to the use of at least one peptide according to the 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 for the treatment of non-small cell lung cancer, in a preferred embodiment the above peptides are used in a combination therapy of non-small cell lung cancer.

Thus, another aspect of the invention relates to the use of at least one peptide according to the 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 the treatment of lymphoma, in a preferred embodiment in the combination therapy of lymphoma. Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 22, 55, 58, 62, 57, 61, 76, 79 and 80 in the treatment of small cell lung cancer, in a preferred embodiment the above in a combination therapy of small cell lung cancer.

Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 17, 56, 76, 82 and 54 in the treatment of renal cell carcinoma, in a preferred embodiment the above in a combination therapy of renal cell carcinoma.

Thus, another aspect of the invention relates to the use of at least one peptide according to the 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 above in a combination therapy of brain cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 31, 33, 35, 36, 37, 38, 39, 41, 42, 45, 46, 48, 50, 52, 53, 54, 55, 63, 68, 70, 75 and 71 for the treatment of gastric cancer, in a preferred embodiment the above in combination therapy of gastric cancer.

Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 26, 34, 40, 74, 80, 88 and 92 in the treatment of colorectal cancer, in a preferred embodiment the above in a combination therapy of colorectal cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 15, 17, 22, 35, 49, 62, 67, 70, 72, 74, 75, 80, 82 and 92 for the treatment of hepatocellular carcinoma, in a preferred embodiment the above in a combination therapy of hepatocellular carcinoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the invention according to any one of SEQ ID nos. 37, 40, 68, 69, 71, 78, 79, 87 and 91 for the treatment of pancreatic cancer, in a preferred embodiment the above in a combination therapy of pancreatic cancer.

Thus, another aspect of the invention relates to the use of at least one peptide of the invention according to any one of SEQ id nos 79 and 91 in the treatment of prostate cancer, in a preferred embodiment the above mentioned in combination therapy of prostate cancer.

Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos. 4, 28, 58, 61, 72, 78, 79, 80, 82, 84, 86, 88, 92 and 85 for the treatment of leukemia, in a preferred embodiment the above in a combination therapy of leukemia.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the 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, in a preferred embodiment the above in a combination therapy of breast cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the 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 for the treatment of melanoma, in a preferred embodiment the above in a combination therapy of melanoma.

Thus, another aspect of the invention relates to the use of at least one peptide according to the invention according to any one of SEQ id nos 8, 62, 70, 78, 79, 80 and 87 in the treatment of ovarian cancer, in a preferred embodiment the above in a combination therapy of ovarian cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the 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, in a preferred embodiment the above in combination therapy of bladder cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the 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, in a preferred embodiment the above in a combination therapy of uterine cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the 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 and bile duct cancer, in a preferred embodiment the above in a combination treatment of gallbladder and bile duct cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the 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, in a preferred embodiment the above in combination therapy of HNSCC.

Thus, another aspect of the invention relates to the use of a peptide of the invention in the treatment, preferably combination treatment, of a proliferative disease selected from the group consisting of esophageal cancer, lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular cancer, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancer and leukemia.

The invention also relates to peptides of the invention having the ability to bind to Major Histocompatibility Complex (MHC) I or to molecules in elongated form, e.g.MHC-class II molecules of varying length.

The invention further relates to a peptide of the invention, wherein the peptide(s) (each peptide) consist(s) of or consist essentially of SEQ ID NO: 1 to SEQ ID NO: 93, or a pharmaceutically acceptable salt thereof.

The invention further relates to a peptide according to the invention, wherein said peptide is modified and/or comprises non-peptide bonds.

The invention further relates to a peptide of the 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.

The invention further relates to a nucleic acid encoding a peptide according to the invention. The invention further relates to a nucleic acid according to the invention, being DNA, cDNA, PNA, RNA, and possibly a combination thereof.

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

The invention further relates to the use of a peptide of the invention, a nucleic acid of the invention or an expression vector of the invention for the treatment of diseases and for the preparation of a medicament, in particular for the treatment of cancer.

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

The invention further relates to T Cell Receptors (TCRs) of the 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.

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

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

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

The invention further relates to a method of the invention wherein an antigen is carried on an MHC class I or II molecule expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by contacting the antigen with the antigen-presenting cell in a sufficient amount.

The invention further relates to a method of the 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 SEQ ID No.1 to SEQ ID No.76, or a variant thereof.

The invention further relates to activated T cells made by the methods of the invention, wherein the T cells selectively recognize a cell that expresses a polypeptide comprising an amino acid sequence of the invention.

The invention further relates to a method of killing a target cell in a patient, wherein the target cell of the patient abnormally expresses a polypeptide comprising any of the amino acid sequences of the invention, the method comprising administering to the patient an effective amount of a T cell made by the method of the invention.

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

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

The invention also relates to the use of the invention, wherein the cancer cell is an esophageal cancer, a lung cancer, a bladder cancer, an ovarian cancer, a melanoma, a uterine cancer, a hepatocellular cancer, a renal cell cancer, a brain cancer, a colorectal cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a gall bladder cancer, a bile duct cancer, a prostate cancer and a leukemia, preferably an esophageal cancer cell.

The invention further relates to a biomarker, herein referred to as "target", based on a peptide of the invention, which can be used for the diagnosis of cancer, preferably esophageal cancer. The marker may be an over-presentation of the peptide itself, or an over-expression of the corresponding gene. The marker may also be used to predict the likelihood of success of a treatment, preferably an immunotherapy, most preferably an 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 to MHC.

Optionally, the antibody has a further effector function, such as an immunostimulatory domain or a toxin.

The invention also relates to the use of these novel targets in the treatment of cancer.

ABHD11 antisense RNA 1(ABHD11-AS1) was described AS a long non-coding RNA, which was shown to be up-regulated in gastric cancer, associated with differentiation and Lauren histological classification. Thus, ABHD11-AS1 may be one potential biomarker for gastric cancer diagnosis (Lin et al, 2014). ABHD11 activity was shown to be associated with the development of metastasis in lung adenocarcinoma distant and therefore could be a potential new biomarker (Wiedl et al, 2011).

ADAMTS2 was shown to be dysregulated in patients with T/myeloid mixed acute leukemia (Tota et al, 2014). ADAMTS2 was described to be associated with the JNK signaling pathway following upregulation by IL-6 in osteosarcoma cells (Alper and Kockar, 2014). ADAMTS2 may be a potential diagnostic marker for follicular thyroid cancer (Fontaine et al, 2009). ADAMTS2 was described as a potential marker for metastasis of squamous cell carcinoma of the tongue (Carinci et al, 2005). ADAMTS2 was shown to be upregulated in renal cell carcinoma, associated with shorter patient survival (Roemer et al, 2004). ADAMTS2 was shown to be regulated by cell proliferation-associated transforming growth factor-. beta.1 (Wang et al, 2003).

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

ANO1 encodes anoctamin 1, a calcium-activated chloride channel associated with small intestinal sarcomas and oral cancers (RefSeq, 2002). ANO1 is amplified in Esophageal Squamous Cell Carcinoma (ESCC), gastrointestinal stromal tumor (GIST), Head and Neck Squamous Cell Carcinoma (HNSCC), pancreatic cancer, and breast cancer (Qu et al, 2014).

ARHGDIA has been shown to be down-regulated in hepatocellular carcinoma as well as in breast cancer development (Liang et al, 2014; Bozzaet al, 2015). ARHGDIA was shown to be associated with tumor invasion, metastasis, overall survival and time to recurrence of hepatocellular carcinoma. Therefore, ARHGDIA may provide a potential therapeutic target for hepatocellular carcinoma (Liang et al, 2014). ARHGDIA was shown to be down-regulated in lung cancer cell line a549 following periplocin treatment. Therefore, periplocin inhibits the growth of lung cancer cells and may be associated with ARHGDIA (Lu et al, 2014). ARHGDIA knockdown was shown to be associated with increased apoptosis in normal and cultured tumor cells of pulmonary origin. ARHGDIA is therefore described as a negative regulator of apoptosis, which may represent a potential therapeutic target (Gordon et al, 2011). ARHGDIA is described as being associated with ovarian clear cells and high grade serous carcinoma stage (Canet et al, 2011). ARHGDIA is described as an apoptosis pathway-associated gene that has been shown to be deregulated in fibrosarcoma HT1080 cells following TRAIL-mediated apoptosis (Daigeler et al, 2008). ARHGDIA was shown to be upregulated in the oxaliplatin-resistant colon cancer cell line THC8307/L-OHP and was 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, which was shown to be regulated by the putative tumor suppressor ACVR2 (a member of the cancer-associated TGFBR2 family), occurred in the MSI-H colon cancer cell line transfected by wild-type ACVR2 carrying the ACVR2 frameshift mutation (Deacu et al, 2004). ARHGDIA is described as a key regulator of Rho gtpase. ARHGDIA depletion was shown to induce constitutive activation of Rho gtpase and the COX-2 pathway that is associated with breast cancer progression in breast cancer xenograft animal models (Bozza et al, 2015). ARHGDIA signaling has been shown to be deregulated in colorectal cancer (Sethi et al, 2015). ARHGDIA was shown to target MEK1/2-ERK after SUMO, which correlates with c-Jun/AP-1 inhibition, cyclin d1 transcription and cell cycle progression. Therefore, ARHGDIA is associated with inhibition of cancer cell growth (Cao et al, 2014). ARHGDIA, described as a novel inhibitor of prostate cancer, may play a key role in the regulation of androgen receptor signaling and prostate cancer growth and progression (Zhu et al,2013 b).

ATIC is described as a potential gene fusion partner of cancer-associated anaplastic lymphoma kinase in anaplastic large cell lymphomas (Cheuk and Chan, 2001). ATIC was demonstrated to appear as a chimeric fusion gene with ALK in inflammatory myofibroblastic tumors of the bladder (Debiec-Rychter et al, 2003). Inhibition of ATIC aminoimidazole carboxamide transformylase (AICAR) activity in breast cancer cell line models has been shown to result in dose-dependent decreases in cell number and cell division rate. Therefore, ATIC may be a potential target for cancer therapy (Spurr et al, 2012).

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

COL6a1 was upregulated in the response stroma of castration against prostate cancer, promoting tumor growth (Zhu et al, 2015). Co16A1 was overexpressed in CD 166-pancreatic cancer cells, which showed greater invasive and migratory activity than CD166+ cancer cells (Fujiwara et al, 2014). COL6a1 was highly expressed in bone metastasis (Blanco et al, 2012). COL6A1 was found to be upregulated in cervical and ovarian cancers (ZHao et al, 2011; Parker et al, 2009). COL6a1 was differentially expressed in astrocytomas and glioblastomas (Fujita et al, 2008).

COL6A2 is associated with cervical cancer, poor global survival of high-grade serous ovarian cancer, B-precursor acute lymphocytic leukemia, hepatocellular carcinoma, primary and metastatic brain tumors, lung squamous cell carcinoma, head and neck squamous cell carcinoma, and is described as a potential DNA methylation of cervical cancer (Cheon et al, 2014; Chen et al,2014 d; Vachani et al, 2007; Liu et al, 2010; Seong et al, 2012; Hogan et al, 2011).

CYFIP1 was shown to be down-regulated during epithelial tumor invasion (silvera et al, 2009). CYFIP1 down-regulation is associated with poor prognosis of epithelial tumors (silvera et al, 2009).

CYP2S1 has been shown to regulate colorectal cancer growth in the cell line HCT116 by correlating with PGE 2-mediated activation of the β -catenin signaling pathway (Yang et al,2015 b). CYP2S1 is described as being upregulated in a variety of epithelium-derived cancers and in hypoxic tumor cells (Nishida et al, 2010; Madanayake et al, 2013). Depletion of CYP2S1 in bronchial epithelial cell lines has been shown to lead to alterations in regulation in major pathways involved in cell proliferation and migration, such as the mTOR signaling pathway (Madanayake et al, 2013). CYP2S1 depletion was demonstrated to correlate with drug sensitivity in colorectal and breast cancers (Tan et al, 2011). CYP2S1 was shown to be associated with breast cancer survival and with a poor prognosis of colorectal cancer (Murray et al, 2010; Kumarakularsingham et al, 2005). CYP2S1 was demonstrated to metabolize BaP-7, 8-diol into 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). In contrast to primary ovarian cancer, CYP2S1 has been shown to be significantly upregulated in ovarian cancer metastasis (Downie et al, 2005).

Expression of DES in the colorectal mesenchyme is associated with advanced disease (Arentz et al, 2011). DES was demonstrated to be upregulated in colorectal cancer (Ma et al, 2009). DES has been shown to be associated with a decrease in the severity and differentiation and survival rate of colorectal cancer (Ma et al, 2009). DES is described as a potential oncofetal serum tumor marker for colorectal cancer (Ma et al, 2009). DES has been shown to be a specific marker of rhabdomyosarcoma (Altmannberger et al, 1985). DES is described as one of three members of a group of proteins that may contribute to staging bladder cancer by using immunohistochemistry (Council and hamed, 2009).

DIS3 has been shown to be frequently mutated in multiple myeloma and to be repeatedly mutated in acute myeloid leukemia (dimeget al, 2012; Lohr et al, 2014). DIS3 mutation in multiple myeloma has been shown to be associated with a short overall survival. The secondary subclonal mutations proved to be associated with a poorer response to treatment compared to patients with the primary subclonal DIS3 mutation (Weissbach et al, 2015). DIS3 was shown to be upregulated in colorectal cancer by 13q increase. DIS3 silence was shown to affect important tumorigenic features such as viability, migration and invasiveness. DIS3 may therefore be a novel candidate gene contributing to colorectal cancer progression (de Groen et al, 2014). DIS3 is described as belonging to a gene panel that can be used in combination with plasma protein-based biomarkers for early diagnosis of epithelial ovarian cancer (Pils et al, 2013). DIS3 may be a potential candidate gene for susceptibility to breast cancer, as numerous polymorphisms were detected in breast cancer mutation screening (Rozenblumet al, 2002).

EEF1A1 was demonstrated to be upregulated in a variety of cancer entities, including colorectal, ovarian, gastric, prostate, glioblastoma and squamous cell carcinoma, 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; Re1man et al, 2012). Mechanistically, EEF1A1 inhibits apoptosis by interacting with p53 and p73, promotes proliferation by transcriptional repression of the cell cycle inhibitor p21 gene and is involved in the regulation of epithelial-to-mesenchymal transition (Blanch et al, 2013; Choi et al, 2009; Husseyet al, 2011).

EEF1A2 is described as being up-regulated in breast, ovarian, lung, pancreatic, gastric, prostate, and TFE3 translocating renal cell carcinoma (Pfleger et al, 2013; Sun et al, 2014; Yang et al,2015 c; Zang et al, 2015; Abbas et al, 2015). EEF1A2 was shown to be associated with poor prognosis in ovarian, gastric, pancreatic ductal, and lung adenocarcinomas (Duanmin et al, 2013; Yang et al,2015 c; Li et al, 2006; Lee and Surh, 2009). EEF1a2 is described as being associated with tumorigenesis in that it stimulates phospholipid signaling and activates Akt-dependent cell migration and actin remodeling, which ultimately favors tumorigenesis (Abbas et al, 2015). EEF1A2 is described to inhibit p53 function in hepatocellular carcinoma by PI 3K/AKT/mTOR-dependent stabilization of MDM 4. Strong activation of the EEF1a2/PI3K/AKT/mTOR/MDM4 signaling pathway has been shown to be associated with short survival of hepatocellular carcinomas and, therefore, may be a therapeutic target for some patients (Longerich, 2014). EEF1a2 was shown to be associated with TNM staging, aggressiveness and survival in pancreatic cancer patients. Thus, EEF1a2 may be a potential target for pancreatic cancer treatment (Zang et al, 2015). EEF1a2 was shown to be associated with prostate cancer development by promoting cell proliferation and apoptosis inhibition, and therefore, could be a potential therapeutic target for prostate cancer (Sun et al, 2014). EEF1a2 was shown to interact with tumor suppressor protein p16, which resulted in the down-regulation of EEF1a2 and was associated with cancer cell growth inhibition (Lee et al, 2013). EEF1A2 was shown to be associated with lymph node metastasis and neuro-infiltration of pancreatic ductal adenocarcinoma (Duansin et al, 2013). EEF1a2 was shown to be associated with breast cancer survival (Kulkarni et al, 2007). EEF1A2 is described as a putative oncogene and tumor suppressor gene in lung adenocarcinoma cell lines and ovarian cancer (Lee, 2003; Zhu et al,2007 a).

EIF2S1 is described as an activator of tumor progression and therapy resistance after phosphorylation. However, EIF2S1 has also been described to be involved in the inhibitory effect of tumorigenesis (Zheng et al, 2014). EIF2S1 is described as a downstream effector of mTOR and reduces survival of cancer cells upon hyperphosphorylation, and thus can be a target for drug development (Tuval-Kochen et al, 2013).

EIF4G2 is described as one of a group of core genes that have been shown to be involved in the elimination of childhood glioma CD133+ cell neoplasia (Baxter et al, 2014). EIF4G2 showed a correlation with the inhibition of the development of diffuse large B-cell lymphoma after down-regulation by miR-520c-3p (Mazan-Mamczarz et al, 2014). EIF4G2 has been shown to promote protein synthesis and cell proliferation by regulating cyclin synthesis (Lee and McCormick, 2006). EIF4G2 was shown to be down-regulated in bladder tumors, and down-regulation was associated with invasive tumors (Buim et al, 2005). EIF4G2 is described as involved in MycN/IFN γ -induced apoptosis and in the viability and death of neuroblastoma cells (Wittke et al, 2001).

F7 in the tissue factor complex was abnormally expressed on the surface of cancer cells, including ovarian cancer, as described. The complex is further described as being associated with the induction of a malignant phenotype of ovarian cancer (Koizume and Miyagi, 2015). The F7-tissue factor complex pathway is described as a mediator of breast cancer progression, which stimulates the expression of a number of malignant phenotypes in breast cancer cells. Thus, the F7-tissue factor pathway is an attractive potential target for breast cancer treatment (Koizume and miyagi, 2014). F7 was shown to be modulated by androgen receptors in breast cancer (Naderi, 2015). F7 was shown to modulate autophagy by mTOR signaling in liver cancer cell lines (Chen et al,2014 a). F7 was shown to be associated with tumor infiltration and metastasis in colorectal and ovarian cancers (Tang et al, 2010; Koizume et al, 2006). F7 was shown to be ectopically upregulated in colorectal cancer (Tang et al, 2009). F7 in tissue factor complex was shown to be associated with chemotherapy resistance in neuroblastoma (Fang et al,2008 a).

FAM115C was upregulated during hypoxia in non-small cell lung cancer (Leithner et al, 2014).

FAM83A is described as a potential biomarker for lung cancer (Li et al, 2005). FAM83A is described as a marker gene that can be used in a plate with NPY1R and KRT19 to detect circulating tumor cells in breast cancer patients (Liu et al,2014 d). Breast cancer cells FAM83A ablation was shown to result in reduced MAPK signaling, significantly inhibiting growth in vitro and tumorigenicity in vivo (cipiano et al, 2014). In addition, the FAM83 protein family is described as a novel oncogene family that modulates MAPK signaling in cancer and is therefore useful in developing cancer treatment methods aimed at inhibiting MAPK signaling (cipiano et al, 2014). FAM83A was shown to be associated with trastuzumab resistance in HER2 positive breast cancer cell lines (Boyer et al, 2013). Generally, FAM83A was demonstrated to be a candidate gene associated with resistance to EGFR tyrosine kinase inhibitors for breast cancer (Lee et al, 2012). FAM83A was described as being associated with poor prognosis of breast cancer (Lee et al, 2012). FAM83A was shown to be upregulated in non-small cell lung cancer (Qu et al, 2010). FAM83A was shown to be a potential specific and sensitive marker for detecting circulating tumor cells in peripheral blood of non-small cell lung cancer patients (Quet al, 2010).

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

FAT1 was described as a significant mutation in head and neck squamous cell carcinoma, with frequent mutations in cervical adenocarcinoma, bladder carcinoma, early T-cell precursor acute lymphocytic leukemia, fludarabine refractory chronic lymphocytic leukemia, glioblastoma and colorectal carcinoma, and 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 was described as being depressed in oral cancer and preferentially down-regulated in invasive breast cancer (Katoh, 2012). FAT1 was described as being upregulated in leukemia, which was associated with a poor prognosis in pre-B acute lymphoblastic leukemia (Katoh, 2012). FAT1 was shown to be upregulated in pancreatic and hepatocellular carcinomas (Valletta et al, 2014; Wojtalewicz et al, 2014). FAT1 was described to inhibit tumor growth through activation of hippopotamus signaling and to promote tumor metastasis through actin polymerization induction (Katoh, 2012). FAT1 proved to be a candidate cancer driver gene for cutaneous squamous cell carcinoma (Pickering et al, 2014). FAT1 is described as a tumor suppressor associated with Wnt signaling and tumorigenesis (Morris et al, 2013).

Filamin a plays a dual role in cancer according to its subcellular localization: in the cytoplasm, filamin a plays a role in different growth signaling pathways in addition to being involved in cell migration and adhesion pathways. Therefore, its overexpression has a tumor-promoting effect. In contrast to full-length filamin a, the C-terminal fragment (released after proteolysis) localizes to the nucleus where it interacts with transcription factors, inhibiting tumor growth and metastasis (Savoy and Ghosh, 2013).

Tumor-specific C-terminal truncations of GBP5 were described as probably responsible for GBP5 dysregulation in lymphoma cells (Wehnerand Herrmann, 2010). GBP5 was described because the restricted expression pattern of three GBP5 splice variants in cutaneous T-cell lymphoma tumor tissues and cell lines, as well as melanoma cell lines, can address possible cancer-related functions (bellenberg et al, 2004).

GJB5 was shown to be down-regulated in non-small cell lung cancer cell lines, laryngeal carcinoma and head and neck squamous cell carcinoma (Zhang et Al, 2012; Broghammer et Al, 2004; Al Moustafa et Al, 2002). GJB5 was 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 was shown to be upregulated in stemless serrated adenomas/polyps, precancerous lesions (probably 20-30% of colon cancers) (Delkeret al, 2014). GJB5 expression was described as significantly altered during skin tumor promotion and development in mouse models (Slagaet al, 1996).

GLS is described as being indirectly regulated by MYC genes to increase glutamine metabolism in cancer cells (Dang et al, 2009). GLS was shown to be inhibited by the tumor suppressor NDRG2 in colorectal cancer (Xu et al, 2015). GLS is described as being up-regulated in pancreatic ductal adenocarcinoma, triple negative breast cancer, hepatocellular carcinoma, oral squamous cell carcinoma, colorectal cancer, and glioblastomas-derived tumors (van Geldermalsen et al, 2015; Szeliga et al, 2014; Huang et al,2014 a; Cetindis et al, 2015; Yu et al,2015 a; Chakrabarti et al, 2015). GLS has been shown to be associated with survival of hepatocellular carcinoma and has also been described as a sensitive and specific biomarker for pathological diagnosis and prognosis of hepatocellular carcinoma (Yu et al,2015 a). Loss of GLS copy was shown to delay tumor progression in hepatocellular carcinoma immune-related MYC-mediated mouse models (Xiang et al, 2015). GLS has been shown to be required for tumorigenesis and tumor-specific inhibition, and is described as a potential approach to cancer treatment (Xiang et al, 2015). GLS has been shown to be associated with paclitaxel resistance in breast cancer (Fu et al,2015 a). GLS overexpression has been shown to be highly correlated with tumor stage and progression in prostate cancer patients (Pan et al, 2015). GLS expression is associated with deeper tumor infiltration and pathological types of tubular adenocarcinoma in colon cancer tumorigenesis. GLS can be targeted for colorectal cancer treatment (Huang et al,2014 a). Silencing of the GLS isoenzyme KGA was shown to lead to decreased survival of the glioma cell lines SFxL and LN229 (Martin-Rufian et al, 2014). Silencing of GLS in glioma cell lines SFxL and LN229 has also been shown to lead to induction of apoptosis by causing lower levels of c-myc and bcl-2 expression and higher pro-apoptotic expression (Martin-Rufian et al, 2014). Activation of ErbB2 was demonstrated to upregulate GLS expression through the NF- κ B pathway, which promotes breast cancer cell proliferation (Qie et al, 2014). Knockdown or inhibition of GLS in breast cancer cells and high levels of GLS have been shown to result in significant reduction in proliferation (qieat al, 2014).

GNA15 was shown to be upregulated in primary and metastatic small intestine neuroendocrine tumors (Zanini et al, 2015). Increased GNA15 expression was shown to be associated with poor survival, suggesting that GNA15 may have a pathobiological role in small intestine neuroendocrine neoplasia and thus may be a potential therapeutic target (Zanini et al, 2015). GNA15 was described as being down-regulated in many non-small cell lung cancer cell lines (Avasarala et al, 2013). High GNA15 expression in normal karyotype acute myelogenous leukemia was shown to be associated with significantly poorer overall survival (de Jonge et al, 2011). GNA15 was shown to be a key downstream effector of non-canonical Wnt signaling as well as a regulator of non-small cell lung cancer cell proliferation and anchorage-independent cell growth. Thus, GNA15 is a potential therapeutic target for non-small cell lung cancer (Avasarala et al, 2013). GNA15 was shown to be associated with tumor signaling in pancreatic cancer (Giovinazzo et al, 2013). GNA15 was shown to stimulate STAT3 via c-Src/JAK and ERK dependent mechanisms following constitutive activation in human embryonic kidney 293 cells (Lo et al, 2003).

Insufficient expression of HAS3 was shown to be associated with advanced tumor stages of upper and bladder urothelial cancer, lymph node metastasis, vascular invasion and poor disease-specific and metastasis-free survival (Chang et al, 2015). Thus, HAS3 is useful as a potential prognostic biomarker and a novel therapeutic target for urothelial cancer (Chang et al, 2015). HAS3 was shown to favor the growth of pancreatic cancer by hyaluronic acid accumulation (Kultti et al, 2014). Inhibition of HAS3 was shown to reduce the viability of colorectal adenocarcinoma cell line SW620 (Heffler et al, 2013). HAS3 inhibition was shown to be associated with differential expression of several genes involved in SW620 colorectal tumor cell survival regulation (Heffler et al, 2013). HAS3 HAS been shown to be associated with the mediation of colon cancer growth by inhibiting apoptosis (Teng et al, 2011). HAS3 was 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 was described as an independent prognostic factor for breast cancer, as HAS3 in the stromal cells of breast cancer patients was shown to be associated with a high recurrence rate and shorter overall survival (auvine et al, 2014). HAS3 was shown to be associated with serous ovarian cancer, renal clear cell carcinoma, endometrial cancer, and osteosarcoma (Nykopp et al, 2010; Weiss et al, 2012; Cai et al, 2011; Tofuku et al, 2006).

