Novel peptides and peptide combinations for use in immunotherapy against hepatocellular carcinoma (HCC) and other cancers

Abstract

To provide peptides particularly for use in the immunotherapy of cancer.SOLUTION: The present invention provides peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods, and furthermore provides tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides, which can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to major histocompatibility complex (MHC) molecules, or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. In particular, the present invention relates to several novel peptide sequences and their variants derived from HLA class I and class II molecules of human tumor cells which can be used in vaccine compositions for eliciting anti-tumor immune responses or as targets for the development of pharmaceutically / immunologically active compounds and cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to peptides, proteins, nucleic acids, and cells for use in immunotherapy. In particular, the present invention relates to cancer immunotherapy. The present invention further relates to tumor-associated T cell peptide epitopes, either alone or in combination with other tumor-associated peptides, which can serve as the active pharmaceutical ingredient of a vaccine composition that, for example, stimulates an anti-tumor immune response or stimulates T cells in vitro for transfer into a patient. The peptide can bind to molecules of the major histocompatibility complex (MHC), or the peptide itself can be a target for antibodies, soluble T cell receptors, and other binding molecules. Specifically, the present invention relates to several novel peptide sequences and their variants derived from HLA class I and class II molecules of human tumor cells, which can be used in a vaccine composition to elicit an anti-tumor immune response or as a development target for pharmacological / immunological active compounds and cells.

Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most frequent tumors in the world, accounting for approximately 6% of all newly diagnosed cancer cases worldwide. In 2012, approximately 782,000 new cases of HCC appeared worldwide, making it the fifth most frequent cancer in men (554,000 cases) and the ninth most frequent cancer in women (228,000 cases). (http: / / globocan.iarc.fr). HCC is the most frequent primary liver malignant lesion, constituting 80% of all adult primary liver cancers.

[0003] The distribution of HCC varies geographically and its incidence is influenced by gender. The age-standardized incidence rate (ASR) of HCC in men is highest in East Asia (31.9) and Southeast Asia (22.2), moderate in Southern Europe (9.5) and North America (9.3), and lowest in Northern Europe and Central Asia (3.7). The incidence rate of HCC in women is lower than the male ASR. The highest ASR in women is in East Asia (10.2) and West Africa (8.1), and the lowest is in Northern Europe (1.9) and Micronesia (1.6).

[0004] The overall prognosis of patients with HCC is not good. The 5-year relative survival rate (5Y-RSR) of HCC is about 15% and is influenced by the stage at diagnosis. For localized HCC where the cancer is still confined to the liver, the 5Y-RSR is about 28%. For local and distant HCC where the cancer has spread to nearby or distant organs, the 5Y-RSR is 7% and 2% respectively.

[0005] The incidence of HCC is associated with several risk factors, with cirrhosis being the most important factor. Cirrhosis often occurs in parallel with alcohol abuse or HBV or HCV infections, but can also be caused by metabolic diseases such as type II diabetes. As a result, healthy liver tissue is replaced by scar tissue that increases the risk of cancer development.

[0006] Disease management is influenced by the tumor stage at diagnosis and the overall condition of the liver. If possible, a part of the liver (partial hepatectomy) or the whole organ (hepatectomy) is removed surgically. In particular, patients with small or completely resectable tumors are eligible for liver transplantation.

[0007] If surgery is not an option for treatment, several other treatment methods are currently available. In tumor ablation, a probe is inserted into the liver and the tumor is destroyed by radio waves or microwaves or cryotherapy. In embolization, the blood supply to the tumor is blocked by mechanical or chemical means. In radiotherapy, high-energy radio waves can be used to destroy the tumor.

[0008] As chemotherapies for HCC, combinations of doxorubicin, 5-fluorouracil, and cisplatin for systemic therapy, and combinations of doxorubicin, floxuridine, and mitomycin C for hepatic arterial infusion can be mentioned. However, most HCCs show high resistance to chemotherapeutic drugs (Enguita-German and Fortes, 2014).

[0009]

[0009] The treatment options for advanced unresectable HCC are limited to sorafenib, a multi-tyrosine kinase inhibitor (Chang et al., 2007; Wilhelm et al., 2004). Sorafenib is the only systemic drug that has been confirmed to increase the survival period by about three months and currently corresponds to the only experimental treatment option for such patients (Chapiro et al., 2014; Llovet et al., 2008).

[0010]

[0010] Recently, a limited number of immunotherapy trials for HCC have been conducted. Cytokines have been used to activate subsets of immune cells and / or increase tumor immunogenicity (Reinisch et al., 2002; Sangro et al., 2004). Other trials have focused on the infusion of tumor-infiltrating lymphocytes or activated peripheral blood lymphocytes (Shi et al., 2004a; Takayama et al., 1991; Takayama et al., 2000).

[0011] To date, a small number of therapeutic vaccination trials have been conducted. Butterfield et al. conducted two trials using peptides derived from alpha-fetoprotein (AFP) as vaccines or dendritic cells (DCs) loaded with AFP peptides in vitro (Butterfield et al., 2003; Butterfield et al., 2006). In two different studies, autologous dendritic cells (DCs) were pulsed in vitro using autologous tumor lysates (Lee et al., 2005) or lysates of the hepatoblastoma cell line HepG2 (Palmer et al., 2009). To date, vaccination trials have shown only limited improvement in clinical outcomes.

Summary of the Invention

Means for Solving the Problems

[0012] In a first aspect of the present invention, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300, or a variant sequence thereof that is at least 80%, preferably at least 90% homologous (preferably at least 80% or at least 90% identical) to SEQ ID NO: 1 to SEQ ID NO: 300, wherein the variant binds to MHC and / or induces a cross-reaction of T cells with the peptide or a pharmaceutically acceptable salt thereof, and wherein the peptide is not the underlying full-length polypeptide.

[0013] The present invention further relates to a peptide of the present invention comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300, or a variant thereof that is at least 80%, preferably at least 88% homologous (preferably at least 80% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 300, wherein the peptide or a variant thereof has a full length of 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0014] The following table shows the peptides according to the invention, their respective SEQ ID NOs, and the genes from which those peptides are expected to originate. All peptides in Table 1 bind to HLA-A * 02, and the peptides in Table 2 bind to the HLA-A * 24 allele. The peptides in Table 3 were previously disclosed in a broad list as a result of high-throughput screening that had a high error rate or was calculated using an algorithm, but have not heretofore been associated with cancer at all. They bind to HLA-A * 02. The peptides in Table 4 are additional peptides that may be useful in combination with other peptides of the invention. The peptides bind to A * 02, or, if indicated, to A * 24. The peptides in Table 5 are further useful in the diagnosis and / or treatment of various malignancies involving overexpression or over-presentation of the respective base polypeptides.

[0015] Table 1: HLA-A * 02 Peptides according to the invention; S * = Phosphoserine

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0016] Table 2: HLA-A * 24 Peptides according to the invention and SEQ ID NOs; S * = Phosphoserine

Table 2

[0017] Table 3: Additional peptides according to the invention with previously unknown cancer relevance; S * = Phosphoserine

Table 3-1

Table 3-2

[0018] Table 4: Peptides useful, for example, for personalized cancer therapy; S * = Phosphoserine

Table 4-1

Table 4-2

[0019] The present invention further relates to peptides according to the invention that are generally used in the treatment of proliferative diseases such as, for example, pancreatic cancer, colorectal cancer, kidney cancer, brain tumors, and / or leukemia.

[0020] Particularly preferred are peptides according to the invention, alone or in combination, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300. More preferably, preferably A * 02 A group consisting of SEQ ID NO: 1 to SEQ ID NO: 124 (see Table 1) for binding, and preferably A * 24 Peptides, alone or in combination, selected from the group consisting of SEQ ID NO: 187 to SEQ ID NO: 218 (see Table 2) for binding, and their use in immunotherapy for HCC, brain tumors, kidney cancer, pancreatic cancer, colorectal cancer or leukemia, preferably HCC.

[0021] As shown in Tables 5A and B below, many of the peptides according to the invention can also be used in immunotherapies for other indications. The tables show additional tumor types in which the peptides were found that showed overexpression (including specific expression) in more than 5% of the measured tumor samples, or were expressed in more than 5% of the measured tumor samples with a geometric mean ratio of tumor to normal tissue greater than 3. Overexpression is defined as expression on higher tumor samples compared to normal samples with the highest expression. Normal tissues in which overexpression was tested against it were adipose tissue, adrenal gland, blood cells, blood vessels, bone marrow, brain, cartilage, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung, lymph nodes, nerve, pancreas, thyroid, peritoneum, pituitary gland, pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid, trachea, ureter, bladder.

[0022] Table 5A: Peptides according to the invention and their specific uses in other cancerous diseases, in particular other proliferative diseases; S * = Phosphoserine

Table 5A-1

Table 5A-2

Table 5A-3

Table 5A-4

Table 5A-5

Table 5B-1

Table 5B-2

Table 5B-3

Table 5B-4

Table 5B-5

Table 5B-6

[0023] Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention as set forth in any one of SEQ ID NOs: 1, 14, 15, 41, 43, 58, 59, 60, 81, 121, 135, 139, 144, 176, 236, 248, 275, 276, 283, 286, 288, 289, 290, 291, 300, 302, 304, 308, 313, 316, 317, 325, 326, 329, 331, 334, 342, and 343, and in a preferred embodiment, to its use in combination therapy for pancreatic cancer.

[0024] Accordingly, another aspect of the present invention relates to the use of at least one peptide according to the present invention as set forth in SEQ ID NO: 6, 15, 16, 22, 26, 30, 34, 36, 47, 59, 65, 69, 70, 77, 80, 81, 88, 121, 123, 125, 127, 133, 137, 139, 169, 172, 176, 181, 186, 221, 223, 229, 230, 231, 232, 233, 234, 236, 237, 238, 244, 247, 249, 250, 251, 255, 260, 261, 266, 269, 271, 274, 275, 282, 285, 289, 290, 291, 293, 297, 301, 302, 304, 306, 310, 313, 316, 317, 319, 327, 328, 329, 330, 331, 332, 334, and 342, preferably for use in combination therapy for colon cancer or renal cancer.

[0025] Accordingly, another aspect of the present invention relates to the use of at least one peptide according to the present invention as set forth in SEQ ID NO: 10, 14, 15, 22, 36, 39, 54, 55, 60, 72, 77, 81, 90, 96, 112, 116, 119, 121, 133, 137, 138, 148, 169, 170, 172, 177, 186, 187, 189, 192, 197, 198, 203, 206, 219, 221, 229, 230, 233, 234, 236, 255, 260, 270, 272, 275, 277, 278, 279, 281, 282, 285, 289, 291, 292, 295, 296, 297, 301, 302, 305, 308, 311, 313, 315, 316, 319, 321, 324, 328, 329, 333, 334, 335, 336, and 346, preferably for use in combination therapy for renal cancer.

[0026] Accordingly, another aspect of the present invention relates to the use of at least one peptide according to the present invention as set forth in SEQ ID NOs: 14, 15, 16, 17, 36, 39, 47, 51, 54, 65, 88, 101, 123, 125, 133, 134, 135, 137, 141, 147, 161, 166, 169, 176, 179, 184, 186, 187, 189, 191, 192, 193, 194, 195, 196, 197, 199, 203, 206, 208, 214, 220, 221, 224, 229, 230, 231, 234, 238, 239, 244, 245, 250, 251, 255, 258, 259, 260, 268, 269, 270, 271, 272, 278, 279, 282, 295, 297, 302, 304, 305, 306, 309, 310, 311, 312, 313, 316, 317, 319, 321, 325, 327, 328, 329, 330, 331, 332, 333, 334, 336, 337, 338, 339, 340, 342, 343, 344, 345, and 347, preferably for use in combination therapy for brain tumors.

[0027] Accordingly, another aspect of the present invention relates to the use of at least one peptide according to the present invention as set forth in SEQ ID NOs: 172, 173, 240, 250, 287, 299, 302, 334, and 335, preferably for use in combination therapy for CLL.

[0028] Similarly, the peptides listed in Table 5B as above may form the basis for combination therapy of the indicated diseases in a preferred embodiment.

[0029] Accordingly, another aspect of the present invention preferably relates to the use of a peptide according to the present invention for combination therapy of a proliferative disease selected from the group of HCC, brain tumor, renal cancer, pancreatic cancer, colon or rectal cancer, and leukemia.

[0030] The present invention further relates to a peptide according to the present invention having the ability to bind to human major histocompatibility complex (MHC) class I molecules or, in extended forms such as length variants, to MHC class II.

[0031] The present invention further relates to a peptide according to the present invention, said peptide consisting of, or consisting essentially of, the amino acid sequences set forth in SEQ ID NOs: 1 to 300 (respectively).

[0032] The present invention further relates to a peptide according to the present invention, said peptide being modified and / or containing non-peptide bonds.

[0033] The present invention further relates to a peptide according to the present invention, said peptide being part of a fusion protein, particularly fused to the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii), or fused to (or within its sequence) an antibody such as an antibody specific for dendritic cells.

[0034] The present invention further relates to a nucleic acid encoding a peptide according to the present invention. The present invention further relates to a nucleic acid according to the present invention which is DNA, cDNA, PNA, RNA, or a combination thereof.

[0035] The present invention further relates to an expression vector capable of expressing and / or expressing a nucleic acid according to the present invention.

[0036] The present invention further relates to a peptide according to the present invention, a nucleic acid according to the present invention, or an expression vector according to the present invention, which is used in the treatment of diseases and in medicine, particularly in the treatment of diseases such as cancer and autoimmune / inflammatory / immunopathological diseases.

[0037] The present invention further relates to an antibody against a peptide according to the present invention, or a complex of said peptide according to the present invention and MHC, and methods for producing these.

[0038] The present invention further relates to a T cell receptor (TCR), particularly a soluble TCR (sTCR) and a cloned TCR, incorporated into autologous or allogeneic T cells, and methods for producing these, and methods for producing NK cells or other cells having or cross-reacting with said TCR.

[0039] Antibodies and TCRs are additional embodiments of the immunotherapeutic use of the peptides according to the invention.

[0040] The invention further relates to host cells comprising a nucleic acid or expression vector according to the invention as described above. The invention further relates to host cells according to the invention which are antigen-presenting cells, preferably dendritic cells.

[0041] The invention further relates to a method for producing a peptide according to the invention, comprising culturing a host cell according to the invention and isolating the peptide from the host cell or its culture broth.

[0042] The invention further relates to a method according to the invention, wherein an antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by contacting the antigen-presenting cell with a sufficient amount of antigen.

[0043] The invention further relates to a method according to the invention, wherein the antigen-presenting cell is capable of expressing or expresses the peptide, preferably comprising SEQ ID NO: 1 to SEQ ID NO: 300, more preferably SEQ ID NO: 1 to SEQ ID NO: 124 and SEQ ID NO: 187 to SEQ ID NO: 218 or a mutated amino acid sequence, and comprises an expression vector.

[0044] The invention further relates to activated T cells produced by the method according to the invention, wherein the T cells selectively recognize cells expressing a polypeptide comprising the amino acid sequence according to the invention.

[0045] The invention further relates to a method for killing target cells that abnormally express a polypeptide comprising any amino acid sequence according to the invention in a patient, comprising administering to the patient an effective number of T cells produced by the method according to the invention.

[0046] The present invention further relates to the use of any peptide described, nucleic acid according to the invention, expression vector according to the invention, cell according to the invention, activated T lymphocyte, T cell receptor or antibody or other peptide and / or peptide-MHC binding molecule as a medicament or in the manufacture of a medicament. Preferably, the medicament is effective against cancer.

[0047] Preferably, the medicament is for cell therapy and is a soluble TCR or antibody-based vaccine or protein.

[0048] The present invention further relates to the use according to the invention, wherein the cancer cells are HCC, brain tumor, kidney cancer, pancreatic cancer, colon or rectal cancer or leukemia, and preferably HCC cells.

[0049] The present invention further relates to specific labeled proteins and biomarkers based on peptides according to the invention, referred to herein as "targets", which can be used in the diagnosis and / or prognostic diagnosis of HCC. The present invention also relates to the use of these novel targets in the context of cancer therapy.

[0050] There are two classes of MHC molecules, MHC class I and MHC class II. MHC molecules are each composed of a heavy chain and either β-2-microglobulin (MHC class I receptor) or α and β chains (MHC class II receptor). Their three-dimensional structures result in a binding groove that is used for non-covalent interactions with peptides. MHC class I molecules are found on most nucleated cells. They present peptides derived mainly from endogenous proteins, defective ribosomal products (DRIP), and proteolytic cleavage of larger peptides. MHC class II molecules are found mostly on professional antigen-presenting cells (APC) and mainly present peptides of exogenous or transmembrane proteins that are taken up by the APC during endocytosis and subsequently processed. While the complex of peptide and MHC class I is recognized by CD8-positive T cells having an appropriate TCR (T cell receptor), the complex of peptide and MHC class II molecule is recognized by CD4-positive helper T cells having an appropriate TCR. As a result, it is well known that TCR, peptide, and MHC are present in stoichiometric amounts of 1:1:1.

[0051] CD4-positive helper T cells play an important role in inducing and maintaining an effective response by CD8-positive cytotoxic T cells. Identification of CD4-positive T cell epitopes derived from tumor-associated antigens (TAAs) is highly important in the development of pharmaceuticals for initiating anti-tumor immune responses (Gnjatic S, et al. Survey of naturally occurring CD4+T cell responses against NY-ESO-1 in cancer patients: correlation with antibody responses. Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8862-7). In the tumor site, T helper cells maintain a cytotoxic T lymphocyte (CTL)-affinity cytokine environment to attract effector cells such as, for example, CTL, NK cells, macrophages, granulocytes (Mortara L, et al. CIITA-induced MHC class II expression in mammary adenocarcinoma leads to a Th1 polarization of the tumor microenvironment, tumor rejection, and specific antitumor memory. Clin Cancer Res. 2006 Jun 1;12(11 Pt 1):3435-43) (Hwang ML, et al. Cognate memory CD4+ T cells generated with dendritic cell priming influence the expansion, trafficking, and differentiation of secondary CD8+ T cells and enhance tumor control. J Immunol. 2007 Nov 1;179(9):5829-38).

[0052] In the absence of inflammation, the expression of MHC class II molecules is mainly limited to immune system cells, particularly professional antigen-presenting cells (APCs) such as, for example, monocytes, monocyte-derived cells, macrophages, dendritic cells, etc. In cancer patients, it has been found that tumor cells express MHC class II molecules (Dengjel J, et al. Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas. Clin Cancer Res. 2006 Jul 15;12(14 Pt 1):4163-70).

[0053] The extended (longer) peptides of the present invention can act as MHC class II active epitopes. T helper cells activated by MHC class II epitopes play an important role in integrating the effector functions of CTLs in anti-tumor immunity. T helper cell epitopes that initiate a TH1-type T helper cell response support the effector functions of CD8-positive killer T cells, which include cytotoxic functions directed against tumor cells presenting tumor-associated peptide / MHC complexes on their cell surface. Thus, tumor-associated T helper cell peptide epitopes can serve as the active pharmaceutical ingredient of a vaccine composition that stimulates an anti-tumor immune response, either alone or in combination with other tumor-associated peptides.

[0054] For example, in mammalian models such as mice, it has been shown that CD4-positive T cells are sufficient to inhibit tumor development through angiogenesis inhibition by secretion of interferon γ (IFNγ) even in the absence of CD8-positive T lymphocytes.

[0055] There is evidence that CD4 T cells are direct anti-tumor effectors (Braumuller et al., 2013; Tran et al., 2014).