HIF1A was shown to be associated with tumor necrosis in aggressive endometrial cancer. HIF1A is further described as a potential target for the treatment of this disease (bredhot et al, 2015). HIF1A was shown to be associated with hepatogenesis, sarcoma metastasis and nasopharyngeal carcinoma (Chen et al,2014 c; El-Naggar et al, 2015; Li et al,2015 b). The single nucleotide polymorphism of HIF1A has been shown to be significantly correlated with clinical outcomes in patients with invasive liver cancer after surgery (Guo et al, 2015). Aberrant HIF1A activity was shown to drive tumor progression in malignant schwannoma cell lines along with aberrant STAT3 activity. 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 resistance in multiple myeloma (Maiso et al, 2015). HIF1A was shown to be expressed asymmetrically in three different cell lines, corresponding to different stages of multiple myeloma pathogenesis, suggesting that HIF1A is involved in tumorigenesis and metastasis of multiple myeloma (Zhao et al,2014 b). Long non-coding HIF1A antisense RNA-2 has been described as upregulated in non-papillary clear cell renal and gastric cancers, and is associated with tumor cell proliferation and poor prognosis in gastric cancers (Chen et al,2015 b). Deregulation of the PI3K/AKT/mTOR pathway by HIF1A has been described as critical to the quiescence, maintenance and survival of prostate cancer stem cells (Marhold et al, 2015). HIF1A is described as a gene in the 4-gene classification that is predictive of stage I lung adenocarcinoma (Okayama et al, 2014). Polymorphisms in HIF1A were shown to be associated with increased susceptibility to digestive tract cancers in the asian population (Xu et al, 2014). HIF1A is described as a prognostic marker for sporadic male breast cancer (Deb et al, 2014).

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

Studies have shown that IGHG1 is overexpressed in human pancreatic cancer tissue compared to adjacent non-cancerous tissue. In contrast, IGHG1 protein was down-regulated in invasive ductal carcinoma tissues (Kabbage et al, 2008; Li et al,2011 b). siRNA targeting silencing of IGHG1 inhibited cell viability and promoted apoptosis (Pan et al, 2013).

Researchers found that IGHG3 was expressed in sauter women affected by breast cancer. Also, increased copy number and elevated levels of IGHG3 were detected in prostate cancer african males. Another report showed that IGHG3 expression was found on squamous non-small cell lung cancer, malignant mesothelioma, and tumor cells, which are occasionally found in MALT lymphomas 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,2013 c; Sugimoto et al, 2014).

IGHG4 encodes immunoglobulin heavy chain constant gamma 4(G4m marker), located on chromosome 14q32.33 (RefSeq, 2002). Recent work detected that rearrangement of IGHG4 was involved in primary testicular diffuse large B-cell lymphoma (Twa et al, 2015).

The IGHM encodes immunoglobulin heavy chain constant μ (RefSeq, 2002). Studies have found that IGHM is down-regulated in chinese patients affected by rhabdomyosarcoma. Others detected expression of IGHM in diffuse large B-cell lymphomas. Another group found that in diffuse large B-cell lymphomas, the IGHM gene was conserved only on the potent IGH allele in most IgM + tumors. Furthermore, epithelial hamartoma samples showed any reactivity to transcription factors or transcription factor EBs bound to IGHM enhancer factor 3 (Kato et al, 2009; Blenk et al, 2007; rumino et al, 2011; Liu et al,2014 b).

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

Decreased INA expression is associated with metastasis, recurrence and shorter overall survival of pancreatic neuroendocrine tumors. Thus, INA may be a useful prognostic biomarker for the invasiveness of pancreatic neuroendocrine tumors (Liu et al,2014 a). INA is described as being upregulated in oligodendrocyte phenotype gliomas, and INA expression was shown to be associated with progression-free survival of oligodendrogliomas and glioblastomas (Suh et al, 2013). INA is described as a marker for neuroblastoma and can be used for differential diagnosis of small round cell tumors in childhood (Willoughby et al, 2008).

ITGA6 expression is up-regulated in different cancer entities (breast, prostate, colon, gastric) 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 effect of the Abeta4 variant of ITGA6 appears to be mediated through the Wnt/β -catenin pathway (grouix et al, 2014). The Abeta4 variant of ITGA6 results in VEGF-dependent activation of the PI3K/Akt/mTOR pathway. This pathway has an important role in the survival of metastatic cancer cells (Chung et al, 2002).

KRT14 is highly expressed in various squamous cell carcinomas (e.g., esophageal, lung, laryngeal, cervical), as well as adenoma odontogenic tumors. However, it is absent in bladder small cell carcinomas, and weaker in lung adenocarcinomas, gastric adenocarcinomas, colorectal adenocarcinomas, hepatocellular carcinomas, pancreatic ductal adenocarcinomas, breast invasive ductal carcinomas, thyroid papillary carcinomas, and endometrioid 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).

KRT16 overexpression was found in basal-like breast cancer cell lines as well as in situ carcinomas. Others did not find a significant difference in the immunohistochemical expression of KRT16 between non-recurrent ameloblasts and recurrent ameloblasts (Joosse et al, 2012; Ida-Yonomechol et al, 2012; Safadi et al, 2016). Furthermore, silicon analysis showed a correlation between KRT16 expression and short recurrence-free survival in metastatic breast cancer (Joosse et al, 2012).

KRT5 was shown to be upregulated in breast cancer in young women (Johnson et al, 2015). KRT5 was shown to correlate with poor disease-free survival of breast cancer in young women and clinical outcomes in hormone receptor positive breast cancer pre-menopausal patients (Johnson et al, 2015; Sato et al, 2014). KRT5 was shown to be regulated by the tumor suppressor BRCA1 in breast cancer cell lines HCC1937 and T47D (Gorski et al, 2010). KRT5 was shown to be dysregulated in malignant pleural mesothelioma (Melaiu et al, 2015). KRT5 is described as a diagnostic mesothelial marker for malignant mesothelioma (Arif and husain, 2015). KRT5 was shown to be associated with endometrial cancer progression (Zhao et al, 2013). KRT5 was shown to be down-regulated in the invasive tumor area of patients with warty carcinoma (Schumann et al, 2012). KRT5 was shown to be part of four protein series, which are differentially expressed in colorectal cancer biopsies compared to normal tissue samples (Yang et al, 2012). KRT5 and the other three proteins of the four protein series are described as novel markers and potential targets for colorectal cancer treatment (Yang et al, 2012). KRT5 was described as being associated with basal cell carcinoma (Depianto et al, 2010). KRT5 was described as a candidate gene for the identification of urothelial cancer stem cells (Hatina and Schulz, 2012).

Activation of the kynurenine pathway involved in KYNU was demonstrated to be significantly higher in glioblastoma, indicating that the kynurenine pathway is involved in the pathophysiology of glioma (Adams et al, 2014). KYNU is described as a cancer syngen whose expression is altered upon aromatic receptor knockdown in the MDA-MB-231 breast cancer cell line (Goode et al, 2014). KNYU was shown to be differentially expressed in highly and non-invasive osteosarcoma cell lines, suggesting that it may play an important role in the process of osteosarcoma tumorigenesis. Therefore, KYNU may also represent a candidate gene for future therapeutic targets (Lauvrak et al, 2013). KYNU was demonstrated to be associated with tumorigenic re-expression of mixed cells of non-tumorigenic HeLa and human skin fibroblasts. Therefore, KYNU may provide a relevant candidate for regulation of tumorigenic expression (Tsujimoto et al, 1999).

Combined transcriptional analysis of LAMB3 with two other genes proved to be beneficial for diagnosis of papillary thyroid carcinoma and prediction of the risk of lymph node metastasis (Barros-Filho et al, 2015). LAMB3 has been shown to be associated with cancer entities in oral squamous cell carcinoma, prostate cancer, gastric cancer, colorectal cancer, Ewing family tumors, lung cancer, breast cancer and ovarian cancer (Volpi et al, 2011; Ii et al, 2011; Reis et al, 2013; Stull et al, 2005; Irifune et al, 2005; Tanis et al, 2014). LAMB3 was shown to be upregulated in cervical squamous cell carcinoma, lung carcinoma, gastric carcinoma, nasopharyngeal carcinoma, and esophageal squamous cell carcinoma (Kwon et al, 2011; Wang et al,2013 a; Yamamoto et al, 2013; Kitaet al, 2009; Fang et al,2008 b). LAMB3 is described as a protein that is 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). Knockdown of LAMB3 was 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 the development and metastasis of lung cancer (Wang et al,2013 a). LAMB3 was shown to be regulated by the tumor suppressor miR-218 in squamous cell carcinoma of the head and neck (Kinoshita et al, 2012). Silence of LAMB3 in squamous cell carcinoma of the head and neck has been shown to lead to inhibition of cell migration and invasion (Kinoshita et al, 2012). LAMB3 expression correlated with depth of and venous infiltration of esophageal squamous cell carcinoma (Kita et al, 2009). Methylation of LAMB3 was shown to be associated with several parameters that are associated with poor prognosis of bladder cancer (sathanaarayana et al, 2004).

Inhibition of LAP3 was shown to result in inhibition of invasion in the ovarian cancer cell line ES-2 by down-regulation of fascin and MMP-2/9. Thus, LAP3 can serve as a potential anti-metastatic therapeutic target (Wang et al,2015 d). High expression of LAMB3 was shown to be associated with poor staging of malignant tumors and prognosis in glioma patients (He et al, 2015). LAP3 was shown to promote glioma progression by modulating cell growth, migration and invasion, and could therefore be a new predictor (He et al, 2015). Frame shift mutations in genes involved in amino acid metabolism, including LAP3, were detected in high microsatellite instability gastric and colorectal cancers (Oh et al, 2014). LAP3 was shown to be up-regulated in hepatocellular carcinoma, esophageal squamous cell carcinoma, and prostate cancer (Zhang et al, 2014; Tian et al, 2014; Lexander et al, 2005). LAP3 was shown to promote hepatoma cell proliferation by regulating the cell cycle and the G1/S phase checkpoint of late cell migration (Tian et al, 2014). Expression of LAP3 was further shown to be associated with prognosis and malignant development of hepatocellular carcinoma (Tianetal, 2014). Silencing of LAP3 in esophageal squamous cell carcinoma cell line ECA109 was demonstrated to reduce cell proliferation and colony formation, whereas LAP3 knockdown resulted in cell cycle arrest (Zhang et al, 2014). Overexpression of LAP3 in the esophageal squamous cell carcinoma cell line TE1 was shown to be beneficial for cell proliferation and invasion (Zhang et al, 2014). Thus, LAP3 was shown to play a role in the malignant development of esophageal squamous cell carcinoma (Zhang et al, 2014).

Researchers have reported that M6PR is expressed in colon cancer cell lines as well as chorionic cells (Braulke et al, 1992; O' Gorman et al, 2002). In breast cancer, low levels of M6PR expression correlate with a poorer prognosis for the patient (Esseghir et al, 2006). Furthermore, overexpression of M6PR resulted in a decreased cell growth rate in vitro and decreased tumor growth in nude mice (O' Gorman et al, 2002).

MAPK6 was shown to play a role in regulating the cell morphology and migration of the breast cancer cell line MDA-MB-231 (Al-Mahdi et Al, 2015). MAPK6 is described as part of a cancer-associated MAPK signaling pathway, associated with BRAF and MEK1/2 signaling in melanoma (Lei et al, 2014; Hoeflich et al, 2006). MAPK6 was shown to be upregulated in lung, gastric, and oral cancers (Long et al, 2012; Rai et al, 2004; Liang et al,2005 a). MAPK6 was shown to promote lung cancer cell invasion by phosphorylation of the oncogene SRC-3. Thus, MAPK6 may be an attractive target for aggressive lung cancer therapy (Long et al, 2012). MAPK6 has been described as a potential target for development of drug resistant breast cancer cell anti-cancer drugs (Yang et al, 2010). Overexpression of MAPK6 in gastric cancer was shown to be associated with TNM staging, serosal infiltration, and lymph node involvement (Liang et al,2005 a). MAPK6 was shown to be a binding partner for the core cell cycle mechanical component cyclin D3, indicating that MAPK6 has potential activity in cell proliferation (Sun et al, 2006).

MNAT1 was shown to be associated with a poor prognosis of estrogen receptor positive/HER 2 negative breast cancer (santapria etal, 2013). The intrinsic debris loss of MNAT1 during granulocytopoiesis was shown to promote the growth and metastasis of leukemic myeloblasts (Lou et al, 2013). MNAT1 was shown to be deregulated in the ovarian cancer cell line OAW42 following knockdown of the putative oncogene ADRM1 (Fejzo et al, 2013). siRNA-mediated silencing of MNAT1 gene was demonstrated to inhibit cell growth of pancreatic cancer cell line BxPC3 in vitro and to achieve anti-tumor effects on subcutaneous transplanted pancreatic tumors in vivo (Liu et al,2007 a). Genetic variants of MNAT1 were described to be associated with susceptibility to lung cancer (Li et al, 2007). Infection of the pancreatic cancer cell line BxPC3 with antisense MNAT1 encoded by recombinant adenovirus was shown to result in decreased expression of MNAT1 and an increased proportion of cells in G0/G1 phase. Thus, MNAT1 was shown to play an important role in the regulation of the cell cycle G1 to S-phase transition in the pancreatic cancer cell line BxPC3 (Zhang et al, 2005). MNAT1 regulation of cyclin-dependent kinase activation kinase activity was shown to cross-regulate neuroblastoma cell G1 phase block and play a key role in the transition from proliferation to differentiation of neuroblastoma (Zhang et al, 2004).

DNA methylation-related silencing of NEFH in breast cancer has proven to be frequent, cancer-specific, and associated with clinical features of disease progression (Calmon et al, 2015). NEFH is further described to be inactivated by DNA methylation of pancreatic, gastric and colon cancers and thus may also contribute to the progression of these malignancies (Calmon et al, 2015). NEFH CpG island methylation was shown to be associated with advanced disease, distant metastasis and prognosis of renal cell carcinoma (Dubrowinskajaet al, 2014). Thus, NEFH methylation may be a candidate prognostic marker for the prognosis of renal cell carcinoma (Dubrowinskajaet al, 2014). NEFH is shown to be upregulated in extra-vulvar mucoid chondrosarcoma (doltic et al, 2014). Overexpression of NEFH in liver cancer cell lines was shown to reduce cell proliferation, while knockdown of NEFH promotes cell invasion and migration in vitro and increases the ability to form tumors in mice. Thus, NEFH acts as a tumor suppressor of hepatocellular carcinoma (Revill et al, 2013). NEFH is demonstrated to be frequently methylated in ewing's sarcoma and therefore may be associated with tumorigenesis (holle et al, 2013).

DNA methylation-mediated silencing of NEFL has been shown to be a frequent event in breast cancer, possibly contributing to the progression of breast cancer and other malignancies (e.g., pancreatic, gastric and colon cancers) (Calmon et al, 2015). NEFL is described as a potential tumor suppressor gene that is associated with cancer in several organs (Huang et al,2014 c). NEFL was described as likely to play a role in cancer cell apoptosis and invasion in head and neck squamous cell carcinoma cell lines (Huang et al,2014 c). NEFL methylation is described as a new mechanism that generates cisplatin resistance in head and neck cancer cell lines by interacting with the mTOR signaling pathway (Chen et al, 2012). NEFL was described as a candidate biomarker that can predict the efficacy and survival of head and neck tumor chemotherapy (Chen et al, 2012). High NEFL expression was described to correlate with better clinical effects of supratentorial ependymomas (Hagel et al, 2013). NEFL was demonstrated to be ectopically expressed in breast cancer, decreased in primary breast cancer with lymph node metastasis compared to lymph node negative cancer (Li et al, 2012). Low NEFL expression has been shown to suggest poor 5-year disease-free survival in patients with early breast cancer and therefore may be a potential prognostic factor in patients with early breast cancer (Li et al, 2012). NEFL was shown to be down-regulated in glioblastoma multiforme (Khalil, 2007). Allelic deletions at chromosome 8p21-23 in NEFL were described as early and frequent events in cancer formation and lung cancer progression, and also in association with breast, prostate and hepatitis B virus positive hepatocellular carcinomas (Seitz et al, 2000; Becker et al, 1996; Haggman et al, 1997; Kurimoto et al, 2001).

NEFM has been described as a gene involved in tumor progression and in metastatic involvement (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 often down-regulated in glioblastoma (Lee et al,2015 a). DNA methylation-related silencing of NEFM in breast cancer has proven to be frequent, cancer-specific, and correlated with clinical features of disease progression (Calmon et al, 2015). NEFM is further described to be inactivated by DNA methylation of pancreatic, gastric and colon cancers, and thus may also contribute to the progression of these malignancies (Calmon et al, 2015). NEFM has been shown to be associated with prostate cancer and astrocytomas (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 was shown to correlate with the prognosis of renal cell carcinoma (Ricketts et al, 2013). NEFM is described as a potential diagnostic marker that has been shown to be differentially expressed in neuroendocrine tumor cell lines compared to non-neuroendocrine tumor cell lines (Hofsli et al, 2008).

NUP155 is described as a potential epigenetic biomarker of white blood cell DNA associated with breast cancer susceptibility (khakpourr et al, 2015). NUP155 is 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 the disease (De et al, 2014).

OAS2 was shown to be associated with impaired expression of the CD 3-zeta chain activated by caspase-3. The lack of CD 3-zeta chain is described as being frequently observed in oral cancer (Dar et al, 2015). OAS2 is described as a sub-pathway involving innate immune and inflammatory pathways associated with a risk of advanced prostate cancer (Kazma et al, 2012). Sub-pathway analysis showed that OAS2 is nominally associated with a risk of advanced prostate cancer (Kazma et al, 2012).

Lower expression of PABPN1 in non-small cell lung cancer is associated with poor prognosis (Ichinose et al, 2014). Loss of PABPN1 was described as potentially promoting tumor invasiveness by releasing cancer cells from non-small cell lung cancer microrna-mediated gene regulation (Ichinose et al, 2014). N-terminal poly (alanine) dilatants of PABPN1 were shown to be associated with apoptosis induction via the p53 pathway in HeLa and HEK-293 cell lines (Bhattacharjee et al, 2012).

PCBP1 was described as being particularly important for cancer stem cell enrichment and function of prostate cancer cells (Chen et al,2015 a). PCBP1 is described as an inhibitor of gastric cancer pathogenesis, with down-regulation associated with a malignant phenotype in cultured and xenografted gastric cancer cells (Zhang et al,2015 e). Based on differential expression between benign and malignant sera and tissue samples from ovarian serous adenocarcinoma patients, PCBP1 was shown to play a role in the pathophysiology of ovarian cancer (Wegdam et al, 2014). PCBP1 was shown to be an important mediator of TGF- β induced epithelial mesenchymal transformation (a prerequisite for tumor metastasis in the gallbladder cancer cell line GBC-SD) (Zhang and Dou, 2014). The expression level of PCBP1 was shown to regulate the in vitro migration and invasion capacity of the gallbladder cancer cell line GBC-SD (Zhang and Dou, 2014). Thus, PCBP1 may be a potential prognostic marker for gallbladder cancer metastasis (Zhang and Dou, 2014). PCBP1 down-regulation was described as likely to be involved in the pathogenesis of cervical cancer (Pathak et al, 2014). PCBP1 was described as a regulator of the tumor suppressor-associated transcription factor p63 (Choet al, 2013). High expression of PCBP1 in complete grapes was demonstrated to be associated with a lower risk of developing gestational trophoblastic tumors, whereas PCBP1 expression was significantly reduced in malignant transformed nevi (Shi et al, 2012). Thus, PCBP1 was shown to play an important role in the pathogenesis of gestational trophoblastic tumors (Shi et al, 2012). PCBP1 overexpression was shown to lead to inhibition of metastasis-associated PRL-3 protein translation and AKT inactivation, whereas PCBP1 knock-down was shown to cause activation of AKT and promote tumorigenesis (Wang et al, 2010). PCBP1 was described to play a negative role in tumor invasion of the liver cancer cell line HepG2 (Zhang et al, 2010). Loss of PCBP1 in human liver tumors was described as contributing to the formation of a metastatic phenotype (Zhang et al, 2010).

PDPN is described as being up-regulated in squamous cell carcinoma, mesothelioma, glioblastoma and osteosarcoma (Fujita anddakugi, 2012). PDPN is described as a regulator of tumor invasion and metastasis, as PDPN is associated with several pathways involved in epithelial-mesenchymal transition, collective cell migration, platelet activation, aggregation, and lymphangiogenesis (Dang et al, 2014). PDPN is described as a marker for oral cancer and epithelial mesothelioma (Swain et al, 2014; Ordonez, 2005). PDPN upregulation is described as being associated with lymph node metastasis and poor prognosis in squamous cell carcinoma of the upper respiratory digestive tract (Chuang et al, 2013). PDPN is described as being expressed in vascular tumors, malignant mesotheliomas, central nervous system tumors, germ cell tumors, squamous cell carcinomas, and aggressive tumors with higher invasive and metastatic potential (Raica et al, 2008). Thus, PDPN can be considered as an attractive therapeutic target for tumor cells (Raica et al, 2008).

PHTF2 was shown to be down-regulated in squamous cell carcinoma of the tongue (Huang et al, 2007).

PKM2 proved to be critical for cancer cell proliferation and tumor growth (Chen et al,2014 b; Li et al, 2014; Delabare et al, 2014). The N-myc gene acts as a transcriptional regulator of PKM2 in medulloblastomas (Techt al, 2015). PKM2 appears to play a role in liver cancer pathogenesis, epithelial-mesenchymal transition, and angiogenesis (Nakao et al, 2014). PKM2 is one of the two key factors for 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 as a transcriptional coactivator and a 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; Tamadaet al, 2012; Venneti and Thompson, 2013; Yang and Lu, 2013; Gupta et al, 2014; Iqbal et al, 2014; Chen et al,2014 b; Warner et al, 2014). Low activity dimer PKM2, which is not an active tetrameric form, may play a role in cancer (Mazurek, 2011; Wong et al, 2015; Iqbal et al, 2014; Mazurek, 2007).

PKP1 was shown to be down-regulated in prostate and esophageal adenocarcinomas (Kaz et al, 2012; Yang et al,2015 a). The knockdown of PKP1 in the non-tumor, prostate BPH-1 cell line resulted in a decrease in apoptosis and differential expression of genes (e.g., the prostate cancer-associated SPOCK1 gene) (Yang et al,2015 a). Overall, altered expression of PKP1 and SPOCK1 appears to be a frequent and serious event in prostate cancer, suggesting that PKP1 has tumor suppressor function (Yang et al,2015 a). The decreased expression of PKP1 was shown to be associated with a significantly shorter time to distant metastasis in oral squamous cell carcinoma (Harriset al, 2015). Loss of PKP1 by activator methylation was described as being associated with Barrett's progression of esophagus to esophageal adenocarcinoma (Kaz et al, 2012). PKP1 was shown to be upregulated in non-small cell lung cancer, and may be a good marker for distinguishing squamous cell carcinoma specimens (Sanchez-Palencia et al, 2011). PKP1 was shown to be up-regulated in the well-differentiated liposarcoma cell line GOT3 (Persson et al, 2008). A decrease in PKP1 expression is described as promoting increased activity in head and neck squamous cell carcinoma (Sobolik-delmarire et al, 2007). Loss of PKP1 was shown to be associated with cervical cancer development (Schmitt-Graeff et al, 2007). PKP1 has been shown to be associated with local recurrence or metastasis and poor prognosis in oropharyngeal squamous cell carcinoma patients (Papagerakis et al, 2003).

The increase of PKP3 mRNA in the blood of patients with gastrointestinal tumors can be used as a biomarker and a prognostic predictor of disease (Valladares-Ayerbes et al, 2010). PKP3 overexpression is associated with poor prognosis in breast, lung and prostate cancer, while down-regulation 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 results in increased levels of MMP7 and PRL3 proteins, which are required for cell migration and tumor formation (Khapare et al, 2012; Basu et al, 2015).

Knockdown of PPP4R1 was shown to inhibit cell proliferation of the breast cancer cell line ZR-75-30 (Qi et al, 2015). Thus, PPP4R1 promotes the proliferation of breast cancer cells and may play a crucial role in the development of breast cancer (Qi et al, 2015). The knockdown of PPP4R1 in the hepatocellular carcinoma cell line HepG2 was shown to result in cell proliferation, colony formation and cell cycle arrest at G2/M (Wu et al, 2015). PPP4R1 knockdown was further shown to lead to inactivation of the p38 and c-Jun N-terminal kinase signaling cascades in HepG2 cells, suggesting that PPP4R1 may promote cell proliferation (Wu etal, 2015). Thus, PPP4R1 plays a key role in promoting growth of hepatocellular carcinoma cells (Wu et al, 2015). PPP4R1 is described as a negative regulator of an inhibitor of lymphocyte NF-kB kinase activity, which down-regulates oncogenic NF-kB signaling in the T-cell lymphoma subgroup (Brechmann et al, 2012).

PRC1 was described as being associated with cervical cancer radiation resistance, as cervical cancer tissue shows high differential expression of PRC1 upon irradiation (Fu et al,2015 b). The genetic locus in intron 14 of PRC1 was described as being associated with breast cancer susceptibility (Cai et al, 2014). PRC1 is described as one gene of a five-gene signature that can be used as a prognostic signature for disease-free survival in breast cancer patients (musacchi et al, 2013). PRC1 was shown to be upregulated in ovarian, cervical and bladder cancers (Epineosa et al, 2013; Ehrlichova et al, 2013; Kanehira et al, 2007). PRC1 was shown to be upregulated during 4-hydroxyestradiol-mediated malignant transformation of the mammary epithelial cell line MCF-10A (Okoh et al, 2013). PRC1 is described as a gene with significant biological significance in tumor pathogenesis that can be used in gene populations to predict prognosis after adjuvant chemotherapy in tumor-resectable non-small cell lung cancer patients (Tang et al, 2013). PRC1 was shown to be negatively regulated by the cell cycle associated kinase Plk1 (Hu et al, 2012). Knockdown of PRC1 in bladder cancer cell line NIH3T3 was shown to result in a significant increase in multinucleated cells and subsequent cell death (Kanehira et al, 2007). Furthermore, PRC1 was shown to interact with the novel cancer-testis antigen MPHOSPH1 in bladder cancer cells, and the MPHOSPH1/PRC1 complex was shown to play a key role in bladder cancer development, possibly as a new therapeutic target (Kanehira et al, 2007). PRC1 was shown to be regulated by p53 (Li et al, 2004).

Expression sequence tag analysis determined PRDM15 to be an up-regulated gene in lymphoma (giallowrakis et al, 2013). PRDM15 was described as a candidate tumor suppressor gene that might contribute to the development or progression of pancreatic cancer (Bashyam et al, 2005).

Different polymorphisms of PTHHLH have been shown to be associated with the risk and prognosis of lung cancer (Manenti et al, 2000). PTHLH upregulation in a spontaneous metastatic breast cancer C57BL/6 mouse-derived model was described as likely to be involved in metastatic transmission of breast cancer (Johnstone et al, 2015). PTHHLH has been shown to be upregulated in oral squamous cell carcinoma, chondroma, adult T-cell leukemia/lymphoma, and renal clear cell carcinoma (Bellon et al, 2013; Yang et al,2013 a; Yao et al, 2014; Lv et al, 2014). PTHLH upregulation has been shown to be associated with poor pathological differentiation and poor prognosis in patients with head and neck squamous cell carcinoma (Lv et al, 2014). PTHLH was demonstrated to be upregulated by p38 signaling, which promotes lung colon cancer cell extravasation by caspase-independent death of endothelial cells in the pulmonary microvasculature (urolevic et al, 2014). PTHLH was shown to be significantly differentially expressed in squamous cell carcinoma compared to normal skin (Prasad et al, 2014). PTHLH is described as part of a four-gene signature that is associated with survival in early non-small cell lung cancer patients (Chang et al, 2012). Disruption of antiproliferative effects by PTHLH frameshift mutations was described as promoting the development of early colorectal cancer in hereditary nonpolyposis colorectal cancer patients (Yamaguchi et al, 2006). PTHLH upregulation has been shown to correlate with poor outcome in overall survival and disease-free survival in patients with renal clear cell carcinoma who received nephrectomy (Yao et al, 2014). PTHHLH was shown to upregulate cell cycle progression and to be involved in cell cycle regulated protein expression through the ERK1/2, p38, MAPK and PI3K signaling pathways of the colorectal adenocarcinoma cell line, Caco-2 (Calvo et al, 2014). RAP1GDS1 was shown to promote pancreatic cancer cell proliferation (Schuld et al, 2014). The simultaneous loss of two splice variants of RAP1GDS1 in a mouse non-small cell lung cancer cell line NCI-H1703 xenograft was shown to result in reduced tumorigenesis (Schuld et al, 2014). RAP1GDS1 was shown to promote cell cycle progression in various types of cancer, making it a valuable target for cancer therapy (Schuld et al, 2014). RAP1GDS1 was 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 was shown to be a unique activator of RhoA and NF-. kappa.B activity, which plays a functional role in breast cancer malignancy (Hauser et al, 2014). High expression of RAP1GDS1 was shown to correlate with poor clinical outcome in breast cancer (Hauser et al, 2014). RAP1GDS1 has been shown to modulate cell proliferation, migration and NF-. kappa.B transcriptional activity of non-small cell lung cancer, thus contributing to the malignant phenotype of the disease. Thus, RAP1GDS1 is an interesting therapeutic target for non-small cell lung cancer (Tew et al, 2008). RAP1GDS1 was shown to be fused to NUP98 in T-cell acute lymphoblastic leukemia (Romana et al, 2006).

RNPEP activity has been shown to be up-regulated in colorectal adenomas, papillary thyroid carcinoma, breast cancer, and renal clear cell carcinoma (Ramirez-Exposito et al, 2012; Larrinaga et al, 2013; Perez et al, 2015; Varonaet al, 2007). RNPEP was shown to be associated with tumor growth of rat C6 glioma implanted in the subcutaneous region (Mayaset al, 2012).

RORA is described as a potential lung cancer oncogene (Wang et al,2015 e). RORA has been shown to be associated with the expression of the potential tumor suppressor OPCML in colon cancer (Li et al,2015 a). Two single nucleotide polymorphisms of RORA have been shown to be associated with breast cancer (trouong et al, 2014). RORA is described as a potential tumor suppressor and therapeutic target for breast cancer (Du and Xu, 2012). RORA has been shown to be down-regulated in colorectal adenocarcinoma and breast cancer (Kottorou et al, 2012; Du and Xu, 2012). Stable over-expression of RORA in the hepatocellular carcinoma cell line HepG2 was shown to affect the expression of genes involved in glucose metabolism and liver cancer formation, suggesting that RORA is associated with carcinogenesis in liver-derived cells (Chauvet et, 2011). RORA is demonstrated to be differentially methylated in gastric cancer compared to normal gastric mucosa (Watanabe et al, 2009). RORA is described as being associated with cell growth and differentiation control and metastatic behavior control of androgen-independent prostate cancer cell line DU 145 (Moretti et al, 2002).

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

RPS26 knockdown was shown to induce p53 stabilization and activation, leading to p 53-dependent cell growth inhibition (Cui etal, 2014). RPS26 further showed a role in DNA damage response by directly affecting the transcriptional activity of p53 (Cuiet al, 2014).

S100A2 was shown to be associated with non-small cell lung cancer and was described as a predictive marker of poor overall survival in patients with squamous cell lung cancer (Hountis et al, 2014; Zhang et al,2015 d). S100a2 is described as a downstream target of the oncogene KRAS, an activator of lung cancer tumor progression (Woo et al, 2015). S100a2 was described as one promising marker for predicting overall survival of pancreatic ductal adenocarcinoma (Jamieson et al, 2011). Altered S100a2 expression by nitrosamine N-nitrosopyrrolidine is described as a potential cause of tumor progression in black south africa esophageal squamous cell carcinoma (piliayer al, 2015). S100A2 was shown to be up-regulated in plasma, laryngeal, gastric, and epidermal tumors of patients with early stage non-small cell lung cancer, nasopharyngeal carcinoma (Zhu et al,2013 a; Lin et al, 2013; Zhang et al,2015 a; ZHa et al, 2015; Wang et al,2015 c). Methylation-related inactivation of S100a2 was shown to occur frequently in head and neck and bladder cancers and therefore may be an important event in tumorigenesis for these diseases (Lee et al,2015 c). Cytoplasmic expression of S100a2 was shown to be up-regulated in oral squamous cell carcinoma, while nuclear expression was down-regulated (Kumar et al, 2015). Cytoplasmic upregulation of S100a2 was shown to be a potential predictor of risk of recurrence in oral squamous cell carcinoma patients (Kumar et al, 2015). S100a2 is described as playing a role in breast cancer metastasis (Naba et al, 2014). S100A2 was shown to be a BRCA1/p63 co-regulated tumor suppressor gene that plays a role in the regulation of the stability of mutant p53 by modulating mutant p53 binding to HSP90 (Buckley et al, 2014). S100a2 is described as a candidate tumor suppressor gene that is down-regulated in recurrent nasopharyngeal carcinoma and therefore may play an important role in the development of recurrent nasopharyngeal carcinoma (Huang et al,2014 b). S100a2 was shown to be down-regulated in gastric cancer, and down-regulation was shown to be associated with depth of infiltration, lymph node metastasis, decreased probability of no recurrence, and decreased overall survival (Liu et al,2014 e). Therefore, S100a2 down-regulation may be an independent negative prognosis biomarker for gastric cancer (Liu et al,2014 e). S100A2 has further been shown to down-regulate the MEK/ERK signaling pathway in MGC-803 cancer cells (Liu et al,2014 e). In immunodeficient mice, overexpression of S100a2 was shown to induce epithelial-mesenchymal transformation of a549 lung cancer cells, followed by increased invasiveness, enhanced Akt phosphorylation, and accelerated tumor growth (Naz et al, 2014). The proto-oncogene behavior of Si00a2 is further described as being involved in PI3/Akt signaling regulation and functional interaction with the TGF β signaling pathway protein Smad3 (Naz et al, 2014). S100a2 expression was shown to be associated with histological grading, lymph node metastasis, clinical staging and poor survival in patients with peri-pulmonic and extrahepatic bile duct cancer (Sato et al, 2013). Thus, S100a2 can serve as a prognostic marker in biliary tract cancer patients (Sato et al, 2013).

S100A8 is described as an important mediator of acute and chronic inflammation, which interacts with myeloid-derived suppressor cells in a positive feedback loop to promote tumor development and metastasis (Zheng et al, 2015). S100A8 is described as a potential diagnostic marker, prognostic indicator and therapeutic target for non-small cell lung cancer (Lim and Thomas, 2013). Overexpression of S100A8 was demonstrated to be associated with stage progression, invasion, metastasis and poor survival of bladder cancer (Yao et al, 2007). S100A8 proved to be a diagnostic marker for invasive bladder cancer (Ismail et al, 2015). S100A8 was shown to be up-regulated in undifferentiated thyroid carcinoma, osteocytic giant cell tumor and colorectal cancer (Reeb et al, 2015; Zhang et al,2015 b; Liao et al,2015 a). In vivo analysis in mice using S100A8 knockdown of undifferentiated thyroid cancer cells suggested that tumor growth and lung metastasis were reduced and animal survival was significantly prolonged (Reeb et al, 2015). S100A8 was shown to promote proliferation of undifferentiated thyroid cancer cells by interacting with RAGE, which activates the p38, ERK1/2 and JNK signaling pathways in tumor cells (Reeb et al, 2015). Thus, S100A8 may represent a relevant therapeutic target for undifferentiated thyroid cancer (Reeb et al, 2015). S100A8 was shown to be associated with high risk chronic lymphocytic leukemia (Alsagaby et al, 2014). S100A8 was shown to be associated with kidney cancer progression and was described as a potential biomarker and therapeutic target for kidney cancer (Mirza et al, 2014). S100A8 is described as part of calprotectin, a heterodimer required for non-inflammatory driven progression of 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,2015 b).

SERPINH1 encodes a serine protease inhibitor, clade H (heat shock protein 47) member 1, (collagen binding protein 1), a serine protease inhibitor. SERPINH1 acts as a collagen-specific chaperone for the endoplasmic reticulum (RefSeq, 2002). SERPINH1 is overexpressed in many human cancers, including gastric, lung, pancreatic ductal adenocarcinoma, glioma, and ulcerative colitis-associated cancers (Zhao et al,2014 a). SERPINH1 is upregulated in hepatocellular carcinoma, esophageal squamous cell carcinoma, cholangiocellular carcinoma, gastric cancer, lung cancer, pancreatic ductal adenocarcinoma, ulcerative colitis-related cancers, and glioma (ZHaoet al,2014 a; Padden et al, 2014; Lee et al,2015 b; Naboulsi et al, 2015). Overexpression of SERPINH1 was shown to be associated with poor prognosis in esophageal squamous cell carcinoma patients, and the levels of immunostaining and pathological staging of SERPINH1 were shown to be significantly associated with overall and relapse-free survival (Lee et al,2015 b). Thus, SERPINH1 may be a potential prognostic marker for esophageal squamous cell carcinoma (Lee et al,2015 b). SERPINH1 knockdown in glioma cells was shown to inhibit glioma cell growth, migration and invasion in vitro, while SERPINH1 knockdown in vivo was shown to inhibit tumor growth and induce apoptosis (Zhao et al,2014 a). Thus, SERPINH1 may be a therapeutic target for gliomas (Zhao et al,2014 a). Compared to the primary oral squamous cell carcinoma tumor with multiple lymph node involvement, SERPINH1 was shown to be down-regulated in metastatic cancers, suggesting that SERPINH1 may be associated with the metastatic potential of these tumors (Nikitakis et al, 2003).

SLC7a11 was shown to be down-regulated in resistant variants of the W1 ovarian cancer cell line, possibly playing a role in cancer cell drug resistance (Januchowski et al, 2013). SLC7a11 was described as regulating the tumor microenvironment, leading to cancer growth advantage (savask and Eyupoglu, 2010). SLC7a11 was described as being involved in the neurodegenerative process of gliomas, making SLC7a11 a potential primary target for cancer treatment (savask et al, 2015). SLC7a11 was shown to be inhibited by p53 in the case of iron death, and the p53-SLC7a11 axis was described as remaining in the p53(3KR) mutant and contributing to its inhibition of tumorigenesis in the absence of classical tumor suppression mechanisms (Jiang et al, 2015). SLC7a11 is described as a functional subunit of the system Xc-, whose function is increased in invasive breast cancer cells (line-Melville et al, 2015). High membrane staining of SLC7a11 in cisplatin-resistant bladder cancer proved to be associated with poor clinical outcome, and SLC7a11 inhibition was described as a promising treatment for this disease (Drayton et al, 2014). SLC7A11 was shown to be differentially expressed in the human promyelocytic leukemia cell line HL-60, which had been exposed to benzene and its metabolites, thus underlining the potential association of SLC7A11 with leukemic development (Sarma et al, 2011). SLC7a11 disruption was described as leading to growth inhibition in a variety of cancers, including lymphoma, glioma, prostate and breast cancer (Chen et al, 2009). SLC7a11 inhibition was demonstrated to inhibit cell infiltration of the esophageal cancer cell line KYSE150 and experimental metastasis in nude mice in vitro, establishing the role of SLC7a11 in tumor metastasis (Chen et al, 2009).

SRPR was shown to be amplified in the case of acute myeloid leukemia with double minichromosomes (Crossen et al, 1999).

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

Dysregulated STK17A expression was associated with different cancer types. Decreased expression in cervical and colorectal cancers is associated with the pro-apoptotic character of STK17A associated with tumor progression. STK17A from glioblastoma and head and neck cancer is overexpressed in a level-dependent manner, possibly caused by effects on other tumor-associated pathways (e.g., TGF-. beta.) (Mao et al,2013 a; Thomas et al, 2013; Park et al, 2015; Bandres et al, 2004). STK17A is a direct target of the tumor suppressor p53 and is a regulator of Reactive Oxygen Species (ROS) (Kerley-Hamilton et al, 2005; Mao et al, 2011).

SYK is described as a regulator of tumorigenesis, acting as a tumor activator in some cells by providing survival function, and as a tumor suppressor in other cells by limiting epithelial-mesenchymal transition and inhibiting migration (Krisenko and Geahlen, 2015). SYK is described as associated with B Cell Receptor (BCR) activation in B cell lymphomas (Seda and Mraz, 2015). Inhibition of BCR pathway-critical kinases (e.g., SYK) has been found to reduce chronic lymphocytic leukemia cell viability in preclinical models (Davids and Brown, 2012). SYK was shown to be upregulated in chronic lymphocytic leukemia (Feng and Wang, 2014). SYK is described as being associated with the pathogenesis of chronic lymphocytic leukemia and may have value in assessing treatment efficacy and prognosis of this disease (Feng and Wang, 2014). SYK is described as a potential tumor suppressor of breast cancer, which is associated with poor outcome in primary breast tumor deficiencies (Navara, 2004). SYK was shown to play a key role in paclitaxel resistance in ovarian cancer (Yu et al,2015 b). SYK down-regulation has been described as being associated with the development of a variety of cancers, including colorectal cancer (Peng et al, 2015). The different gene polymorphisms of the SYK activator have been shown to be independent risk factors for colorectal cancer development in the Han population in southern China (Peng et al, 2015). SYK proved to be frequently methylated in hepatocellular carcinoma, and SYK methylation proved to determine a poorly prognostic subset of hepatocellular carcinomas (Shin et al, 2014).

The TP63 translocation was described as an event in a subset of anaplastic lymphoma kinase positive anaplastic large cell lymphomas, which was associated with the invasive process of the disease (happoood and Savage, 2015). TP63 was 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 isomer TAp63 was described as being overexpressed in hematological malignancies, while TP63 missense mutations were reported to occur in squamous carcinoma and at TP63 translocations in lymphomas and some lung adenocarcinomas (orizol et al, 2015). Aberrant splicing leading to overexpression of the TP63 isoform, δ Np63, is often found in human cancers (e.g., cutaneous squamous cell carcinoma), which are likely to contribute to tumor development and progression (Missero and Antonini, 2014; Inoue and Fry, 2014).

TPM1 was shown to be down-regulated in renal cell carcinoma, esophageal carcinoma cell line squamous cell carcinoma, metastatic canine breast carcinoma, and neuroblastoma cell lines (Klopfleisch et al, 2010; Yager et al, 2003; Zare et al, 2012; Wang et al,2015 b). TPM1 expression was demonstrated to be associated with tumor size, Fuhrman stratification and prognosis in renal cell carcinoma patients. Transfection of TPM1 into renal cell carcinoma cell lines OSRC-2 and 786-0 was shown to reduce migration and invasion capacity while enhancing apoptosis (Wang et al,2015 b). Thus, TPM1 was described as being able to display the characteristics of tumor suppressor genes, while being overexpressed in renal cell carcinoma cells (Wang et al,2015 b). The RAS/PI3K/AKT and RAS/MEK signaling pathways have been described as involved in TPM1 regulation and inhibition in the intrahepatic cholangiocarcinoma cell line HuCCT1 and the esophageal squamous cell carcinoma cell line (Zare et al, 2012; Yang et al,2013 b). TPM1 is described as a tumor suppressor whose overexpression in the breast cancer cell line MCF-7 inhibits anchorage-independent cell growth (Zhu et al,2007 b). Epigenetic inhibition of TPM1 is described as being associated with altered TGF β tumor suppressor function and may contribute to the development of metastatic properties of tumor cells (Varga et al, 2005).

Tryptase has been shown to be upregulated in certain acute myeloid leukemia patients (Jin et al, 2014). Tryptase expression was described as regulated by SCF/c-KIT signaling via ERK1/2 and p38MAPK pathways (Jin et al, 2014). Mast cell tryptase, described as being involved in colorectal cancer angiogenesis, was shown to be more highly expressed in serum of colorectal cancer patients before and after radical surgical resection (amendola et al, 2014).

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

TSPAN10 was demonstrated to be a gene differentially expressed between metastatic melanoma samples and normal skin samples, which could be a potential biomarker for metastatic melanoma treatment (Liu et al,2014 c). Among other genes, TSPAN10 was shown to be upregulated in uterine leiomyosarcoma metastases compared to primary leiomyosarcoma, thus helping to distinguish these diseases and possibly helping to understand the tumor progression of the cancer (Davidson et al, 2014).

TTPAL was described as a candidate oncogene showing mutations in microsatellite-unstable colorectal cancer (Tuupanenet al, 2014).

TUBGCP2 was shown to be upregulated in the paclitaxel-resistant ovarian cancer cell line and was described as being associated with the sensitivity of the non-small cell lung cancer cell line NCI-H1155 to paclitaxel (Huang and Chao, 2015). TUBGCP2 was shown to be upregulated in glioblastoma tumors, with overexpression antagonizing the inhibitory effect of CDK5 regulatory subunit-related tumor suppressor protein 3 on DNA damage G2/M checkpoint activity (Draberova et al, 2015).

VIM is described as a downstream target of STAT3, which is associated with breast tumor progression after deregulation by STAT3 (Banerjee and resot, 2015). VIM is described as a potential nasopharyngeal carcinoma-associated protein (Chen et al,2015 c). The negative methylation status of vimentin was demonstrated to be predictive of an improved prognosis in pancreatic cancer patients (Zhou et al, 2014). VIM was shown to be upregulated in non-small cell lung cancer by C6orf106 and was described to be subsequently associated with enhanced cancer cell invasiveness (Zhang et al, 2015C). VIM is described as an independent predictor of overall survival in patients with squamous cell lung carcinoma (Che et al, 2015). VIM is described as a biomarker that can potentially distinguish melanoma subtypes and possibly predict the aggressiveness of melanoma in different subgroups of melanoma (qendero et al, 2014). VIM was shown to be upregulated in clear cell renal cell carcinoma (Shi et al, 2015). High VIM expression is described as an independent predictor of clear cell renal cell carcinoma (Shi etal, 2015). VIM was shown to act as a scaffold to recruit Slug to ERK and promote Slug phosphorylation, which is described as required for the development processes employed during tumorigenesis to activate epithelial-mesenchymal transition, promoting metastatic capacity (virtakovuet al, 2015).

WDR1 was shown to be up-regulated in interstitial fluid from ovarian cancer and poor prognosis of high grade canine cutaneous mast cell tumors compared to well-prognosed low grade mast cell tumors (Schlieben et al, 2012; Haslene-Hox et al, 2013). WDR1 was shown to be down-regulated in chemotherapy-resistant advanced serous epithelial ovarian cancer (Kim et al, 2011). Down-regulation of WDR1 in chemotherapy-resistant advanced serous epithelial ovarian cancer was shown to be associated with poor overall survival (Kim et al, 2011). WDR1 was shown to be upregulated in the region between the anterior of the breast cancer invasive tumor and normal tissue (interface region), and thus, may be involved in the progression and metastasis of breast cancer (Kang et al, 2010).

YWHAE fusion with NUTM2B/NUTM2E was described as an event observed in a few renal clear cell sarcomas (Karlsson et al, 2015). YWHAE-NUTM2 fusion was described as a common event in high-grade endometrial interstitial sarcoma (Ali et al, 2014). High-grade endometrial-stromal sarcomas containing YWHAE-NUTM2 fusions are described as a subset of endometrial-stromal sarcomas with aggressive clinical behavior and poor prognosis (Kruse et al, 2014). Lesions at three sites, including ywboe, were described as potential factors for the development of uterine sarcoma (Suzuki et al, 2014). YWHAE was shown to be down-regulated in gastric cancer, and decreased levels of YWHAE were associated with diffuse gastric cancer and the early onset of this pathology, suggesting that YWHAE may play a role in the development of gastric cancer (Leal et al, 2012). YWHAE has been shown to be differentially expressed in breast cancer patient tissues with and without recurrence, and to be associated with both disease-free survival and overall survival (Ciminoethanol, 2008). Therefore, YWHAE can be used as an independent prognostic marker and a potential drug target for breast cancer (Cimino et al, 2008).

Changes in ZNF292 are described as chronic lymphocytic leukemia driven changes (Puente et al, 2015). ZNF292 is described as a tumor suppressor gene for colorectal cancer (Takeda et al, 2015). ZNF292 is described as an immunogenic antigen with clinical relevance in head and neck squamous cell carcinoma (Heubeck et al, 2013).

Detailed description of the invention

Whether or not an immune response is stimulated depends on the presence of antigens that are recognized as foreign by the host immune system. The discovery that the presence of tumor associated antigens increases the likelihood of using the host immune system to interfere with tumor growth. Currently, various mechanisms of utilizing the humoral and cellular immune systems for immunization are being explored for cancer immunotherapy.

Specific elements of the cellular immune response specifically recognize and destroy tumor cells. T-cells isolated from tumor infiltrating cell populations or peripheral blood suggest that these cells play an important role in the natural immune defense of cancer. In particular, CD8 positive T cells play an important role in this response, TCD8+ recognizes class I molecules contained in peptides carried by the Major Histocompatibility Complex (MHC) of typically 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRIP) located in the cytoplasm. Human MHC molecules are also known as human leukocyte-antigens (HLA).

Unless otherwise indicated, all terms used herein are defined as follows.

The term "T cell response" refers to the specific spread and activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, the effector function may be lysis of peptide pulsed, peptide precursor pulsed or native peptide presented target cells, secretion of cytokines, preferably peptide induced interferon- γ, TNF- α or IL-2, secretion of effector molecules, preferably peptide induced granzyme or perforin, or degranulation.

The term "peptide" as used herein refers to a series of amino acid residues, typically joined by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. These peptides are preferably 9 amino acids in length, but may be 8 amino acids in length to as short as 10, 11, 12, 13 or 14 amino acids in length or longer, and in the case of MHC class II peptides (elongate variants of the peptides of the invention) may be 14, 15, 16, 17, 18, 19 or 20 amino acids in length or longer.

Thus, the term "peptide" shall include salts of a series of amino acid residues, typically linked by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, for example: chloride or acetic acid (trifluoroacetic acid) salt. It must be noted that the salts of the peptides of the invention are substantially different from the peptides in their in vivo state, since they are not salts in vivo.

The term "peptide" shall also include "oligopeptides". The term "oligopeptide" as used herein refers to a series of amino acid residues, typically linked by peptide bonds between the alpha-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. Generally, oligopeptides are less than about 30 amino acid residues in length and longer than about 15 amino acids in length.

The term "polypeptide" refers to a series of amino acid residues, typically joined by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, so long as the correct epitope is maintained. The term "polypeptide" as opposed to the term peptide or oligopeptide refers to a molecule comprising more than about 30 amino acid residues.

A peptide, oligopeptide, protein or nucleotide encoding such a molecule is "immunogenic" (and thus an "immunogen" in the present invention) if it induces an immune response. In the context of the present invention, a more specific definition of immunogenicity is the ability to induce a T cell response. An "immunogen" is therefore a molecule capable of inducing an immune response, and in the context of the present invention, a molecule capable of inducing a T cell response. In another aspect, the immunogen may be a peptide, a complex of a peptide with MHC, and/or a protein for increasing specific antibody or TCR resistance.

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

In humans, there are three distinct genetic 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-A01, HLA-A02 and HLA-B07 are examples of different MHC class I alleles that can be expressed from these gene loci.

Table 5: HLA-A02 and HLA-A24 and the frequency of expression F of the most common HLA-DR serotypes. Frequency was adapted from the haplotype frequency within the U.S. population according to Hardy-Weinberg formula F ═ 1- (1-Gf)2 used by Mori et al (Mori et al, 1997). Due to linkage disequilibrium, a 02 or a 24 combinations within certain HLA-DR alleles may be concentrated or less frequent than their expected single frequency. For details, see Chanock et al (Chanock et al, 2004).

The peptide of the invention, preferably when incorporated into the vaccine of the invention as described herein, is conjugated to a x 02. Vaccines may also include pan-bound MHC class II peptides. Thus, the vaccines of the invention can be used to treat cancer in patients positive for a x 02, but not because of the extensive tuberculous nature of these peptides, MHC class II allotypes must be selected.

If an a 02 peptide of the invention is combined with a peptide that binds to another allele, for example a 24, a higher proportion of the patient population can be treated than the MHC class I allele alone. Although less than 50% of patients in most populations are resolved by individual alleles, a vaccine of the invention containing both HLA-A24 and HLA-A02 epitopes can treat at least 60% of patients in any relevant population. In particular, in each region, at least one of these alleles of the patient in the following ratios has a positive effect: 61% in the united states, 62% in western europe, 75% in china, 77% in korea, and 86% in japan (calculated according to www.allelefrequencies.net).

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

The nucleotide sequence encoding a particular peptide, oligopeptide or polypeptide may be a natural nucleotide sequence or a synthetic nucleotide sequence. Generally, the DNA segments encoding the peptides, polypeptides and proteins of the invention are composed of eDNA segments and short oligonucleotide linkers, or a series of oligonucleotides, to provide a synthetic gene that can be expressed in a recombinant transcription unit comprising regulatory elements derived from a microbial or viral operon.

The term "nucleotide encoding of a peptide" as used herein refers to encoding a peptide with a nucleotide sequence, wherein the peptide comprises artificial (man-made) activation and stop codons compatible with the biological system that will express the sequence by the dendritic cell or another cellular system used to generate the TCR.

Reference herein to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acids. Thus, unless otherwise indicated herein, for example with respect to DNA, a particular sequence is a single-stranded DNA of that sequence, a duplex (double-stranded DNA) of that sequence with its complement, and the complement of that sequence.

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, i.e., the region that encodes the natural expression product of the gene in vivo.

The coding region may be derived from a non-mutated ("normal") gene, a mutated gene or an aberrant gene, and may even be derived from a DNA sequence, and may well be synthesized in the laboratory using DNA synthesis methods well known in the art.

The term "expression product" refers to a polypeptide or protein that is the translation product of any nucleic acid sequence encoded equivalent by the degeneracy of genes and genetic codes and thus encoding the same amino acids.

The term "fragment," when referring to a coding sequence, refers to a portion of DNA that contains 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.

The term "DNA fragment" refers to a DNA polymer, either in the form of individual fragments or as a component of a larger DNA construct, which is obtained in substantially pure form, i.e., free of contaminating endogenous material, from DNA that has been isolated at least once, and in quantities or concentrations that enable the identification, manipulation and recovery of the fragment and its component nucleotide sequences using standard biochemical methods, e.g., using cloning vectors. Such fragments exist in the form of open reading frames (not interrupted by internal untranslated sequences) or introns (usually present in eukaryotic genes). The untranslated DNA sequence may be present downstream of the open reading frame where it does not interfere with the manipulation or expression of the coding region.

The term "primer" refers to a short nucleic acid sequence that can pair with a DNA strand and provide a free 3' -OH terminus where DNA polymerase begins to synthesize a strand of deoxyribonucleic acid.

The term "activator" refers to a region of DNA that is involved in the binding of RNA polymerase to activate transcription.

The term "isolated" means that a substance is removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a native nucleotide or polypeptide in a living animal is not isolated, but a nucleotide or polypeptide isolated from some or all of the coexisting materials in the native system is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides and polypeptides may be part of a composition, and as the vector or composition is not part of its natural environment, it remains isolated.

The polynucleotides and recombinant or immunogenic polypeptides disclosed in the present invention may also be present in "purified" form. The term "purified" does not require absolute purity; it is a relative definition and may include highly purified or partially purified preparations, as those skilled in the relevant art will understand. For example, each clone isolated from a cDNA library that has been purified by conventional methods to have an electrophoretic isotype. It is expressly contemplated that the starting material or natural substance may be purified by at least one order of magnitude, preferably two or three orders of magnitude, more preferably four or five orders of magnitude. Furthermore, it is expressly contemplated that the purity of the polypeptide is preferably 99.999%, or at least 99.99% or 99.9%; even more suitably 99% by weight or more.