[0056] Constitutive expression of HLA class II molecules is usually limited to immune cells, so it was not considered possible to directly isolate class II peptides from primary tumors. However, Dengjel et al. successfully identified several MHC class II epitopes directly from tumors (WO 2007 / 028574 pamphlet, EP 1760088B1 specification).

[0057] Antigens recognized by tumor-specific cytotoxic T lymphocytes, i.e., their epitopes, can be molecules derived from all protein classes such as enzymes, receptors, transcription factors, etc., which are expressed in each tumor cell and are usually upregulated compared to non-transformed cells of the same origin.

[0058] Since both CD8- and CD4-dependent types of responses contribute synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens recognized by either CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4-positive T helper cells (ligand: MHC class II molecule + peptide epitope) are important for the development of tumor vaccines.

[0059] For MHC class I peptides to initiate (induce) a cellular immune response, they must also bind to MHC molecules. This process depends on the alleles of the MHC molecule and the specific polymorphism of the amino acid sequence of the peptide. MHC class I-binding peptides are usually 8 - 12 amino acid residues long and usually contain two conserved residues ("anchors") in those sequences that interact with the corresponding binding groove of the MHC molecule. In this way, each MHC allele has a "binding motif" that determines which peptides can specifically bind to the binding groove.

[0060] In MHC class I-dependent immune responses, peptides not only have to be able to bind to specific MHC class I molecules expressed by tumor cells, but they also have to be subsequently recognized by T cells with specific T cell receptors (TCRs).

[0061] The current classification of tumor - associated antigens consists of the following major groups: a) Cancer - testis antigens: The first TAAs identified that can be recognized by T cells belong to this class and were originally called cancer - testis (CT) antigens because their members are expressed in histologically different human tumors and are present only in spermatocytes / spermatogonia of the testis and sometimes in the placenta in normal tissues. Since testicular cells do not express class I and II HLA molecules, these antigens cannot be recognized by T cells of normal tissues and are thus considered immunologically tumor - specific. Well - known examples of CT antigens are members of the MAGE family or NY - ESO - 1.

[0062] b) Differentiation antigens: These TAAs are shared between the tumor and the normal tissue from which the tumor arises; most are found in melanoma and normal melanocytes. Many of these melanocyte - related proteins are involved in melanin biosynthesis and are thus not tumor - specific, yet are still widely used for cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan - A / MART - 1 for melanoma, or PSA for prostate cancer.

[0063] c) Overexpressed TAAs: Genes encoding TAAs that are widely expressed are detected at generally lower expression levels in histologically different tumor types as well as in many normal tissues. Many of the epitopes processed and potentially presented by normal tissues may be below the T - cell recognition threshold level, while their overexpression in tumor cells can trigger an anti - cancer response by disrupting pre - established immune tolerance. Notable examples of this class of TAAs are Her - 2 / neu, survivin, telomerase, or WT1.

[0064] d) Tumor-specific antigens: These unique TAAs result from mutations in normal genes (such as β-catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and / or progression. Tumor-specific antigens can usually induce a strong immune response without the risk of autoimmune reactions against normal tissues. On the other hand, these TAAs are, in most cases, only related to the very tumor in which they are identified and are usually not shared among many individual tumors. In the case of proteins with tumor-specific (related) isoforms, the tumor specificity (or relatedness) of the peptide may also occur when the peptide is derived from a tumor (related) exon.

[0065] e) TAAs arising from abnormal post-translational modifications: Such TAAs may arise from proteins that are not specific or overexpressed in tumors, but still become tumor-associated antigens mainly by active post-translational processes in tumors. Examples of this class result from modified glycosylation patterns, leading to new epitopes such as MUC1 in tumors, or events such as protein splicing during degradation, which may or may not be tumor-specific.

[0066] f) Tumor viral proteins: These TAAs are viral proteins that may play an important role in the carcinogenic process and, since they are exogenous (not of human origin), can induce a T cell response. Examples of such proteins are the human papillomavirus type 16 viral proteins E6 and E7 expressed in cervical cancer.

[0067] For a protein to be recognized as a tumor-specific or tumor-associated antigen by cytotoxic T lymphocytes and utilized in therapy, certain requirements must be met. The antigen should be expressed primarily by tumor cells and not, or only relatively minimally, by healthy tissues. In a preferred embodiment, the peptide should be overpresented by tumor cells compared to healthy tissues. It is further desirable that each antigen not only be present in one tumor type but also be present at a high density (i.e., the copy number of each peptide per cell). Tumor-specific and tumor-associated antigens often derive from proteins that are directly involved in the transformation of normal cells into tumor cells, for example, due to their function in cell cycle control or apoptosis inhibition. Furthermore, downstream targets of the protein that is the direct cause of transformation may be upregulated and thus (und) indirectly tumor-associated. Such indirectly tumor-associated antigens may also be targets of vaccination approaches. (Singh-Jasuja et al., 2004). For such a peptide (an "immunogenic peptide") derived from a tumor-associated antigen to reliably elicit an in vitro or in vivo T cell response, it is essential that the epitope be present within the amino acid sequence of the antigen.

[0068] Basically, any peptide that can bind to MHC molecules may function as a T cell epitope. Requirements for inducing an in vitro or in vivo T cell response are the presence of T cells with the corresponding TCR and the absence of immune tolerance to this specific epitope.

[0069] Therefore, TAAs are the starting point for the development of T cell-based therapies, including but not limited to tumor vaccines. Methods for identifying and characterizing TAAs are based on the use of T cells that can be isolated from patients or healthy individuals, or they are based on the generation of differential transcriptional profiles or differential peptide expression patterns between tumors and normal tissues.

[0070] However, the identification of genes that are overexpressed in tumor tissue or human tumor cell lines, or selectively expressed in such tissue or cell lines, does not provide accurate information regarding the use of antigens transcribed from these genes in immunotherapy. This is because only individual subpopulations of epitopes of these antigens are suitable for such uses, since there must be T cells with the corresponding TCR, and immune tolerance to this specific epitope must be absent or minimal. Thus, in highly preferred embodiments of the invention, it is important to select only peptides that are over- or selectively presented and for which there are functional and / or proliferative T cells. Such functional T cells are defined as T cells that can clonally expand upon stimulation with a specific antigen and can perform effector functions ("effector T cells").

[0071] In the case of the TCRs and antibodies according to the invention, the immunogenicity of the underlying peptide is secondary. In the TCRs and antibodies according to the invention, presentation is the determining factor.

[0072] Both therapeutic and diagnostic uses for additional cancerous diseases are disclosed in the more detailed description below regarding the base proteins (polypeptides) of the peptides according to the invention.

[0073] Differential expression of COL18A1 has been reported in bladder cancer, rhabdoid tumor tumors, and ovarian cancer, and specific polymorphisms within the gene have been shown to increase the risk of sporadic breast cancer (Fang et al., 2013; Gadd et al., 2010; Peters et al., 2005; Lourenco et al., 2006).

[0074] Changes in COPA gene expression and RNA editing have been shown to be associated with hepatocellular carcinoma, and experimental studies have revealed the anti-apoptotic effect of COPA in mesothelial cells (Sudo et al., 2010; Qi et al., 2014; Wong et al., 2003).

[0075] The activity of CPB2 has been shown to be significantly decreased in acute promyelocytic leukemia (Meijers et al., 2000).

[0076] CRP, an acute-phase protein synthesized in the liver, has been shown to be a prognostic marker in various cancer types, particularly renal cell carcinoma and multiple myeloma (Ljungberg, 2007; Fassas and Tricot, 2004).

[0077] CRYZ is a target gene of the tumor suppressor p53 (Bansal et al., 2011). Its encoded protein, ζ-crystallin, has been shown to directly interact with the mRNA of the anti-apoptotic molecule bcl-2 and stabilize the overexpression of bcl-2 in T-cell acute lymphoblastic leukemia (Lapucci et al., 2010).

[0078] Overexpression of CSRP2 is associated with the dedifferentiation of hepatocellular carcinoma (Midorikawa et al., 2002).

[0079] CYB5A encodes an enzyme that detoxifies carcinogenic molecules and is a prognostic factor for pancreatic cancer (Blanke et al., 2014; Giovannetti et al., 2014).

[0080] Increased expression levels of CYP27A1 are associated with endometrial cancer, breast cancer, and colorectal cancer (Bergada et al., 2014; Nelson et al., 2013; Matusiak and Benya, 2007).

[0081] Overexpression of CYP2E1 has been reported in colorectal cancer, and specific polymorphisms are associated with bladder and lung cancers and breast cancer cells (Ye et al., 2014; Patel et al., 2014; Deng et al., 2014; Leung et al., 2013).

[0082] CYP2J2 is an enzyme that has been shown to be overexpressed in a variety of human cancers, including esophageal, lung, breast, gastric, liver, and colon cancers (Jiang et al., 2005; Narjoz et al., 2014).

[0083] CYP4F8 has been shown to be highly expressed in prostate cancer (Vainio et al., 2011). Both CYP4F2 and CYP4F3 have been shown to be overexpressed in pancreatic ductal adenocarcinoma, and only CYP4F2 has been shown to be overexpressed in ovarian cancer (Gandhi et al., 2013; Alexanian et al., 2012).

[0084] The expression of CYP4F11 has been shown to be regulated by NF-κB and p53 (Kalsotra et al., 2004; Bell and Strobel, 2012; Goldstein et al., 2013).

[0085] Genetic variations in CYPAF12 are significantly associated with gemcitabine response in pancreatic cancer patients (Goldstein et al., 2013; Harris et al., 2014).

[0086] High levels of DAP3 correlate with a better response to chemotherapy in gastric cancer and a better clinical outcome in breast cancer, while on the other hand, overexpression of DAP3 has been reported in thyroid follicular cell tumors and invasive glioblastoma multiforme (Jia et al., 2014; Wazir et al., 2012; Jacques et al., 2009; Mariani et al., 2001).

[0087] PEX19 is essential for peroxisome biogenesis, but has also been shown to interact directly with 19ARF, ultimately resulting in the retention of this factor in the cytoplasm and the inactivation of the p53 tumor suppressor function (Sugihara et al., 2001).

[0088] DDX11, which belongs to the DEAH family of DNA helicases, is highly expressed in advanced melanoma (Bhattacharya et al., 2012).

[0089] NME4 is a nucleoside diphosphate kinase that is overexpressed in colon and gastric cancers, as well as in myelodysplastic syndromes, and the latter disease is associated with a poor prognosis (Kracmarova et al., 2008; Seifert et al., 2005).

[0090] DENND5B acts as a GDP-GTP exchange factor and activates Rab-GTPases (Yoshimura et al., 2010).

[0091] DIEXF has been shown to mediate the non-proteasomal degradation of the tumor suppressor p53 (Tao et al., 2013).

[0092] DOCK7 is a guanine nucleotide exchange factor that is overexpressed in glioblastoma and has been shown to increase glioblastoma cell invasion in response to HGF by activating Rac-1 (Murray et al., 2014).

[0093] In hepatocellular carcinoma cell lines, DRG2 is downregulated during chemotherapy agent-induced apoptosis, and overexpression of DRG2 has been shown to inhibit doxorubicin-induced apoptosis in these cells (Chen et al., 2012a).

[0094] DROSHA, one of two important enzymes in microRNA biosynthesis, is overexpressed in several cancers, including gastrointestinal tumors, breast cancer, and cervical cancer, and appears to promote tumor cell proliferation, colony formation, and migration (Avery-Kiejda et al., 2014; Havens et al., 2014; Zhou et al., 2013b).

[0095] SNPs in the DUSP14 gene are associated with altered melanoma risk (Yang et al., 2014a; Liu et al., 2013b).

[0096] Exome sequencing studies have revealed somatic mutations in the DYNC1H1 gene in patients with intraductal papillary mucinous neoplasms of the pancreas (Furukawa et al., 2011).

[0097] EEF2 protein has been shown to be overexpressed in lung, esophageal, pancreatic, breast, and prostate cancers, glioblastoma multiforme, and non-Hodgkin lymphoma and to play an oncogenic role in cancer cell proliferation (Oji et al., 2014; Zhu et al., 2014a).

[0098] Mutations within the EFR3A gene were identified in colorectal adenoma samples (Bojjireddy et al., 2014; Zhou et al., 2013a).

[0099] EIF2B5 encodes one subunit of translation initiation factor B. Single nucleotide polymorphisms in this gene have been described as being associated with survival in ovarian cancer (Goode et al., 2010).

[0100] EIF3A, which is eukaryotic translation initiation factor 3 subunit A, has been shown to be overexpressed in breast, lung, cervical, esophageal, gastric, and colon cancers and to be involved in cell cycle regulation (Dong and Zhang, 2006).

[0101] EIF4E is a potent oncogene that is elevated in the top 30% of human malignancies, including breast, prostate, lung, head, and neck cancers, as well as in numerous leukemias and lymphomas (Carroll and Borden, 2013).

[0102] ELOVL2 has been shown to be overexpressed in hepatocellular carcinoma (Jakobsson et al., 2006; Zekri et al., 2012).

[0103] EPRS encodes a multifunctional aminoacyl-tRNA synthetase that has been reported to be a tumor-associated antigen in colorectal cancer (Line et al., 2002).

[0104] EXOSC4 promoter activity is increased in hepatocellular carcinoma due to DNA hypomethylation. EXOSC4 effectively and specifically inhibits cancer cell proliferation and cell invasion ability (Drazkowska et al., 2013; Stefanska et al., 2014).

[0105] The hydrolase FUCA2 was found to be essential for the attachment of H. pylori to human gastric cancer cells (Liu et al., 2009a).

[0106] GABRQ encodes the θ subunit of the GABAA receptor. GABA has been shown to stimulate human hepatocellular carcinoma growth through the overexpressed θ subunit of the GABAA receptor (Li et al., 2012).

[0107] In squamous cell carcinoma, overexpression of GALNT2 has been reported to enhance tumor cell invasiveness by modifying O-glycosylation and GFR activity (Lin et al., 2014; Hua et al., 2012a; Wu et al., 2011).

[0108] High levels of GGH are associated with cellular resistance to antifolates, especially methotrexate, and poor prognosis in invasive breast cancer and lung endocrine tumors (Schneider and Ryan, 2006; Shubbar et al., 2013; He et al., 2004).

[0109] GLUL is overexpressed in human breast cancer cells and gliomas (Zhuang et al., 2011; Collins et al., 1997; Christa et al., 1994; Cadoret et al., 2002).

[0110] GNPAT has been reported to be involved in growth inhibition and apoptosis induction in metastatic melanoma (Ofman et al., 2001; Qin et al., 2013).

[0111] Deletions in the chromosomal region of GOLGA4 have been reported in cervical cancer, and an in-frame mRNA fusion of GOLGA4 and PDGFRB has been reported in myeloproliferative neoplasms (Senchenko et al., 2003; Hidalgo-Curtis et al., 2010).

[0112] GPAM is expressed in human breast cancer, which is associated with changes in cell metabolism and better overall survival (Brockmoller et al., 2012).

[0113] High serum levels of GPT have been reported to increase the risk of gastrointestinal cancer and are associated with carcinogenesis and recurrence in hepatitis C virus-induced hepatocellular carcinoma (Kunutsor et al., 2014; Tarao et al., 1997; Tarao et al., 1999).

[0114] GRB14 has been shown to be upregulated in breast cancer, and high expression was significantly associated with better disease-free and overall survival (Huang et al., 2013; Balogh et al., 2012).

[0115] Single nucleotide polymorphisms in the GTF2H4 gene have been reported to increase the risk of developing smoking-related lung cancer and papillomavirus-induced cervical cancer (Mydlikova et al., 2010; Buch et al., 2012; Wang et al., 2010).

[0116] Genetic polymorphisms that suggest an important role of HSPA2 in the disease progression of cervical cancer, renal cell cancer, and bladder cancer have been associated with the development of gastric cancer (Singh and Suri, 2014; Ferrer-Ferrer et al., 2013; Garg et al., 2010a; Garg et al., 2010b).

[0117] HSPA8 has been shown to be overexpressed in esophageal squamous cell carcinoma. Furthermore, HSPA8 is overexpressed in multiple myeloma and colorectal cancer, and BCR-ABL1-induced HSPA8 expression promotes cell survival in chronic myeloid leukemia (Dadkhah et al., 2013; Wang et al., 2013a; Chatterjee et al., 2013; Kubota et al., 2010; Jose-Eneriz et al., 2008).

[0118] MDN1 is considered a candidate tumor suppressor gene and is mutated in luminal B breast cancer (Cornen et al., 2014).

[0119] MIA3, also known as trafficking and Golgi organization protein 1 (TANGO), is downregulated in intestinal and hepatocellular carcinomas and has been reported to play a tumor-suppressive role in these entities (Arndt and Bosserhoff, 2007). In contrast, studies in oral squamous cell carcinoma suggest an association between tumor progression, metastasis formation, and clinical stage and MIA3 expression, indicating an oncogenic role of MIA3 (Sasahira et al., 2014).

[0120] CPSF6 has been identified as one of the genes within a "transcriptional readiness gene cassette" associated with significant differences in the metastatic and invasive properties of several tumor types, such as breast, colon, liver, lung, esophageal, and thyroid cancers (Yu et al., 2008).

[0121] Low-level expression of MPDZ has been reported to be associated with poor prognosis in breast cancer patients (Martin et al., 2004).

[0122] NAA35, also known as MAK10, encodes N(α)-acetyltransferase 35, a NatC auxiliary subunit. In patients with esophageal squamous cell carcinoma, cancer highly enriched chimeric GOLM1-MAK10 RNA, which encodes a secreted fusion protein and is potentially useful as a molecular marker, was detected (Zhang et al., 2013b).

[0123] NAV2 has been shown to be specifically expressed in a group of colon cancers, and treatment of colon cancer cells with antisense oligonucleotides of NAV2 induced apoptosis (Ishiguro et al., 2002).

[0124] Overexpression of NCSTN is a sign of poor overall survival in estrogen receptor-negative breast cancer patients, high levels of nicastrin and Notch4 are detected in endocrine therapy-resistant breast cancer cells, and their activation ultimately drives invasive behavior (Sarajlic et al., 2014; Lombardo et al., 2014).

[0125] In non-small cell lung cancer, the NKD1 protein is decreased, while NKD1 mRNA is increased, and the former correlates with increased invasiveness and poor prognosis (Zhang et al., 2011). NKD1 mRNA was also found to be increased in cells from human colon tumors (Yan et al., 2001; Zhang et al., 2011).

[0126] In esophageal cancer, NUDC has been reported to be associated with lymph node metastasis, while overexpression of NUDC in prostate cancer cells results in cell division arrest (Hatakeyama et al., 2006; Lin et al., 2004).

[0127] Studies investigating the role of the Notch signaling pathway in ovarian cancer have reported a higher incidence of RFNG expression in adenomas compared to cancer (Gu et al., 2012; Hopfer et al., 2005).

[0128] RINT1 has been identified as an oncogene in glioblastoma multiforme and as a moderately penetrant cancer susceptibility gene found in breast cancer as well as Lynch syndrome-related cancers (Ngeow and Eng, 2014; Quayle et al., 2012).

[0129] High expression of RORC has been found to be associated with a longer metastasis-free survival in breast cancer. Attenuation of RORC expression was associated with increased tumor size in growth hormone-secreting cell adenomas and blunted the clinical response to somatostatin therapy (Cadenas et al., 2014; Lekva et al., 2013).

[0130] RPL17 has been reported to promote multidrug resistance by suppressing drug-induced apoptosis (Shi et al., 2004b).

[0131] Increased expression of RPS29 has been reported in gastric and colorectal cancers (Takemasa et al., 2012; Sun et al., 2005).