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

The terms "portion", "segment" and "fragment" as used herein when used in relation to a polypeptide refer to a contiguous sequence of residues, such as amino acid residues, the sequence of which forms a subset of a larger sequence. For example, if a polypeptide is treated with any endopeptidase (e.g., trypsin or chymotrypsin), the oligopeptide resulting from the treatment will represent a portion, segment, or fragment of the starting polypeptide. When used in relation to a polynucleotide, these terms refer to the product resulting from treatment of the polynucleotide with any endonuclease.

According to the present invention, the term "percent identity" or "percent identity", if referring to a sequence, means that the sequence to be compared ("the compared sequence") is compared to the sequence or sequences of claims after alignment of the sequence to be compared ("the reference sequence"). The percent equivalence is then calculated according to the following formula:

percent equivalence of 100[1- (C/R) ]

Wherein C is the number of differences between the reference sequence and the compared sequence over the alignment length between the reference sequence and the compared sequence, wherein

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

(ii) each gap in the reference sequence, an

(iii) Each aligned base or amino acid in the reference sequence is different from the aligned base or amino acid in the aligned sequence, i.e., a difference is formed and

(iiii) alignment must begin at position 1 of the alignment sequence;

and R is the number of bases or amino acids in the reference sequence that produce any gaps in the reference sequence over the length of alignment of the reference sequence to the compared sequence, also counted as one base or amino acid.

If there is an alignment between the "aligned sequence" and the "reference sequence" that is approximately equal to or greater than the specified minimum percent of identity as calculated above, then the aligned sequence has the specified minimum percent of identity with the reference sequence, although there may be alignments where the percent of identity as calculated above herein is less than the specified percent of identity.

Thus, as mentioned above, the present invention proposes a peptide comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 93, or a sequence identical to SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof which induces a T cell cross-reaction with the peptide, having 88% homology. The peptides of the invention have the ability to bind to a class II molecule of the Major Histocompatibility Complex (MHC) I or an elongated version of the peptide.

In the present invention, the term "homology" refers to the degree of identity between two amino acid sequences (see percent identity above, e.g., peptide or polypeptide sequences. the "homology" as described above is determined by aligning two sequences adjusted under ideal conditions with the sequences to be compared.

One skilled in the art can assess whether T cells induced by a particular peptide variant are cross-reactive with the peptide itself (Apay et al, 2006; Colombetti et al, 2006; Fong et al, 2001; Zarmemba et al, 1997).

By "variant" of a given amino acid sequence, the inventors mean that the side chain of one or two amino acid residues, etc., is altered by substitution with the side chain of another natural amino acid residue or other side chain, so that such a peptide can still bind 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 to at least maintain (e.g., not increase) its ability to interact with and bind to the binding groove of a suitable MHC molecule such as HLA-a 02 or-DR, and to at least maintain (e.g., not increase) its ability to bind to a TCR of an activated T cell.

These T cells can then cross-react with cells expressing the polypeptide (which comprises the natural amino acid sequence of the homologous peptide as defined in the present invention) and with killer cells. As described in the scientific literature and databases (Rammensee et al, 1999; Godkin et al,1997), certain sites of HLA-A binding peptides are usually anchor residues, which form a core sequence commensurate with the binding motif of HLA-binding grooves, whose definition is determined by the polarity, electrophysical, hydrophobic and steric properties of the polypeptide chains that make up the binding grooves. Thus, one skilled in the art can modify seq id No: 1 to SEQ ID NO: 93, and determining whether the variants retain the ability to bind to MHC class I or II molecules. The variants of the invention retain the ability to bind to the TCR of activated T cells, which can then cross-react with and kill cells expressing a polypeptide comprising the native amino acid sequence of the cognate peptide as defined herein.

The original (unmodified) peptides disclosed herein may be modified by substitution of one or more residues at different (possibly selective) positions within the peptide chain, if not otherwise indicated. Preferably, these substitutions are at the end of the amino acid chain. Such substitutions may be conservative, for example, where one amino acid is substituted for another amino acid of similar structure and characteristics, such as where one hydrophobic amino acid is substituted for another. More conservative substitutions are those between amino acids of the same or similar size and chemical nature, for example, leucine substituted with isoleucine. In the study of sequence variations in the native homologous protein family, certain amino acid substitutions tend to be more tolerant than others, and these amino acids tend to show similarity correlations between the size, charge, polarity and hydrophobicity of the original amino acid, which underlies the identification of "conservative substitutions".

Herein, conservative substitutions are defined as exchanges within one of the following five groups: group 1 small aliphatic, apolar 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 residue (His, Arg, Lys); group 4-the bulky aliphatic nonpolar residue (Met, Leu, Ile, Val, Cys) and group 5-the bulky aromatic residue (Phe, Tyr, Trp).

Less conservative substitutions may involve the substitution of one amino acid with another having similar characteristics but differing in size, such as: alanine is substituted with an isoleucine residue. Highly non-conservative substitutions may involve the substitution of one acidic amino acid with another amino acid having polar or even basic properties. However, such "aggressive" substitutions cannot be considered ineffective and are not considered because the chemical action is not fully predictable, and aggressive substitutions may bring about unexpected contingent effects in their simple chemical principles.

Of course, such substitutions may involve structures other than the normal L-amino acid. Thus, D-amino acids may be substituted by L-amino acids commonly found in the antigenic peptides of the present invention and remain within the scope of this disclosure. In addition, non-standard amino acids (i.e., in addition to the common natural proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

If substitutions at more than one position are found to result in a peptide having an antigenic activity substantially equal to or greater than the value defined below, the combination of substitutions is tested to determine whether the combined substitutions produce a additive or synergistic effect on the antigenicity of the peptide. The number of positions within the peptide that are simultaneously substituted cannot exceed 4 at the most.

A peptide consisting essentially of the amino acid sequence referred to herein may have one or two non-anchor amino acids (see below in relation to the anchor motif) exchanged, without the situation that the ability to interact with human Major Histocompatibility Complex (MHC) -class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide. In another embodiment, one or two amino acids may be exchanged with their conserved exchange partners (see below) in a peptide consisting essentially of the amino acid sequences described herein, without the situation that the ability to human Major Histocompatibility Complex (MHC) -class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide.

These amino acid residues that do not substantially interact with the T cell receptor can be modified by substituting other amino acids that hardly affect the T cell response and do not interfere with the binding to the relevant MHC. Thus, except for certain limiting conditions, a peptide of the invention may be any peptide comprising a given amino acid sequence or portion or variant thereof (this term as used by the inventors includes oligopeptides or polypeptides).

Table 6: according to SEQ ID NO: 4. 9 and 18 peptide variants and motifs

Longer (elongated) peptides may also be suitable. MHC class I epitopes (typically 8 to 11 amino acids in length) may result from processing of peptides from longer peptides or proteins containing the actual epitope. Residues flanked by actual epitopes are preferably residues that hardly affect the proteolytic cleavage required to expose the actual epitope during processing.

The peptides of the invention may be elongated by up to four amino acids, i.e. 1, 2, 3 or 4 amino acids, and may be present in a ratio of 4: 0 and 0: 4 to either end. The inventive combinations of elongations are shown in table 7.

Table 7: elongated combinations of the peptides of the invention

The stretched/extended amino acid may be the pro-sequence peptide of the protein or any other amino acid. Elongation may be used to enhance the stability or solubility of the peptide.

Thus, the epitope described herein may be identical to a native tumor-associated epitope or tumor-specific epitope, and may also include different peptides of no more than four residues from a reference peptide, so long as they have substantially the same antigenic activity.

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

Thus, the invention proposes peptides and variants of MHC class I epitopes, wherein the total length of the peptide or antibody is between 8 and 100, preferably between 8 and 30, most preferably between 8 and 14 amino acids in length (i.e. 10, 11, 12, 13, 14 amino acids, and if a class II binding peptide is elongated, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids in length).

Of course, the peptides or variants of the invention are capable of binding to human Major Histocompatibility Complex (MHC) class I or II molecules. Binding of the peptide or variant to the MHC complex can be tested using methods known in the art.

Preferably, when peptide-specific T cells of the invention are tested in comparison to the substituted peptide, the peptide concentration is no more than about 1mM, preferably no more than about 1. mu.M, more preferably no more than about 1nM, even more preferably no more than about 100pM, and most preferably no more than about 10pM, if the solubility of the substituted peptide increases to half of the maximum relative to the background peptide. Also preferably, the substituted peptide is recognized by more than one T cell, a minimum of 2, more preferably 3.

In a particularly preferred embodiment of the invention, the peptide consists or consists essentially of a sequence according to SEQ ID NO: 1 to SEQ ID NO: 93, and (b) 93, or a pharmaceutically acceptable salt thereof.

Substantially consisting of "means a peptide of the invention, except that according to SEQ ID NO: 1 to SEQ ID NO: 93 or a variant thereof, and further comprises amino acids located in other N-and/or C-terminal extensions, which are not necessarily capable of forming peptides that are epitopes of MHC molecules.

However, these extended regions are important for efficiently introducing the peptide of the present invention into cells. In one embodiment of the present invention, the peptide is a part of a fusion protein, and contains 80N-terminal amino acids of HLA-DR antigen-associated invariant chain (p33, hereinafter referred to as "Ii") derived from NCBI and GenBank accession number X00497, and the like. In other fusions, the peptides of the invention may be fused to the antibodies described herein, or functional portions thereof, in particular to the sequences of the antibodies, so that the antibodies perform a specific targeting action, or, for example, into dendritic cell-specific antibodies as described herein.

In addition, the peptide or variant may 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.

In the trans peptide bond amino acid, the peptide (-CO-NH-) is not linked to its residue, but its peptide bond is reversed. Such reverse-reverse mimetic peptides (retro-inverso peptidomimetics) can be prepared by methods known in the art, for example: the method described in Meziere et al (Meziere et al,1997) is incorporated herein by reference. This method involves the preparation of a mimetic peptide comprising a change in the backbone (rather than in the side chains). Studies by Meziere et al (Meziere et al,1997) showed that these mimetics are favorable for MHC binding and helper T cell responses. The reverse peptide with NH-CO bond to replace CO-NH peptide bond has greatly raised hydrolysis resistance.

Non-peptide bonds are-CH 2-NH, -CH2S-, -CH2CH2-, -CH ═ CH-, -COCH2-, -CH (oh) CH2-, and-CH 2SO-, etc. U.S. Pat. No. 4897445 proposes a non-solid phase synthesis of non-peptide bonds (-CH2-NH) in polypeptide chains involving polypeptides synthesized according to standard procedures and non-peptide bonds synthesized by the interaction of an amino aldehyde and an amino acid containing NaCNBH 3.

Peptides containing the above sequences may be synthesized with other chemical groups at the amino and/or carboxy terminus of the peptide, thereby improving stability, bioavailability, and/or affinity of the peptide. For example, a hydrophobic group such as benzyloxycarbonyl or dansyl, or a tert-butoxycarbonyl group may be added to the amino terminus of the peptide. Similarly, acetyl or 9-fluorenylmethyloxycarbonyl may be located at the amino terminus of the peptide. Furthermore, a hydrophobic group, a t-butyloxycarbonyl group, or an amino group may be added to the carboxy terminus of the peptide.

In addition, all peptides of the present invention may be synthesized to change their spatial configuration. For example, it is possible to use the right-hand forms of one or more amino acid residues of these peptides, usually not the left-hand forms thereof. Further, at least one amino acid residue of the peptide of the present invention may be substituted with a known non-natural amino acid residue. Such changes may contribute to increased stability, bioavailability and/or binding of the peptides of the invention.

Also, the peptide or variant of the present invention may be chemically modified by reaction of specific amino acids before or after synthesis of the peptide. Examples of such modifications are well known in the art, and are summarized, for example, in "chemical reagents for Protein Modification" (3rd ed. crc Press, 2004) (Lundblad, 2004) by r.lundblad, which is incorporated herein by reference. Although there is no limitation to the chemical modification method of amino acids, it includes (but is not limited to) modification by the following methods: acylation, amidination, lysine pyridoxylation, reductive alkylation, trinitrophenylation of amino groups with 2, 4, 6-trinitrobenzenesulfonic acid (TNBS), amino modification of carboxyl groups and sulfhydryl groups by oxidation of cysteine performic acid 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, carbamoylation with cyanate at basic pH. In this connection, the skilled worker refers to the extensive methods described In chapter 15 of Current Protocols In Protein Science (eds. Coligan et al (John Wiley and Sons NY1995-2000)) (Coligan et al,1995) In connection with the chemical modification of proteins.

Briefly, the arginyl residues of modified proteins and the like are often based on the reaction of ortho-dicarbonyl compounds (e.g., benzaldehyde, 2, 3-butanedione, and 1, 2-alkenylhexanedione) to form adducts. Another example is the reaction of methylglyoxal with an arginine residue. Cysteine can be modified without concomitant modification at nucleophilic sites such as lysine and histidine. Thus, there are a number of reagents available for cysteine modification. The website of Sigma-Aldrich (http:// www.sigma-Aldrich. com) etc. contains information on the specific reagents.

Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds may be formed and oxidized in biopharmaceutical heat treatments. Wood wade reagent K can be used to modify specific glutamic acid residues. N- (3-dimethylaminopropyl) -N' -ethyl-carbodiimide can be used to form intramolecular cross-links of lysine and glutamic acid residues. For example: diethylpyrocarbonate is an agent that modifies histidine residues in proteins. Histidine can also be modified with 4-hydroxy-2-nonenal. The reaction of lysine residues with other alpha-amino groups, for example, facilitates peptide binding to the surface or cross-linking of the protein/peptide. Lysine poly is the attachment point for poly (ethylene) glycol and is also the primary modification site for protein glycosylation. Methionine residues of proteins can be modified by iodoacetamide, bromoethylamine, chloramine T, and the like.

Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosine residues. The crosslinking via the di-tyrosine can be accomplished by hydrogen peroxide/copper ions.

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

Successful modification of therapeutic proteins and peptides containing polyethylene glycol often results in prolonged circulation half-lives when crosslinking of the protein with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used to formulate hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamoylation of potassium cyanate.

A peptide or variant wherein the peptide is modified or contains non-peptide bonds, preferably an embodiment of the invention. In general, peptides and variants (containing at least peptide linkages between amino acid residues) can be synthesized using the solid phase peptide synthesis Fmoc-polyamide model disclosed by Lukas et al (Lukas et al,1981) and the references cited therein. The fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection for the N-amino group. The cleavage was repeated using the highly base-sensitive protecting group in 20% dimethylpiperidine in N, N-dimethylformamide. The side chain function may be protected due to their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), t-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). As long as glutamine and asparagine are C-terminal residues, the side chain amino function protection is provided by a 4, 4' -dimethoxydiphenyl group. The solid support is based on a polydimethylacrylamide polymer, which is composed of three monomers, dimethylacrylamide (backbone monomer), bisacryloylethylene diamine (crosslinker) and N-acryloylsarcosine methyl ester (functional agent). The peptide-resin coupling agent used is an acid-sensitive 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives were added as their preformed symmetrical anhydride derivatives, except 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 test procedures. After completion of the synthesis, the peptide was cleaved from the resin support with concomitant removal of the side chain protecting groups using trifluoroacetic acid at a concentration of 95% containing 50% scavenger mix. Commonly used scavenger mixtures include ethanedithiol, phenol, anisole and water, the exact choice being based on the amino acid composition of the synthetic peptide. Furthermore, it is possible to synthesize peptides using a combination of solid-phase and liquid-phase methods (see, for example, Bruckdorfer et al,2004 and references cited therein)

Trifluoroacetic acid was removed by evaporation in vacuo, followed by titration with diethyl ether bearing the crude peptide. Any scavenger mixture present was purged using a simple extraction procedure (after lyophilization of the aqueous phase, which produced peptides without scavenger mixture). Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nonburg, England).

Purification can be carried out by any one or a combination of the following techniques, such as: recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (typically) reversed phase high performance liquid chromatography (e.g., using an acetonitrile/water gradient separation).

Peptide analysis can be performed using thin layer chromatography, electrophoresis, particularly capillary electrophoresis, solid phase extraction (CSPE), reverse 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.

To select for over-presented peptides, a presentation graph is calculated showing the amount of bit-present as well as the variation in replication in the sample. This feature allows the baseline values of the relevant tumor entity samples to be aligned with those of normal tissue samples. Each of the above features can be incorporated into the over-presentation score by calculating the p-value that adjusts the linear mixture effect model (Pinheiro et al,2015) to adjust the multiple test by the false discovery rate (Benjamini and Hochberg, 1995) (see example 1).

For identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock frozen tissue samples are purified and HLA-related peptides are isolated. The isolated peptides were separated and identified by an on-line nano-electrospray-ionization (nanoESI) liquid chromatography-spectroscopy (LC-MS) experiment. The peptide sequence thus generated was verified by comparing the pattern of fragments of the native tumor associated peptide (TUMAP) recorded in esophageal cancer samples (N16 a 02 positive samples) with the pattern of fragments of the corresponding synthetic reference peptide of the same sequence. Since these peptides were directly identified as ligands for HLA molecules of primary tumors, these results provide direct evidence for the natural processing and presentation of certain peptides on primary cancer tissues from 16 patients with esophageal cancer.

Discovering a pipelinev2.1 (see, e.g., US 2013-. This is achieved by the following method: the label-free differential quantification method is developed using LC-MS acquisition data processed by proprietary data analysis pipelines, in combination with sequence recognition algorithms, spectral clustering, calculating ions, retention time adjustment, state of charge convolution and normalization.

Levels of presentation were established for each peptide and sample, including error estimates. Peptides that are abundantly presented in tumor tissues and peptides that are excessively presented in tumor and non-tumor tissues and organs have been identified.

HLA peptide complexes from esophageal cancer tissue samples were purified and HLA-related peptides were isolated and analyzed using LC-MS (see examples). All TUMAPs contained in this application were identified using the method of primary esophageal cancer specimens, confirming their presentation on primary esophageal cancer.

TUMAP determined on multiple esophageal cancer and normal tissues was quantified using an ion counting method with no marker LC-MS data. This method assumes that the LC-MS signal region of the peptide correlates with its abundance in the sample. All quantitative signals for peptides in various LC-MS experiments were normalized on a central trend basis, averaged per sample, and combined into a histogram (referred to as a presentation graph). The presentation graph integrates different analytical methods, such as: protein database retrieval, spectral clustering, state of charge convolution (neutralization) and retention time calibration and normalization.

In addition to over-presenting the peptides, mRNA expression of potential genes was also tested. mRNA data were obtained by RNA sequencing analysis of normal and cancerous tissues (see example 2). An additional source of normal tissue data is a database of RNA expression data publicly obtained from 3000 normal tissue samples (Lonsdale, 2013). Peptides obtained from protein-encoding mrnas are highly expressed in cancer tissues, but are very low or absent in important normal tissues, and these peptides are preferably incorporated into the present invention.

In addition, a pipeline is foundX direct absolute quantification of MHC-peptides (preferably HLA restricted peptides) on cancer or other infected tissues. Briefly, the total cell count is calculated from the total DNA content of the tissue sample being analyzed. The total peptide amount of TUMAP in tissue samples was determined by nanoLC-MS/MS as the ratio of native TUMAP and known amount of isotopically labeled version of TUMAP, referred to as internal standard. TUMAP separation efficiency determination method: the peptide: MHC for all selected TUMAPs were added to tissue lysates at the earliest time point of the TUMAP isolation procedure and detected by nanoLC-MS/MS after completion of peptide isolation. Total cell counts and total peptide amounts were calculated from three measurements per tissue sample. The peptide-specific isolation efficiency was calculated as the average of 10 spiking experiments measured in triplicate (see example 6 and table 12).

The present invention proposes to be useful in the treatment of cancer/tumors, preferably in the treatment of esophageal cancer presenting either an excess or only the peptide of the invention. These peptides were directly revealed by mass spectrometry, but were naturally presented by HLA molecules in primary human esophageal cancer samples.

Many of the genes/proteins of origin (also designated as "full-length proteins" or "potential proteins") of peptides are highly overexpressed in cancer compared to normal tissues-the "normal tissues" to which the invention relates are healthy esophageal cells or other normal cells, indicating a high association of the tumor with these genes of origin (see example 2). Furthermore, these peptides themselves are also over-presented in tumor tissue ("tumor tissue" in connection with the present invention refers to a sample from a patient with esophageal cancer), but not in normal tissue (see example 1).

HLA-binding peptides are recognized by the immune system, particularly T lymphocytes. T cells can destroy cells presenting the recognized HLA/peptide complex (e.g., esophageal cancer cells presenting peptide derived therefrom).

All peptides of the invention have been shown to have the ability to stimulate a T cell response and are presented in excess and thus can be used to prepare antibodies and/or TCRs of the invention, e.g., soluble TCRs (see example 3 and example 4). Furthermore, peptides, when combined with the corresponding MHC, may also be used to prepare antibodies and/or TCRs, particularly stcrs, of the invention. The respective methods are well known to the skilled worker and can be found in the respective literature. Thus, the peptides of the invention can be used to generate an immune response in a patient, thereby enabling the destruction of tumor cells. The immune response in a patient can be induced by direct administration of the peptide or precursor (e.g., an elongated peptide, protein, or nucleic acid encoding such peptide) to the patient, preferably in combination with an agent that enhances immunogenicity. The immune response derived from this therapeutic vaccine is expected to be highly specific against tumor cells, since the target peptides of the invention present a smaller number of replications on normal tissues, preventing the risk of the patient of an adverse autoimmune response against normal cells.

The present specification also relates to T Cell Receptors (TCRs) comprising one alpha chain and one beta chain ("alpha/beta TCRs"). HAVCR1-001 peptides that bind to TCR and antibodies when presented by MHC molecules are also provided. The specification also relates to nucleic acids, vectors, and host cells for expressing the TCR and the peptides of the specification; and methods of using them. The term "T cell receptor" (abbreviated TCR) refers to a heterodimeric molecule comprising one alpha polypeptide chain (α chain) and one beta polypeptide chain (β chain), wherein the heterodimeric receptor is capable of binding a peptide antigen presented by an HLA molecule. The term also includes so-called γ/δ TCRs.

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

In another aspect, the present disclosure relates to a method according to the present disclosure, wherein the antigen is loaded into class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by binding to a sufficient amount of antigen containing the antigen-presenting cell, or the antigen is loaded into class I or II MHC tetramer/class I or II MHC complex monomer by tetramerization.

The α and β chains of α/β TCRs and the γ and δ chains of γ/δ TCRs are generally considered to have two "domains," respectively, variable and constant domains. The variable domain consists of a combination of a variable region (V) and a linking region (J). The variable domain may also comprise a leader region (L). The beta and delta chains may also include a diversity region (D). The alpha and beta constant domains may also include a C-terminal Transmembrane (TM) domain that anchors the alpha and beta chains to the cell membrane. The term "TCR γ variable domain" as used herein refers to the combination of a TCR γ v (trgv) region without the leader region (L) and a TCR γ (TRGJ) region, and the term TCR γ constant domain refers to an extracellular TRGC region, or a C-terminally truncated TRGC sequence, relative to the TCR of γ/δ. Similarly, the term "TCR δ variable domain" refers to the combination of TCR δ v (trdv) and TCR δ D/J (TRDD/TRDJ) regions without leader (L), and the term "TCR 6 constant domain" refers to the extracellular TRDC region, or a C-terminally truncated TRDC sequence.

The TCRs of the specification preferably bind to the HAVCR1-001 peptide HLA molecule complex with a binding affinity (KD) of about 100. mu.M or less, about 50. mu.M or less, about 25. mu.M or less, or about 10. mu.M or less. More preferred are high affinity TCRs having a binding affinity of about 1 μ M or less, about 100nM or less, about 50nM or less, or about 25nM or less. Non-limiting examples of preferred binding affinity ranges for the inventive TCR include from about 1nM to about 10 nM; about 10nM to about 20 nM; about 20nM to about 30 nM; about 30nM to about 40 nM; about 40nM to about 50 nM; about 50nM to about 60 nM; about 60nM to about 70 nM; about 70nM to about 80 nM; about 80nM to about 90 nM; and about 90nM to about 100 nM.

In connection with the TCRs of the present specification, "specific binding" and grammatical variants thereof are used herein to refer to TCRs having binding affinity (KD) for HAVCR1-001 peptide-HLA molecule complexes of 100 μ M or less.

The α/β heterodimeric TCRs of the present specification may have an introduced disulfide bond 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, unless threonine 48 of TRAC and serine 57 of TRBC1 or TRBC2 are substituted with cysteine residues which form a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant region sequence of the TCR.

The α/β heterodimeric TCRs of the present specification, with or without the introduction of interchain linkages as described above, 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.

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

In one embodiment, a 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 a non-mutated TCR.

In one embodiment, a TCR comprising at least one mutation in the TCR α chain and/or the TCR β chain has a binding affinity and/or binding half-life for the HAVCR1-001 peptide HLA molecule complex that is at least twice the binding affinity of a TCR comprising a non-mutated TCR α chain and/or a non-mutated TCR β chain. Tumor-specific TCR affinity enhancement and its development rely on the window where the optimal TCR affinity exists. The presence of such windows is based on observations: HLA-A2-restricted pathogen-specific TCRs generally have KD values approximately 10-fold lower than HLA-A2-restricted tumor-associated autoantigen-specific TCRs. It is now known that, although tumor antigens may be immunogenic, because tumors are derived from the individual's own cells, only mutant proteins or proteins with altered translational processing will be considered foreign by the immune system. Antigens that are up-regulated or overexpressed (so-called autoantigens) do not necessarily induce a functional immune response against the tumor: t cells expressing TCRs that are highly reactive against these antigens are selected against the thymus in a procedure called central tolerance, i.e. only cells with low affinity TCRs for self-antigens remain. Thus, the affinity of the TCRs or variants of the specification for HAVCR1-001 may be enhanced by methods well known in the art.

The present specification also relates to a method of identifying and isolating a TCR of the present invention, the method comprising: PBMCs were incubated with a2/HAVCR1-001 peptide monomers from HLA-a x 02 negative healthy donors, with tetramer-Phycoerythrin (PE) and analyzed for isolation of high affinity T cells by fluorescence activated cell sorting (FAGS) -Calibur method.

The present specification also relates to a method of identifying and isolating a TCR of the present invention, the method comprising: transgenic mice containing the entire human TCR α β gene locus (1.1and 0.7Mb) were obtained whose T cells express diversified human TCRs to compensate for mouse TCR deficiency, mice were immunized with HAVCR1-001, PBMCs obtained from the transgenic mice were incubated with tetramer-Phycoerythrin (PE), and high affinity T cells were isolated by fluorescence activated cell sorting (FAGS) -Calibur assay.

In one aspect, to obtain a T cell expressing a TCR of the specification, a nucleic acid encoding a TCR-a and/or a TCR- β chain of the specification is cloned into an expression vector, such as a gamma retrovirus or lentivirus. Recombinant viruses are produced and then tested for functions such as antigen specificity and functional avidity. Aliquots of the final product are then used to transduce a population of target T cells (typically PBMCs purified from the patient) and are expanded prior to infusion into the patient. On the other hand, to obtain T cells expressing TCRs of the present specification, TCRRNA is synthesized by techniques known in the art (e.g., in vitro transcription systems). In vitro synthesized TCR RNA is then introduced into primary CD8+ T cells obtained from healthy donors by electroporation to re-express tumor specific TCR-alpha and/or TCR-beta chains.