[0132] SAMM50 encodes a component of the sorting and assembly machinery (SAM) of the mitochondrial outer membrane that functions in the integration of β-barrel proteins into the mitochondrial outer membrane. A growth-promoting chimeric mRNA (SAMM50-PARVB) was detected in breast and ovarian cancer cells and in some samples from breast, gastric, colon, kidney, and uterine cancers (Plebani et al., 2012).

[0133] SERPINF2 encodes the major inhibitor of plasmin, which degrades fibrin and various other proteins. Plasma levels of the plasmin-α2-plasmin inhibitor complex have been shown to be predictors of survival in non-small cell lung cancer, and low activity of α2-antiplasmin has been observed in the blood of patients with prostate cancer (Zietek et al., 1996; Taguchi et al., 1996).

[0134] Overexpression of SF3B3 significantly correlates with overall survival and endocrine resistance in estrogen receptor-positive breast cancer (Gokmen-Polar et al., 2014).

[0135] The protein level of SHC1 is elevated in prostate, metastatic breast, ovarian, and thyroid cancers, and in different isoforms, and it is thought to function as a major adapter protein that mediates steroid mitogenic signals at the non-genomic level (Alam et al., 2009; Rajendran et al., 2010).

[0136] Since AMACR is highly overexpressed in prostate cancer, it is used as a biomarker in this entity (Wu et al., 2014). Furthermore, it is used as an immunohistochemical marker for the diagnosis of renal cell carcinoma (Ross et al., 2012).

[0137] Experimental data suggest that C1QTNF3 expression may have a role in osteosarcoma tumor growth associated with activation of the ERK1 / 2 signaling pathway, and that it is a novel anti-apoptotic adipokine that protects mesenchymal stem cells from hypoxia / serum deprivation-induced apoptosis through the PI3K / Akt signaling pathway (Hou et al., 2014; Akiyama et al., 2009).

[0138] GPC3 is expressed by most hepatocellular carcinomas. Two therapeutic approaches for HCC targeting GPC3, a humanized GPC3 monoclonal antibody and a vaccine consisting of two GPC3-derived peptides, are currently being tested in phase II clinical trials. The peptides used in the latter study are different from those reported in this document. GPC3 expression has been identified in all yolk sac tumors, some squamous cell lung cancers, and ovarian clear cell carcinomas (Filmus and Capurro, 2013; Kandil and Cooper, 2009).

[0139] MAGEB2 is classified as a cancer testis antigen as it is expressed in the testis and placenta and in a significant proportion of various histological types of tumors, particularly multiple myeloma and head and neck squamous cell carcinomas (Pattani et al., 2012; van et al., 2011).

[0140] MAPKAPK5 encodes a tumor suppressor and a member of the serine / threonine kinase family. MAPKAPK5 is underexpressed in colorectal cancer, leading to increased activity of the myc oncoprotein, and has been shown to reduce carcinogenesis by suppressing oncogenic ras activity in a mouse model of hematopoietic cancer (Yoshizuka et al., 2012; Kress et al., 2011).

[0141] Overexpression of USP14 is associated with increased tumor cell proliferation and poor prognosis in epithelial ovarian, non-small cell lung, and colorectal cancers (Wang et al., 2015; Wu et al., 2013a; Shinji et al., 2006).

[0142] C4A has been proposed as a biomarker for polycystic ovary syndrome and endometrial cancer, and experimental data suggest that C4 may mediate cancer growth (Galazis et al., 2013; Rutkowski et al., 2010).

[0143] CAPZB has been reported to be overexpressed in human papillomavirus 18-positive oral squamous cell carcinoma and was identified as a prostate cancer susceptibility locus (Lo et al., 2007; Nwosu et al., 2001).

[0144] Single nucleotide polymorphisms within the CFHR5 gene are associated with disease-free survival in follicular lymphoma (Charbonneau et al., 2012).

[0145] CLIP1 encodes CAP-GLY domain-containing linker protein 1, which binds endocytic vesicles to microtubules. This gene is highly expressed in Reed-Sternberg cells of Hodgkin's disease and breast cancer, and appears to be involved in the migration and invasion of breast and pancreatic cancer cells (Sun et al., 2013; Suzuki and Takahashi, 2008; Li et al., 2014a; Sun et al., 2012).

[0146] CLU may inhibit tumor progression in advanced neoplasms, while it may provide a significant survival advantage to tumors by suppressing a number of therapeutic stressors and promoting metastasis. CLU plays an important role in prostate cancer development and has been shown to control the aggressive behavior of human renal clear cell carcinoma through regulation of ERK1 / 2 signaling and MMP-9 expression, conferring treatment resistance at advanced stages of lung cancer (Trougakos, 2013; Panico et al., 2009; Takeuchi et al., 2014; Wang et al., 2014).

[0147] The fusion gene SEC16A-NOTCH1 was reported as the first recurrent fusion gene in breast cancer (Edwards and Howarth, 2012).

[0148] Recurrent deletions of the SHQ1 gene have been observed in prostate and cervical cancers, suggesting a tumor-suppressive role for SHQ1 (Krohn et al., 2013; Lando et al., 2013).

[0149] In clear cell renal cell carcinoma and bladder cancer, high SLC16A1 expression is associated with poor prognostic factors and predicts tumor progression. In colorectal cancer, single nucleotide polymorphisms of the SLC16A1 gene may affect clinical outcomes and can be used to predict response to adjuvant chemotherapy (Kim et al., 2015; Fei et al., 2014a; Fei et al., 2014a).

[0150] Glioblastoma has been shown to release glutamate at high levels, which may stimulate tumor cell proliferation, promote tumor invasion, and downregulate SLC1A2, suggesting its potential role in glioma progression, which correlates with higher tumor malignancy. Furthermore, in gastric cancer, a fusion gene of SLC1A2 and CD44 has been detected, which may correspond to a class of gene fusions that establish a carcinogenic-promoting metabolic environment and act favorably on tumor growth and survival (Tao et al., 2011; deGroot et al., 2005).

[0151] High expression of SLC3A2 is associated with tumor growth, biological malignancy, and survival of patients with cholangiocarcinoma, and significantly contributes to poor prognosis of non-small cell lung cancer patients through promoting cell proliferation via the PI3K / Akt pathway. Furthermore, overexpression of SLC3A2 is associated with the progression and liver metastasis of colorectal cancer together with integrin s1, integrin s3, and Fak (Kaira et al., 2014; Fei et al., 2014b; Sun et al., 2014).

[0152] Evidence that SLC9A3R1 is involved in carcinogenesis exists in hepatocellular carcinoma, schwannoma, glioblastoma, colorectal cancer, and particularly breast cancer (Saponaro et al., 2014).

[0153] NFYC has been reported to promote the expression of cancer genes in gastric and prostate cancer cells (Zhang et al., 2014a; Gong et al., 2013).

[0154] THY1 is a candidate tumor suppressor gene in nasopharyngeal carcinoma with anti-invasive activity (Lung et al., 2010).

[0155] TIMM17A is overexpressed in 21T breast cancer cells, and its mRNA expression in breast cancer tissues correlates with tumor progression (Xu et al., 2010).

[0156] TMEM209 is widely expressed in lung cancer (Fujitomo et al., 2012).

[0157] TNK2, also known as ACK1 tyrosine kinase, is activated, amplified or mutated in a variety of human cancers. The deregulated kinase is oncogenic and its activation is associated with progression to the metastatic stage. ACK1 inhibitors have shown promise in preclinical trials (Mahajan and Mahajan, 2013).

[0158] TRIM55 encodes a RING zinc finger protein that transiently binds to microtubules, myosin, and titin during muscle sarcomere assembly and is also involved in signal transduction from the sarcomere to the nucleus (Pizon et al., 2002).

[0159] RNA interference of the Ufd1 protein can sensitize the hydroxycamptothecin-resistant colon cancer cell line SW1116 / HCPT to hydroxylcamptothecin (Chen et al., 2011a; Chen et al., 2011c).

[0160] In colorectal cancer, the UGT1A1 gene is silenced through methylation and is thus considered a target point for the study of the regulatory mechanisms for irinotecan (CPT-11) drug resistance and drug resistance reversal (Xie et al., 2014).

[0161] UGT1A10 is expressed in the stomach and bile duct tissues (Strassburg et al., 1997), and its overexpression significantly increased the cytotoxicity of the antitumor agent 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone C-130 (Pawlowska et al., 2013). Furthermore, UGT1A10 catalyzes the glucuronidation of xenobiotics, mutagens, and reactive metabolites, and thus plays an indirect antioxidant role. Xenobiotic (XRE) and antioxidant (ARE) response elements were detected in the UGT1A8, UGT1A9, and UGT1A10 promoters (Kalthoff et al., 2010).

[0162] UGT1A8 is mainly expressed in the gastrointestinal tract (Gregory et al., 2003), and its mRNA expression is upregulated during treatment with the chemopreventive agent sulforaphane (SFN) (Wang et al., 2012).

[0163] The UGT1A7 haplotype is associated with an increased risk of hepatocellular carcinoma in hepatitis B carriers (Kong et al., 2008).

[0164] UGT1A6 is overexpressed in methotrexate-resistant breast cancer cells (de Almagro et al., 2011) and is presumably induced by the chemopreventive agent β-naphthoflavone (Hanioka et al., 2012).

[0165] UGT1A9 is mainly expressed in the liver and kidneys (Gregory et al., 2003). UGT1A9 germline polymorphisms are potential predictors of prostate cancer recurrence after prostatectomy (Laverdiere et al., 2014).

[0166] UGT1A4 promoter and coding region polymorphisms result in variability in the glucuronidation of anastrozole, an aromatase inhibitor for breast cancer patients (Edavana et al., 2013).

[0167] UPF1 is part of the nonsense-mediated mRNA decay (NMD) machinery and may have a functional role in prostate cancer progression and metastasis (Yang et al., 2013). Furthermore, the UPF1 RNA surveillance gene is commonly mutated in pancreatic adenocarcinoma (Liu et al., 2014).

[0168] UQCRB is a subunit of mitochondrial complex III. Inhibition of UQCRB in tumor cells suppresses hypoxia-induced tumor angiogenesis (Jung et al., 2013). Two SNPs within the 3’ untranslated region of UQCRB are candidate prognostic markers for colorectal cancer (Lascorz et al., 2012).

[0169] Copy number changes in USO1 correlate with differential gene expression in superficial spreading melanoma compared to nodular melanoma (Rose et al., 2011).

[0170] Significant decreases in both USP10 and SIRT6 protein expression were detected in human colon cancer (Lin et al., 2013).

[0171] UTP18 also alters translation, promotes stress resistance and proliferation, and is frequently increased and overexpressed in cancer (Yang et al., 2014b).

[0172] The VARS rs2074511 polymorphism is associated with the survival of patients with triple-negative breast cancer and may therefore be considered a prognostic factor for the survival of patients with early breast cancer (Chae et al., 2011).

[0173] VMP1, a protein related to stress-induced autophagy, is also induced by the oncogene KRAS (Lo Re et al., 2012). VMP1 is overexpressed in poorly differentiated human pancreatic cancer as a response to chemotherapeutic agents (Gilabert et al., 2013). Marked downregulation of VMP1 is seen in human HCC tissues and is closely correlated with multiple tumor nodules, absence of capsule formation, venous invasion, and poor prognosis of HCC (Guo et al., 2012).

[0174] WDR26 protects cardiomyocytes from oxidative stress (Feng et al., 2012).

[0175] ZC3H7A is a member of the CCCH zinc finger protein family known as a regulator of macrophage activation (Liang et al., 2008). ZC3H7A was found to have a higher allele frequency of functional mutations in metastatic tumors of pancreatic ductal adenocarcinoma (Zhou et al., 2012).

[0176] FASN is fatty acid synthase and is involved in promoting lipid synthesis in different cancer types including breast, pancreas, prostate, liver, ovary, colon, and endometrial cancers (Wu et al., 2014; Zhao et al., 2013).

[0177] FGG is upregulated in hepatocellular carcinoma as well as in prostate, lung, and breast cancers (Vejda et al., 2002; Zhu et al., 2009).

[0178] FMO5 is a monooxygenase, a dominant liver-specific FMO, which is upregulated in estrogen receptor α-positive breast tumors (Bieche et al., 2004; Zhang and Cashman, 2006).

[0179] HADHA mRNA decreases with the progression of HCC dedifferentiation (Tanaka et al., 2013) and in estrogen receptor α-negative breast tumors (Mamtani and Kulkarni, 2012).

[0180] Genetic diversity of the HAL gene may have a role in the development of skin cancer (Welsh et al., 2008).

[0181] HLTF is a member of the SWI / SNF family of transcriptional regulators with helicase and E3 ubiquitin ligase activities and has been found to be inactivated by hypermethylation in colon, stomach, uterus, bladder, and lung tumors (Debauve et al., 2008; Castro et al., 2010; Garcia-Baquero et al., 2014).

[0182] HDAC10 is a histone deacetylase and transcriptional regulator. The expression of HDAC10 was significantly decreased in gastric cancer tissues compared to adjacent tissues (Jin et al., 2014). HDAC10 is inversely correlated with lymph node metastasis in human patients with cervical squamous cell carcinoma (Song et al., 2013). HDAC10 is hypermethylated in malignant adrenal cortical tumors (Fonseca et al., 2012). HDAC10 levels are increased in chronic lymphocytic leukemia (Wang et al., 2011). The HDAC10-589C>T promoter polymorphism was significantly associated with HCC development in chronic HBV patients and HCC acceleration in chronic HBV patients (Park et al., 2007). Decreased expression of class II histone deacetylase genes is associated with poor prognosis in lung cancer patients (Osada et al., 2004).

[0183] Low HIP1R expression is strongly associated with poor outcome in diffuse large B-cell lymphoma patients (Wong et al., 2014).

[0184] HM13 is a signal peptide peptidase that has been shown to affect cell survival in colorectal adenomas (Sillars-Hardebol et al., 2012).

[0185] Serum HPR levels are significantly higher in patients with malignant lymphoma than in non-diseased controls, and HPR expression increases with disease progression (Epelbaum et al., 1998). HPR expression parallels the increase in malignancy of breast cancer, and HPR-positive breast cancer is more likely to recur after primary resection and is associated with a shorter disease-free period (Shurbaji et al., 1991).

[0186] A variant (rs932335) in the HSD11B1 gene is associated with colorectal and breast cancer (Feigelson et al., 2008; Wang et al., 2013b).

[0187] HSD17B6 expression in tissues from prostate cancer patients who have received androgen deprivation therapy (ADT) is significantly higher than in tissues from untreated individuals (Ishizaki et al., 2013).

[0188] HSPE1 is a mitochondrial chaperonin with functions in protein folding and cell signaling (NF-κB and WNT signaling). Increased levels of Hsp10 have been found in tumor cells of colorectal cancer, cervical cancer, prostate cancer, mantle cell lymphoma, and serous ovarian cancer. A decrease in the level of Hsp10 has been reported in bronchogenic carcinoma (David et al., 2013).

[0189] Ovarian cancer xenografts transplanted into the flanks of nude mice and treated with paclitaxel showed a decrease in IDI1 expression compared to untreated xenografts (Bani et al., 2004).

[0190] IGFBPL1 is a regulator of insulin growth factor and is downregulated by abnormal hypermethylation in breast cancer cell lines. Methylation of IGFBPL1 is clearly associated with worse overall survival and disease-free survival (Smith et al., 2007).

[0191] The androgen-sensitive microsomal-related protein IKBKAP regulates the expression of prostate epithelial and neuronal markers, attenuates proliferation through an androgen receptor-dependent mechanism, and co-regulates androgen receptor-mediated transcription in LNCaP prostate cancer cells (Martinez et al., 2011).

[0192] INTS8 is part of a marker panel that discriminates gastric cancer from adjacent non-cancerous tissue (Cheng et al., 2013).

[0193] The IRS2-derived peptide pIRS-21097-1105 has been reported on HLA-A2(+) melanoma and breast, ovarian, and colorectal cancer tumors (Zarling et al., 2014). The IRS-21057DD genotype and the D allele were significantly associated with HCC risk (Rashad et al., 2014).

[0194] ITGA7 is the α chain of the laminin-1 receptor dimer integrin α-7 / β-1. ITGA7 is a tumor suppressor gene important for the growth inhibition of malignant tumors. Mutation analysis has revealed ITGA7 mutations in prostate cancer, hepatocellular carcinoma, soft tissue leiomyosarcoma, and glioblastoma multiforme. ITGA7 was downregulated in non-metastatic prostate cancer and leiomyosarcoma (Tan et al., 2013).

[0195] ITIH4 is downregulated in several tumor tissues, including colon, stomach, ovary, lung, kidney, rectum, and prostate (Hamm et al., 2008). Low serum ITIH4 levels are associated with shorter survival in HBV-related HCC patients (Noh et al., 2014). A significant increase in ITIH4 serum concentration was observed in breast cancer, and the serum level of ITIH4 significantly decreased after surgery (van, I et al., 2010).

[0196] A missense mutation was identified in SHKBP1 that acts downstream of FLT3, and the receptor tyrosine kinase was mutated in approximately 30% of AML cases (Greif et al., 2011). SHKBP1 is one of several possible protein biomarker candidates for classifying well-differentiated small intestinal neuroendocrine tumors (WD-SI-NET) at different stages (Darmanis et al., 2013).

[0197] KLB expression is increased in HCC tissues compared to corresponding non-tumor tissues (Poh et al., 2012).

[0198] The LBP polymorphism rs2232596 is associated with a significant increase in colorectal cancer risk in Han Chinese (Chen et al., 2011b). LBP is a candidate serum biomarker in ovarian cancer (Boylan et al., 2010). LBP significantly decreased after chemotherapy treatment in small cell lung cancer patients (Staal-vanden Brekel AJ et al., 1997).

[0199] LBR mRNA expression is directly related to tumor malignancy and Nottingham Prognostic Index in breast cancer (Wazir et al., 2013). LBR is highly expressed in papillary thyroid cancer cells, but abnormal protein folding may explain the lack of its immunohistochemical reactivity and may be related to abnormal folding of the nuclear membrane (Recupero et al., 2010).

[0200] LEPR dysregulation has been reported in diverse malignant cells including colon cancer, hepatocellular carcinoma, endometrial cancer, thyroid cancer, breast cancer, and lung cancer (Ntikoudi et al., 2014; Surmacz, 2013; Uddin et al., 2011).

[0201] LIG1 single nucleotide polymorphisms are associated with lung cancer, endometrial cancer, and glioma risk (Doherty et al., 2011; Lee et al., 2008; Liu et al., 2009b).

[0202] LRPPRC expression in gastric cancer tissue is significantly higher than in paired control tissue (Li et al., 2014b). LRPPRC levels serve as a prognostic diagnostic marker for patients with prostate adenocarcinoma (PCA), and patients with high LRPPRC levels survive for a shorter period after surgery than those with low LRPPRC levels (Jiang et al., 2014). LRPPRC is highly expressed in various types of tumors such as lung adenocarcinoma, esophageal squamous cell carcinoma, gastric, colon, breast and endometrial adenocarcinoma, and lymphoma (Tian et al., 2012).

[0203] MANEA expression is controlled by androgens in prostate cancer cells (Romanuik et al., 2009).

[0204] OPLAH is expressed in normal and tumor tissues of the lung, breast, kidney, colon, and ovary, and OPLAH levels are significantly higher in normal specimens than in tumors in individual patients (Srivenugopal and Ali-Osman, 1997).

[0205] The ORM2 glycoform provides valuable information regarding the discrimination of primary and secondary liver cancers (Mackiewicz and Mackiewicz, 1995). Plasma ORM2 levels have been confirmed to be significantly elevated in patients with colorectal cancer compared to controls (Zhang et al., 2012). Fucosylated glycoform ORM2 levels were significantly higher in adenocarcinoma lung cancer cases compared to controls (Ahn et al., 2014). ORM2 is a putative biomarker for the early diagnosis of cholangiocarcinoma (Rucksaken et al., 2012).

[0206] Increased tetrahydrobiopterin levels result in increased PAH activity and PAH protein within human liver tumor cells (McGuire, 1991).

[0207] PARP14 is highly expressed in multiple myeloma plasma cells and is associated with disease progression and low survival rates. PARP14 is deeply involved in JNK2-dependent survival duration. PARP14 has been found to promote the survival of multiple myeloma cells by binding to and inhibiting NK1 (Barbarulo et al., 2013).

[0208] PC levels are elevated in liver tumors and lung cancer (Chang and Morris, 1973; Fan et al., 2009).

[0209] Increased PCNT levels and centrosome abnormalities have been described in a variety of hematological malignancies and solid tumors, including AML, CML, mantle cell lymphoma, breast cancer, and prostate cancer (Delaval and Doxsey, 2010).

[0210] PIGN is a cancer chromosomal instability (CIN) suppressor gene that frequently undergoes copy number loss in CIN(+) colorectal cancer (Burrell et al., 2013).

[0211] PIPOX expression varies according to breast cancer subtypes, with HER-2 type tumors showing increased expression and triple-negative breast cancer subtypes showing decreased expression. Tumor PIPOX negativity was associated with shorter disease-free survival (Yoon et al., 2014). PIPOX was decreased in prostate tumors and reduced the potential of prostate cell carcinogenesis by metabolizing sarcosine (Khan et al., 2013).

[0212] Increased PSMD4 levels were detected in colorectal cancer, myeloma, and hepatocellular carcinoma (Arlt et al., 2009; Midorikawa et al., 2002; Shaughnessy, Jr. et al., 2011).

[0213] PLIN2 is significantly increased in patients with clear cell and papillary renal cell carcinoma compared to controls. The preoperative urine concentration of PLIN2 reflects tumor size and stage (Morrissey et al., 2014). PLIN2 expression is significantly higher in lung adenocarcinoma specimens than in normal tissues and lung squamous cell carcinoma (Zhang et al., 2014b).

[0214] PLK4 is frequently rearranged or deleted in human cancers, particularly at a high rate in hepatocellular carcinoma, but also in colorectal cancer and head and neck cancer (Swallow et al., 2005). PLK4 is overexpressed in breast cancer (Marina and Saavedra, 2014).

[0215] QARS is a member of the aminoacyl-tRNA synthetase (ARS) and loads glutamine onto tRNA. ARS expression and polymorphism are associated with breast cancer and glioblastoma (He et al., 2014b; Kim et al., 2012).

[0216] The methylated PMF1 gene is a diagnostic and predictive biomarker for patients with bladder cancer (Kandimalla et al., 2013).

[0217] Several human tumors and hematological malignancies, including thyroid, prostate, pancreas, testis, endometrium / uterus, liver and kidney cancers, lymphoid tissues, bladder tumors, ALL and CML, upregulate PON2, and such overexpression confers resistance to different chemotherapeutic drugs (imatinib, doxorubicine, staurosporine, or actinomycin) (Witte et al., 2011).

[0218] PRKAR2A is a regulatory subunit of protein kinase A. PRKAR2A significantly increased the survival of prostate cancer cell lines treated with taxol and docetaxel (Zynda et al., 2014). PRKAR2A is overexpressed in lung adenocarcinoma (Bidkhori et al., 2013).

[0219] PRPF6 is a component of the tri-snRNP (small ribonucleoprotein) spliceosome complex that drives colon cancer growth by preferential splicing of genes related to growth control (Adler et al., 2014). PRPF6 is overexpressed in lung adenocarcinoma (Bidkhori et al., 2013).

[0220] PSMC4 was significantly and consistently upregulated in prostate cancer cells compared to corresponding adjacent normal prostate tissue (Hellwinkel et al., 2011).

[0221] QPRT expression increases with malignant lesions in gliomas, and in recurrent glioblastomas, QPRT expression after chemoradiotherapy is associated with poor prognosis (Sahm et al., 2013). QPRT is a potential marker for immunohistochemical screening of follicular thyroid nodules (Hinsch et al., 2009).

[0222] RABGGTB is overexpressed in chemotherapy-refractory diffuse large B-cell lymphoma (Linderoth et al., 2008).

[0223] RAD21 is overexpressed in gastrointestinal tumors, colorectal cancer, advanced endometrial cancer, prostate cancer, and breast cancer (Atienza et al., 2005; Deb et al., 2014; Porkka et al., 2004; Supernat et al., 2012; Xu et al., 2014).

[0224] RAD23B has a potential role in breast cancer progression (Linge et al., 2014). The single nucleotide polymorphism RAD23B rs1805329 was significantly associated with the development and recurrence of HCC in Japanese patients with HCV (Tomoda et al., 2012).

[0225] RASAL2 is a RAS-GTPase activating protein that has tumor suppressor functions in estrogen receptor-positive breast cancer, ovarian cancer, and lung cancer (Li and Li, 2014; Huang et al., 2014). In contrast, RASAL2 is oncogenic in triple-negative breast cancer and drives mesenchymal invasion and metastasis (Feng et al., 2014a).

[0226] Depletion of RNMT effectively and specifically inhibits cancer cell proliferation and cell invasion ability in different cancer types, including liver cancer (Stefanska et al., 2014).

[0227] Overexpression or mutation of ROCK1, which leads to increased kinase activity, has been reported in several cancers, including lung cancer, gastric cancer, CML, and AML (Rath and Olson, 2012).

[0228] RPL10A is a c-Myc target gene and may contribute to hepatocyte transformation (Hunecke et al., 2012).

[0229] In particular, the Inv(3) and t(3;3) breakpoint associated with poor prognosis in myeloid leukemia or myelodysplastic syndrome cluster within regions located centromeric and downstream of the RPN1 gene (Wieser, 2002).

[0230] RRBP1 is overexpressed in lung cancer and breast cancer (Telikicherla et al., 2012; Tsai et al., 2013).

[0231] SCFD1 expression increases in erosive gastritis associated with gastric cancer (Galamb et al., 2008).

[0232] ABCB1 encodes P-glycoprotein (P-gp) that is expressed in normal cells of various organs such as the intestine, liver, kidney, brain, and placenta. Overexpression and genetic polymorphisms of P-gp have been detected in colorectal cancer, adrenal-derived tumors, lung cancer, and ALL (Zhang et al., 2013a; Fojo et al., 1987; Gervasini et al., 2006; Jamroziak et al., 2004).

[0233] ABCB10 encodes an ABC transporter of subfamily B (MDR / TAP). ABCB10 has been shown to be involved in cisplatin resistance in KCP-4 human squamous carcinoma cells (Oiso et al., 2014).

[0234] The expression of ABCB11 has been shown to be upregulated in pancreatic ductal adenocarcinoma, one of the most drug-resistant cancers. Therefore, it may contribute to the generally poor treatment response of this cancer (Mohelnikova-Duchonova et al., 2013).

[0235] The upregulated expression of ABCC2 in primary fallopian tube carcinoma is associated with poor prognosis (Halon et al., 2013).

[0236] ABCC6 was downregulated in colorectal cancer patients who were non - responders to palliative chemotherapy (Hlavata et al., 2012). In contrast, it was upregulated in gemcitabine - resistant human NSCLC A549 cells (Ikeda et al., 2011).

[0237] The expression of ACACA was upregulated in a number of human cancers such as breast, prostate, and liver cancers, and was shown to correlate with the promotion of lipidogenesis in cancer cells. Various ACACA inhibitors have shown therapeutic effects in the treatment of cancer cell lines by suppressing cell proliferation and inducing cell death through apoptosis (Zu et al., 2013).

[0238] ACLY was abnormally expressed in various tumors such as breast, liver, colon, lung, and prostate cancers, and was inversely correlated with tumor stage and differentiation (Zu et al., 2012).

[0239] ACSL3 was overexpressed in lung cancer and, based on pre - clinical studies, is a promising new therapeutic target in lung cancer (Pei et al., 2013). The overexpressed ACSL3 expression may play a role as a possible biomarker for estrogen receptor - specific breast cancer risk (Wang et al., 2013c).

[0240] ACSL4 was overexpressed in estrogen receptor - negative breast tumors and androgen receptor - negative breast and prostate tumors, and the loss of steroid hormone sensitivity was associated with the induction of ACSL4 expression (Monaco et al., 2010). The onset of ACSL4 upregulation was shown to occur during the transformation from adenoma to adenocarcinoma (Cao et al., 2001).

[0241] The methylation of ACSS3 was found to be associated with at least one of the classical risk factors in neuroblastoma, namely age, stage, or MYCN status (Decock et al., 2012).

[0242] The deletion of ADSSL1 is frequently observed in carcinogen-induced murine primary lung adenocarcinoma, murine and human lung adenocarcinoma cell lines, and is associated with a more extensive chromosomal instability phenotype in primary murine lung tumors (Miller et al., 2009).

[0243] AGFG2 was identified as one of 14 predictive gene candidates in the identification of cases of hormone receptor-negative or triple-negative breast cancer that presumably remain free of metastatic recurrence (Yau et al., 2010).

[0244] AGT is a very potent anti-angiogenic factor and has been shown to produce anti-tumor effects in vitro and in vivo (Bouquet et al., 2006). In transgenic mice, overexpression of human AGT has been shown to reduce angiogenesis in liver cancer and thus delay tumor progression (Vincent et al., 2009).

[0245] AKR1C4 encodes human aldo-keto reductase family 1 member C4 and catalyzes the reduction of retinaldehyde to retinol (Ruiz et al., 2011). Thus, depletion of retinaldehyde downregulates the biosynthesis of retinoic acid, followed by blockade of retinoid signaling that favors tumor progression (Tang and Gudas, 2011; Ruiz et al., 2012) The expression of ALDH1L1 has been shown to be downregulated in HCC and glioma. Downregulation of ALDH1L1 in these cancers was associated with an unfavorable prognosis and a more invasive phenotype (Rodriguez et al., 2008; Chen et al., 2012b) The expression of ALG3 has been shown to be promoted in esophageal squamous cell carcinoma and cervical cancer (Shi et al., 2014; Choi et al., 2007). In esophageal squamous cell carcinoma, increased expression of ALG3 correlated with lymph node metastasis (Shi et al., 2014).

[0246] ANKS1A has been identified as a novel target of Src family kinases, which is known to be involved in the development of some colorectal cancers (Emaduddin et al., 2008).

[0247] APOA1 encodes apolipoprotein A-I, which is the major protein component of high-density lipoprotein (HDL) in plasma. In multiple animal tumor models, APOA1 has been shown to play a potent immunomodulatory role in tumorigenesis, suppressing tumor growth and metastasis by supporting innate and adaptive immune processes (Zamanian-Daryoush et al., 2013).

[0248] APOA2 has been shown to be significantly decreased in pancreatic cancer patients (Honda et al., 2012). In contrast, increased expression of APOA2 has been associated with HCC (Liu et al., 2007).

[0249] In α-fetoprotein-negative HBV-related HCC, APOB was found to be one of 14 differentially expressed proteins that may be associated with HCC progression (He et al., 2014a). In advanced breast cancer, APOB was found to be one of 6 differentially expressed proteins that can predict the responsiveness of patients to neoadjuvant chemotherapy and the survival period without recurrence (Hyung et al., 2011).

[0250] In stage III colorectal cancer patients and human melanoma cells, AQP9 was associated with increased chemoresistance (Dou et al., 2013; Gao et al., 2012).

[0251] ARG1 has been shown to be a highly sensitive and specific marker in differentiating HCC from other metastatic tumors in the liver (Sang et al., 2013). ARG1 may also contribute to local immunosuppression in NSCLC (Rotondo et al., 2009).

[0252] It was found that phosphorylated, and thus more highly active forms of the ARSB protein, increase in peripheral white blood cells derived from patients with chronic myeloid leukemia compared to healthy donors (Uehara et al., 1983).

[0253] Downregulation of ASNA1 in ovarian cancer cells has been shown to increase sensitivity to the chemotherapeutic drugs cisplatin, carboplatin, oxaliplatin, and arsenite (Hemmingsson et al., 2009).

[0254] ASPH has been shown to be overexpressed in various cancers and cancer cell lines (Yang et al., 2010). Immunization with ASPH-loaded dendritic cells produced cytotoxicity against cholangiocarcinoma cells in vitro and significantly suppressed tumor growth and metastasis in the liver (Noda et al., 2012).

[0255] ATP1A2 was found among 31 proteins significantly upregulated in glioblastoma (Com et al., 2012). In contrast, ATP1A2 has been shown to be downregulated in bone marrow-infiltrating metastatic neuroblastoma (Morandi et al., 2012).

[0256] ATP1A3 was found among 31 proteins significantly upregulated in glioblastoma (Com et al., 2012).

[0257] ATP6V1C1 may promote breast cancer growth and bone metastasis through regulation of V-ATPase activity within lysosomes. ATP6V1C1 knockdown significantly inhibited the growth, metastasis, and osteolytic lesions of transplanted tumors of mouse 4T1 breast cancer cell line in vivo (Feng et al., 2013). ATP6V1C1 has been shown to be overexpressed in oral squamous cell carcinoma and is associated with tumor cell motility (Otero-Rey et al., 2008).

[0258] ATP7B is associated with cancer resistance to cisplatin, a widely used anticancer agent (Dmitriev, 2011).

[0259] AXIN2 encodes Axin (axis inhibition)-related protein 2, which likely plays an important role in controlling the stability of β-catenin in the Wnt signaling pathway (Salahshor and Woodgett, 2005). Furthermore, AXIN2 has been shown to suppress the expression of the oncogene c-MYC (Rennoll et al., 2014).

[0260] In HCC, low BAAT expression was associated with a poor survival period compared to patients with higher BAAT expression (Furutani et al., 1996).

[0261] A strong decrease in transcripts of BHMT and BHMT2 was shown in HepG2 cells and HCC samples compared to normal liver tissue (Pellanda et al., 2012).

[0262] C12orf44 is essential for autophagy and has been shown to interact with ULK1 and Atg13 in a dependent-like manner (Mercer et al., 2009). Autophagy has a dual role in cancer, acting as a tumor suppressor by preventing the accumulation of damaged proteins and organelles, and also acting as a cell survival mechanism that can promote the growth of established tumors (Yang et al., 2011b).

[0263] C17orf70 is a component of the Fanconi anemia core complex and is essential for the stability of the complex. The Fanconi anemia core complex plays a central role in the DNA damage response network. The Fanconi anemia core complex-mediated DNA damage response is involved in breast cancer susceptibility gene products, BRCA1 and BRCA2 (Ling et al., 2007).

[0264] C19orf80 encodes the hepatocellular carcinoma-related gene TD26 and was shown to be one of five loci with the highest methylation levels in HCC and the lowest in control tissues (Ammerpohl et al., 2012).

[0265] CCT7 was found to be part of a protein subnet that can significantly discriminate advanced human colorectal cancer (Nibbe et al., 2009).

[0266] CDK6 has been shown to regulate the activity of the tumor suppressor protein Rb. CDK6 can exert its tumor-promoting function by promoting proliferation and stimulating angiogenesis (Kollmann et al., 2013). Pharmacological inhibition of CDK6 has been shown to inhibit the proliferation and differentiation of abnormal leukemia cells (Placke et al., 2014).

[0267] CFH may have a role in cutaneous squamous cell carcinoma progression (Riihila et al., 2014). CFH may play an important role in complement-mediated lysis resistance in various cancer cells and has been shown to be overexpressed in NSCLC, which is associated with a poor prognosis (Cui et al., 2011).

[0268] Inactivating mutations of CLPTM1 were discovered in prostate cancer cells (Rossi et al., 2005).

[0269] CMAS encodes cytidine monophosphate N-acetylneuraminic acid synthetase, which catalyzes the activation of sialic acid and its conversion to cytidine monophosphate diester. Activated sialic acid is used for N-glycosylation, a common post-translational modification in cell differentiation. Increased expression of sialic acid sugars on the cancer cell surface is one of the well-known tumor characteristics (Bull et al., 2014).

[0270] Transferrin (TF) receptor (TFR) is overexpressed on malignant cells and plays an important role in cellular iron uptake through its interaction with TF. Therefore, TF is one of the most widely used tumor-targeting ligands (Biswas et al., 2013). The expression level of TFR has been suggested to correlate with tumor stage or cancer progression (Tortorella and Karagiannis, 2014).

[0271] TH1L may play an important role in the regulation of the proliferation and invasion of human breast cancer and may be a potential target for the treatment of human breast cancer (Zou et al., 2010).

[0272] THTPA hydrolysis may be involved in the anti-proliferative effect of Ndrg-1. Ndrg-1 has been shown to reduce invasion and metastasis in breast, colon, prostate, and pancreatic cancers (Kovacevic et al., 2008).

[0273] SMYD3 promotes cancer invasion through the epigenetic upregulation of the metalloproteinase MMP-9 (Medjkane et al., 2012). The expression of SMYD3 is undetectable or very weak in many types of normal human tissues, whereas the overexpression of SMYD3 has been associated with the development and progression of gastric, colorectal, hepatocellular, prostate, and breast cancers (Hamamoto et al., 2006; Liu et al., 2014; Liu et al., 2013a).

[0274] The association between STAT2 and tumorigenesis has been observed in genetically engineered mice lacking STAT2 (Yue et al., 2015) or constantly expressing IFN-α in the brain (Wang et al., 2003).

[0275] TACC3 is overexpressed in a number of human cancers, including ovarian cancer, breast cancer, squamous cell carcinoma, and lymphoma (Ma et al., 2003; Jacquemier et al., 2005; Lauffart et al., 2005).

[0276] SPB has also been shown to suppress the transcriptional activity of estrogen receptor α (ERα). Overexpression of SPBP inhibited the growth of ERα-dependent breast cancer cell lines (Gburcik et al., 2005). In the cell nucleus, SPBP exhibits relatively low motility and is rich in regions of high chromatin density, clearly suggesting that it is a chromatin-binding protein (Darvekar et al., 2012). TCF20 is important for promoting the induction of proteins involved in the cellular defense program against oxidative stress (Darvekar et al., 2014).

[0277] C3 is a prominent element of the inflammatory tumor microenvironment (Rutkowski et al., 2010), and activation can confer a growth advantage to tumors (Markiewski et al., 2008). Enzymatic cleavage of C3 results in the production of the inflammatory mediator and chemoattractant anaphylatoxin C3a and C3b (Sahu et al., 1998).

[0278] CLN3 is an anti-apoptotic gene in NT2 neural progenitor cells and a few cancer types (Zhu et al., 2014b). It is involved in intracellular trafficking and regulation in both neuronal and non-neuronal cells (Rakheja et al., 2008; Getty and Pearce, 2011), and it is involved in several important signaling pathways (Persaud-Sawin et al., 2002). CLN3 mRNA and protein are overexpressed in several cancer cell lines, including breast, colon, melanoma, prostate, ovarian, neuroblastoma, and glioblastoma multiforme, but not in lung or pancreatic cancer cell lines (Rylova et al., 2002).

[0279] SLC13A5 is one of the seven CIMP marker genes. The CIMP (CpG island methylation phenotype) in clear cell renal cell carcinomas (ccRCCs) is characterized by the accumulation of DNA methylation in CpG islands and poor patient outcomes (Tian et al., 2014; Arai et al., 2012).