To increase expression, the nucleic acids encoding the TCRs of the specification may be operably linked to strong activators such as retroviral Long Terminal Repeats (LTRs), Cytomegalovirus (CMV), Murine Stem Cell Virus (MSCV) U3, phosphoglycerate kinase (PGK), beta actin, ubiquitin proteins, and simian virus 40(SV40)/CD43 complex activators, Elongation Factor (EF) -1a, and Spleen Focus Forming Virus (SFFV) activators. In a preferred embodiment, the activator is heterologous to the nucleic acid being expressed. In addition to strong activators, the TCR expression cassettes of the present specification may contain additional elements that increase transgene expression, including a central polypurine tract (CPPT) that promotes nuclear translocation of lentiviral constructs (Follenzi et al,2000), and woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE) that increase transgene expression levels by increasing RNA stability (Zufferey et al, 1999).

The α and β chains of the inventive TCR may be encoded by separate vector nucleic acids, or may be encoded by polynucleotides located on the same vector.

Achieving high levels of TCR surface expression requires high levels of transcription of the TCR-a and TCR- β chains into which the TCR is introduced. To achieve this, the TCR- α and TCR- β chains of the present specification can be cloned into a bicistronic construct in a single vector, which has been shown to overcome this obstacle. The use of a viral internosomal entry site (IRES) between the TCR-alpha and TCR-beta chains results in the coordinated expression of both chains, since both the TCR-alpha and TCR-beta chains are produced from a single transcript that splits into two proteins during translation, thereby ensuring that an equal molar ratio of TCR-alpha and TCR-beta chains is produced. (Schmitt et al 2009).

The nucleic acid encoding the TCR of the specification may be codon optimized for increased expression from the host cell. The genetic code redundancy allows some amino acids to be encoded by more than one codon, but some codons are not "optimized" by others because of the relative availability of matching tRNAs and other factors (Gustafsson et al, 2004). Modification of TCR-a and TCR- β gene sequences such that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as elimination of mRNA instability motifs or cryptic splice sites, has been shown to significantly increase TCR-a and TCR- β gene expression (Scholten et al, 2006).

Furthermore, mismatches between the introduced and endogenous TCR chains may lead to specificity being obtained, which constitutes a significant risk of autoimmunity. For example, the formation of mixed TCR dimers may reduce the number of CD3 molecules available to form the correct paired TCR complex, and thus, may significantly reduce the functional avidity of cells expressing the introduced TCR (Kuball et al, 2007).

To reduce mismatches, the C-terminal domain of the TCR chains introduced in the present specification can be modified to promote interchain affinity while reducing the ability of the introduced chain to pair with endogenous TCRs. These strategies may include replacement of the human TCR- α and TCR- β C-terminal domains (the murinized C-terminal domains) with murine counterparts; generating a second interchain disulfide bond of 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 interacting residues of the C-terminal domains of the TCR-alpha and TCR-beta chains ("knob and hole structures"); direct fusion of TCR-alpha and TCR-beta chain variable domains to CD3 zeta (CD3 zeta fusion) (Schmitt et al 2009).

In one embodiment, the host cell is structurally altered to express a TCR of the specification. In a preferred embodiment, the host cell is a human T cell or T cell progenitor cell. In some embodiments, the T cells or T cell progenitors are obtained from a cancer patient. In other embodiments, the T cells or T cell progenitors are obtained from a healthy donor. The host cells of the present specification may be allogeneic or autologous with respect to the patient to be treated. In one embodiment, the host is a γ/δ T cell transformed to express an α/β TCR.

"pharmaceutical composition" refers to a composition suitable for use in a medical facility for the human body. Preferably, the pharmaceutical compositions are sterile and manufactured according to GMP guidelines.

Pharmaceutical compositions include the peptide 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 peptide wherein the peptide is modified by making acid or base salts of pharmaceutical agents. For example, acid salts are prepared with the free base (usually where the neutral drug has a neutral-NH 2 group) reacted with a suitable acid. Suitable acids for preparing the acid salts include organic acids such as: acetic, propionic, hydroxy, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, benzenesulfonic, salicylic, and the like, as well as inorganic acids such as: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. In contrast, base salt formulations of acidic groups that may be presented on a peptide are prepared using pharmaceutically acceptable bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

In a particularly preferred embodiment, the pharmaceutical composition comprises the peptide in the form of acetic acid (acetate), trifluoroacetate or hydrochloric acid (chloride).

The agent of the invention is preferably an immunotherapeutic agent, for example, a vaccine. The vaccine may be administered directly to the affected organ of the patient, either systemically by i.d., i.m., s.c., i.p., and i.v. injection, or applied in vitro to cells from the patient or a cell line thereof (which are then injected into the patient), or ex vivo to a subpopulation of cells from immune cells from the patient (which are then re-administered to the patient). If the nucleic acid is injected into the cells in vitro, it may be beneficial to transfect the cells to co-express an immunostimulatory cytokine (e.g., interleukin-2). The peptides may be administered entirely alone, in combination with an immunostimulating adjuvant (see below), or in combination with an immunostimulating cytokine, or in a suitable delivery system (e.g. liposomes). The peptide may also be conjugated to form a suitable carrier, such as Keyhole Limpet Hemocyanin (KLH) or mannoprotein (see WO 95/18145 and (Longenecker et al, 1993)). The peptide may also be labeled, may be a fusion protein, or may be a hybrid molecule. Peptides of the sequences given in the present invention are expected to stimulate CD4 or CD 8T cells. However, CD 8T cell stimulation was more effective with the help of CD 4T-helper cells. Thus, for stimulation of the MHC class I epitope of CD 8T cells, a fusion partner or fragment of a hybrid molecule provides an appropriate epitope for stimulation of CD4 positive T cells. CD 4-and CD 8-stimulating epitopes are well known in the art and include the epitopes identified in the present invention.

In one aspect, the vaccine comprises a polypeptide comprising at least SEQ ID NO: 1 to SEQ ID NO: 93, preferably 2 to 50, more preferably 2 to 25, even more preferably 2 to 20, most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. Peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

In another aspect, the invention features a nucleic acid (e.g., a polynucleotide) encoding a peptide or peptide variant of the invention. A polynucleotide may be, for example, DNA, cDNA, PNA, RNA, or combinations thereof, which may be single-and/or double-stranded, or a native or stabilized form of a polynucleotide (e.g., a polynucleotide having a phosphorothioate backbone), and which may or may not contain an intron so long as it encodes a peptide. Of course, polynucleotides can only encode peptides that incorporate natural peptide bonds and contain natural amino acid residues. In another aspect, the invention features an expression vector for expressing a polypeptide according to the invention.

For the ligation of polynucleotides, various methods have been developed, particularly for DNA, ligation can be performed by a method of supplementing a vector with a ligatable end or the like. For example, a complementary homopolymer track can be added to the DNA fragment, after which the DNA fragment is inserted into the vector DNA. The vector and DNA fragment are then bound by hydrogen bonding of the complementary homopolymer tail, thereby forming a recombinant DNA molecule.

Synthetic linkers containing one or more cleavage sites provide an alternative method for ligating DNA fragments into vectors. Synthetic linkers containing various restriction endonucleases are commercially available through a variety of tubes, including from International Biotechnology Inc., New Haven, CN, USA.

A desirable modification method of the DNA encoding the polypeptide of the present invention is to use the polymerase chain reaction method employed by Saiki et al (Saiki et al, 1988). This method can be used to introduce the DNA into a suitable vector (e.g., by designing appropriate cleavage sites), and can also be used to modify the DNA by other useful methods known in the art. If viral vectors are used, either poxvirus vectors or adenoviral vectors are preferred.

The DNA (or RNA in the case of retroviral vectors) may then be expressed in a suitable host to produce a polypeptide comprising a peptide or variant of the invention. Thus, a DNA encoding a peptide or variant of the invention may be used according to known techniques, suitably modified by the methods described herein, to construct an expression vector, which is then used to transform a suitable host cell, thereby expressing and producing a 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.

DNA (or RNA in the case of retroviral vectors) encoding a polypeptide comprising a compound of the invention may be added 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 needs to be maintained episomally or bound to each other.

In general, the DNA can be attached to an expression vector (e.g., a plasmid) in the proper orientation and correct expression reading frame. If necessary, the DNA may be linked to corresponding transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control functions are typically present in expression vectors. The vector is then introduced into the host by standard methods. In general, not all hosts will be transformed by the vector. Therefore, it is necessary to select transformed host cells. The selection method involves the insertion of a DNA sequence encoding a selectable attribute (e.g., antibiotic resistance) in the transformed cell into the expression vector using any necessary control elements.

Alternatively, the gene having such selective properties may be on another vector which is used to co-transform the desired host cell.

Host cells transformed with the recombinant DNA of the invention are then cultured under suitable conditions, as described herein, familiar to those skilled in the art, for a time sufficient to express the peptide that can be recovered thereafter.

There are many known expression systems, including bacteria (e.g., E.coli and Bacillus subtilis), yeasts (e.g., yeast), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The system may preferably be mammalian cells, such as CHO cells from the ATCC Cell Biology Collection (Cell Biology Collection).

Typical mammalian cell constitutive expression vector plasmids include CMV or SV40 activator with a suitable poly-A tail and resistance markers (e.g., neomycin). An example is pSVL obtained from Pharmacia (Piscataway, New Jersey, USA). An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, generally available from Stratagene cloning Systems, Inc. (La Jol1a, CA 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrative plasmids (YIp) with the insertion of the yeast selectable markers HIS3, TRP1, LEU2 and URA 3. The pRS413-416 plasmid is a yeast centromere plasmid (Ycp). CMV activator-based vectors (e.g., from Sigma-Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, as well as N-terminal or C-terminal markers in various combinations of FLAG, 3xFLAG, C-myc, or MATN. These fusion proteins can be used for detection, purification and analysis of recombinant proteins. Dual label fusion provides flexibility for detection.

The potent human Cytomegalovirus (CMV) activator regulatory region allows expression of constitutive proteins in COS cells at levels as high as 1 mg/L. For weaker cell lines, protein levels are generally below 0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication competent COS cells. For example, CMV vectors may comprise the origin of pMB1 (a derivative of pBR 322) replication in bacterial cells, the calcium-lactamase gene for ampicillin resistance selection in bacteria, the origin of hGH polyA and f 1. Vectors containing preproinsulin leader (PPT) sequences can be secreted into the medium for purification using anti-FLAG antibodies, resins and plate-directed FLAG fusion proteins. Other vectors and expression systems for use with a variety of host cells are well known in the art.

In another embodiment, two or more peptides or peptide variants of the invention are encoded and, thus, expressed in a sequential order (similar to a "string of beads" construct). To achieve this, the peptides or peptide variants may be linked or fused together by extensions of linker amino acids (e.g., LLLLLL), or may be linked without any additional peptide between them. These constructs are also useful in cancer therapy, and induce immune responses involving MHC class I and MHC class II molecules.

The invention also relates to a host cell transformed with the polynucleotide vector construct of the invention. The host cell may be a prokaryotic cell or a eukaryotic cell. In some instances, bacterial cells are preferred prokaryotic host cells, typically E.coli strains, e.g., E.coli strain DH5 (obtained from Bethesda Research Laboratories, Inc. (Bethesda, Md., USA)) and RR1 (obtained from 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: mouse, rat, monkey or human fibroblasts and colon cancer cell lines. Yeast host cells include YPH499, YPH500 and YPH501, and are generally available from Stratagene Cloning Systems, Inc. (La Jol1a, CA92037, USA). The preferred mammalian host cells include CCL61 cells from ATCC as Chinese Hamster Ovary (CHO) cells, CRL 1658 cells from ATCC as NIH Swiss mouse embryo cells NIH/3T3, CRL1650 cells from ATCC as monkey kidney-derived COS-1 cells, and 293 cells from human embryonic kidney cells. The preferred insect cell is Sf9 cell, which can be transfected with baculovirus expression vector. A summary of the selection of suitable host cells for expression can be found in textbooks (Paulina Balb. sup. s and Argelia Lorence. Methods in Molecular Biology recombination Gene expression, Reviews and Protocols, Part One, Second Edition, ISBN 978-1-58829-262-9) and other documents known to the skilled worker.

Transformation of a suitable host cell containing a DNA construct of the invention may be accomplished using well known methods, generally depending on the type of vector used. For the transformation of prokaryotic host cells, see, for example, Cohen et al (Cohen et al,1972) and (Green and Sambrook, 2012). Transformation of yeast cells is described in Sherman et al (Sherman et al, 1986). The methods described in Beggs (Beggs, 1978) are also useful. For vertebrate cells, reagents for transfecting these cells, etc., e.g., calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, Inc. or Life Technologies, Inc. (Gaithersburg, MD20877, USA). Electroporation may also be used to transform and/or transfect cells, and is a well-known method used in the art for transforming yeast cells, bacterial cells, insect cells, and vertebrate cells.

Successfully transformed cells (i.e.cells containing the DNA construct of the invention) can be identified by well-known methods, such as PCR. Alternatively, proteins present in the supernatant may be detected using antibodies.

It will be appreciated that certain host cells of the invention are useful for the production of peptides of the invention, such as bacterial cells, yeast cells and insect cells. However, other host cells may be useful for certain therapeutic approaches. For example, antigen presenting cells (e.g., dendritic cells) can be used to express the peptides of the invention, allowing them to be loaded with the corresponding MHC molecules. Thus, the present invention provides a host cell comprising a nucleic acid or expression vector of the invention.

In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell or an antigen-presenting cell. On 29/4/2010, the U.S. Food and Drug Administration (FDA) approved recombinant fusion proteins loaded with Prostatic Acid Phosphatase (PAP) for use in the treatment of asymptomatic or mildly symptomatic metastatic HRPC (Rini et al, 2006; Smallet al, 2006).

In another aspect, the invention features a method of formulating a peptide and variants thereof, the method including culturing a host cell and isolating the peptide from the host cell or culture medium thereof.

In another embodiment, the peptide, nucleic acid or expression vector of the invention is used in medicine. For example, the peptide or variant thereof may be prepared as an intravenous (i.v.) injection, a subcutaneous (s.c.) injection, an intradermal (i.d.) injection, an intraperitoneal (i.p.) injection, an intramuscular (i.m.) injection. Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. injections. Preferred methods of DNA injection are i.d., i.m., s.c., i.p., and i.v. injection. For example, 50. mu.g to 1.5mg, preferably 125. mu.g to 500. mu.g, of peptide or DNA is administered, depending on the particular peptide or DNA. The above dose ranges were successfully used in previous trials (Walter et al, 2012).

Polynucleotides used for active immunization may be in substantially purified form, or may be coated onto a carrier or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for the design and introduction of such nucleic acids are well known in the art. For example, there is a summary thereof in the literature (Teufel et al, 2005). Polynucleotide vaccines are readily prepared, but the mode of action of these vectors to induce an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems based on adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus, or mixed viruses containing more than one viral element. Non-viral delivery systems, including cationic liposomes and cationic polymers, are well known in the art for DNA delivery. Physical delivery systems, such as by "gene gun," can also be used. The peptide or nucleic acid encoding the peptide can be a fusion protein, e.g., containing an epitope that stimulates T cells to perform the above-described CDRs.

The agents of the invention may also include one or more adjuvants. Adjuvants are those substances that non-specifically enhance or potentiate an immune response (e.g., an immune response to an antigen mediated by CD 8-positive T cells and helper T (TH) cells and are therefore considered useful for the agents of the invention AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimodresiquimod、ImuFact IMP321、Interleukins IL-2, IL-13, IL-21, interferon alpha or beta, or polyethylene glycol derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs,Lipovac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide eISA 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, and,Carrier system, polylactide-based composite glycolide [ PLG]And dextran microparticles, recombinant human lactoferrin SRL172, viral and other virus-like particles, YF-17D, VEGFtrap, R848, β -glucan, Pam3Cys, QS21 stimulators from Aquila corporation derived from saponin, mycobacterial extracts, and bacterial cell wall synthesis mimics, as well as other proprietary adjuvants such as: ribi's Detox, Quil or Superfos. Preferred adjuvants are, for example: freund's adjuvant or GM-CSF. Several dendritic cell-specific immunoadjuvants (e.g. MF59) and methods for their preparation have been described (Allison and Krummel, 1995). Cytokines may also be used. Some cytokines directly affect dendritic cell migration to lymphoid tissues (e.g., TNF-), accelerate dendritic cell maturation to effective antigen presenting cells of T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5849589, specifically incorporated herein by reference in its entirety), and act as immunological adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN- α, IFN- β) (Gabrilovich et al, 1996).

CpG immunostimulatory oligonucleotides are reported to enhance the effect of adjuvants in vaccines. Without being bound by theory, CpG oligonucleotides can function by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), mainly TLR 9. CpG-induced 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. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and enhanced Cytotoxic T Lymphocyte (CTL) production, even with the loss of CD 4T cell repertoire. The activation-induced TH1 bias of TLR9 was maintained even in the presence of vaccine adjuvants such as: alum or Freund's incomplete adjuvant (IFA) which normally promotes TH2 migration. CpG oligonucleotides, when prepared or co-administered with other adjuvants or formulations such as microparticles, nanoparticles, fat emulsions or the like, exhibit enhanced adjuvant activity, which is particularly necessary to induce a strong response when the antigen is relatively weak. They also accelerated the immune response, reducing antigen dose by about two orders of magnitude, and in some experiments, produced similar antibody responses to full dose vaccines without CpG (Krieg, 2006). U.S. Pat. No. 4, 6406705, 1 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to elicit an antigen-specific immune response. One CpG TLR9 antagonist is dlim (dual stem-loop immunomodulator) from Mologen, berlin, germany, which is a preferred ingredient of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as: the RNA binds TLR7, TLR8, and/or TLR 9.

Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), dsRNA mimetics, e.g., 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 immunologically active small molecules and antibodies, e.g.: cyclophosphamide, sumicitizumab,Celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA 4, other antibody targeting major structures of the immune system (e.g., anti-CD 40, anti-TGF β, anti-TNF α receptor) and SC58175, which may have therapeutic and/or adjuvant effects. The skilled artisan can readily determine the amounts and concentrations of adjuvants and additives useful in the present invention without undue experimentation.

Preferred adjuvants are particulate formulations of anti-CD 40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon alpha, CpG oligonucleotides and derivatives, poly (I: C) and derivatives, RNA, sildenafil and PLG or viral particles.

In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is selected from the group consisting of colony stimulating factor-containing preparations, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargrastim), cyclophosphamide, imiquimod, resiquimod and interferon- α.

In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is selected from the group consisting of colony stimulating factor-containing preparations, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargrastim), cyclophosphamide, imiquimod and resimiquimod. In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. More preferred adjuvants are Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, poly-ICLCAnd anti-CD 40 mAB or a combination thereof.

The composition can be administered by parenteral injection, such as subcutaneous injection, intradermal injection, intramuscular injection, or oral administration. For this purpose, the peptides and other selective molecules are dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. In addition, the composition may comprise adjuvants such as: buffers, binders, impactors, diluents, flavorants, lubricants, and the like. These peptides may also be used in combination with immunostimulatory substances, such as: a cytokine. Further adjuvants which can be used in such compositions are known from Handbook of pharmaceutical excipients (Kibbe, 2000) et al, by a. The combination is useful for the prevention, prophylaxis and/or treatment of adenoma or cancerous disease. For example, there are exemplified formulations in EP 2112253.

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. And different cancer-related signaling pathways are attacked. This has the advantage over other vaccines that target only one or a few targets, which may lead to easy adaptation of the tumor to attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Thus, the combination of several tumor-associated peptides ensures that each tumor bears at least some of the targets. The composition is designed in such a way that it is expected that each tumor can express several antigens and cover several independent pathways required for tumor growth and maintenance. Thus, the vaccine can be readily "off-the-shelf" for use in a larger patient population. This means that patients pre-selected for vaccine treatment can be restricted to HLA typing without any additional biomarker assessment of antigen expression, but still ensure that multiple targets are simultaneously challenged by an induced immune response, which is important for therapeutic efficacy (Banchereau et al, 2001; Walter et al, 2012).

The term "scaffold" as used herein refers to a molecule that specifically binds to a (e.g. antigenic) determinant. In one embodiment, the scaffold is capable of directing the entity (e.g., the (second) antigen-binding moiety) to which it is attached to a target of interest, e.g., to a specific type of tumor cell or tumor substrate bearing an antigenic determinant (e.g., a complex of a peptide and MHC according to the present 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 antibody heavy chain variable regions and antibody light chain variable regions, bound proteins including at least one ankyrin repeat motif and Single Domain Antigen Binding (SDAB) molecules, aptamers, (soluble) TCRs, and (modified) cells, such as allogeneic or autologous T cells. To assess whether a certain molecule is a scaffold bound to a target, a binding assay can be performed.

By "specific" binding is meant that the scaffold binds better to the peptide-MHC complex of interest than to other native peptide-MHC complexes to the extent that a scaffold possessing an active molecule capable of killing cells bearing a particular target is not capable of killing another cell not bearing the particular target but presenting one or more other peptide-MHC complexes. If the cross-reactive peptide-MHC peptide is not native, i.e., not from the human HLA-polypeptide group, binding to other peptide-MHC complexes is not critical. Assays to assess target cell killing are well known in the art. They should be carried out with target cells (primary cells or cell lines) or cells loaded with peptides, which contain unaltered peptide-MHC presentation, in order to reach the level of native peptide-MHC.

Each scaffold may include a label that detects binding to the scaffold by determining the presence or absence of a signal provided by the tag. For example, the scaffold may be labeled with a fluorescent dye or any other suitable cellular marker molecule. Such marker molecules are well known in the art. For example, fluorescent labeling by a fluorescent dye may provide visualization of the bound aptamer by fluorescence or laser scanning microscopy or flow cytometry.

Each scaffold can be conjugated to a second active molecule (e.g., IL-21, anti-CD 3, anti-CD 28).

For further information on polypeptide scaffolds, see, e.g., in the background section of WO2014/071978a1, and incorporated by reference.

The invention also relates to aptamers. Aptamers (see, e.g., 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 suitable alternatives for the development of targeted therapies. Aptamers have been shown to selectively bind complex targets with high affinity and specificity.

Aptamers that recognize cell surface molecules have been identified over the past decade and provide a means for developing diagnostic and therapeutic methods. Aptamers are promising candidates for biomedical applications, since they have been shown to be almost non-toxic and immunogenic. In fact, aptamers, such as prostate specific membrane antigen recognition aptamers, have been successfully used for targeted therapy and have been shown to function in xenografts in vivo models. In addition, it is recognized that aptamers to specific tumor cell lines have also been identified.

DNA aptamers can be selected to reveal a broad spectrum of signature properties of various cancer cells, particularly those from solid tumors, while non-tumorigenic and predominantly healthy cells are not recognized. If the aptamers identified not only recognize tumor-specific subtypes, but also interact with a range of tumors, this makes the aptamers suitable as so-called broad-spectrum diagnostic and therapeutic tools.

Furthermore, studies of cell binding behavior with flow cytometry showed that aptamers displayed good affinity in the nanomolar range.

Aptamers are used for diagnostic and therapeutic purposes. In addition, it may also be shown that some aptamers are taken up by tumor cells and thus can enter tumor cells as molecular excipients for targeted delivery of anticancer agents, such as siRNA.

Aptamers can be selected against targets of complexes such as cells and tissues and peptide complexes and MHC molecules according to the current invention comprising, preferably including, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 93 using the cellular SELEX (systematic evolution of ligands by exponential enrichment) technique.

The peptides of the invention are useful for the generation and development of specific antibodies against MHC/peptide complexes. These antibodies are useful in therapy to target toxins or radioactive substances to diseased tissue. Another use of these antibodies is to target radionuclides to diseased tissue for imaging purposes (e.g., PET). This may help to detect small metastases or to determine the size and accurate location of diseased tissue.

Thus, another aspect of the invention is directed to a method of generating a recombinant antibody that specifically binds to class I or II human Major Histocompatibility Complex (MHC) complexed with an HLA-restricted antigen, the method comprising: immunizing a genetically engineered non-human mammal comprising a molecule expressing said Major Histocompatibility Complex (MHC) class I or II with a soluble form of a (MHC) class I or II molecule complexed with an HLA-restricted antigen; separating the mRNA molecules from antibodies raised to said non-human mammalian cells; generating a phage display library displaying protein molecules encoded by said mRNA molecules; and separating at least one bacteriophage from said phage display library, said at least one bacteriophage displaying said antibody specifically binding to said human Major Histocompatibility Complex (MHC) class I or II complexed to an HLA-restricted antigen.

Another aspect of the invention provides an antibody that specifically binds to a class I or II human Major Histocompatibility Complex (MHC) complexed to an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and/or a chimeric antibody.

Corresponding methods for producing such antibodies and single chain class I major histocompatibility complexes, as well as other tools for producing such antibodies, are disclosed in WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and publications (Cohen et al,2003 a; Cohen et al,2003 b; Denkberg et al,2003), all references being incorporated herein by reference in their entirety for the purposes of the present invention.

Preferably, the binding affinity of the antibody to the complex is less than 20 nanomolar, preferably less than 10 nanomolar, which is also considered to be "specific" in the context of the present invention.

The present invention relates to a peptide comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 93 or a sequence of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93, or a variant which induces a T cell cross-reaction with said variant peptide, wherein said peptide is not a full-length polypeptide.

The invention further relates to a peptide comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 93, or a sequence identical to SEQ ID NO: 1 to SEQ ID NO: 93, wherein the total length of the peptide or variant is from 8 to 100, preferably from 8 to 30, most preferably from 8 to 14 amino acids.

The invention further relates to peptides of the invention having the ability to bind to a Major Histocompatibility Complex (MHC) class I or II molecule.

The invention further relates to a peptide of the invention, wherein the peptide consists or consists essentially of SEQ ID NO: 1 to SEQ ID NO: 93, or a pharmaceutically acceptable salt thereof.

The invention further relates to a peptide of the invention, wherein the peptide is (chemically) modified and/or comprises non-peptide bonds.

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

Another embodiment of the invention relates to a non-natural peptide, wherein said peptide consists or consists essentially of a peptide 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. The salts of the peptides of the invention are substantially different from peptides in their in vivo state, since these in vivo produced peptides are not salts. The non-native salt form of the peptide mediates the solubility of the peptide, particularly in the case of pharmaceutical compositions comprising the peptide, e.g., the peptide vaccines disclosed herein. In order to effectively provide a peptide to a subject in need of treatment, it is desirable that the peptide has sufficient, at least substantial, solubility. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. These salts of the invention include alkali and alkaline earth salts, such as salts of the Hofmeister series, comprising anionic PO ₄ ^3-、SO₄ ^2-、CH₃COO^-、Cl^-、Br^-、NO3^-、ClO4^-、I^-、SCN^-And the cation NH4⁺、Rb⁺、K⁺、Na⁺、Cs⁺、Li⁺、Zn²⁺、Mg²⁺、Ca²⁺、Mn²⁺、Cu²⁺And Ba²⁺. In particular, the salt is selected from (NH)₄)₃PO₄、(NH₄)₂HPO₄、(NH₄)H₂PO₄、(NH₄)₂SO₄、NH₄CH₃COO、NH₄Cl、NH₄Br、NH₄NO₃、NH₄CIO₄、NH₄I、NH₄SCN、Rb₃PO₄、Rb₂HPO₄、RbH₂PO₄、Rb₂SO₄、Rb₄CH₃COO、Rb₄Cl、Rb₄Br、Rb₄NO₃、Rb₄CIO₄、Rb₄I、Rb₄SCN、K₃PO₄、K₂HPO₄、KH₂PO₄、K₂SO₄、KCH₃COO、KCl、KBr、KNO₃、KClO₄、KI、KSCN、Na₃PO₄、Na₂HPO₄、NaH₂PO₄、Na₂SO₄、NaCH₃COO、NaCl、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、BaI₂And Ba (SCN)₂. NH acetic acid and MgCl are particularly preferable₂、KH₂PO₄、Na₂SO₄KCl, NaCl and CaCl₂For example: chloride or BAcid (trifluoroacetic) acid salt.