[0280] SLC35B2 is involved in the co-transcriptional regulation during the induction of sialylsulfoglucosamine biosynthesis in acute inflammation (Huopaniemi et al., 2004) and the sulfation of 6-sulfo-lactosamine epitopes within human colorectal cancer cell lines (Kamiyama et al., 2006). Colorectal cancer cell lines as well as human colorectal tissues express SLC35B2 (Kamiyama et al., 2011).

[0281] PLOD1 expression is associated with human breast cancer progression (Gilkes et al., 2013).

[0282] PRDX5 is upregulated in a number of malignancies, and inhibition of PRDX5 (Urig and Becker, 2006) may prevent tumor initiation and progression, suggesting that PRDX5 is a promising target for cancer therapy. Its highly nucleophilic and accessible selenocysteine active site may be the most important target for drug design (Liu et al., 2012).

[0283] Increased expression of PSMD8 in the lung periphery may provide potential information regarding which important cell populations are involved in the development of invasive cancer (Zhou et al., 1996).

[0284] SNRPD1 is a core spliceosome protein that is upregulated in malignancies.

[0285] Reduced expression of SPTBN1 is associated with poor prognosis in pancreatic cancer (Jiang et al., 2010).

[0286] SQSTM1 functions as a signaling hub for various signal transduction pathways such as NF-κB signaling, apoptosis, and Nrf2 activation, whose dysregulation is associated with Paget's bone disease and tumorigenesis (Komatsu et al., 2012).

[0287] PCNA expression predicts survival in anorectal malignant melanoma (Ben-Izhak et al., 2002). Within PCNA, a cancer-related isoform of PCNA (caPCNA) has been identified that contains an abnormal pattern of methyl ester groups on a number of glutamine and asparagine residues (Hoelz et al., 2006).

[0288] Depletion of SRP54 in several tumor cell lines did not result in a dominant cell phenotype such as growth arrest or death, even in cells selected for a stable reduction in SRP components (Ren et al., 2004).

[0289] At the molecular level, STAT1 inhibits the proliferation of both mouse and human tumor cells treated with IFN-γ through its ability to increase the expression of the cyclin-dependent kinase inhibitor p21Cip1 or decrease c-myc expression (Ramana et al., 2000). The antitumor activity of STAT1 is further supported by its ability to inhibit angiogenesis and tumor metastasis in mouse models (Huang et al., 2002). An increase in STAT1 mRNA levels has been shown to be part of a molecular signature associated with a better prediction of metastatic outcome in patients with hormone receptor-negative and triple-negative breast cancer (Yau et al., 2010).

[0290] Fine needle aspiration samples from follicular neoplasms demonstrated that malignant nodules overexpress STT3A compared to benign diseases (Patel et al., 2011).

[0291] Meta-analysis showed that the STXBP4 / COX11 rs6504950 polymorphism was significantly correlated with breast cancer risk (Tang et al., 2012).

[0292] A peptide consisting of or consisting essentially of an amino acid sequence as shown herein may have one or two non-anchor amino acids that are exchanged without substantially changing or being adversely affected in its ability to bind to human major histocompatibility complex (MHC) class I or II molecules when compared to an unmodified peptide (see below for the anchor motif). In another embodiment, in a peptide consisting essentially of an amino acid sequence as shown herein, one or two amino acids may be exchanged with their conservative exchange partners (see below) without substantially changing or being adversely affected in its ability to bind to human major histocompatibility complex (MHC) class I or II molecules as compared to an unmodified peptide.

[0293] The present invention further relates to a peptide according to the present invention, which is modified and / or contains non-peptide bonds as described below.

[0294] The present invention further relates to a peptide according to the present invention, wherein the peptide is part of a fusion protein, in particular fused to the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii), or fused to an antibody (or in its sequence) specific for dendritic cells, i.e., an antibody that binds to dendritic cells.

[0295] The present invention further relates to a nucleic acid encoding a peptide according to the present invention. The present invention further relates to a nucleic acid according to the present invention, which is DNA, cDNA, PNA, RNA, or a combination thereof.

[0296] The present invention further relates to an expression vector capable of expressing, expressing, and / or presenting a nucleic acid according to the present invention.

[0297] The present invention further relates to a peptide according to the invention, a nucleic acid according to the invention, or an expression vector according to the invention for use in medicine.

[0298] The present invention further relates to antibodies, and methods for producing them, which are described in more detail below. Preferred are antibodies that are specific for the peptides of the invention and / or for the peptides of the invention upon binding to their MHC. Preferred antibodies can be monoclonal.

[0299] The present invention further relates to T cell receptors (TCRs), in particular soluble TCRs (sTCRs) that target the peptides according to the invention and / or their peptide-MHC complexes, and methods for producing them.

[0300] The present invention further relates to antibodies or other binding molecules that target the peptides according to the invention and / or their peptide-MHC complexes, and methods for producing them.

[0301] The present invention further relates to host cells comprising a nucleic acid or an expression vector according to the invention as described above. The present invention further relates to host cells according to the invention that are antigen-presenting cells. The present invention further relates to host cells according to the invention wherein the antigen-presenting cell is a dendritic cell.

[0302] The present invention further relates to aptamers. Aptamers (see, for example, WO 2014 / 191359 pamphlet and the documents cited therein) are short single-stranded nucleic acid or peptide molecules that fold into a defined three-dimensional structure and can recognize a specific target structure. They seem to be a suitable alternative for developing targeted therapies. Aptamers have been shown to bind selectively with high affinity and specificity to a variety of complex targets.

[0303] Aptamers that recognize molecules located on the cell surface have been identified within the past decade and provide a means for developing diagnostic and therapeutic approaches. Since aptamers have been shown to have little to no toxicity and immunogenicity, they are promising candidates for biomedical applications. Indeed, aptamers such as, for example, prostate-specific membrane antigen recognition aptamers have been successfully used for targeted therapy and have been shown to be functional in xenograft in vivo models. Furthermore, aptamers that recognize specific tumor cell lines have been identified.

[0304] DNA aptamers can be selected to exhibit broad-spectrum recognition properties against various cancer cells, particularly those derived from solid tumors, while not recognizing non-tumorigenic and primary healthy cells. When the identified aptamers not only recognize specific tumor subtypes but rather interact with a series of tumors, this enables the application of aptamers as so-called broad-spectrum diagnostic and therapeutic agents.

[0305] Furthermore, studies of cell-binding behavior by flow cytometry have shown that aptamers exhibit very good apparent affinity in the nanomolar concentration range.

[0306] Aptamers are useful for diagnostic and therapeutic purposes. Furthermore, it can be shown that some aptamers are taken up by tumor cells and can thus function as molecular vehicles for the targeted delivery of anticancer agents such as siRNA into tumor cells.

[0307] Aptamers can be selected using cell SELEX (in vitro evolution method) technology against complex targets such as cells and tissues and against complexes of peptides comprising and preferably consisting of the sequences described in any of SEQ ID NO: 1 to SEQ ID NO: 300 according to the invention and MHC molecules.

[0308] As used herein, the term "scaffold" refers to a molecule that specifically binds to a (e.g., antigenic) determinant. In one embodiment, the scaffold can also direct the entity to which it attaches (e.g., a (second) antigen-binding moiety) to a type-targeted site such as a specific tumor cell or tumor stroma having an antigen determinant (e.g., the peptide complex described in this application), for example. In another embodiment, the scaffold can activate signal transduction via its target antigen, such as a T cell receptor complex antigen. Scaffolds include, but are not limited to, antibodies and their fragments, antigen-binding domains of antibodies comprising antibody heavy chain variable regions and antibody light chain variable regions, binding proteins comprising at least one ankyrin repeat motif and a single domain antigen-binding (SDAB) molecule, aptamers, (soluble) TCRs, and (modified) cells such as allogeneic or autologous T cells.

[0309] Each scaffold can comprise a label, which provides for the detection of a bound scaffold by determining the presence or absence of a signal provided by the label. For example, the scaffold can be labeled with a fluorescent dye or any other applicable cell marker molecule. Such marker molecules are well known in the art. For example, a fluorescent label provided by a fluorescent dye can provide for visualization of a bound aptamer by fluorescence or laser scanning microscopy or flow cytometry.

[0310] Each scaffold can be conjugated to a second active molecule such as, for example, IL-21, anti-CD3, anti-CD28. Each polypeptide scaffold is described, for example, in the background art section of WO 2014 / 071978A1 pamphlet and the references cited therein.

[0311] The invention further relates to a method for producing a peptide according to the invention, comprising culturing a host cell according to the invention and isolating the peptide from the host cell and / or its culture broth.

[0312] The present invention further relates to an in vitro method for producing activated T lymphocytes, comprising the step of contacting T cells with antigen-loaded human class I or II MHC molecules expressed on the surface of appropriate antigen-presenting cells for a time sufficient to activate said T cells in an antigen-specific manner, wherein said antigen is at least one peptide according to the present invention. The present invention further relates to a method of loading an antigen onto class I or II MHC molecules expressed on the surface of appropriate antigen-presenting cells by contacting the antigen-presenting cells with a sufficient amount of the antigen.

[0313] The present invention further relates to a method according to the present invention, wherein the antigen-presenting cell comprises an expression vector capable of expressing said peptide containing the amino acid sequences of SEQ ID NOs: 1 to 300, or a mutant amino acid sequence thereof.

[0314] The present invention further relates to activated T cells produced by the method according to the present invention, which selectively recognize cells that abnormally express a polypeptide comprising the amino acid sequence according to the present invention.

[0315] The present invention further relates to a method of killing target cells that abnormally express a polypeptide comprising any amino acid sequence according to the present invention in a patient, comprising the step of administering to the patient an effective number of T cells according to the present invention.

[0316] The present invention further relates to the use of any of the peptides described, nucleic acids according to the present invention, expression vectors according to the present invention, cells according to the present invention, or activated T cells according to the present invention as a medicament or in the manufacture of a medicament.

[0317] The present invention further relates to the use according to the present invention, wherein said medicament is a vaccine, a cell, for example, a cell population such as a cell line, sTCR, and a monoclonal antibody.

[0318] The present invention further relates to the use according to the present invention, wherein the medicament is effective against cancer.

[0319] The present invention further relates to the use according to the present invention, wherein the cancer cells are cells of HCC.

[0320] The present invention further relates to specific labeled proteins and biomarkers based on peptides according to the present invention, which can be used in the diagnosis and / or prognostic diagnosis of HCC.

[0321] Furthermore, the present invention relates to the use of these novel targets for cancer treatment.

[0322] Furthermore, the present invention relates to a method for manufacturing an individualized anti-cancer vaccine for an individual patient using a database of pre-screened tumor-related peptides (also referred to herein as a "repository").

[0323] Stimulation of the immune response depends on the presence of antigens recognized as foreign by the host immune system. The discovery of the presence of tumor-associated antigens has increased the possibility of intervening in tumor growth by utilizing the host immune system. Various mechanisms that utilize both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

[0324] Certain elements of the cellular immune response have the ability to specifically recognize and destroy tumor cells. Isolation of T cells from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in the natural immune defense against cancer. In particular, CD8-positive T cells that recognize class I molecules of major histocompatibility complex (MHC) peptides, usually 8-10 amino acid residues, derived from proteins located within the cytoplasmic sol or defective ribosomal products (DRiPs), play an important role in this response. Human MHC molecules are also referred to as human leukocyte antigens (HLA).

[0325] The term "peptide" is used herein to name a series of amino acid residues that are linked to each other by peptide bonds between the α-amino group and the carbonyl group of adjacent amino acids. Peptides are preferably 9 amino acids in length, but can be as short as about 8 amino acids in length or as long as about 10, 11, 12, or 13 amino acids in length. In the case of MHC class II peptides (extended variants of the peptides of the present invention), they can be as long as about 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0326] Furthermore, the term "peptide" is typically intended to include salts of a series of amino acid residues that are linked to each other by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. Preferably, the salts are pharmaceutically acceptable salts of the peptide, such as, for example, chloride or acetate (trifluoroacetate). It should be noted that since peptides are not salts in vivo, the salts of the peptides according to the present invention are substantially different from the peptides in their in vivo state.

[0327] The term "peptide" is also intended to include "oligopeptide". The term "oligopeptide" is used herein to name a series of amino acid residues that are linked to each other by peptide bonds between the α-amino group and the carbonyl group of adjacent amino acids. The length of the oligopeptide is not important for the present invention as long as the correct epitope or epitopes are retained therein. Oligopeptides are typically less than about 30 amino acid residues in length and more than about 15 amino acids in length.

[0328] The term "peptides of the present invention" is intended to include peptides consisting of or comprising the peptides set forth in SEQ ID NOs: 1 to 300 as defined above.

[0329] The term "polypeptide" typically refers to a continuous series of amino acid residues linked to one another by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, provided that the correct epitope is retained. In contrast to the terms peptide or oligopeptide, the term polypeptide is intended to refer to a molecule containing more than about 30 amino acid residues.

[0330] A peptide, oligopeptide, protein or polynucleotide encoding such a molecule is "immunogenic" (and thus an "immunogen" within the present invention) if it has the ability to induce an immune response. In the present invention, immunogenicity is more specifically defined as the ability to induce a T cell response. Thus, an "immunogen" is a molecule capable of inducing an immune response, and in the present invention, a molecule capable of inducing a T cell response. In another aspect, an immunogen can be a peptide, a peptide-MHC complex, an oligopeptide, and / or a protein that is used to generate specific antibodies or TCRs thereto.

[0331] Class I T cell "epitopes" require short peptides that bind to Class I MHC receptors to form a three-component complex (MHC Class I α-chain, β-2-microglobulin, and peptide), which can be recognized by T cells bearing a compatible T cell receptor that binds to the MHC / peptide complex with appropriate affinity. Peptide binding to MHC Class I molecules is typically 8-14 amino acids in length, most typically 9 amino acids in length.

[0332] In humans, there are three different loci, HLA-A, HLA-B, and HLA-C, that encode MHC Class I molecules (human MHC molecules also referred to as human leukocyte antigens (HLA)). HLA-A * 01, HLA-A * 02, and HLA-B * 07 are examples of different MHC Class I alleles that can be expressed from these loci.

[0333] Table 6: HLA-A * 02 and HLA-A * The expression frequencies F of 24, and the most frequent HLA-DR serotypes. The frequencies were estimated from the haplotype frequencies Gf in the US population adapted from Mori et al. (Mori M, et al. HLA gene and haplotype frequencies in the North American population: the National Marrow Donor Program Donor Registry. Transplantation. 1997 Oct 15;64(7):1017-27) using the Hardy-Weinberg formula, F = 1-(1-Gf). 2 Due to linkage disequilibrium, the combinations of A * 02 or A * 24 with specific HLA-DR alleles may be more or less abundant than predicted from their single frequencies. For details, see Chanock et al. (S.J. Chanock, et al(2004) HLA-A,-B,-Cw,-DQA1 and DRB1 in an African American population from Bethesda, USA Human Immunology, 65:1223-1235).

Table 6-1

Table 6-2

[0334] The peptides of the present invention preferably bind to A * 02 or A * 24 when included in the vaccine of the present invention described herein. The vaccine may also contain pan-binding MHC class II peptides. Thus, using the vaccine of the present invention, A * 02 positive, A * 24 positive, or A * 02 and A* In patients who are positive for 24, the cancer can be treated, but due to the pan-binding properties of these peptides, there is no need to select an MHC class II allotype.

[0335] For example, A * 02 and A * Combining the 02 and 24 peptides in one vaccine has the advantage that a higher percentage of any patient population can be treated compared to dealing with only one of the MHC class I alleles. In most populations, less than 50% of patients are dealt with by only one of the alleles, while the vaccines of the present invention can treat at least 60% of patients in any relevant population. Specifically, in various regions, the following percentages of patients are positive for at least one of these alleles: 61% in the USA, 62% in Western Europe, 75% in China, 77% in South Korea, 86% in Japan (calculated from www.allelefrequencies.net).

[0336] As used herein, references to DNA sequences include both single-stranded and double-stranded DNA. Thus, a specific sequence refers to single-stranded DNA of such a sequence, double-stranded (double-stranded DNA) of such a sequence and its complement, and the complement of such a sequence, unless the context clearly suggests a different meaning. The term "coding region" refers to the portion of a gene that, in its native genomic context, naturally or normally encodes the expression product of the gene, i.e., the region that encodes the native expression product of the gene in vivo.

[0337] The coding region can be derived from a non-mutated ("normal"), mutated or modified gene, or even from a DNA sequence or gene that has been completely synthesized in the laboratory using methods well known to those skilled in the art of DNA synthesis technology.

[0338] In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

[0339] Nucleotide sequences encoding specific peptides, oligopeptides, or polypeptides may be of natural origin or they may be synthetically constructed. Generally, DNA fragments encoding the peptides, polypeptides, and proteins of the present invention are constructed from cDNA fragments and short oligonucleotide linkers or from a continuous series of oligonucleotides to provide synthetic genes that can be expressed in recombinant transcription units that include regulatory elements derived from microbial or viral operons.

[0340] As used herein, the term "peptide-coding (or encoding) nucleotide" refers to a nucleotide sequence that encodes a peptide that includes artificial (man-made) start and stop codons and is compatible with a biological system in which the sequence is expressed, for example, by dendritic cells or another cell line useful for the production of TCRs.

[0341] The term "expression product" means a polypeptide or protein that is the natural translation product of a gene and any equivalent thereof that encodes a nucleic acid sequence that encodes the same amino acid due to genetic code degeneracy.

[0342] When referring to a coding sequence, the term "fragment" means a portion of DNA that includes a coding region that is less than complete and whose expression product retains an essentially identical biological function or activity as the expression product of the complete coding region.

[0343] The term "DNA fragment" refers to a DNA polymer in the form of separate fragments or as a component of a larger DNA construct, which is in a substantially pure form, i.e., free of contaminating endogenous substances, and is isolated at least once in an amount or concentration such that the fragment and its constituent nucleotide sequences can be identified, manipulated, and recovered by standard biochemical methods using, for example, a cloning vector. Such fragments are provided in the form of an open reading frame, which is typically not interrupted by internal non-translated sequences or introns that are present within eukaryotic genes. The non-translated DNA sequences may be present downstream of the open reading frame, where they do not interfere with the manipulation or expression of the coding region.

[0344] The term "primer" means a short nucleic acid sequence that can pair with a single strand of DNA and provides a free 3'-OH end at which DNA polymerase can initiate deoxyribonucleotide strand synthesis.

[0345] The term "promoter" means a region of DNA that is involved in the binding of RNA polymerase to initiate transcription.

[0346] The term "isolated" means that a substance has been removed from its original environment (e.g., its natural environment if it is of natural origin). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the substances that coexist in the natural system is isolated. Such a polynucleotide can be part of a vector and / or such a polynucleotide or polypeptide can be part of a composition, and still be isolated in the sense that such a vector or composition is not part of its natural environment.

[0347] The polynucleotides and recombinant or immunogenic polypeptides disclosed by the present invention may be in a "purified" form. The term "purified" does not necessarily mean completely pure; rather, it is intended to be a relative definition and may include highly purified preparations or preparations that are only partially purified, as these terms are understood by those skilled in the art. For example, individual clones isolated from a cDNA library are electrophoretically homogeneous and purified by conventional methods. Purification of the starting material or natural substance to at least one digit, preferably two or three digits, more preferably four or five digits is explicitly contemplated. Further, the claimed polypeptides having a purity of preferably 99.999%, or at least 99.99% or 99.9% by weight; more desirably 99% or more are explicitly contemplated.

[0348] The nucleic acid and polypeptide expression products disclosed by the present invention, and expression vectors containing such nucleic acids and / or such polypeptides may be in an "enriched form". As used herein, the term "enriched" means a substance concentration that is at least about 2, 5, 10, 100, or 1000 times its natural concentration, and advantageously is 0.01% by weight, preferably at least about 0.1% by weight. Enriched preparations at about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The sequences, constructs, vectors, clones, and other substances that make up the present invention may advantageously be in an enriched or isolated form.

[0349] The term "active fragment" usually refers to a fragment that, when administered alone or optionally with a suitable adjuvant or within a vector, to an animal such as a rabbit or mouse and also mammals including humans, elicits an immune response (i.e., is immunogenic), and such an immune response takes a form that stimulates a T cell response within the recipient animal such as a human. Alternatively, an "active fragment" may also be used to induce an in vitro T cell response.

[0350] As used herein, when used in connection with a polypeptide, the terms "portion", "fragment", and "segment" refer to a sequence of contiguous residues, such as amino acid residues, that forms a subset of a larger sequence. For example, if a polypeptide is treated with any of the common endopeptidases such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would correspond to portions, fragments or segments of the starting polypeptide. When used in connection with a polynucleotide, these terms refer to the products resulting from the treatment of said polynucleotide with any of the endonucleases.

[0351] As used in the present invention, when referring to sequences, the terms "percent homology", "percentage of identity", or "percent identical" mean that after alignment of the sequences being compared (the "comparison sequence") with the sequence described or claimed (the "reference sequence"), the sequence is compared with the sequence described or claimed. The percentage of identity is then determined according to the following formula: Percent identity = 100 [1 - (C / R)] where C is the number of differences between the reference sequence and the comparison sequence over the length of the alignment between the reference sequence and the comparison sequence being compared, (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence, and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence that is different from the aligned base or amino acid in the comparison sequence constitutes a difference, (iiii) the alignment must start at position 1 of the aligned sequences; R is the number of bases or amino acids in the reference sequence over the length of the alignment with the comparison sequence, and any gap occurring in the reference sequence is also counted as a base or amino acid.

[0352] If there is an alignment between a comparison array and a reference array for which the percent identity is calculated as above that is approximately the same as or greater than a particular minimum percent identity, then even if there is an alignment in which the percent identity calculated as above is less than the particular percent identity, the comparison array has a particular minimum percent identity with the reference array.

[0353] The original (unmodified) peptides disclosed herein may be modified by substitution of one or more residues at different, perhaps selective, sites within the peptide chain, unless otherwise specified. Preferably these substitutions are located at the termini of the amino acid chain. Such substitutions may be conservative in nature, for example, an amino acid is substituted by another amino acid with similar structure and characteristics, such as a hydrophobic amino acid being substituted by another hydrophobic amino acid. Even more conservative substitutions are substitutions of amino acids of the same or similar size and chemical properties, such as substitution of leucine by isoleucine. In studies of sequence diversity of naturally occurring homologous protein families, certain amino acid substitutions are more often tolerated than others, and these often show a correlation with the similarity in size, charge, polarity, and hydrophobicity between the original amino acid and its substitute, which is the basis for the definition of "conservative substitution".

[0354] Conservative substitutions are defined herein as exchanges within one of the following five groups: Group 1 - small aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2 - polar negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3 - polar positively charged residues (His, Arg, Lys); Group 4 - large aliphatic nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5 - large aromatic residues (Phe, Tyr, Trp).

[0355] Less conservative substitutions may involve substitution with another amino acid having similar characteristics but a somewhat different size, such as substitution of an alanine residue with an isoleucine residue. Highly non-conservative substitutions may involve substitution of a polar amino acid with an acidic amino acid, or substitution of an amino acid that is even basic. However, since the chemical effects are not completely predictable and free radical substitutions can result in accidental effects that cannot be predicted from simple chemical principles, such "radical" substitutions cannot be rejected as potentially ineffective.

[0356] Of course, such substitutions may involve structures other than the normal L-amino acids. Thus, D-amino acids may substitute for the L-amino acids normally found in the antigenic peptides of the present invention and still be encompassed by the disclosure herein. In addition, amino acids retaining non-standard R groups (i.e., R groups other than those found in the normal 20 amino acids of natural proteins) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

[0357] If substitutions at two or more positions are found to result in a peptide having substantially equivalent or greater antigenic activity as defined below, combinations of these substitutions are tested to determine whether the combination of substitutions results in an additive or synergistic effect on the antigenicity of the peptide. At most, four or fewer positions within the peptide are substituted simultaneously.

[0358] The peptides of the present invention can be extended by up to four amino acids, i.e., in any combination between 4:0 and 0:4, with 1, 2, 3, or 4 amino acids added to either end.

[0359] Combinations of extensions according to the present invention can be shown from Table 7.

Table 7

[0360] The amino acids for elongation can be peptides of the original protein sequence or any other amino acids. Elongation can be utilized to enhance the stability or solubility of the peptide.

[0361] The term "T cell response" means the specific proliferation and activation of effector functions induced by a peptide, either in vitro or in vivo. In MHC class I-restricted CTLs, the effector functions are lysis of peptide-pulsed, peptide precursor-pulsed or native peptide-presenting target cells; preferably secretion of cytokines that are interferon-γ, TNF-α, or IL-2 induced by the peptide; preferably secretion of effector molecules that are granzymes or perforin induced by the peptide; or degranulation may also be involved.

[0362] Preferably, when testing T cells specific for a peptide according to the present invention against a replacement peptide, the peptide concentration at which the replacement peptide achieves half of the maximum lysis increase relative to the background is about 1 mM or less, preferably about 1 μM or less, more preferably about 1 nM or less, even more preferably about 100 pM or less, and most preferably about 10 pM or less. It is also preferred that the replacement peptide is recognized by T cells from two or more, at least two, more preferably three individuals.

[0363] Thus, the epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes, or may contain epitopes that differ from the reference peptide by four or fewer residues as long as they have substantially the same antigenic activity.

[0364] MHC class I molecules are found on the majority of nucleated cells that present peptides, which are mainly derived from the proteolytic cleavage of endogenous cytoplasmic or nuclear proteins, DRIPs, and larger peptides. However, peptides derived from the endosomal compartment or of exogenous origin are also frequently found on MHC class I molecules. This non-classical mode of class I presentation is referred to in the literature as cross-presentation.

[0365] Since both CD8- and CD4-dependent types of responses contribute jointly and synergistically to the antitumor effect, the identification and characterization of tumor-associated antigens recognized by either CD8-positive T cells (MHC class I molecules) or CD4-positive T cells (MHC class II molecules) are important in the development of tumor vaccines. Accordingly, it is an object of the present invention to provide a peptide composition containing peptide binding to either class of MHC complexes.

[0366] Given the serious side effects and costs associated with cancer treatment, better prognostic and diagnostic methods are urgently needed. Accordingly, there is a need to identify other factors corresponding to biomarkers for cancer in general and HCC in particular. Further, there is a need to identify factors that can be used in cancer treatment methods in general and HCC in particular.

[0367] The present invention provides peptides useful for treating cancer / tumors that preferentially or exclusively present the peptides of the present invention, preferably CLL. These peptides have been shown by mass spectrometry to be naturally presented by HLA molecules on primary human HCC samples.

[0368] The originating gene / protein from which the peptide is derived (also referred to as the "full-length protein" or "basal protein") has been shown to be highly overexpressed in cancer compared to normal tissue, demonstrating a high tumor association of the originating gene, where "normal tissue" is meant to be either healthy hepatocytes or other normal tissue cells in the context of the present invention (see Example 2). Further, the peptide itself is strongly overpresented on tumor tissue, where "tumor tissue" is meant to be a sample from a patient suffering from HCC but not normal tissue in the context of the present invention (see Example 1).

[0369] HLA-binding peptides can be recognized by the immune system, specifically T lymphocytes. T cells can destroy cells presenting recognized HLA / peptide complexes, such as HCC cells presenting the inducing peptide.

[0370] The peptides of the present invention are capable of stimulating T cell responses and / or have been shown to be overexpressed, and can thus be used for the production of antibodies and / or TCRs, particularly TCRs according to the present invention (see Example 3). Furthermore, the peptides, when complexed with their respective MHCs, can likewise be utilized for the production of antibodies and / or TCRs, particularly TCRs according to the present invention. Each method is well known to those skilled in the art and can also be found in their respective references. Thus, the peptides of the present invention are useful for generating an immune response in a patient that can thereby destroy tumor cells. The immune response in a patient can preferably be induced by administering the described peptides, or suitable precursors (e.g., extended peptides, proteins, or nucleic acids encoding those peptides) directly to the patient, in combination with an agent that enhances immunogenicity (i.e., an adjuvant). Since the target peptides of the present invention are not presented at equivalent copy numbers on normal tissues, the immune response resulting from such therapeutic vaccination can be predicted to be highly specific for tumor cells and prevent the risk of unwanted autoimmune reactions against the patient's normal cells.

[0371] Preferably, a "pharmaceutical composition" is a composition that is preferably suitable for administration to humans in a medical situation. Preferably, the pharmaceutical composition is sterile and manufactured in accordance with GMP guidelines.

[0372] The pharmaceutical composition comprises a peptide either in free form or in the form of a pharmaceutically acceptable salt. (See also above). In the context of this specification, "pharmaceutically acceptable salt" refers to derivatives of the disclosed peptides, where the peptide is modified by making acidic or basic salts of the agent. For example, acidic salts are prepared from the free base (typically where the neutral form of the agent has a neutral NH2 group) by reaction with an appropriate acid. Suitable acids for preparing acidic salts include, for example, organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc., as well as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Conversely, preparations of basic salts of acidic moieties that may be present on the peptide are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, etc.

[0373] In a particularly preferred embodiment, the pharmaceutical composition comprises the peptide as a salt of acetic acid (acetate), trifluoroacetic acid or hydrochloric acid (chloride).

[0374] Particularly preferred are the composition and / or the use of said composition, where the composition is, for example, in the form of a vaccine and comprises a peptide having the sequences set forth in SEQ ID NOs: 1, 2, 7, 225, 228, 301, 303, and 312, or a scaffold reactive with a peptide having the sequences set forth in SEQ ID NOs: 1, 2, 7, 225, 228, 301, 303, and 312, and complexes thereof with MHC molecules.

[0375] In addition to being useful for treating cancer, the peptides of the present invention are also useful as diagnostic agents. Since the peptides are generated from HCC and these peptides have been determined to be absent or present at lower levels in normal tissue, these peptides can be utilized to diagnose the presence of cancer.

[0376] The presence of the claimed peptides on histopathology in a blood sample can assist the pathologist in cancer diagnosis. Detection of specific peptides by means of antibodies, mass spectrometry or other methods known in the art can inform the pathologist that the tissue sample is malignant or inflammatory or generally diseased, or can be used as a biomarker for HCC. The presence of peptide groups can enable the classification or sub-classification of diseased tissue.

[0377] Detection of peptides on a diseased tissue specimen allows determination of the benefits of immunotherapy treatments, particularly when T lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a well-described mechanism by which infected malignant cells evade immune surveillance. Thus, the presence of peptides indicates that this mechanism is not being utilized by the cells analyzed.

[0378] The peptides of the present invention may be used to analyze lymphocyte responses to these peptides, such as T cell responses or antibody responses to peptides complexed with peptides or MHC molecules. These lymphocyte responses can be used as prognostic markers to determine further treatment steps. These responses can also be used as surrogate markers in immunotherapy approaches aimed at inducing lymphocyte responses by different means, such as vaccination with proteins, nucleic acids, self-materials, and adoptive immunotransfer of lymphocytes. In the context of gene therapy, lymphocyte responses to peptides can be considered in the assessment of side effects. Monitoring of lymphocyte responses may also be a useful means for follow-up examinations in transplantation therapy, such as detection of graft-versus-host disease and host-versus-graft disease.

[0379] Using the peptides of the present invention, specific antibodies against MHC / peptide complexes can be created and developed. These can be used for therapeutic methods that target toxin or radioactive substances to diseased tissues. Another use of these antibodies can be the targeting of radionuclides for imaging purposes, such as PET, to diseased tissues. This use can assist in the detection of small metastases or the determination of the size and exact location of diseased tissues.

[0380] Accordingly, it is a further aspect of the present invention to provide a method for producing a recombinant antibody that specifically binds to human major histocompatibility complex (MHC) class I or II complexed with an HLA-restricted antigen, the method comprising immunizing a genetically engineered non-human mammal comprising cells expressing said human major histocompatibility complex (MHC) class I or II with a soluble form of MHC class I or II molecule complexed with said HLA-restricted antigen; isolating mRNA molecules from the antibody-producing cells of said non-human mammal; creating a phage display library presenting protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, wherein said at least one phage presents said antibody that specifically binds to said human major histocompatibility complex (MHC) class I or II complexed with said HLA-restricted antigen.

[0381] It is also a further aspect of the present invention to provide an antibody that specifically binds to human major histocompatibility complex (MHC) class I or II complexed with an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and / or a chimeric antibody.

[0382] Another aspect of the present invention relates to a method for producing the antibody that specifically binds to human major histocompatibility complex (MHC) class I or II complexed with an HLA-restricted antigen. The method comprises immunizing a genetically engineered non-human mammal comprising cells expressing the human major histocompatibility complex (MHC) class I or II with a soluble form of an MHC class I or II molecule complexed with the HLA-restricted antigen; isolating mRNA molecules from antibody-producing cells of the non-human mammal; creating a phage display library presenting protein molecules encoded by the mRNA molecules; and isolating at least one phage from the phage display library, wherein the at least one phage presents the antibody capable of specifically binding to the human major histocompatibility complex (MHC) class I or II complexed with the HLA-restricted antigen.Such antibodies and the respective methods of making single-chain class I major histocompatibility complexes, as well as other tools for making these antibodies, are for the purposes of the present invention hereby expressly incorporated by reference in their entireties: WO 03 / 068201, WO 2004 / 084798, WO 01 / 72768, WO 03 / 070752, and Cohen CJ, et al. Recombinant antibodies with MHC-restricted, peptide-specific, T-cell receptor-like specificity: new tools to study antigen presentation and TCR-peptide-MHC interactions. J Mol Recognit. 2003 Sep-Oct;16(5):324-32.; Denkberg G, et al. Selective targeting of melanoma and APCs using a recombinant antibody with TCR-like specificity directed toward a melanoma differentiation antigen. J Immunol. 2003 Sep 1;171(5):2197-207; and Cohen CJ, et al. Direct phenotypic analysis of human MHC class I antigen presentation: visualization, quantitation, and in situ detection of human viral epitopes using peptide-specific, MHC-restricted human recombinant antibodies. J Immunol. 2003 Apr 15;170(8):4349-61.

[0383] Preferably, the antibody binds to the complex with a binding affinity of less than 20 nanomolar, preferably less than 10 nanomolar, which is considered "specific" in the context of the present invention.

[0384] It is also a further aspect of the present invention to provide a method for producing a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex. Such soluble T cell receptors can be produced from specific T cell clones, and their affinity can be increased by mutagenesis targeting complementarity-determining regions. Phage display can be utilized for the selection of T cell receptors (U.S. Patent No. 2010 / 0113300; Liddy N, et al. Monoclonal TCR-redirected tumor cell killing. Nat Med 2012 Jun;18(6):980-987). In phage display and for practical use as a drug, for the purpose of stabilizing the T cell receptor, the α and β chains can be linked, for example, by non-natural disulfide bonds, other covalent bonds (single-chain T cell receptors), or dimerization domains (Boulter JM, et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng 2003 Sep;16(9):707-711.; Card KF, et al. A soluble single-chain T-cell receptor IL-2 fusion protein retains MHC-restricted peptide specificity and IL-2 bioactivity. Cancer Immunol Immunother 2004 Apr;53(4):345-357; and Willcox BE, et al. Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding. Protein Sci 1999 Nov;8(11):2418-2423). The T cell receptor can be linked to an effector cell mobilizing domain such as a toxin, a drug, a cytokine (see, for example, U.S. Patent No. 2013 / 0115191), an anti-CD3 domain, etc. in order to perform a specific function on the target cell.Furthermore, it can be expressed in T cells used for adoptive immunotransfer. Further information can be found in WO 2004 / 033685 A1 pamphlet and WO 2004 / 074322 A1 pamphlet. Combinations of TCRs are described in WO 2012 / 056407 A1 pamphlet. Further manufacturing methods are disclosed in WO 2013 / 057586 A1 pamphlet.

[0385] Furthermore, cancer diagnosis based on a pathologist's biopsy sample can be confirmed using the peptides and / or TCRs or antibodies or other binding molecules of the present invention.

[0386] To select overpresented peptides, a presentation profile showing median sample presentation as well as replicate assay variation is calculated. The profile juxtaposes samples of the tumor entity of interest against a baseline of normal samples. Next, by calculating the p-value of a linear mixed effects model, each of these profiles can be integrated into an overpresentation score (J. Pinheiro, et al. The nlme Package: Linear and Nonlinear Mixed Effects Models. 2007) adjusting for multiple testing by False Discovery Rate (Y. Benjamini and Y. Hochberg. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society. Series B (Methodological), Vol. 57 (No. 1): 289 - 300, 1995).

[0387] For the identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules were purified from shock-frozen samples and HLA-associated peptides were isolated. The isolated peptides were separated and their sequences were identified by online nanoelectrospray ionization (nanoESI) liquid chromatography mass spectrometry (LC-MS) experiments. The resulting peptide sequences were confirmed by comparison with the fragmentation patterns of corresponding synthetic standard peptides with the same sequences as the fragmentation patterns of native TUMAP recorded from HCC samples (N = 16 A * 02-positive samples, 13 A * 02:01-positive samples, including N = 15 A * 24-positive samples). Since the peptides were directly identified as ligands of HLA molecules in primary tumors, these results provide direct evidence of the natural processing and presentation of the identified peptides on primary cancer tissues obtained from 31 HCC patients.

[0388] The discovery pipeline XPRESIDENT® v2.1 (see, for example, US Patent No. 2013-0096016, the entire contents of which are incorporated herein by reference) enables the identification and selection of valid overpresented peptide vaccine candidates based on the direct relative quantification of HLA-restricted peptide levels on cancer tissues compared to several different non-cancerous tissues and organs. This was achieved by the development of label-free differential quantification using acquired LC-MS data processing by a labeled data analysis pipeline, a combination of algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, and charge state deconvolution and normalization.

[0389] Presentation levels were established, including error estimates for each peptide and sample. Peptides that were exclusively presented on tumor tissues and peptides that were overpresented within tumors were identified in comparison with non-cancerous tissues and organs.

[0390] HLA peptide complexes from HCC tissue samples were purified to isolate HLA-binding peptides, which were analyzed by LC-MS (see Examples). All TUMAPs included in this application were identified on primary HCC samples by this approach, and their presentation on primary HCC was confirmed.

[0391] TUMAPs identified on multiple HCC tumors and normal tissues were quantified using ion counts of label-free LC-MS data. The method assumes that the LC-MS signal area of a peptide correlates with its abundance in the sample. All quantitative signals of peptides in various LC-MS experiments were normalized based on central tendency, averaged for each sample, and merged into a bar graph called a presentation profile. The presentation profile integrates different analytical methods such as protein database search, spectral clustering, charge state deconvolution (desalting), and retention time alignment and normalization.

[0392] The present invention relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 300, or a variant thereof that is at least 90% homologous (preferably identical) to SEQ ID NOs: 1 to 300, or a variant thereof that cross-reacts T cells with the peptide, wherein the peptide is not the underlying full-length polypeptide.

[0393] The present invention further relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 300, or a variant thereof that is at least 90% homologous (preferably identical) to SEQ ID NOs: 1 to 300, wherein the peptide or variant has a full length of 8 to 100, preferably 8 to 30, and most preferably 8 to 14 amino acids.