In general, peptides and variants (containing at least peptide linkages between amino acid residues) can be synthesized using the solid phase peptide synthesis Fmoc-polyamide model disclosed by Lukas et al (Lukaset al,1981) and the references cited therein. The fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection for the N-amino group. The cleavage was repeated using the highly base-sensitive protecting group in 20% dimethylpiperidine in N, N-dimethylformamide. The side chain function may be protected due to their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), t-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). As long as glutamine and asparagine are C-terminal residues, the side chain amino function protection is provided by a 4, 4' -dimethoxydiphenyl group. The solid support is based on a polydimethylacrylamide polymer, which is composed of three monomers, dimethylacrylamide (backbone monomer), bisacryloylethylene diamine (crosslinker) and N-acryloylsarcosine methyl ester (functional agent). The peptide-resin coupling agent used is an acid-sensitive 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives were added as their preformed symmetrical anhydride derivatives, except 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 test procedures. After completion of the synthesis, the peptide was cleaved from the resin support with concomitant removal of the side chain protecting groups using trifluoroacetic acid at a concentration of 95% containing 50% scavenger mix. Commonly used scavenger mixtures include ethanedithiol, phenol, anisole and water, the exact choice being based on the amino acid composition of the synthetic peptide. Furthermore, it is possible to use solid-phase and liquid-phase methods in combination for the synthesis of peptides (see, for example, Bruckdorfer et al,2004 and references cited therein).

Trifluoroacetic acid was removed by evaporation in vacuo, followed by titration with diethyl ether bearing the crude peptide. Any scavenger mixture present was purged using a simple extraction procedure (after lyophilization of the aqueous phase, which produced peptides without scavenger mixture). Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nonburg, England).

Purification can be carried out by any one or a combination of the following techniques, such as: recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (typically) reversed phase high performance liquid chromatography (e.g., using an acetonitrile/water gradient separation).

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

The invention further relates to a nucleic acid according to the invention, being DNA, cDNA, PNA, RNA or a combination thereof.

The invention further relates to an expression vector capable of expressing the nucleic acid of the invention.

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

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

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

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

The invention further relates to methods of the invention wherein an antigen is loaded onto an MHC class I or II molecule expressed on the surface of a suitable antigen-presenting cell by contacting the antigen with the antigen-presenting cell in a sufficient amount.

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

The invention further relates to activated T cells made by the methods of the invention, wherein the T cells selectively recognize a cell that aberrantly expresses a polypeptide comprising an amino acid sequence of the invention.

The invention further relates to a method of killing target cells in a patient, wherein the target cells in the patient abnormally express a polypeptide comprising any of the amino acid sequences of the invention, the method comprising administering to the patient an effective amount of a T cell of the invention.

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

The invention further relates to the use of the invention, wherein the medicament is a vaccine. The invention further relates to a use according to the invention, wherein the medicament is effective against cancer.

The invention also relates generally to the use of the invention, wherein the cancer cell is an esophageal cancer cell or other solid or hematological tumor cell, such as: lung cancer, bladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellular cancer, renal cell carcinoma, brain cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, prostate cancer, and leukemia.

The invention further relates to a specific marker protein and biomarker, herein referred to as "target", based on the peptides of the invention, which can be used for the diagnosis and/or prognosis of esophageal cancer. The invention also relates to these novel targets for use in cancer therapy.

The term "antibody" is defined herein in a broad sense to include both polyclonal and monoclonal antibodies. In addition to intact or "whole" immunoglobulin molecules, the term "antibody" also includes fragments (e.g., CDR, Fv, Fab, and Fc fragments) or polymers of these immunoglobulin molecules and humanized immunoglobulin molecules, so long as they exhibit any of the desired properties of the invention (e.g., specific binding of an esophageal cancer marker (poly) peptide, delivery of a toxin to esophageal cancer cells at increased levels of expression of a cancer marker gene, and/or inhibition of the activity of an esophageal cancer marker polypeptide).

Antibodies of the invention may be purchased from commercial sources whenever possible. The antibodies of the invention may also be prepared using known methods. The skilled person will appreciate that full length esophageal cancer marker polypeptides, or fragments thereof, may be used to prepare antibodies of the invention. The polypeptides used to produce the antibodies of the invention may be partially or wholly purified from natural sources or may be produced using recombinant DNA techniques.

For example, a cDNA of the invention encoding a peptide, e.g., a peptide according to SEQ ID NO: 1 to SEQ ID NO: 93, or a variant or fragment thereof, may be expressed in prokaryotic (e.g., bacterial) or eukaryotic cells (e.g., yeast, insect or mammalian cells), after which the recombinant protein may be purified and used to produce a monoclonal or polyclonal antibody preparation that specifically binds to the esophageal cancer marker polypeptide used to produce the antibodies of the invention.

One skilled in the art will recognize that two or more different sets of monoclonal or polyclonal antibodies maximize the likelihood of obtaining an antibody with the specificity and affinity (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy) required for its intended use. Depending on the use of the antibody, its desired activity is tested by known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance in producing and testing antibodies, see, e.g., Greenfield, 2014). For example, the antibody can be detected by 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 detected according to known clinical test methods.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a large homogeneous population of antibodies, i.e., a population of antibodies consisting of identical antibodies, except for natural mutations that may be present in minor amounts. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chains are identical (homogeneous) to the corresponding sequences of antibodies obtained from a particular species or antibodies belonging to a particular antibody type and class, while the remaining chains are identical (homogeneous) to the corresponding sequences of antibodies obtained from other species or antibodies belonging to a particular antibody type and sub-class, and fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. patent No. 4816567, which is incorporated herein in its entirety).

The monoclonal antibodies of the invention may be made using hybridoma methods. In the hybridoma method, a mouse or other appropriate host animal is typically primed with an immunizing agent to elicit or produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

Monoclonal antibodies can also be made by recombinant DNA methods, such as: as described in U.S. patent No. 4816567. 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).

In vitro methods are also suitable for the production of monovalent antibodies. Digestion of antibodies to produce fragments of antibodies, particularly Fab fragments, can be accomplished using conventional techniques known in the art. Digestion may be accomplished, for example, by using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4342566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments (each having an antigen binding site) and a residual Fc fragment. Pepsin treatment produced an F (ab ') 2 fragment and a pFc' fragment.

Antibody fragments, whether attached to other sequences or not, may include insertions, deletions, substitutions, or other selective modifications of particular regions or particular 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 some additional attributes, such as: amino acids that can bind to disulfide bonds are deleted/added to increase their biological life, alter their secretory properties, etc. In any case, the antibody fragment must possess bioactive properties such as: binding activity, modulating binding capacity of the binding domain, and the like. The functional or active region of an antibody can be determined by genetic mutation of a particular region of the protein, subsequent expression and testing of the expressed polypeptide. Such methods are well known to those skilled in the art and may include site-specific genetic mutations in the nucleic acids encoding the antibody fragments.

The antibody of the present invention may further include a humanized antibody or a human antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibody immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab' or other antigen binding sequences of an antibody) which contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are substituted with residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit having the specificity, affinity and capacity therefor. In some cases, Fv Framework (FR) residues of the human immunoglobulin are substituted for corresponding non-human residues. Humanized antibodies may also include residues found in neither the recipient antibody nor the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of the same sequence of a human immunoglobulin. Ideally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. In general, humanized antibodies have one or more amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and are typically obtained from an "import" variable domain. Humanization can be essentially accomplished by substituting rodent CDRs or CDR sequences with corresponding human antibody sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567) in which substantially less than an entire human variable domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are immunized to produce fully human antibodies in the absence of endogenous immunoglobulin can be used. For example, it is described that homozygous deletion of antibody heavy chain junction region genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in such germline variant mice will result in the production of human antibodies following antigen challenge. Human antibodies can also be produced in phage display libraries.

The antibodies of the invention are preferably administered to a subject in the form of a pharmaceutically acceptable carrier. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable 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, the carrier may also include sustained release formulations such as: semipermeable matrices of solid hydrophobic polymers containing the antibody, wherein the matrices are in the form of shaped articles, 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, for example, on the route of administration and the concentration of the antibody.

The antibody can be administered to a subject, patient, or cell by injection (e.g., intravenously, intraperitoneally, subcutaneously, intramuscularly) or by other means such as infusion, ensuring that it is delivered to the blood in an effective form. These antibodies can also be administered by intratumoral or peritumoral routes, thereby exerting local and systemic therapeutic effects. Topical or intravenous injection is preferred.

Effective dosages and schedules for administration of the antibodies 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 antibody that must be administered will vary depending on factors such as: the subject receiving the antibody, the route of administration, the antibody used, and the particular type of drug being used. Typical daily dosages of antibody used alone may range from about 1. mu.g/kg up to 100mg/kg body weight or more, depending on the factors mentioned above. After administration of the antibody, preferably for the treatment of esophageal cancer, the efficacy of the therapeutic antibody can be assessed by various methods well known to the skilled artisan. For example: the size, amount, and/or distribution of cancer in a subject receiving treatment can be monitored using standard tumor imaging techniques. An antibody administered as a result of treatment prevents tumor growth, causes tumor shrinkage, and/or prevents the development of new tumors, as compared to the course of disease in the absence of administration of the antibody, and is an antibody effective for the treatment of cancer.

In another aspect of the invention, a method of making a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex is provided. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by complementarity determining region-targeted mutagenesis. For the purpose of T cell receptor selection, phage display can be used (us 2010/0113300, (litdy et al, 2012)). For the purpose of stabilizing T cell receptors during phage display and when actually used as drugs, the alpha and beta chains can be linked by non-native 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 may be linked to toxins, drugs, cytokines (see US 2013/0115191), domain recruitment effector cells such as anti-CD 3 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 2004/033685a1 and WO 2004/074322a 1. Combinations of stcrs are described in WO2012/056407a 1. Further methods of preparation are disclosed in WO 2013/057586a 1.

In addition, the peptides and/or TCRs or antibodies or other binding molecules of the invention can be used to validate the pathologist's diagnosis of cancer on the basis of biopsy samples.

The antibodies or TCRs may also be used in vivo diagnostic assays. Generally, antibodies are labeled with radionuclides (e.g., 111In, 99Tc, 14C, 131I, 3H, 32P, or 35S) to localize tumors by immunoscintigraphy. In one embodiment, the antibody or fragment thereof binds to two or more target extracellular domains of proteins selected from the group consisting of the proteins described above, and has an affinity value (Kd) of less than 1X 10. mu.M.

Diagnostic antibodies can be labeled by various imaging methods using probes suitable for detection. Probe detection methods include, but are not limited to, fluorescence, light, confocal and electron microscopy methods; magnetic resonance imaging and spectroscopy techniques; 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 earths, paramagnetic iron, fluorine-18, and other positron emitting radionuclides. In addition, the probe may be bifunctional or multifunctional, and detection may be performed using more than one of the methods described above. These antibodies can be labeled with the probes directly or indirectly. The linking of antibody probes, including covalent attachment of probes, fusion of probes into antibodies, and covalent attachment of chelating compounds to bind probes, among other methods well known in the art. For immunohistochemistry, diseased tissue samples may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin. The fixed or embedded sections include samples contacted with labeled primary and secondary antibodies, wherein the antibodies are used to detect in situ protein expression.

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

Preferably, the TAP peptide transporter is absent or reduced in level or function in mammalian cells. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter associated with antigen processing.

Human peptide-loaded defective cell line T2, catalog number CRL1992, from American type culture Collection (ATCC, 12301ParklawnDrive, Rockville, Maryland 20852, USA); the ATCC catalogue CRL 19863, which is subordinate to the Drosophila cell line Schneider No. 2 strain; mouse RMA-S cell line Ljunggren et al (Ljunggren and Karre, 1985) has been described.

Preferably, the host cell does not substantially express MHC class I molecules prior to transfection. The stimulator cells also preferably express molecules that play an important role in T cell costimulatory signaling, e.g., any of B7.1, B7.2, ICAM-1, and LFA 3. Nucleic acid sequences for a number of MHC class I molecules and co-stimulatory molecules are publicly available from GenBank and EMBL databases.

When MHC class I epitopes are used as one antigen, the T cells are CD8 positive T cells.

If the antigen presenting cell is transfected to express such an epitope, preferred cells include an expression vector capable of expressing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 93, or a variant amino acid sequence.

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) prepared T cells using autologous peripheral blood lymphocytes (PLB). Alternatively, it is also possible to pulse dendritic cells with peptides or polypeptides or to make autologous T cells by infection with recombinant viruses. In addition, B cells can be used to prepare autologous T cells. In addition, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses may be used to formulate autologous T cells. Walter et al (Walter et al,2003) describe in vitro activation of T cells by using artificial antigen presenting cells (aapcs), which is also a suitable method for generating T cells that act on selected peptides. In the present invention, according to biotin: streptomycin biochemical method streptomycin is prepared by mixing preformed MHC: peptide complexes were coupled to polystyrene particles (microspheres) to generate aapcs. The system enables precise regulation of MHC density on aapcs, which allows for the selective priming of high-potency antigen-specific T cell responses of high or low avidity in blood samples. In addition to MHC: in addition to peptide complexes, aapcs should also carry other proteins containing co-stimulatory activity, such as anti-CD 28 antibodies coupled to the surface. In addition, such aAPC-based systems often require the addition of appropriate soluble factors, e.g., cytokines such as interleukin 12.

T cells can also be prepared from allogeneic cells, a process which is described in detail in WO 97/26328, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present peptides, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells. Furthermore, plant viruses can also be used (see, for example, Porta et al (1994) which describes the development of cowpea mosaic virus as a highly productive system for the presentation of foreign peptides.

Activated T cells are directed against the peptides of the invention, contributing to the treatment. Thus, in another aspect of the invention, activated T cells produced by the methods of the invention described above are provided.

Activated T cells made as described above will selectively recognize abnormally expressed cells containing SEQ ID NO: 1 to seq id NO 93.

Preferably, the T cell recognizes the cell by interacting with (e.g., binding to) the TCR of its HLA/peptide-containing complex. T cells are cells useful in a method of killing a target cell in a patient, wherein the target cell abnormally expresses a polypeptide comprising an amino acid sequence of the invention. Such patients are administered 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. By "healthy individual" we mean a person who is generally in good condition, preferably has a qualified immune system, and more preferably is free of any disease that can be easily tested or detected.

According to the present invention, the in vivo target cells of CD 8-positive T cells may 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)).

The T cells of the invention are useful as active ingredients in therapeutic compositions. Accordingly, the present invention also provides a method of killing target cells in a subject, wherein the target cells in the subject aberrantly express a polypeptide comprising an amino acid sequence of the invention, the method comprising administering to the subject an effective amount of a T cell as described above.

The meaning of "aberrantly expressed" as used by the inventors also includes that the polypeptide is overexpressed compared to the expression level in normal (healthy) tissue, or that the gene is not expressed in tissue from the tumor but is expressed in the tumor. "overexpression" refers to a level of polypeptide that is at least 1.2 fold higher than in normal tissue; preferably at least 2-fold, more preferably at least 5 or 10-fold, that of normal tissue.

T cells can be prepared by methods known in the art (e.g., as described above).

T cell secondary transfer protocols are well known in the art. A review can be found in: gattioni et al, and Morgan et al (Gattinone et al, 2006; Morgan et al, 2006).

Another aspect of the invention includes the use of peptides complexed with MHC to generate T cell receptors, the nucleic acids of which are cloned and introduced into host cells, preferably T cells. The genetically engineered T cells can then be delivered to a patient for cancer therapy.

Any of the molecules of the invention (i.e., peptides, nucleic acids, antibodies, expression vectors, cells, activated T cells, T cell receptors, or encoding nucleic acids) is useful in treating diseases characterized by cellular escape from the immune response. Thus, any of the molecules of the present invention may be used as a medicament or in the manufacture of a medicament. Such molecules may be used alone or in combination with other or known molecules of the present invention.

The invention also relates to a kit comprising:

(a) a container containing the above pharmaceutical composition in the form of a solution or lyophilized powder;

(b) optionally a second container containing a diluent or reconstitution fluid in the form of a lyophilized powder; and

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

The kit may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a filtrate, (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.

The kit of the invention preferably comprises a lyophilized formulation in a suitable container together with recombinant and/or instructions for use. Suitable containers include, for example, bottles, vials (e.g., dual chamber bottles), syringes (e.g., 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 a container or instructions for the container indicating the direction of reconstitution and/or use. For example, the label may indicate that the lyophilized dosage form will reconstitute to the peptide concentration described above. The label may further indicate that the formulation is for subcutaneous injection.

The container holding the formulation may use a multi-purpose vial of penicillin such that the reconstituted dosage form may be administered repeatedly (e.g., 2-6 times). The kit may further comprise a second container containing a suitable diluent, such as sodium bicarbonate solution.

After mixing the dilution and the lyophilized formulation, the final concentration of peptide in the recombinant formulation is preferably at least 0.15 mg/mL/peptide (═ 75 μ g), and not more than 3 mg/mL/peptide (═ 1500 μ g). The kit may also include other materials that are commercially and user-friendly, including other buffers, diluents, filtrates, needles, syringes, and package inserts with instructions for use.

The kit of the present invention may have a separate container containing a formulation of a pharmaceutical composition of the present invention with or without other ingredients (e.g., other compounds or pharmaceutical compositions thereof), or with separate containers for each ingredient.

Preferably, the kits of the invention comprise a formulation of the 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 or inhibitory agent, an apoptosis inducing agent or a chelator) or a pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be placed in a separate container prior to administration to a patient. The components of the kit may be one or more solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit may also be in solid form, converted to a liquid by addition of a suitable solvent, and preferably placed in a separate container.

The container of the therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means for holding a solid or liquid. Typically, when more than one component is present, the kit will contain a second vial or other container of penicillin, so that it can be dosed separately. The kit may also comprise another container for holding a medicinal liquid. Preferably, the therapeutic kit will contain a device (e.g., one or more needles, syringes, eye droppers, pipettes, etc.) that allows for the injection of the medicament of the invention (the composition of the kit).

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. Preferably, the administration is subcutaneous, most preferably intradermal, and may also be by infusion pump.

Since the peptide of the present invention is isolated from esophageal cancer, the agent of the present invention is preferably used for the treatment of esophageal cancer.

The invention further relates to a method for preparing a personalized medicine for an individual patient, comprising: manufacturing a pharmaceutical composition comprising at least one peptide selected from a pre-screened TUMAP reservoir, wherein the at least one peptide used in the pharmaceutical composition is selected to be suitable for an individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be modified to produce T cell clones for downstream applications such as: TCR spacers or soluble antibodies and other therapeutic options.

"personalized medicine" refers to treatments that are specific to an individual patient and will only be used for that individual patient, including personalized active cancer vaccines and adoptive cell therapies using autologous tissue.

As used herein, "depot" shall refer to a group or series of peptides that have been subjected to immunogenic pre-screening and/or over-presented in a particular tumor type. The term "depot" does not imply that the particular peptide included in the vaccine has been pre-manufactured and stored in physical equipment, although such a possibility is contemplated. It is expressly contemplated that the peptides may be used in the new manufacture of each individual vaccine, and may also be pre-manufactured and stored. The repository (e.g., database format) consists of tumor-associated peptides that are highly overexpressed in tumor tissues of various HLA-A HLA-B and HLA-C allele esophageal cancer patients. It may comprise peptides including MHC class I and MHC class II or elongated MHC class I peptides. In addition to tumor-associated peptides collected from several esophageal cancer tissues, the pool may also contain HLA-a 02 and HLA-a 24 marker peptides. These peptides allow quantitative comparison of the TUMAP-induced T cell immunity, leading to important conclusions about the capacity of vaccines to respond to tumors. Second, they can serve as important positive control peptides from "non-self" antigens in the absence of any vaccine-induced T cell responses from the patient's "self" antigen TUMAP. Third, it may also allow conclusions to be drawn on the immune function status of the patient.

TUMAP from the repository was identified using a functional genomics approach combining gene expression analysis, mass spectrometry and T-cell immunologyThis approach ensures that only TUMAPs that are actually present in a high percentage of tumors but are not or only rarely expressed in normal tissues are selected for further analysis. For the initial peptide selection, the patient esophageal cancer samples and blood of healthy donors were analyzed in a progressive manner:

1. HLA ligands for malignant material are determined using mass spectrometry;

2. use of a genome-wide messenger ribonucleic acid (mRNA) expression assay for determining genes that are overexpressed in malignant tumor tissue (esophageal cancer) compared to a range of normal organs and tissues;

3. the determined HLA ligands are compared to gene expression data. Considering peptides that are over-or selectively presented on tumor tissue, preferably encoded by the selectively expressed or over-expressed genes detected in step 2, as suitable candidate TUMAPs for polypeptide vaccines;

4. literature search to determine more evidence to support the relevance of peptides identified as TUMP;

correlation of mRNA level overexpression was determined by the re-detection of TUMAP on tumor tissue and absence (or low frequency) on healthy tissue selected by step 3;

6. To assess whether the selected peptides induce a T cell response in vivo, in vitro immunogenicity assays were performed using human T cells from healthy donors as well as esophageal cancer patients.

In one aspect, the peptides are screened for immunogenicity prior to being added to the repository. For example, but not limited to, methods for determining the immunogenicity of peptides incorporated into a depot include in vitro T cell activation, specifically: CD8+ T cells from healthy donors were repeatedly stimulated with artificial antigen presenting cells loaded with peptide/MHC complexes and anti-CD 28 antibodies.

This method is preferred for rare cancers and patients with rare expression profiles. In contrast to the cocktail containing polypeptides currently developed as fixed components, the depot allows a higher degree of matching of the actual expression of antigens in tumors to the vaccine. In a multi-objective approach, each patient will use a selected single peptide or combination of several "off-the-shelf" peptides. Theoretically, one approach based on selecting, for example, 5 different antigenic peptides from a 50 antigenic peptide library could provide about 170 million possible pharmaceutical product (DP) components.

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

HLA phenotypic, transcriptional and peptidological data were collected from tumor material and blood samples of patients to determine the peptides most appropriate for each patient and containing a "repository" and patient-unique (i.e., mutated) TUMAP. Those peptides selected are selectively or over-expressed in patient tumors and, possibly, exhibit strong in vitro immunogenicity if tested with patient individual PBMCs.

Preferably, a method of determining the peptide comprised by the vaccine comprises: (a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample from an individual patient; (b) aligning the peptides identified in (a) with a repository (database) of such peptides; and (c) selecting from a repository (database) at least one peptide that is associated with the identified tumor associated peptide in the patient. For example, TUMAPs presented in tumor samples can be identified by: (a1) comparing expression data from a tumor sample to expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are over-or aberrantly expressed in the tumor tissue; and (a2) correlating the expression data with MHC ligand sequences bound to MHC class I and/or class II molecules in the tumor sample to determine MHC ligands derived from proteins overexpressed or abnormally expressed by the tumor. Preferably, the sequence of the MHC ligand is determined by: the MHC molecules isolated from the tumor sample are eluted with the peptide and the eluted ligands are sequenced. Preferably, the tumor sample and the normal tissue are obtained from the same patient.

In addition to the use of a repository (database) model for peptide selection, as an alternative, TUMAPs can be identified in vitro in patients and then included in vaccines. As an example, candidate TUMAPs in a patient may be identified by: (a1) comparing expression data from a tumor sample to expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are over-or aberrantly expressed in the tumor tissue; and (a2) correlating the expression data with MHC ligand sequences bound to MHC class I and/or class II molecules in the tumor sample to determine MHC ligands derived from proteins overexpressed or abnormally expressed by the tumor. As another example, the protein may be identified as comprising a mutation that is unique to a tumor sample relative to the corresponding normal tissue of the individual patient, and the TUMAP may be identified as specifically targeting the variation. For example, the genome of a tumor and corresponding normal tissue can be sequenced by whole genome sequencing methods: to find non-synonymous mutations in the protein coding region of the gene, genomic DNA and RNA were extracted from tumor tissue and normal non-mutated genomic germline DNA was extracted from Peripheral Blood Mononuclear Cells (PBMC). The NGS method used is limited to the re-sequencing of protein coding regions (exome re-sequencing). For this purpose, exon DNA from human samples was captured using a target sequence enrichment kit supplied by the supplier, followed by sequencing using HiSeq2000 (Illumina). In addition, mRNA from the tumor was sequenced to directly quantify gene expression and confirm that the mutant gene was expressed in the patient's tumor. Millions of sequence reads are obtained and processed through software algorithms. The output list contains mutations and gene expression. Tumor specific somatic mutations were determined by comparison to PBMC-derived germline changes and optimized. Then, for the purpose of storage it is possible to test newly identified peptides for immunogenicity as described above and select candidate TUMAPs with appropriate immunogenicity for use in vaccines.

In an exemplary embodiment, the peptides contained in the vaccine are determined by: (a) identifying a tumor associated peptide (TUMAP) presented from a tumor sample of an individual patient using the method described above; (b) comparing the peptides identified in (a) to a pool of peptides in tumors (compared to corresponding normal tissues) that have been pre-screened for immunogenicity and over-presentation; (c) selecting from a repository at least one peptide that is associated with a tumor associated peptide identified in the patient; and (d) optionally selecting at least one of the peptides newly identified in (a) for confirmation of immunogenicity.

In an exemplary embodiment, the peptides contained in the vaccine are determined by: (a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample of an individual patient; and (b) selecting at least one of the peptides newly identified in (a) and confirming its immunogenicity.

Once the peptides for the individualized 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, e.g., about 33% DMSO.

Each peptide listed in the product was dissolved in DMSO. The choice of the concentration of the individual peptide solutions depends on the amount of peptide to be included in the product. The single peptide-DMSO solutions were mixed equally to achieve a solution containing all peptides at a concentration of 2.5mg/ml per peptide. The mixed solution was then diluted 1: 3 with water for injection to a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution was filtered through a 0.22 μm sterile screening procedure. Thereby obtaining the final bulk solution.

The final bulk solution was filled into vials and stored at-20 ℃ prior to use. One vial contained 700 μ L of solution with 0.578mg of each peptide. Of which 500 μ L (about 400 μ g of each peptide) will be used for intradermal injection.

The peptides of the invention are useful in diagnosis as well as in the treatment of cancer. Since peptides are produced by esophageal cancer cells and it has been determined that these peptides are absent or present at low levels in normal tissues, these peptides can be used to diagnose the presence or absence of cancer.

A tissue biopsy from a blood sample containing the peptide of claim, useful for a pathologist in diagnosing cancer. Detection of certain peptides by antibodies, mass spectrometry, or other methods known in the art allows a pathologist to determine whether a tissue sample is malignant or inflammatory or diseased in general, and may also be used as a biomarker for esophageal cancer. Presentation of the peptide groups allows for classification or further subclassification of the diseased tissue.

Detection of peptides in diseased specimens allows for the determination of the benefit of immune system treatment methods, particularly if T-lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a mechanism that adequately accounts for which infected malignant cells evade immune surveillance. Thus, presentation of the peptide indicated that the analyzed cells did not utilize this mechanism.