[0394] The present invention further relates to a peptide according to the present invention that has the ability to bind to a human major histocompatibility complex (MHC) class I or II molecule.

[0395] The present invention further relates to a peptide according to the present invention, wherein the peptide consists of or consists essentially of the amino acid sequences set forth in SEQ ID NOs: 1 to 300.

[0396] The present invention further relates to a peptide according to the present invention, wherein the peptide is (chemically) modified and / or contains non-peptide bonds.

[0397] The present invention further relates to a peptide according to the present invention, wherein the peptide is part of a fusion protein, particularly comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii), or the peptide is fused to (and into) an antibody such as a dendritic cell-specific antibody.

[0398] The present invention further relates to a nucleic acid encoding a peptide according to the present invention, provided that the peptide is not a full (full-length) human protein.

[0399] The present invention further relates to a nucleic acid according to the present invention, which is DNA, cDNA, PNA, RNA or a combination thereof.

[0400] The present invention further relates to an expression vector capable of expressing a nucleic acid according to the present invention.

[0401] The present invention further relates to a peptide according to the present invention, a nucleic acid according to the present invention or an expression vector according to the present invention for use in medicine, particularly for the treatment of HCC.

[0402] The present invention further relates to a host cell comprising a nucleic acid according to the present invention or an expression vector according to the present invention.

[0403] The present invention further relates to a host cell according to the present invention, which is an antigen-presenting cell, preferably a dendritic cell.

[0404] The present invention further relates to a method according to the present invention, wherein an antigen is loaded onto class I or II MHC molecules expressed on the surface of appropriate antigen-presenting cells by contacting the antigen-presenting cells with a sufficient amount of the antigen.

[0405] The present invention further relates to a method according to the present invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing the peptides of SEQ ID NOs: 1 to 300, or said peptides containing the heterologous amino acid sequences.

[0406] The present invention further relates to the use of any of the peptides described, the nucleic acids according to the present invention, the expression vectors according to the present invention, the cells according to the present invention, or the activated cytotoxic T lymphocytes according to the present invention, as a medicament or in the manufacture of a medicament. The present invention further relates to the use according to the present invention, wherein the medicament is effective against cancer.

[0407] The present invention further relates to the use according to the present invention, wherein the medicament is a vaccine. The present invention further relates to the use according to the present invention, wherein the medicament is effective against cancer.

[0408] The present invention further relates to the use according to the present invention, wherein the cancer cells are HCC cells or other solid or hematological tumor cells such as pancreatic cancer, brain tumor, kidney cancer, colon or rectal cancer, or leukemia.

[0409] The present invention further relates to specific peptide-based labeled proteins and biomarkers according to the present invention, referred to herein as "targets", which can be used in the diagnosis and / or prognostic diagnosis of HCC. The present invention also relates to the use of these novel targets for cancer treatment.

[0410] The term "antibody(ies)" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to untreated or "complete" immunoglobulin molecules, the term "antibody" also includes fragments (e.g., CDRs, Fv, Fab, and Fc fragments), or polymers of these immunoglobulin molecules and humanized versions of immunoglobulin molecules, so long as they exhibit any of the desired properties according to the present invention (e.g., specific binding to an HCC marker polypeptide, delivery of a toxin to HCC cells that express the cancer marker gene at elevated levels, and / or inhibition of the activity of an HCC marker polypeptide).

[0411] Whenever possible, the antibodies of the present invention may be purchased from commercial suppliers. Alternatively, the antibodies of the present invention may be produced using well-known methods. Those skilled in the art will understand that either the full-length CLL marker polypeptide or a fragment thereof may be used to produce the antibodies of the present invention. The polypeptide used to produce the antibodies of the present invention may be partially or fully purified from a natural source or may be produced using recombinant DNA technology.

[0412] For example, a cDNA encoding a peptide according to the present invention, such as a peptide described in SEQ ID NO:1 to SEQ ID NO:300 polypeptide, or a variant or fragment thereof, can be expressed in a prokaryotic cell (e.g., a bacterium) or a eukaryotic cell (e.g., a yeast, insect, or mammalian cell), and then the recombinant protein is purified and used to produce monoclonal or polyclonal antibody products that specifically bind to the HCC marker polypeptide used to produce the antibodies according to the present invention.

[0413] One of ordinary skill in the art will understand that generating two or more different sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining antibodies with the specificity and affinity required for their intended use (e.g., ELISA, immunohistochemical assays, in vivo imaging, immunotoxin therapy). Antibodies are tested for their desired activity by known methods according to the purpose for which the antibody is to be used (e.g., ELISA, immunohistochemical assays, immunotherapy, etc.; for further guidance on antibody production and testing, see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988, new 2nd edition 2013). For example, antibodies may be tested in ELISA assays, Western blots, immunohistochemical staining of formalin-fixed cancer or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested by known clinical trial methods.

[0414] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. As used herein, “monoclonal antibody” expressly includes “chimeric” antibodies and fragments of such antibodies, provided that they exhibit the desired antagonistic activity, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass (U.S. Patent No. 4,816,567, the entire contents of which are incorporated herein by reference).

[0415] The monoclonal antibodies of the present invention may be prepared using the hybridoma method. In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to generate lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. As an alternative, the lymphocytes may be immunized in vitro.

[0416] The monoclonal antibodies may also be produced by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of a mouse antibody).

[0417] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of an antibody to produce antibody fragments, particularly Fab fragments, can be accomplished using conventional techniques known in the art. For example, the digestion can be carried out using papain. Examples of papain digestion are described in WO 94 / 29348 pamphlet and U.S. Patent No. 4,342,566. Papain digestion of an antibody typically produces two identical antigen-binding fragments, designated Fab fragments, each having a single antigen-binding site, and the remaining Fc fragment. Pepsin treatment yields F(ab’)2 fragments and pFc’ fragments.

[0418] Antibody fragments can also include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment, regardless of whether it attaches to other sequences. These modifications can provide several additional properties, such as removal / addition of amino acids capable of forming disulfide bonds, increasing its biological lifespan, modifying its secretion characteristics, etc. In any case, the antibody fragment must have bioactive properties such as binding activity and regulation of binding in the binding region. The functional or active region of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily understood by those skilled in the art and may include site-directed mutagenesis of the nucleic acid encoding the antibody fragment.

[0419] The antibody of the present invention may further comprise a humanized antibody or a human antibody. Humanized forms such as non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as the Fv, Fab, Fab’ of the antibody or other antigen-binding partial sequences) that contain the minimum sequences derived from non-human immunoglobulins. As a humanized antibody, residues derived from the recipient's complementarity-determining regions (CDRs) therein are residues from the CDRs of non-human species (donor antibodies) such as mice, rats or rabbits that have the desired specificity, affinity, and ability. Substituted by residues, human immunoglobulins (recipient antibodies) are mentioned. In some cases, the Fv framework (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. The humanized antibody may also comprise residues that are not found in either the recipient antibody or the transplanted CDR or framework sequence. Generally, a humanized antibody comprises substantially all of at least one, typically two variable regions, in which all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin consensus sequences. The humanized antibody preferably also comprises at least a part of the immunoglobulin constant region (Fc), which is typically the human immunoglobulin constant region.

[0420] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from non-human origins. These non-human amino acid residues are often referred to as "transplanted" residues, which are typically obtained from the "transplanted" variable region. Humanization can be basically carried out by replacing the rodent CDR group or CDR sequence with the corresponding human antibody sequence. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which less than substantially intact human variable regions are replaced by the corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies, in which some CDR residues and perhaps some FR residues are replaced by residues derived from similar sites in rodent antibodies.

[0421] When immunized, a genetically engineered animal (e.g., a mouse) that can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production can be used. For example, it has been described that homozygous deletion of the antibody heavy chain joining region gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transcription of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge. Human antibodies can also be generated in phage display libraries.

[0422] The antibodies of the present invention are preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Further carriers include sustained release formulations of a semipermeable matrix of a solid hydrophobic polymer containing the antibody, the matrix being in the form of shaped articles such as, for example, films, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending, for example, on the route of administration and concentration of the antibody being administered.

[0423] The antibody can be administered to a subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular) or by other methods such as infusion that ensure delivery of the antibody in its active form to the bloodstream. The antibody can also be administered by the intratumoral or peritumoral route to exert local as well as systemic therapeutic effects. Local injection or intravenous injection is preferred.

[0424] The effective dosage and schedule for administering an antibody may be determined empirically, and the conduct of such measurements is within the skill of the art. One of ordinary skill in the art will understand that the dosage of antibody to be administered will vary depending, for example, on the subject to whom the antibody is administered, the route of administration, the particular antibody type being used, and other agents being administered. The typical daily dose of an antibody used alone may range from about 1 μg / kg to up to 100 mg / kg body weight or more per day, depending on the above factors. Preferably, following antibody administration for treating HCC, the efficacy of the therapeutic antibody can be evaluated in a variety of ways well known to the skilled practitioner. For example, standard tumor imaging techniques may be used to monitor the size, number, and / or distribution of tumors in the subject being treated. A therapeutically administered antibody that halts tumor growth, causes tumor shrinkage, and / or prevents the development of new tumors, compared to the disease progression that occurs in the absence of antibody administration, is an effective antibody for cancer treatment.

[0425] The peptides mentioned in the above table of the invention, and thus their underlying polypeptides, are highly expressed in HCC and are expressed at considerably low to extremely low levels in normal cells. Therefore, inhibition of the proteins selected from the group consisting of the protein products of the following genes, and the expression or activity of these markers may preferably be incorporated, for example, into treatment strategies for treating or preventing HCC. For inhibition, and still preferred for antibodies and / or TCRs thereto, GLUL, GPAM, PLIN2, SLC16A1, SLC9A3R1, PCBD1, SEC16A, AKR1C4, ABCB11, HAL, CYP2E1, C4A, C4B, ALDH1L1, CRP, ACSL4, EEF2, HLTF, FBXO22, GALK1, TMCO1, TMEM33, ZNF318, IPO9, AMACR, C1QTNF3, CYP4F8, CYP4F3, CYP4F11, CYP4F12, CYP4F2, MOCOS, A1CF, COL18A1, HPR, LBP, C19orf80, CFHR5, ITIH4, TMEM110, LARP4, LMF2, SLC10A5, and SLC16A11; still preferred for inhibition, and for antibodies and / or TCRs thereto, ANKFY1, C12orf44, C16orf58, CPSF1, DCAF8, PEX19, DDX11, DDX12P, DECR2, NME4, DENND5B, DYM, EDC4, ERI3, FAM20A, FNDC3A, GPR107, GYG2, HEATR2, IFT81, KCTD3, SHKBP1, KIAA1324L, KLHL24, MARCH6, MBTPS2, MIR1279, CPSF6, NOC4L, NXF1, PANK2, PCNXL3, PIPSL, PSMD4, PSMD14, SLC35B1, TCP11L2, THNSL2, THOC2, TOMM5, TRAPPC6B, TRIM54, TRIM55, TRIM63, UGGT2, URB1, VPS54, WIZ, ZNF451, RFTN2, SCFD1, SERINC5, CCT7P2, CMAS, ANKS1A, C17orf70, CCT7, CDK5RAP2, CLPTM1; most preferred for inhibition, and for antibodies and / or TCRs thereto, APOB, FASN, and / or COPA.

[0426] The principle of antisense therapy is based on the hypothesis that sequence-specific inhibition of gene expression (via transcription or translation) may be achieved by intracellular hybridization between genomic DNA or mRNA and a complementary antisense species. The formation of such hybrid nucleic acid duplexes interferes with the transcription of the genomic DNA encoding the target tumor antigen, or the processing / transport / translation and / or stability of the target tumor antigen mRNA.

[0427] Antisense nucleic acids can be delivered by a variety of approaches. For example, antisense oligonucleotides or antisense RNA can be administered directly to a subject (e.g., by intravenous injection) in a form that enables uptake by tumor cells. Alternatively, a viral or plasmid vector encoding antisense RNA (or an RNA fragment) can be introduced into cells in vivo. The antisense effect can also be induced by sense sequences; however, the degree of phenotypic change is highly variable. Phenotypic changes induced by effective antisense therapy are evaluated, for example, by changes in target mRNA levels, target protein levels, and / or target protein activity levels.

[0428] In a specific example, targeting / inhibition of the marker function of HCC by antisense gene therapy may be achieved by direct administration of antisense tumor marker RNA to a subject. Antisense tumor marker RNA may be produced and isolated by any standard technique, but is most easily produced by in vitro transcription using an antisense tumor marker cDNA under the control of a highly efficient promoter (e.g., the T7 promoter). Administration of antisense tumor marker RNA to cells can be carried out by any of the direct nucleic acid administration methods described below.

[0429] Alternative strategies for inhibiting the function of a protein selected from the group consisting of the above proteins, most preferably APOB, FASN, and / or COPA, involve the use of nucleic acids (e.g., siRNA, or a nucleic acid encoding an anti-protein antibody or a portion thereof that can be introduced into cancer cells or other cells to effect intracellular antibody expression and secretion), proteins or small molecules, or any other compound that targets the expression, translation, and / or biological function of this protein.

[0430] In the methods described above, which include the administration and uptake of exogenous DNA into target cells (i.e., gene conversion or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be within a vector that delivers the nucleic acids to the cells in order to inhibit HCC marker protein expression. The vector can be a commercially available preparation such as an adenoviral vector (e.g., from Quantum Biotechnologies, Inc., Laval, Quebec, Canada). Delivery of the nucleic acid or vector to the cells can occur via a variety of mechanisms. As one example, delivery can occur via liposomes, and commercially available liposome preparations such as Lipofectin, Lipofectamine (GIBCO-25 BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc., Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis., US) as well as other liposomes developed according to standard procedures in the art are used. Additionally, the nucleic acids or vectors of the present invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc., San Diego, US., as well as by means of a Sonoporation device (ImaRx Pharmaceutical Corp., Tucson, Arizona, US). As one example, vector delivery can occur via a viral system such as a retroviral vector system that can package a recombinant retroviral genome. The recombinant retrovirus is then used to infect the cells, whereby an antisense nucleic acid that inhibits the expression of a protein selected from the group consisting of the proteins described above is delivered to the infected cells. The exact method of introducing the modified nucleic acids into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques, including the use of adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, pseudotyped retroviral vectors, are generally available for this procedure. Physical transfection techniques such as liposome delivery and receptor-mediated and other endocytosis mechanisms can also be used. The present invention can be used in combination with any of these or other commonly used gene transfer methods.

[0431] Antibodies may also be used for in vivo diagnostic assays. Typically, antibodies are radiolabeled with radioactive nucleotides ( 111 In, 99 Tc, 14 C, 131 I, 3 H, 32 P or 35 S, etc.) so that tumors can be located using immunoscintiography. In one embodiment, the antibody or fragments thereof bind to the extracellular domains of two or more targets of a protein selected from the group consisting of the proteins described above, and the affinity (Kd) is less than 1×10 μM.

[0432] Diagnostic antibodies may be labeled with probes suitable for detection by various imaging methods. Detection methods for probes include, but are not limited to, fluorescence, optical, confocal, and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin, and other fluorophores, radioisotopes, gold, gadolinium, and other lanthanides, paramagnetic iron, fluorine-18, and other positron-emitting radionuclides. Further, the probe may be bifunctional or polyfunctional and detectable by one or more of the listed methods. These antibodies may be labeled directly or indirectly with the probe. Attachment of the probe to the antibody, which is well approved in the technical field, includes covalent bonding of the probe, incorporation of the probe into the antibody, and covalent bonding of a chelate compound for probe binding. In immunohistochemical examinations, disease tissue samples may be fresh or frozen, or paraffin-embedded and fixed with a preservative such as formalin. Fixed or embedded sections containing the sample are contacted with labeled primary and secondary antibodies, and in situ protein expression is detected using the antibodies.

[0433] Therefore, as described above, the present invention provides a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300, or a variant thereof that is 90% homologous to SEQ ID NO: 1 to SEQ ID NO: 300, or a variant thereof that induces a cross-reaction between a T cell and the peptide. The peptide of the present invention has the ability to bind to a human major histocompatibility complex (MHC) class I molecule or an extended version of the peptide to class II.

[0434] In the present invention, the term "homologous" refers to the degree of identity (see the percent identity above) between the sequences of two amino acid sequences, i.e., peptide or polypeptide sequences. The aforementioned "homology" is determined by comparing two sequences aligned under optimal conditions over the sequences to be compared. Such sequence homology can be calculated, for example, by creating an alignment using the ClustalW algorithm. Commonly available sequence analysis software, more specifically, Vector NTI, GENETYX or other analysis tools are provided by public databases.

[0435] One skilled in the art will be able to evaluate whether T cells induced by a variant of a particular peptide can cross-react with the peptide itself (Fong L, et al. Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc Natl Acad Sci USA. 2001 Jul 17;98(15):8809-14; Zaremba S, et al. Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. Cancer Res. 1997 Oct 15;57(20):4570-7; Colombetti S, et al. Impact of orthologous melan-A peptide immunizations on the anti-self melan-A / HLA-A2 T cell cross-reactivity. J Immunol. 2006 Jun 1;176(11):6560-7; Appay V, et al. Decreased specific CD8+ T cell cross-reactivity of antigen recognition following vaccination with Melan-A peptide. Eur J Immunol. 2006 Jul;36(7):1805-14).

[0436] By "variant" of a given amino acid sequence, we mean that the peptide can still bind to HLA molecules, substantially in the same way as a peptide consisting of a given amino acid sequence consisting of SEQ ID NOs: 1 to 300, for example, by substituting them with side chains of another natural amino acid residue or other side chains, such that the side chains of one or two residues of the amino acid are changed. For example, the peptide is such that it is HLA-A *It may be modified to at least maintain, without improving, the ability to bind to the binding groove of a suitable MHC molecule such as MHC class II or -DR, and in so doing it at least maintains, without improving, the ability to bind to the TCR of an activated CTL.

[0437] These T cells can subsequently cross-react with cells and kill cells expressing a polypeptide containing the native amino acid sequence of a cognate peptide as defined in aspects of the present invention. As can be derived from the scientific literature (Godkin A, et al. Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2). Int Immunol. 1997 Jun;9(6):905-11), and the database (Rammensee H.et al. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3-4):213-9), certain positions of HLA-binding peptides typically form a core sequence that is compatible with the binding motif of the HLA receptor defined by the polar, electrophysical, hydrophobic, and steric properties of the polypeptide chains that make up the binding groove. Thus, one of ordinary skill in the art can modify the amino acid sequences set forth in SEQ ID NOs: 1 to 300 by retaining known anchor residues and determine whether such variants maintain the ability to bind to MHC class I or II molecules. The variants of the present invention maintain the ability to bind to the TCR of activated T cells, which can subsequently cross-react with cells and kill cells expressing a polypeptide containing the native amino acid sequence of a cognate peptide as defined in aspects of the present invention.

[0438] Amino acid residues that do not substantially contribute to the interaction with the T cell receptor can be modified by substitution with another amino acid such that their incorporation does not substantially affect T cell reactivity and does not preclude binding to the relevant MHC. Thus, except for the given provisos, the peptides of the invention may be any peptide (including oligopeptides or polypeptides in that term as used by the inventors) that includes an amino acid sequence as given, or portions or variants thereof.

[0439] Amino acid residues that do not substantially contribute to the interaction with the T cell receptor can be modified by substitution with another amino acid such that their incorporation does not substantially affect T cell reactivity and does not preclude binding to the relevant MHC. Thus, except for the given provisos, the peptides of the invention may be any peptide (including oligopeptides or polypeptides in that term as used by the inventors) that includes an amino acid sequence as given, or portions or variants thereof.