The peptides of the invention can be used to assay lymphocyte responses to peptides (e.g., T cell responses), or antibody responses to peptides complexed with peptides or MHC molecules. These lymphocyte responses can be used as prognostic indicators to determine whether further treatment should be undertaken. These responses can also be used as surrogate indicators in immunotherapy aimed at inducing lymphocyte responses in different ways, such as vaccination, nucleic acids, autologous material, adoptive transfer of lymphocytes. In gene therapy, the reaction of lymphocytes to peptides can be taken into account in the assessment of side effects. Lymphocyte response monitoring may also be a valuable tool in follow-up examinations of transplantation therapies, e.g., for detecting graft-versus-host and host-versus-graft disease.

The invention will now be illustrated by the following examples describing preferred embodiments, and with reference to the accompanying drawings, but without being limited thereto. All references cited herein are incorporated by reference for the purposes of this invention.

Drawings

FIGS. 1A to 1V show the over-presentation of various peptides in normal tissues (white bars) and esophageal cancer (black bars). A) The gene symbols are as follows: KRT14/KRT16, peptide: STYGGGLSV (SEQ ID NO: 1) from left to right tissue: adipose tissue 1, adrenal gland 3, artery 8, bone marrow 5, brain 7, breast 5, cartilage 2, central nerve 1, colon 13, duodenum 1, gallbladder 2, heart 5, kidney 14, liver 21, lung 44, lymph node 4, leukocyte sample 4, ovary 3, pancreas 8, peripheral nerve 5, peritoneum 1, pituitary gland 3, placenta 4, pleura 3, prostate 3, rectus 6, salivary gland 7, skeletal muscle 4, skin 6, small intestine 2, spleen 4, stomach 5, testis 6, thymus 3, thyroid 3, trachea 7, ureter 2, urinary bladder 6, uterus 2, vein 2, esophagus 6, esophagus 16. This peptide was also detected in 4/91 lung cancer. FIG. 1B) Gene symbol: GJB5, peptide: SIFEGLLSGV (SEQ ID NO: 7). Organization from left to right: adipose tissue 1, adrenal gland 3, artery 8, bone marrow 5, brain 7, breast 5, cartilage 2, central nerve 1, colon 13, duodenum 1, gallbladder 2, heart 5, kidney 14, liver 21, lung 44, lymph node 4, leukocyte sample 4, ovary 3, pancreas 8, peripheral nerve 5, peritoneum 1, pituitary gland 3, placenta 4, pleura 3, prostate 3, rectus 6, salivary gland 7, skeletal muscle 4, skin 6, small intestine 2, spleen 4, stomach 5, testis 6, thymus 3, thyroid 3, trachea 7, ureter 2, urinary bladder 6, uterus 2, vein 2, esophagus 6, esophagus 16. The peptide was also detected in 1/43 prostate cancer, 1/3 gallbladder cancer, 1/20 ovarian cancer, 5/91 lung cancer and 1/4 bladder cancer. Fig. 2C) gene symbol: PKP3, peptide: SLVSEQLEPA (SEQ ID NO: 34). Organization from left to right: adipose tissue 1, adrenal gland 3, artery 8, bone marrow 5, brain 7, breast 5, cartilage 2, central nerve 1, colon 13, duodenum 1, gallbladder 2, heart 5, kidney 14, liver 21, lung 44, lymph node 4, leukocyte sample 4, ovary 3, pancreas 8, peripheral nerve 5, peritoneum 1, pituitary gland 3, placenta 4, pleura 3, prostate 3, rectus 6, salivary gland 7, skeletal muscle 4, skin 6, small intestine 2, spleen 4, stomach 5, testis 6, thymus 3, thyroid 3, trachea 7, ureter 2, urinary bladder 6, uterus 2, vein 2, esophagus 6, esophagus 16. The peptide was also detected in 8/24 colorectal cancer, 1/20 ovarian cancer, 1/46 gastric cancer, 5/91 lung cancer and 2/4 bladder cancer. Fig. 2D) gene symbols: RNPEP, peptide: YTQPFSHYGQAL (SEQ ID NO: 37). Organization from left to right: adipose tissue 1, adrenal gland 3, artery 8, bone marrow 5, brain 7, breast 5, cartilage 2, central nerve 1, colon 13, duodenum 1, gallbladder 2, heart 5, kidney 14, liver 21, lung 44, lymph node 4, leukocyte sample 4, ovary 3, pancreas 8, peripheral nerve 5, peritoneum 1, pituitary gland 3, placenta 4, pleura 3, prostate 3, rectus 6, salivary gland 7, skeletal muscle 4, skin 6, small intestine 2, spleen 4, stomach 5, testis 6, thymus 3, thyroid 3, trachea 7, ureter 2, urinary bladder 6, uterus 2, vein 2, esophagus 6, esophagus 16. The peptide was also detected in 1/19 pancreatic cancer, 7/46 gastric cancer and 1/91 lung cancer. Fig. 4E) gene symbols: 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 cancer, 2 breast cancer, 1 colon cancer, 2 esophageal cancer, 1 chronic leukemia, 2 liver cancer, 22 lung cancer, 7 ovarian cancer, 4 prostate cancer, 1 rectal cancer). Fig. 5F) gene symbols: KRT5, peptide: SLYNLGGSKRISI (SEQ ID NO: 2). Organization from left to right: 20 cancer tissue (9 head and neck cancer, 2 esophageal cancer, 1 esophageal and gastric cancer, 7 lung cancer, 1 bladder cancer). Fig. 6G) gene symbol: KRT5, peptide: TASAITPSV (SEQ ID NO: 3). Organization from left to right: 17 cancer tissue (2 esophageal cancer, 6 head and neck cancer, 7 lung cancer, 2 bladder cancer). Fig. 7H) gene symbols: s100a2, peptide: SLDENSDQQV (SEQ ID NO: 10). Organization from left to right: and 7 cancer tissues (3 head and neck cancer, 2 esophageal cancer, 1 lung cancer, 1 bladder cancer). Fig. 8I) gene symbol: LAMB3, peptide: ALWLPTDSATV (SEQ ID NO: 11). Organization from left to right: 12 cancer tissue (2 esophageal cancer, 1 gallbladder cancer, 8 lung cancer, 1 skin cancer). Fig. 9J) gene symbol: IL36RN, peptide: SLSPVILGV (SEQ ID NO: 13). Organization from left to right: 26 cancer tissue (8 head and neck cancer, 3 esophageal cancer, 10 lung cancer, 3 skin cancer, 1 bladder cancer, 1 uterine cancer). Fig. 10K) gene symbols: AN01, peptide: LLANGVYAA (SEQ ID NO: 15). Organization from left to right: cancer tissue 8 (2 esophageal cancer, 1 gallbladder cancer, 1 liver cancer, 1 lung cancer, 1 stomach cancer, 1 bladder cancer, 1 uterine cancer). Fig. 11L) gene symbols: f7, IGHV4-31, IGHG1, IGHG2, IGHG3, IGHG4, IGHM, peptide: MISRTPEV (SEQ ID NO: 17). Organization from left to right: 19 cancer tissue (2 esophageal cancer, 2 kidney cancer, 2 liver cancer, 9 lung cancer, 1 lymph node cancer, 1 testicular cancer, 2 bladder cancer). Fig. 12M) gene symbols: QSER1, peptide: SLNGNQVTV (SEQ ID NO: 30). Organization from left to right: 1 cell line (1 pancreas), 14 cancer tissue (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 cancer, 3 bladder cancer, 2 uterine cancer). Fig. 13N) gene symbols: HAS3, peptide: YMLDIFHEV (SEQ ID NO: 32). Organization from left to right: normal tissue (1 uterus), 15 cancer tissue (1 brain cancer, 2 esophageal cancer, 1 gallbladder cancer, 3 head and neck cancer, 4 lung cancer, 4 bladder cancer). Fig. 140) gene symbols: PKP3, peptide: SLVSEQLEPA (SEQ ID NO: 34). Organization from left to right: 1 cell line (1 pancreas), 1 normal tissue (1 colon), 28 cancerous tissue (6 head and neck cancer, 1 breast cancer, 1 caecum cancer, 3 colon cancer, 1 colorectal cancer, 3 esophageal cancer, 6 lung cancer, 1 ovarian cancer, 3 rectal cancer, 3 bladder cancer). Fig. 15P) gene symbols: SERPINH1, peptide: GLAFSLYQA (SEQ ID NO: 40). Organization from left to right: 3 cell lines (1 kidney, 2 pancreas), 4 normal tissues (1 adrenal gland, 1 lung, 2 placenta), 41 cancer tissues (3 head and neck cancer, 3 breast cancer, 2 colon cancer, 2 esophageal cancer, 1 gall bladder cancer, 1 liver cancer, 15 lung cancer, 1 ovarian cancer, 1 pancreatic cancer, 3 rectal cancer, 2 skin cancer, 1 stomach cancer, 4 bladder cancer, 2 uterine cancer). Fig. 16Q) gene symbol: TMEM132A, peptide: ALVEVTEHV (SEQ ID N0: 56). Organization from left to right: 7 normal tissue (5 lung, 1 thyroid, 1 trachea), 64 cancerous tissue (6 head and neck cancer, 12 brain cancer, 4 breast cancer, 3 esophageal cancer, 1 gall bladder cancer, 5 kidney cancer, 21 lung cancer, 1 lymph node cancer, 7 ovarian cancer, 1 pancreatic cancer, 1 skin cancer, 2 uterine cancer). Fig. 17R) gene symbols: PRC1, peptide: GLAPNTPGKA (SEQ ID NO: 57). Organization from left to right: 14 cancer tissue (1 head and neck cancer, 1 breast cancer, 2 esophageal cancer, 6 lung cancer, 1 ovarian cancer, 1 skin cancer, 1 bladder cancer, 1 uterine cancer). Fig. 18S) gene symbol: MAPK6, peptide: LILESIPVV (SEQ ID NO: 58). Organization from left to right: cell line 2 (1 blood cell, 1 skin), 25 cancerous tissue (5 head and neck cancer, 1 colon cancer, 2 esophageal cancer, 1 leukemia cancer, 8 lung cancer, 2 lymph node cancer, 3 skin cancer, 2 bladder cancer, 1 uterine cancer). Fig. 19T) gene symbols: PPP4R1, peptide: SLLDTLREV (SEQ ID NO: 59). Organization 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). Fig. 12U) gene symbols: TP63, peptide: VLVPYEPPQV (SEQ ID NO: 77). Organization from left to right: 2 normal tissue (1 esophagus, 1 trachea), 47 cancer tissue (8 head and neck cancer, 4 esophagus cancer, 1 gallbladder cancer, 14 lung cancer, 7 lymph node cancer, 2 prostate cancer, 1 skin cancer, 8 bladder cancer). Fig. 21V) gene symbols: KIAA0947, peptide: AVLPHVDQV (SEQ ID NO: 81). Organization from left to right: 3 cell lines (1 blood cell, 1 pancreas), 12 cancer tissues (5 brain cancer, 2 esophageal cancer, 1 lung cancer, 3 lymph node cancer, 1 uterine cancer).

FIGS. 2A to 2D show representative expression profiles of the source genes of the invention, which are highly over-or exclusively expressed in esophageal cancer in a range of normal tissues (white bars) and in 11 esophageal cancer samples (black bars). Organization from left to right: 7 arterial, 1 brain, 1 heart, 2 liver, 2 lung, 2 vein, 1 adipose tissue, 1 adrenal gland, 4 bone marrow, 1 colon, 2 esophagus, 2 gall bladder, 1 kidney, 6 lymph node, 1 pancreas, 1 pituitary, 1 rectum, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thymus, 1 thyroid, 5 trachea, 1 bladder, 1 breast, 3 ovaries, 3 placenta, 1 prostate, 1 testis, 1 uterus, 11 esophageal cancer samples. Fig. 2A) CT45a1, CT45A3, CT45a5, CT45a6, CT45a2, RP11-342L5.1, gene symbols: (ii) a PTHLH; fig. 2B) CLDN16, gene symbol: KRT 14; fig. 2C) ESR1, gene signature: FAM 83A; fig. 2D) IDO1, gene signature: PDPN.

Fig. 3A to E show exemplary results of peptide-specific CD8+ T cell in vitro responses of healthy HLA-a x 02+ donors, i.e. exemplary immunogenicity data: flow cytometry results after staining of peptide specific multimers. Fig. 3A) gene symbol: SF3B3, peptide: ELDRTPPEV (SEQ ID NO: 97); fig. 3B) gene symbols: TNC, peptide: AMTQLLAGV (SEQ ID NO: 101). In addition, the CD8+ T cells were prepared by the method of: artificial APCs coated with anti-CD 28mAb and HLA-a x 02 were compared to SEQ ID NO: 5 peptide (C, left panel), SEQ ID NO: 2 peptide (D, left panel) and SEQ ID NO: 77 peptide (E, left panel) synthesis. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A.about.02/SeqID No 5(C), A.about.02/SeqID No 2(D), A.about.02/SeqID No 77 (E). Right panels (C, D and E) show control staining of cells stimulated with irrelevant a × 02/peptide complexes. Viable single cells were gated on CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells and CD8+ lymphocytes is suggested.

Examples

Example 1

Identification and quantification of cell surface presented tumor associated peptides

Tissue sample

Tumor Tissue from patients was obtained from Asterand (Detroit, USA and Royston, Herts, UK), ProteGenex Inc, (silver City, CA, USA), Tissue Solutions Ltd. (Glasgow, UK), Dibingen university Hospital. Normal tissues were obtained from Asterand (Detroit, USA and Royston, Herts, UK), Bio-OptionsInc. (CA, USA), BioServe (Beltsville, MD, USA), Capita Bioscience Inc. (Rockville, Md., USA), Geneticist Inc. (Glendale, Calif., Japan university Hospital, Heidelberg university Hospital, Kyoto Fuli medical university Hospital (KPUM), Munich university Hospital, ProteoGenex Inc. (Culver City, Calif., USA), German Detroit Binggen university Hospital, Tissue Solutions Ltd. (Glasgow, UK). All patients received written informed consent prior to surgery or autopsy. Immediately following resection, the tissue was cold shocked and stored at-70 ℃ or below prior to isolation of the TUMAP.

Isolation of HLA peptides from tissue samples

With minor modifications to the protocol (Falk et al, 1991; Seeger et al,1999), HLA peptide libraries of frozen tissue samples were obtained from solid tissues by immunoprecipitation using HLA-A x 02-specific antibody BB7.2, HLA-A, HLA-B, HLAC-specific antibody W6/32, CNBr-activated sepharose, acid treatment and ultrafiltration.

Mass spectrometric analysis

The obtained HLA peptide library was separated by reverse phase chromatography (Waters) according to its hydrophobicity, and the eluted peptides were analyzed by LTQ-velos fusion hybridization Mass Spectrometry (ThermoElectron) equipped with an electrospray source. The peptide library was loaded directly into an analytical fused silica microcapillary column (75 μm id x250mm) filled with 1.7 μm c18 reverse phase material (Waters) using a flow rate of 400nL per minute. Subsequently, the peptides were separated using a two-step 180 min binary gradient from solvent B at a concentration of 10% to 33% at a flow rate of 300nL per minute. The gradient was made from solvent a (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoliter electrospray source. The LTQ-Orbitrap mass spectrometer was operated in a data dependent mode using the TOP 5(TOP5) strategy. In short, a scan cycle is first started at orbitrap with a high accurate mass complete scan (R30000), followed by MS/MS scanning of the 5 most abundant precursor ions in orbitrap with the dynamic exclusion technique of the previously selected ions (R7500). Tandem mass spectra were interpreted in sequence and another manual controller. The resulting fragmentation pattern of the natural peptide was compared to the fragmentation pattern of a reference peptide of the same synthetic sequence, ensuring the identified peptide sequence.

Label-free relative LC-MS quantification was performed by ion counting (i.e. by LC-MS functional extraction and analysis) (Mueller et al, 2007). This method assumes that the LC-MS signal region of the peptide correlates with its abundance in the sample. The extracted features are further processed by state-of-charge deconvolution and retention time calibration (Mueller et al, 2008; Sturmet al, 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to combine the quantitative data of different samples and tissues with peptide presentation features. Quantitative data were normalized in a two-layer fashion from the pooled data to account for both technical and biological replicative variation. Thus, each identified peptide can be correlated with quantitative data, thereby allowing a relative quantification between the sample and the tissue. In addition, all quantitative data obtained for candidate peptides were manually checked to ensure data consistency and to verify the accuracy of automated analysis. For each peptide, a presentation graph is calculated showing the mean presentation of the sample as well as the variation in replication. These features allow baseline values for esophageal cancer samples versus normal tissue samples to be juxtaposed.

The presentation profile of an exemplary over-presenting peptide is shown in fig. 1. The scores for exemplary peptides are shown in Table 8.

Table 8: and (5) presenting scores. The table lists peptides that are highly over-represented (+++) on tumors compared to a series of normal tissues, (++) on tumors compared to a series of normal tissues, or (+) on tumors compared to a series of normal tissues. The series of normal tissues include: adipose tissue, adrenal gland, arteries, veins, bone marrow, brain, central and peripheral nerves, colon, rectum, small intestine (including duodenum), esophagus, gall bladder, heart, kidney, liver, lung, lymph nodes, mononuclear leukocytes, pancreas, peritoneum, pituitary, pleura, salivary glands, skeletal muscle, skin, spleen, stomach, thymus, thyroid, trachea, ureters, bladder.

Example 2

Expression profiling of genes encoding the peptides of the invention

An over-presentation or specific presentation of one peptide on tumor cells compared to normal cells is sufficient for its effective use in immunotherapy, some peptides are tumor-specific, although their source proteins are also present in normal tissues. However, mRNA expression profiling increases the safety of other levels in the selection of immunotherapeutic target peptides. Particularly for treatment 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 does not appear in normal tissues.

RNA source and preparation

Surgically excised tissue specimens were provided as described above (see example 1) after obtaining written informed consent from each patient. Tumor tissue specimens were snap frozen immediately after surgery, and then homogenized with a pestle and mortar in liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, germany) followed by clearance with RNeasy (QIAGEN, Hilden, germany); both methods were performed according to the manufacturer's protocol.

Total RNA from tumor tissue used for RNASeq experiments was obtained from: ProteoGenex Inc. (silver City, Calif., USA), Tissue Solutions Ltd. (Glasgow, UK).

Total RNA from healthy human tissue used for RNASeq experiments was obtained from: asterand (Detroit, USA and Royston, Herts, UK), ProteoGenex Inc. (silver City, CA, USA), Geneticist Inc. (Glendale, CA, USA), Istituto Nazionale Tumori "Pascal", Molecular Biology and viral Oncology Unit (IRCCS) (Naples, Italy), university of Heidelberg, BioCat GmbH (Heidelberg, Germany).

The quality and quantity of all RNA samples were evaluated on an Agilent 2100Bioanalyzer analyzer (Agilent, Waldbronn, Germany) using the RNA 6000Pico LabChip Kit (Agilent).

RNAseq experiment

RNA samples from tumor and normal tissues were analyzed for gene expression by CeGaT (Tubingen, Germany) by a new generation sequencing technique (RNAseq). Briefly, sequencing libraries were prepared using the Illumina HiSeq v4 kit according to the supplier's protocol (Illumina Inc, San Diego, CA, USA), including RNA fragmentation, cDNA transformation and addition of sequencing adapters. Libraries obtained from multiple samples were equimolar mixed according to the manufacturer's instructions and sequenced on an illumina hiseq 2500 sequencer, yielding single-ended reads of 50 bp. The processed reads were mapped to the human genome using STAR software (GRCh 38). According to the instructions of the ENSEMBL sequence database (Ensembl77), expression data were set at the transcription level to RPKM (reads per million mapped per kilobase, generated by Cufflinks software) and at the exonic level (total reads, generated by Bedtools software). Exon readings were classified into exon length and calibration size to obtain RPKM values.

The expression profile of the representative source gene of the present invention highly overexpressed in esophageal cancer is shown in FIG. 2. Further representative gene expression scores are shown in table 9.

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

Example 3

In vitro immunogenicity of MHC-I presenting peptides

To obtain information on the immunogenicity of the TUMAPs of the present invention, the inventors performed 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-CD 28 antibodies. In this way, the inventors could 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).

In vitro activation of CD8+ T cells

For in vitro stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD 28 antibodies, the inventors first obtained healthy donor CD8 microbeads from University clinics Mannheim, Germany (Miltenyi Biotec, Bergisch-Gladbach, Germany) and isolated CD8+ T cells by positive selection of fresh HLA-A02 product after leukopheresis.

PBMC and isolated CD8+ lymphocytes were cultured in T cell culture medium (TCM) before use, including RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100U/ml penicillin/100. mu.g/ml streptomycin (Cambrex, Cologne, Germany), 1mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20. mu.g/ml gentamycin (Cambrex). At this step, 2.5ng/ml IL-7(Promocell, Heidelberg, Germany) and 10U/ml IL-2(Novartis Pharma, N ü rnberg, Germany) were also added to the TCM.

Generation of pMHC/anti-CD 28 coated beads, stimulation and readout of T cells were performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition. Purified co-stimulatory mouse IgG2a anti-human CD28 antibody 9.3(Jung et al,1987) was chemically biotinylated using N-hydroxysuccinimide biotin recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were streptavidin-coated polystyrene particles (Bangs laboratories, Ill., USA) of 5.6 μm.

The pMHC used for the positive and negative control stimulators were A.times.0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO: 102) modified from Melan-A/MART-1) and A.times.0201/DDX 5-001 (YLLPAIVHI (SEQ ID NO: 103) obtained from DDX 5), respectively.

800.000 beads/200. mu.l were packed in 96-well plates containing 4X 12.5ng of different biotin-pMHC, washed, and then 600ng of biotin anti-CD 28 was added in a volume of 200. mu.l. Stimulation was activated by co-culturing 1x106CD8+ T cells with 2x105 wash-coated beads in 200 μ l TCM containing 5ng/ml IL-12(Promocell) for 3 days at 37 ℃. Thereafter, half of the medium was exchanged with fresh TCM supplemented with 80U/ml IL-2 and incubated at 37 ℃ for 4 days. This stimulation cycle was performed a total of 3 times. For pMHC multimer readout using 8 different pMHC molecules per condition, the two-dimensional combinatorial coding approach was used as described previously (Andersen et al,2012), slightly modified, encompassing coupling to 5 different fluorescent dyes. Finally, multimer analysis was performed with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1(BD, Heidelberg, Germany) and fluorescent pMHC multimers. For the analysis, a BD LSRII SORP cytometer equipped with a suitable laser and screening procedure 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). The in vitro priming of specific multimers + CD8+ lymphocytes was tested in comparison to the negative control stimulation group. Immunogenicity of a given antigen is tested if at least one evaluable in vitro stimulated well in a healthy donor is found to contain a specific CD8+ T cell strain after in vitro stimulation (i.e. the well contains at least 1% of specific multimers + CD8+ T cells and the percentage of specific multimers + is at least 10 times the median of negative control stimulation).

In vitro immunogenicity of esophageal cancer peptides

For the tested HLA class I peptides, their immunogenicity in vitro can be demonstrated by the generation of peptide-specific T cell lines. Exemplary results of flow cytometry assays of TUMAP-specific multimers after staining 2 peptides of the invention (SEQ ID No 97 and SEQ ID No 101) are shown in FIG. 3, along with corresponding negative control information. The results for the 5 peptides of the invention are summarized in Table 10A.

Table 10A: in vitro immunogenicity of HLA class I peptides of the invention. Exemplary results of in vitro immunogenicity experiments performed by the applicants on the peptides of the invention. Plus < 20% >; 20% -49% ++; 50% -69% ++; 70% ═ 70% +++

SEQ ID NO：	Sequence of	Hole(s)	Donor
				94	TLLQEQGTKTV	+	++
95	LIQDRVAEV	+	++
				97	ELDRTPPEV	++	++++
98	VLFPNLKTV	+	++++
				101	AMTQLLAGV	++	+++

Table 10B: in vitro immunogenicity of HLA-class I peptides of the invention.

Exemplary results of in vitro immunogenicity experiments performed by the applicants on the peptides of the invention. The results of in vitro immunogenicity experiments are suggested. The percentage of positive wells and donors (other evaluable) was summarized as < 20% >; 20% -49% ++; 50% -69% ++; 70% ═ 70% +++

Example 4

Synthesis of peptides

All peptides were synthesized by standard, well-accepted solid-phase peptide synthesis using the Fmoc strategy. The identity and purity of each peptide has been determined using mass spectrometry and RP-HPLC analysis. White to off-white peptides were obtained with > 50% purity by lyophilization (trifluoroacetate). All TUMAPs are preferably administered as the trifluoroacetate or acetate salt, although other pharmaceutically acceptable salt forms are possible.

Example 5

MHC binding assay

The candidate peptides of the present invention based on T cell therapy were further tested for their MHC binding capacity (affinity). The individual peptide-MHC complexes are generated by UV-ligand exchange, wherein the UV-sensitive peptides are cleaved after UV irradiation and exchanged with the relevant peptide for analysis. Only candidate peptides that can effectively bind and stabilize a peptide-accepting MHC molecule can prevent dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA assay based on the detection of the stable MHC complex light chain (. beta.2m) was performed. The assays were generally performed according to the method described by Rodenko et al (Rodenko et al, 2006).

96-well Maxisorp plates (NUNC) were coated overnight at 2ug/ml pronase in PBS at room temperature, washed 4-fold and blocked in 2% BSA with blocking buffer for 1 hour at 37 ℃. Folded HLA-a 02: 01/MLA-001 monomer as standard substance, covering 15500ng/ml range. The peptide-MHC monomer of the uv exchange reaction was diluted 100-fold in blocking buffer. The samples were incubated at 37 ℃ for 1 hour, washed four times, incubated at 37 ℃ for 1 hour with 2ug/ml HRP conjugated anti-. beta.2m, washed again, and detected with NH2SO4 blocked TMB solution. The absorption was measured at 450 nm. Candidate peptides that exhibit high exchange yields (preferably greater than 50%, most preferably greater than 75%) are generally preferred when generating and producing antibodies or fragments thereof and/or T cell receptors or fragments thereof, because they exhibit sufficient affinity for MHC molecules and prevent dissociation of MHC complexes.

Table 11: MHC class I binding score. HLA class I restricted peptides are similar to HLA-a 02: 01 according to the peptide exchange yield classification: more than or equal to 10% +; more than or equal to 20% ++; more than or equal to 50 ++; greater than or equal to 75% +++

Example 6

Absolute quantification of cell surface presented tumor associated peptides

The production of adhesives, such as antibodies and/or TCRs, is a laborious process that can be performed against only a few selected targets. In the case of tumor-associated and specific peptides, selection criteria include, but are not limited to, exclusion of the presentation and concentration of peptides presented on the cell surface. Quantitation of the copy number of TUMAPs per cell in solid tumor samples requires absolute quantitation of the isolated TUMAPs, efficiency of TUMAP isolation, and cell counts of the analyzed tissue samples.

NanoLC-MS/MS peptide quantitation

For accurate quantification of peptides by mass spectrometry, an internal standard method was used to generate a calibration curve for each peptide. Internal standards are dual isotopically labeled variants of each peptide, i.e., 2 isotopically labeled amino acids were incorporated in the TUMAP synthesis. It differs only qualitatively from tumor-associated peptides, but not in other physicochemical properties (Anderson et al, 2012). An internal standard was incorporated into each MS sample and all MS signals were normalized to the internal standard MS signal to balance potential technical differences between MS experiments.

The calibration curves were plotted with at least three different matrices, i.e., HLA peptide eluate from a natural sample similar to a conventional MS sample, and each preparation was measured in duplicate MS runs. For evaluation, MS signals were normalized to internal standard signals and calibration curves were calculated by logistic regression.

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

Efficiency of peptide/MHC separation

For any protein purification process, the isolation of proteins from a tissue sample is associated with some loss of the relevant proteins. To determine the efficiency of the TUMAP separation, peptide/MHC complexes were generated for all TUMAPs selected as absolute quantification. To enable native peptide MHC/complex and loading, a monoisotopically labeled version of TUMAP was used, i.e. 1 isotopically labeled amino acid was incorporated during TUMAP synthesis. These complexes are incorporated into freshly prepared tissue lysates, e.g., at the earliest possible time point during the TUMAP isolation, and then captured as native peptide MHC/complexes in subsequent affinity purifications. Therefore, measuring the recovery of single-marker TUMAPs can lead to conclusions regarding the separation efficiency of individual TUMAPs.

Separation efficiency was analyzed using a small number of samples, and these tissue samples were comparable. In contrast, the separation efficiency differs between individual peptides. This indicates that the separation efficiency, although measured in only a limited number of samples, can be extrapolated to any other tissue preparation. However, since separation efficiency cannot be extrapolated from one peptide to the other, it is necessary to analyze each TUMAP separately.

Determination of cell count in solid, frozen tissue

To determine the cell number of tissue samples quantified by absolute peptides, the inventors used DNA content analysis. This method is applicable to a wide range of samples from different sources, most importantly frozen samples (Alcoser et al, 2011; Forseyand Chaudhuri, 2009; Silva et al, 2013). During the peptide isolation protocol, tissue samples were processed into homogeneous lysates from which a small aliquot of the lysate was taken. The samples were aliquoted in triplicate and DNA isolated therefrom (QiaAmp DNA MiniKit, Qiagen, Hilden, Germany). The total DNA content of each DNA isolation was quantified at least twice using a fluorescence-based DNA quantification Assay (Qubit dsDNAHS Assay Kit, Life Technologies, Darmstadt, Germany).

To calculate the cell number, a standard curve of DNA from a single aliquot of healthy blood cells was generated, using a series of assigned cell numbers. The standard curve was used to calculate the total cell content of the total DNA content per DNA isolation. The average total cell count of the tissue samples used for peptide isolation was calculated taking into account the known volume of lysate aliquots and the total lysate volume.

Peptide copy number per cell

Using the data from the previous experiments, the inventors calculated the number of copies of TUMAP per cell as the total peptide amount divided by the total cell count of the sample, and then divided by the separation efficiency. The cell copy number of the selected peptides is shown in table 12.

Table 12: absolute copy number. The table lists the results of absolute peptide quantification in NSCLC tumor samples. Median copy number per cell for each peptide is expressed as: < 100 ═ plus; 100 ═ 100 ++; 1, 000+ + +; the "> - + + + + +.000. indicates the number of samples, which provides the evaluated high quality MS data.

In summary, the present application includes, but is not limited to, the following:

1. a peptide comprising an amino acid sequence selected from SEQ ID No.1 to SEQ ID No.93, and variant sequences thereof which are at least 88% homologous to SEQ ID No.1 to SEQ ID No.93, wherein the variant peptide binds to Major Histocompatibility Complex (MHC) and/or induces T cells cross-reacting with the variant peptide, wherein the peptide is not a full-length polypeptide, and pharmaceutically acceptable salts thereof.

2. The peptide of claim 1, wherein the peptide has the ability to bind to an MHC class-I or-II molecule, wherein the peptide, when bound to the MHC, is capable of being recognized by CD4 and/or CD8T cells.

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

4. The peptide or variant thereof according to any one of claims 1 to 3, wherein the total length of the peptide or variant thereof is from 8 to 100 amino acids, preferably from 8 to 30 amino acids, more preferably from 8 to 16 amino acids, 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.

5. The peptide or variant thereof according to any one of claims 1 to 4, wherein said peptide is modified, and/or comprises non-peptide bonds.

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

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

8. An expression vector expressing the nucleic acid of item 7.

9. A recombinant host cell comprising the peptide of item 1 to 6, the nucleic acid of item 7, the expression vector of item 8, wherein the host cell is preferably an antigen presenting cell, such as a dendritic cell.

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.

11. A method of making the peptide of any one of claims 1 to 6 or a variant thereof, comprising culturing the host cell of claim 9 which presents the peptide of claims 1 to 6, or expresses the nucleic acid of claim 7, or carries the expression vector of claim 8, and isolating the peptide or variant thereof from the host cell or the culture medium thereof.

12. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial mimetic of an antigen-presenting cell structure for a time sufficient to activate the T cells in an antigen specific manner, wherein the antigen is a peptide as described in any one of items 1 to 4.

13. An activated T lymphocyte, produced by the method of item 12, that selectively recognizes a cell that presents a polypeptide comprising an amino acid sequence given in any one of items 1 to 4.

14. A method of killing a target cell in a patient, wherein the target cell presents a polypeptide comprising an amino acid sequence set forth in any one of items 1 to 4, comprising administering to the patient an effective amount of an activated T cell of item 13.

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

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

17. The use according to item 16, wherein the cancer is selected from the group consisting of esophageal cancer, non-small cell lung cancer, renal cell carcinoma, brain cancer, gastric cancer, colorectal cancer, hepatocellular cancer, pancreatic cancer, prostate cancer, breast cancer, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder cancer, bile duct cancer and other tumors that overexpress a protein from which a peptide of SEQ ID No.1 to SEQ ID No.93 is derived.

18. A kit, comprising:

(a) a container comprising a pharmaceutical composition comprising a peptide or variant of any one of items 1 to 6, a nucleic acid of item 7, an expression vector of item 8, a cell of item 10, an activated T lymphocyte of item 13, or an antibody of item 15, in solution or lyophilized form;

(b) Optionally, a second container containing a diluent or reconstituting fluid for the lyophilized formulation;

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

(d) optionally (i) instructions for using the solution or (ii) reconstituting and/or using the lyophilized formulation.

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

20. The kit of claim 18 or 19, wherein the peptide is selected from SEQ ID No.1 to SEQ ID No. 93.

21. A method for producing a personalized anti-cancer vaccine for use as a compound-based and/or cellular therapy in an individual patient, the method comprising:

a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample of an individual patient;

b) comparing the peptides identified in a) to a library of peptides that have been pre-screened for immunogenicity and/or presentation in tumors relative to normal tissue;

c) selecting at least one peptide in the pool that matches a TUMAP identified in the patient; and

d) preparing a personalized vaccine based on step c).

22. The method of clause 21, wherein the TUMAP is identified by:

a1) comparing the expression data of the tumor sample with expression data of a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are over-or aberrantly expressed in the tumor sample; and

a2) Correlating the expression data with the sequence 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 abnormally expressed by the tumor.

23. The method of claim 21 or 22, wherein the sequence of the MHC ligand is identified by eluting a binding peptide from an MHC molecule isolated from the tumor sample and sequencing the eluted ligand.

24. The method of any one of items 21 to 23, wherein a normal tissue sample corresponding to the tissue type of the tumor sample is obtained from the same patient.

25. A method according to any one of claims 21 to 24, wherein the peptides comprised in the library are identified based on the following steps:

performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue;

ab. selecting the peptide encoded by the gene selectively expressed or overexpressed detected in step aa, and

determining induction of a T cell response in vivo by a selected peptide, comprising an in vitro immunogenicity assay using human T cells from a healthy donor or the patient; or

ba. identifying HLA ligands from the tumor sample by mass spectrometry;

performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue;

comparing the identified HLA ligands to the gene expression data;

bd. selecting the peptide encoded by the selectively expressed or overexpressed gene detected in step bc;

be. re-detecting TUMAP selected by step bd on tumor tissue and absent or infrequently detected on healthy tissue and determining the correlation of overexpression at the mRNA level; and

bf. the induction of a T cell response in vivo is determined by selected peptides, including in vitro immunogenicity assays using human T cells from healthy donors or the patient.

26. The method according to any one of claims 21 to 25, wherein the immunogenicity of the peptides contained in the repertoire is determined by a method comprising in vitro immunogenicity detection, patient immune monitoring for individual HLA binding, MHC multimer staining, ELISPOT analysis and/or intracellular cytokine staining.

27. The method according to any one of claims 21 to 26, wherein the library comprises a plurality of peptides selected from SEQ ID No.1 to SEQ ID No. 101.

28. The method of any one of claims 21 to 27, further comprising identifying at least one mutation that is unique to the tumor sample as compared to the corresponding normal tissue of the individual patient, and selecting a peptide associated with the mutation for inclusion in a vaccine or for use in generating cell therapy.

29. The method of item 28, wherein the at least one mutation is identified by whole genome sequencing.

30. A T cell receptor, preferably a soluble or membrane bound T cell receptor reactive with an HLA ligand, wherein said ligand has at least 75% homology to an amino acid sequence selected from the group consisting of SEQ ID No.1 to SEQ ID No. 93.

31. The T cell receptor of claim 30, wherein the amino acid sequence has at least 88% homology with SEQ ID No.1 to SEQ ID No. 93.

32. The T cell receptor according to claim 30 or 31, wherein the amino acid sequence consists of any one of SEQ ID No.1 to SEQ ID No. 93.

33. The T cell receptor according to any one of claims 30 to 32, wherein the T cell receptor is provided as a soluble molecule and optionally has other effector functions, such as an immunostimulatory domain or a toxin.

34. A nucleic acid encoding a TCR according to any one of claims 30 to 33, optionally linked to a heterologous activator sequence.

35. An expression vector capable of expressing the nucleic acid of item 34.

36. A host cell comprising the nucleic acid of item 34, or a nucleic acid encoding the antibody of item 15, or the expression vector of item 35, wherein the host cell is preferably a T cell or an NK cell.

37. A method of making the T cell receptor of any one of claims 30 to 33, the method comprising culturing the host cell of claim 36, and isolating the T cell receptor from the host cell and/or its culture medium.

38. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of:

a) a peptide selected from SEQ ID No.1 to SEQ ID No.93 or a pharmaceutically acceptable salt thereof;

b) t cell receptors reactive with the peptides and/or peptide MHC complexes in a);

c) a fusion protein comprising the peptide of a) and the 1 st to 80 th N-terminal amino acids of the HLA-DR antigen associated invariant chain (Ii);

d) a nucleic acid encoding any one of a) to c) or an expression vector comprising the nucleic acid;

e) comprising the host cell expressing the vector in d),

f) an activated T lymphocyte obtainable by a method comprising contacting T cells in vitro with a peptide of a) for a time sufficient to activate the T cells in an antigen-specific manner and expressing the peptide on the surface of a suitable antigen-presenting cell, and transferring the activated T cells to an autologous or other patient;

g) Antibodies or soluble T cell receptors which react with the peptides of a) and/or peptide-MHC complexes, and/or cells presenting the peptides of a), and which may be modified by fusion, for example, with an immune activation domain or toxin;

h) an aptamer recognizing a peptide selected from SEQ ID No.1 to SEQ ID No.93 and/or a complex of a peptide selected from SEQ ID No.1 to SEQ ID No.93 and an MHC molecule;

i) a conjugated or labelled peptide or scaffold according to any of a) to h), and a pharmaceutically acceptable carrier, and optionally a pharmaceutically acceptable excipient and/or stabilizer.

39. An aptamer that specifically recognizes the peptide or the variant thereof according to any one of items 1 to 5, preferably the peptide or the variant thereof according to any one of items 1 to 5 that binds to an MHC molecule.

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Sequence listing

<110> Imamatix Biotechnology Ltd

<120> novel peptides and peptide compositions for immunotherapy of esophageal and other cancers

<130> I32873WO

<150> US62/188,870

<151> 2015-07-06

<150> GB1511792.2

<151> 2015-07-06

<160> 103

<170> PatentIn version 3.5

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Ser Thr Tyr Gly Gly Gly Leu Ser Val

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Ser Leu Tyr Asn Leu Gly Gly Ser Lys Arg Ile Ser Ile

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Thr Ala Ser Ala Ile Thr Pro Ser Val

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Ala Leu Phe Gly Thr Ile Leu Glu Leu

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Asn Leu Met Ala Ser Gln Pro Gln Leu

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Leu Leu Ser Gly Asp Leu Ile Phe Leu

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<213> Intelligent people

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Ser Ile Phe Glu Gly Leu Leu Ser Gly Val

1 5 10

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<213> Intelligent people

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Ala Leu Leu Asp Gly Gly Ser Glu Ala Tyr Trp Arg Val

1 5 10

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<213> Intelligent people

<400> 9

His Leu Ile Ala Glu Ile His Thr Ala

1 5

<210> 10

<211> 10

<212> PRT

<213> Intelligent people

<400> 10

Ser Leu Asp Glu Asn Ser Asp Gln Gln Val

1 5 10

<210> 11

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<213> Intelligent people

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Ala Leu Trp Leu Pro Thr Asp Ser Ala Thr Val

1 5 10

<210> 12

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<213> Intelligent people

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Gly Leu Ala Ser Arg Ile Leu Asp Ala

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<400> 13

Ser Leu Ser Pro Val Ile Leu Gly Val

1 5

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<213> Intelligent people

<400> 14

Arg Leu Pro Asn Ala Gly Thr Gln Val

1 5

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<213> Intelligent people

<400> 15

Leu Leu Ala Asn Gly Val Tyr Ala Ala

1 5

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<213> Intelligent people

<400> 16

Val Leu Ala Glu Gly Gly Glu Gly Val

1 5

<210> 17

<211> 8

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<213> Intelligent people

<400> 17

Met Ile Ser Arg Thr Pro Glu Val

1 5

<210> 18

<211> 10

<212> PRT

<213> Intelligent people

<400> 18

Phe Leu Leu Asp Gln Val Gln Leu Gly Leu

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Intelligent people

<400> 19

Gly Leu Ala Pro Phe Leu Leu Asn Ala Val

1 5 10

<210> 20

<211> 12

<212> PRT

<213> Intelligent people

<400> 20

Ile Ile Glu Val Asp Pro Asp Thr Lys Glu Met Leu

1 5 10

<210> 21

<211> 9

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<213> Intelligent people

<400> 21

Ile Val Arg Glu Phe Leu Thr Ala Leu

1 5

<210> 22

<211> 9

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<213> Intelligent people

<400> 22

Lys Leu Asn Asp Thr Tyr Val Asn Val

1 5

<210> 23

<211> 9

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<213> Intelligent people

<400> 23

Lys Leu Ser Asp Ser Ala Thr Tyr Leu

1 5

<210> 24

<211> 9

<212> PRT

<213> Intelligent people

<400> 24

Leu Leu Phe Ala Gly Thr Met Thr Val

1 5

<210> 25

<211> 9

<212> PRT

<213> Intelligent people

<400> 25

Leu Leu Pro Pro Pro Pro Pro Pro Ala

1 5

<210> 26

<211> 9

<212> PRT

<213> Intelligent people

<400> 26

Met Leu Ala Glu Lys Leu Leu Gln Ala

1 5

<210> 27

<211> 9

<212> PRT

<213> Intelligent people

<400> 27

Asn Leu Arg Glu Gly Asp Gln Leu Leu

1 5

<210> 28

<211> 9

<212> PRT

<213> Intelligent people

<400> 28

Ser Leu Asp Gly Phe Thr Ile Gln Val

1 5

<210> 29

<211> 9

<212> PRT

<213> Intelligent people

<400> 29

Ser Leu Asp Gly Thr Glu Leu Gln Leu

1 5

<210> 30

<211> 9

<212> PRT

<213> Intelligent people

<400> 30

Ser Leu Asn Gly Asn Gln Val Thr Val

1 5

<210> 31

<211> 9

<212> PRT

<213> Intelligent people

<400> 31

Val Leu Pro Lys Leu Tyr Val Lys Leu

1 5

<210> 32

<211> 9

<212> PRT

<213> Intelligent people

<400> 32

Tyr Met Leu Asp Ile Phe His Glu Val

1 5

<210> 33

<211> 12

<212> PRT

<213> Intelligent people

<400> 33

Gly Leu Asp Val Thr Ser Leu Arg Pro Phe Asp Leu

1 5 10

<210> 34

<211> 10

<212> PRT

<213> Intelligent people

<400> 34

Ser Leu Val Ser Glu Gln Leu Glu Pro Ala

1 5 10

<210> 35

<211> 9

<212> PRT

<213> Intelligent people

<400> 35

Leu Leu Arg Phe Ser Gln Asp Asn Ala

1 5

<210> 36

<211> 10

<212> PRT

<213> Intelligent people

<400> 36

Phe Leu Leu Arg Phe Ser Gln Asp Asn Ala

1 5 10

<210> 37

<211> 12

<212> PRT

<213> Intelligent people

<400> 37

Tyr Thr Gln Pro Phe Ser His Tyr Gly Gln Ala Leu

1 5 10

<210> 38

<211> 9

<212> PRT

<213> Intelligent people

<400> 38

Ile Ala Ala Ile Arg Gly Phe Leu Val

1 5

<210> 39

<211> 10

<212> PRT

<213> Intelligent people

<400> 39

Leu Val Arg Asp Thr Gln Ser Gly Ser Leu

1 5 10

<210> 40

<211> 9

<212> PRT

<213> Intelligent people

<400> 40

Gly Leu Ala Phe Ser Leu Tyr Gln Ala

1 5

<210> 41

<211> 12

<212> PRT

<213> Intelligent people

<400> 41

Gly Leu Glu Ser Glu Glu Leu Glu Pro Glu Glu Leu

1 5 10

<210> 42

<211> 9

<212> PRT

<213> Intelligent people

<400> 42

Thr Gln Thr Ala Val Ile Thr Arg Ile

1 5

<210> 43

<211> 9

<212> PRT

<213> Intelligent people

<400> 43

Lys Val Val Gly Lys Asp Tyr Leu Leu

1 5

<210> 44

<211> 14

<212> PRT

<213> Intelligent people

<400> 44

Ala Thr Gly Asn Asp Arg Lys Glu Ala Ala Glu Asn Ser Leu

1 5 10

<210> 45

<211> 9

<212> PRT

<213> Intelligent people

<400> 45

Met Leu Thr Glu Leu Glu Lys Ala Leu

1 5

<210> 46

<211> 11

<212> PRT

<213> Intelligent people

<400> 46

Tyr Thr Ala Gln Ile Gly Ala Asp Ile Ala Leu

1 5 10

<210> 47

<211> 9

<212> PRT

<213> Intelligent people

<400> 47

Val Leu Ala Ser Gly Phe Leu Thr Val

1 5

<210> 48

<211> 10

<212> PRT

<213> Intelligent people

<400> 48

Ser Met His Gln Met Leu Asp Gln Thr Leu

1 5 10

<210> 49

<211> 9

<212> PRT

<213> Intelligent people

<400> 49

Gly Leu Met Lys Asp Ile Val Gly Ala

1 5

<210> 50

<211> 11

<212> PRT

<213> Intelligent people

<400> 50

Gly Met Asn Pro His Gln Thr Pro Ala Gln Leu

1 5 10

<210> 51

<211> 9

<212> PRT

<213> Intelligent people

<400> 51

Lys Leu Phe Gly His Leu Thr Ser Ala

1 5

<210> 52

<211> 12

<212> PRT

<213> Intelligent people

<400> 52

Val Ala Ile Gly Gly Val Asp Gly Asn Val Arg Leu

1 5 10

<210> 53

<211> 9

<212> PRT

<213> Intelligent people

<400> 53

Val Val Val Thr Gly Leu Thr Leu Val

1 5

<210> 54

<211> 9

<212> PRT

<213> Intelligent people

<400> 54

Tyr Gln Asp Leu Leu Asn Val Lys Met

1 5

<210> 55

<211> 9

<212> PRT

<213> Intelligent people

<400> 55

Gly Ala Ile Asp Leu Leu His Asn Val

1 5

<210> 56

<211> 9

<212> PRT

<213> Intelligent people

<400> 56

Ala Leu Val Glu Val Thr Glu His Val

1 5

<210> 57

<211> 10

<212> PRT

<213> Intelligent people

<400> 57

Gly Leu Ala Pro Asn Thr Pro Gly Lys Ala

1 5 10

<210> 58

<211> 9

<212> PRT

<213> Intelligent people

<400> 58

Leu Ile Leu Glu Ser Ile Pro Val Val

1 5

<210> 59

<211> 9

<212> PRT

<213> Intelligent people

<400> 59

Ser Leu Leu Asp Thr Leu Arg Glu Val

1 5

<210> 60

<211> 9

<212> PRT

<213> Intelligent people

<400> 60

Val Val Met Glu Glu Leu Leu Lys Val

1 5

<210> 61

<211> 9

<212> PRT

<213> Intelligent people

<400> 61

Thr Gln Thr Thr His Glu Leu Thr Ile

1 5

<210> 62

<211> 10

<212> PRT

<213> Intelligent people

<400> 62

Ala Leu Tyr Glu Tyr Gln Pro Leu Gln Ile

1 5 10

<210> 63

<211> 10

<212> PRT

<213> Intelligent people

<400> 63

Leu Ala Tyr Thr Leu Gly Val Lys Gln Leu

1 5 10

<210> 64

<211> 9

<212> PRT

<213> Intelligent people

<400> 64

Gly Leu Thr Asp Val Ile Arg Asp Val

1 5

<210> 65

<211> 11

<212> PRT

<213> Intelligent people

<400> 65

Tyr Val Val Gly Gly Phe Leu Tyr Gln Arg Leu

1 5 10

<210> 66

<211> 9

<212> PRT

<213> Intelligent people

<400> 66

Leu Leu Asp Glu Lys Val Gln Ser Val

1 5

<210> 67

<211> 9

<212> PRT

<213> Intelligent people

<400> 67

Ser Met Asn Gly Gly Val Phe Ala Val

1 5

<210> 68

<211> 12

<212> PRT

<213> Intelligent people

<400> 68

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

1 5 10

<210> 69

<211> 11

<212> PRT

<213> Intelligent people

<400> 69

Gly Leu Leu Val Gly Ser Glu Lys Val Thr Met

1 5 10

<210> 70

<211> 9

<212> PRT

<213> Intelligent people

<400> 70

Phe Val Leu Asp Thr Ser Glu Ser Val

1 5

<210> 71

<211> 12

<212> PRT

<213> Intelligent people

<400> 71

Ala Ser Asp Pro Ile Leu Tyr Arg Pro Val Ala Val

1 5 10

<210> 72

<211> 9

<212> PRT

<213> Intelligent people

<400> 72

Phe Leu Pro Pro Ala Gln Val Thr Val

1 5

<210> 73

<211> 9

<212> PRT

<213> Intelligent people

<400> 73

Lys Ile Thr Glu Ala Ile Gln Tyr Val

1 5

<210> 74

<211> 9

<212> PRT

<213> Intelligent people

<400> 74

Ile Leu Ala Ser Leu Ala Thr Ser Val

1 5

<210> 75

<211> 10

<212> PRT

<213> Intelligent people

<400> 75

Gly Leu Met Asp Asp Val Asp Phe Lys Ala

1 5 10

<210> 76

<211> 9

<212> PRT

<213> Intelligent people

<400> 76

Lys Val Ala Asp Tyr Ile Pro Gln Leu

1 5

<210> 77

<211> 10

<212> PRT

<213> Intelligent people

<400> 77

Val Leu Val Pro Tyr Glu Pro Pro Gln Val

1 5 10

<210> 78

<211> 9

<212> PRT

<213> Intelligent people

<400> 78

Lys Val Ala Asn Ile Ile Ala Glu Val

1 5

<210> 79

<211> 9

<212> PRT

<213> Intelligent people

<400> 79

Gly Gln Asp Val Gly Arg Tyr Gln Val

1 5

<210> 80

<211> 9

<212> PRT

<213> Intelligent people

<400> 80

Ala Leu Gln Glu Ala Leu Glu Asn Ala

1 5

<210> 81

<211> 9

<212> PRT

<213> Intelligent people

<400> 81

Ala Val Leu Pro His Val Asp Gln Val

1 5

<210> 82

<211> 9

<212> PRT

<213> Intelligent people

<400> 82

His Leu Leu Gly His Leu Glu Gln Ala

1 5

<210> 83

<211> 10

<212> PRT

<213> Intelligent people

<400> 83

Ala Leu Ala Asp Gly Val Val Ser Gln Ala

1 5 10

<210> 84

<211> 9

<212> PRT

<213> Intelligent people

<400> 84

Ser Leu Ala Glu Ser Leu Asp Gln Ala

1 5

<210> 85

<211> 9

<212> PRT

<213> Intelligent people

<400> 85

Asn Ile Ile Glu Leu Val His Gln Val

1 5

<210> 86

<211> 9

<212> PRT

<213> Intelligent people

<400> 86

Gly Leu Leu Thr Glu Ile Arg Ala Val

1 5

<210> 87

<211> 9

<212> PRT

<213> Intelligent people

<400> 87

Phe Leu Asp Asn Gly Pro Lys Thr Ile

1 5

<210> 88

<211> 9

<212> PRT

<213> Intelligent people

<400> 88

Gly Leu Trp Glu Gln Glu Asn His Leu

1 5

<210> 89

<211> 9

<212> PRT

<213> Intelligent people

<400> 89

Ser Leu Ala Asp Ser Leu Tyr Asn Leu

1 5

<210> 90

<211> 9

<212> PRT

<213> Intelligent people

<400> 90

Ser Ile Tyr Glu Tyr Tyr His Ala Leu

1 5

<210> 91

<211> 9

<212> PRT

<213> Intelligent people

<400> 91

Lys Leu Ile Asp Asp Val His Arg Leu

1 5

<210> 92

<211> 9

<212> PRT

<213> Intelligent people

<400> 92

Ser Ile Leu Arg His Val Ala Glu Val

1 5

<210> 93

<211> 9

<212> PRT

<213> Intelligent people

<400> 93

Val Leu Ile Asn Thr Ser Val Thr Leu

1 5

<210> 94

<211> 11

<212> PRT

<213> Intelligent people

<400> 94

Thr Leu Leu Gln Glu Gln Gly Thr Lys Thr Val

1 5 10

<210> 95

<211> 9

<212> PRT

<213> Intelligent people

<400> 95

Leu Ile Gln Asp Arg Val Ala Glu Val

1 5

<210> 96

<211> 10

<212> PRT

<213> Intelligent people

<400> 96

Gly Ala Ala Val Arg Ile Gly Ser Val Leu

1 5 10

<210> 97

<211> 9

<212> PRT

<213> Intelligent people

<400> 97

Glu Leu Asp Arg Thr Pro Pro Glu Val

1 5

<210> 98

<211> 9

<212> PRT

<213> Intelligent people

<400> 98

Val Leu Phe Pro Asn Leu Lys Thr Val

1 5

<210> 99

<211> 10

<212> PRT

<213> Intelligent people

<400> 99

Arg Val Ala Pro Glu Glu His Pro Val Leu

1 5 10

<210> 100

<211> 10

<212> PRT

<213> Intelligent people

<400> 100

Gly Leu Tyr Pro Asp Ala Phe Ala Pro Val

1 5 10

<210> 101

<211> 9

<212> PRT

<213> Intelligent people

<400> 101

Ala Met Thr Gln Leu Leu Ala Gly Val

1 5

<210> 102

<211> 10

<212> PRT

<213> Intelligent people

<400> 102

Glu Leu Ala Gly Ile Gly Ile Leu Thr Val

1 5 10

<210> 103

<211> 9

<212> PRT

<213> Intelligent people

<400> 103

Tyr Leu Leu Pro Ala Ile Val His Ile

1 5