[0440] Table 8A: Variants and motifs of the peptides set forth in SEQ ID NOs: 1, 117, and 246:

Table 8A-1

Table 8A-2

Table 8A-3

[0441] Longer peptides may also be appropriate. MHC class I epitopes are typically 8 - 11 amino acids in length, but can also be generated by peptide processing from longer peptides or proteins that contain the actual epitope. Residues located flanking the actual epitope are preferably residues that do not substantially affect the proteolytic cleavage necessary to expose the actual epitope during processing.

[0442] Accordingly, the present invention provides peptides and variants of MHC class I epitopes, the peptides or variants having a full length of 8 to 100, preferably 8 to 30, most preferably 8 to 14, i.e., 8, 9, 10, 11, 12, 13, 14 amino acids, and in the case of extended class II binding peptides, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

[0443] Of course, the peptides or variants according to the present invention have the ability to bind to molecules of human major histocompatibility complex (MHC) class I or II. The binding of the peptides or variants to the MHC complex may be tested by methods known in the art.

[0444] In a particularly preferred embodiment of the present invention, the peptide consists of, or consists essentially of, the amino acid sequences set forth in SEQ ID NOs: 1 to 300.

[0445] "Consists essentially of" means that the peptide according to the present invention does not necessarily constitute a part of a peptide that functions as an epitope of the MHC molecular epitope in addition to the sequence set forth in any of SEQ ID NOs: 1 to 300 or a variant thereof, and contains a sequence of amino acids located at the additional N- and / or C-terminals.

[0446] Still, these sequences may be important in providing efficient introduction of the peptides according to the present invention into cells. In one embodiment of the present invention, the peptide is, for example, a part of a fusion protein (p33, hereinafter "Ii") comprising 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain, derived from, for example, NCBI, GenBank accession number X00497. In other fusions, the peptides of the present invention may be fused to an antibody as described herein, or a functional part thereof, particularly within the sequence of the antibody, so as to be specifically targeted by said antibody, or may be fused to, for example, an antibody specific for dendritic cells as described herein, or within it.

[0447] Furthermore, the peptide or variant may be further modified to improve stability and / or binding to MHC molecules in order to elicit a stronger immune response. Such methods of optimizing peptide sequences are well known in the art and include, for example, the introduction of retro-inverse peptide bonds or non-peptide bonds.

[0448] In retro-inverse peptide bonds, the amino acid residues are not linked by peptide (-CO-NH-) bonds and the peptide bond is reversed. Such retro-inverso peptide mimetics may be produced using methods known in the art, such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237, which is incorporated herein by reference. This approach involves the generation of peptidomimetics that contain changes involving the backbone rather than the direction of the side chains. Meziere et al (1997) show that these peptidomimetics are useful for MHC binding and T helper cell responses. Retro-inverse peptides containing an NH-CO bond instead of a CO-NH peptide bond are much more resistant to proteolysis.

[0449] Non-peptide bonds are, for example, -CH2-NH, -CH2S-, -CH2CH2-, -CH=CH-, -COCH2-, -CH(OH)CH2-, and -CH2SO-. U.S. Patent No. 4,897,445 provides a method for solid-phase synthesis of non-peptide bonds (-CH2-NH) in a polypeptide chain, involving polypeptides synthesized by standard procedures and non-peptide bonds synthesized by reacting aminoaldehydes with amino acids in the presence of NaCNBH3.

[0450] Peptides comprising the above arrays may be synthesized with additional chemical groups present at their amino and / or carboxy termini to improve peptide stability, bioavailability, and / or affinity. For example, hydrophobic groups such as a carbobenzoxyl, dansyl, or t-butyloxycarbonyl group may be added to the amino terminus of the peptide. Similarly, an acetyl group or a 9-fluorenylmethoxycarbonyl group may be placed at the amino terminus of the peptide. Further, a hydrophobic group, t-butyloxycarbonyl, or amide group may be added to the carboxy terminus of the peptide.

[0451] Furthermore, the peptides of the present invention may be synthesized to modify their configurations. For example, instead of the normal L-isomers, one or more D-isomers of the amino acid residues of the peptide may be used. Still further, at least one of the amino acid residues of the peptides of the present invention may be replaced with one of the well-known non-naturally occurring amino acid residues. Such changes may serve to increase the stability, bioavailability, and / or binding action of the peptides of the present invention.

[0452] Similarly, the peptides or variants of the present invention may be chemically modified by reacting specific amino acids either before or after peptide synthesis. Examples of such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2005, which is incorporated herein by reference. Chemical modifications of amino acids include, although without limitation thereto, acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification and sulfhydryl modification of carboxyl groups by performic acid oxidation of cysteine to cysteic acid, mercury derivative formation, mixed disulfide formation with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and modification by carbamoylation with cyanate at alkaline pH, but is not limited to these. In this regard, those skilled in the art are referred to Chapter 15 of Current Protocols In Protein Science, Eds. Coligan et al. (John Wiley and Sons NY 1995 - 2000) for more detailed procedures regarding chemical modification of proteins.

[0453] Briefly stated, for example, the modification of arginyl residues in proteins is often based on the reaction of adjacent dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form adducts. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without simultaneous modification of other nucleophilic sites such as lysine and histidine. As a result, a number of reagents are available for cysteine modification. Websites of companies such as Sigma-Aldrich (http: / / www.sigma-aldrich.com) provide information on specific reagents.

[0454] The selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Specific glutamic acid residues may be modified using Woodward's reagent K. Intramolecular cross-links can be formed between lysine and glutamic acid residues using N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide. For example, diethyl pyrocarbonate is a reagent for the modification of histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. Reactants for lysine residues and other α-amino groups are useful, for example, for the attachment of peptides to the surface or for protein / peptide cross-linking. Lysine is an attachment site for poly(ethylene) glycol and a major modification site for protein glycosylation. Methionine residues in proteins can be modified, for example, by iodoacetamide, bromoethylamine, and chloramine T.

[0455] Tyrosyl residues can be modified using tetranitromethane and N-acetylimidazole. Cross-linking through the formation of dityrosine can be achieved by hydrogen peroxide / copper ions.

[0456] In recent studies on the modification of tryptophan, N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skator) have been used.

[0457] The successful modification of therapeutic proteins and peptides by PEG is often associated with an extended circulation half-life, while the cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used for hydrogel preparation. The chemical modification of allergens for immunotherapy is often achieved by carbamylation using potassium cyanate.

[0458] Peptides or variants that are modified or contain non-peptide bonds are preferred embodiments of the present invention. Generally, peptides and variants (those containing peptide bonds at least between amino acid residues) may be synthesized by Fmoc polyamide-type solid-phase peptide synthesis as disclosed in Lukas et al. (Solid-phase peptide synthesis under continuous-flow conditions. Proc Natl Acad Sci U S A. May 1981;78(5):2791-2795) and the references cited therein. Temporary N-amino group protection is provided by 9-fluorenylmethyloxycarbonyl (Fmoc) groups. Repeated cleavage of this highly base-labile protecting group is carried out using 20% piperidine in N,N-dimethylformamide. Side-chain functional groups may be protected as their butyl ethers (in the case of serine, threonine, and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), 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). When glutamine or asparagine is the C-terminal residue, a 4,4'-dimethoxybenzhydryl group is utilized to protect the side-chain amide functional group. The solid-phase support is based on a polydimethylacrylamide polymer composed of three monomers: dimethylacrylamide (main-chain monomer), bisacryloylethylenediamine (cross-linking agent), and acryloylsarcosine methyl ester (functionalizing agent). The cleavable binder between the peptide and the resin used is an acid-labile 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives except asparagine and glutamine, which are added using a reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated conjugation procedure, are added as their preformed symmetric anhydride derivatives. All conjugation and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isatin test procedures.Upon completion of synthesis, the peptide is cleaved from the resin support and the side-chain protecting groups are simultaneously removed by treatment with 95% trifluoroacetic acid containing 50% scavenger mixture. Commonly used scavengers include ethanedithiol, phenol, anisole, and water, and the exact choice depends on the constituent amino acids of the peptide being synthesized. Combinations of solid-phase and solution-phase methods for peptide synthesis are also possible (see, for example, Bruckdorfer et al., 2004 and references cited therein).

[0459] Trifluoroacetic acid is removed by vacuum evaporation, and subsequent trituration with diethyl ether yields the crude peptide. Any scavenger present is removed by a simple extraction procedure, which gives a scavenger-free crude peptide upon lyophilization of the aqueous phase. Reagents for peptide synthesis are usually available, for example, from Calbiochem-Novabiochem (Nottingham, UK).

[0460] Purification may be carried out by any one or combination of techniques such as recrystallization, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, and, usually, reverse-phase high-performance liquid chromatography using, for example, an acetonitrile / water gradient separation.

[0461] Analysis of the peptide may be carried out 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.

[0462] A further aspect of the present invention provides a nucleic acid (e.g., polynucleotide) encoding a peptide or peptide variant of the present invention. The polynucleotide may be, for example, either single-stranded and / or double-stranded DNA, cDNA, PNA, RNA, or a combination thereof, as long as it encodes a peptide, or may be an unmodified or stabilized form of a polynucleotide, such as a polynucleotide having a phosphorothioate backbone, and it may or may not contain introns. Of course, only peptides containing natural amino acid residues linked by natural peptide bonds can be encoded by a polynucleotide. Yet a further aspect of the present invention provides an expression vector capable of expressing a polypeptide according to the present invention.

[0463] For example, through complementary attachment ends, various methods have been developed for ligating a polynucleotide, particularly DNA, to a vector. For example, a complementary homopolymer sequence can be added to a DNA fragment to be inserted into vector DNA. Next, the vector and the DNA fragment are ligated by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0464] Synthetic linkers containing one or more restriction enzyme recognition sites provide an alternative method for ligating a DNA fragment to a vector. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from several suppliers, including International Biotechnologies Inc., New Haven, CN, USA.

[0465] A desirable method for modifying the DNA encoding the polypeptide of the present invention uses the polymerase chain reaction as disclosed by Saiki RK, et al. (Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes. N Engl J Med. 1988 Sep 1;319(9):537-41). This method may be used, for example, to introduce the DNA into an appropriate vector by modifying appropriate restriction enzyme recognition sites, or it may be used to modify the DNA in other useful manners known in the art. If a viral vector is used, a poxvirus or adenovirus vector is preferred.

[0466] Next, the DNA (or RNA in the case of a retroviral vector) may be expressed in an appropriate host to produce a polypeptide comprising the peptide or variant of the present invention. In this way, an expression vector may be constructed using the DNA encoding the peptide or variant of the present invention, according to known techniques appropriately modified in view of the teachings contained herein, and then it may be used to transform an appropriate host cell for the expression and production of the polypeptide of the present invention. Such techniques include, for example, those disclosed in U.S. Patent No. 4,440,859, U.S. Patent No. 4,530,901, U.S. Patent No. 4,582,800, U.S. Patent No. 4,677,063, U.S. Patent No. 4,678,751, U.S. Patent No. 4,704,362, U.S. Patent No. 4,710,463, U.S. Patent No. 4,757,006, U.S. Patent No. 4,766,075, and U.S. Patent No. 4,810,648.

[0467] The DNA (or RNA in the case of a retroviral vector) encoding the polypeptide constituting the compound of the present invention may be ligated to a wide variety of other DNA sequences for introduction into a suitable host. The companion DNA depends on the nature of the host, the mode of introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

[0468] Generally, the DNA is inserted into an expression vector such as a plasmid in the appropriate orientation and correct reading frame for expression. If necessary, the DNA may be ligated to appropriate transcriptional and translational control regulatory nucleotide sequences recognized by the desired host, but such regulation is generally available within the expression vector. Next, the vector is introduced into the host through standard techniques. Generally, not all of the host is transformed by the vector. Therefore, it is necessary to select the transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence having any necessary control elements encoding a selectable trait within the transformed cells, such as antibiotic resistance.

[0469] Alternatively, the gene for such a selectable trait may be on another vector used to co-transform the desired host cells.

[0470] Next, considering the teachings disclosed herein, the host cells transformed with the recombinant DNA of the present invention can be cultured for a sufficient time under appropriate conditions known to those skilled in the art to allow expression of the polypeptide, and then it can be recovered.

[0471] A number of expression systems are known, including bacteria (e.g., Escherichia coli (E. coli) and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. Preferably, the system can be mammalian cells such as CHO cells available from the ATCC Cell Biology Collection.

[0472] Typical mammalian cell vector plasmids for constitutive expression comprise a CMV or SV40 promoter with an appropriate polyA tail and a resistance marker such as neomycin. One example is pSVL available from Pharmacia, Piscataway, NJ, USA. pMSG, an example of an inducible mammalian expression vector, is also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, usually available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405, and pRS406 are yeast integrating plasmids (YIps) with the yeast selectable markers HIS3, TRP1, LEU2, and URA3 integrated. Plasmids pRS413-416 are yeast centromere plasmids (Ycps). CMV promoter-based vectors (e.g., from Sigma-Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, and N-terminal or C-terminal labeling in various combinations of FRAG, 3xFLAG, c-myc, or MAT. These fusion proteins enable the detection, purification, and analysis of recombinant proteins. Double-label fusions provide flexibility in detection.

[0473] A strong human cytomegalovirus (CMV) promoter regulatory region raises the constitutive protein expression level to about 1 mg / L in COS cells. In cell lines with lower efficacy, the protein level is typically about 0.1 mg / L. The presence of the SV40 origin of replication results in high-level DNA replication in SV40 replication-permissive COS cells. For example, a CMV vector can contain the pMB1 (a derivative of pBR322) origin of replication in bacterial cells, the β-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and the f1 origin. Vectors containing a preprotrypsin leader (PPT) sequence can induce the secretion of a FRAG fusion protein into the culture medium for purification using anti-FRAG antibodies, resins, and plates. Other vectors and expression systems for use in a variety of host cells are well known in the art.

[0474] In another embodiment, two or more peptides or peptide variants of the invention are encoded and thus sequentially expressed (similar to a "bead-on-a-string" construct). In this case, the peptides or peptide variants may be linked or fused together, for example, by a stretch of linker amino acids such as LLLLLL, or may be linked without any additional peptides between them. These constructs can also be used for cancer treatment and may induce an immune response involving both MHC I and MHC II.

[0475] The present invention also relates to a host cell transformed with the polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some situations and are typically, for example, the E. coli DH5 strain available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and E. coli strains such as RR1 (ATCC number 31343) available from the American Type Culture Collection (ATCC), Rockville, MD, USA. Preferred eukaryotic host cells include yeast, insect, and mammalian cells, preferably vertebrate cells such as those derived from mouse, rat, monkey, or human fibroblast and colon cell lines. Examples of yeast host cells include YPH499, YPH500, and YPH501, which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available as CCL61 from the ATCC, NIH Swiss mouse embryo cells NIH / 3T3 available as CRL1658 from the ATCC, simian kidney-derived COS-1 cells available as CRL1650 from the ATCC, and 293 cells, which are human fetal-derived kidney cells. Preferred insect cells are Sf9 cells that can be transfected with a baculovirus expression vector. An overview of the selection of appropriate host cells for expression can be found, for example, in the textbook Paulina Balbas and Argelia Lorence”Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,”Part One,Second Edition,ISBN 978-1-58829-262-9, and other literature known to those skilled in the art.

[0476] Transformation of a suitable cell host with the DNA construct of the present invention is achieved by well-known methods that depend on the type of vector typically used. For transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110, and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. For vertebrate cells, reagents such as calcium phosphate and DEAE-dextran or liposome formulations, which are useful for transfecting such cells, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation is also useful for transforming and / or transfecting cells and is well known in the art of transforming yeast cells, bacterial cells, insect cells, and vertebrate cells.

[0477] Cells that have been successfully transformed, i.e., cells containing the DNA construct of the present invention, can be identified by well-known techniques such as PCR. Alternatively, the presence of the supernatant protein can be detected using an antibody.

[0478] For example, certain host cells of the present invention such as bacteria, yeast, and insect cells will be understood to be useful in the preparation of the peptides of the present invention. However, other host cells may be useful in certain therapeutic methods. For example, antigen-presenting cells such as dendritic cells may be usefully employed to express the peptides of the present invention so that they may be loaded within appropriate MHC molecules. Accordingly, the present invention provides a host cell comprising a nucleic acid or expression vector according to the present invention.

[0479] In a preferred embodiment, the host cell is an antigen-presenting cell, particularly a dendritic cell or an antigen-presenting cell. APCs loaded with a recombinant fusion protein containing prostate acid phosphatase (PAP) were approved by the US Food and Drug Administration (FDA) on April 20, 2010, for the treatment of asymptomatic or minimally symptomatic metastatic HRPC (Sipuleucel-T) (Small EJ, et al. Placebo-controlled phase III trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. J Clin Oncol. 2006 Jul 1;24(19):3089-94. Rini et al. Combination immunotherapy with prostatic acid phosphatase pulsed antigen-presenting cells (provenge) plus bevacizumab in patients with serologic progression of prostate cancer after definitive local therapy. Cancer. 2006 Jul 1;107(1):67-74).

[0480] A further aspect of the present invention comprises culturing a host cell and isolating a peptide from the host cell or its culture broth. A method for producing a peptide or a variant thereof is provided.

[0481] In another embodiment, the peptide, nucleic acid or expression vector of the present invention is used in medicine. For example, the peptide or a variant thereof may be formulated for intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. Preferred methods of DNA injection include i.d., i.m., s.c., i.p., and i.v. For example, a dose of 50 μg to 1.5 mg, preferably 125 μg to 500 μg of peptide or DNA may be administered, depending on each peptide or DNA. Doses in this range have been successfully used in previous clinical trials (Walter et al Nature Medicine 18, 1254-1261 (2012)).

[0482] Another aspect of the present invention comprises a step of contacting in vitro T cells with antigen-loaded human MHC molecules expressed on the surface of appropriate antigen-presenting cells for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide according to the present invention. Preferably, a sufficient amount of antigen is used together with the antigen-presenting cells.

[0483] Preferably, the mammalian cells are devoid of or have reduced levels or function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter involved in antigen processing.

[0484] The human peptide-deficient cell line T2 can be obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, under catalog number CRL1992; the Drosophila cell line Schneider line 2 can be obtained from the ATCC under catalog number CRL19863; the mouse RMA-S cell line is described by Karre et al. (Ljunggren, H.-G., and K. Karre. 1985. J. Exp. Med. 162:1745).

[0485] Preferably, prior to transfer, the host cell does not substantially express MHC class I molecules. It is also preferred that the stimulator cells express important molecules in providing co-stimulatory signals for T cells, such as any of B7.1, B7.2, ICAM-1, and LFA3. Nucleic acid sequences of a number of MHC class I molecules and co-stimulatory factor molecules are publicly available from the GenBank and EMBL databases.

[0486] When an MHC class I epitope is used as an antigen, the T cells are CD8-positive T cells.

[0487] When antigen-presenting cells are transfected to express such epitopes, preferably the cells comprise an expression vector capable of expressing a peptide containing the amino acid sequences of SEQ ID NOs: 1 to 300, or variant amino acid sequences thereof.

[0488] For the in vitro production of T cells, several other methods may be used. For example, autologous tumor-infiltrating lymphocytes can be used to produce CTLs. Plebanski et al. (Induction of peptide-specific primary cytotoxic T lymphocyte responses from human peripheral blood. Eur J Immunol. 1995 Jun;25(6):1783-7) utilize autologous peripheral blood lymphocytes (PLB) in the preparation of T cells. Additionally, it is possible to produce autologous T cells by pulsing dendritic cells with peptides or polypeptides, or infecting them with recombinant viruses. B cells can also be used in the production of autologous T cells. Furthermore, macrophages pulsed with peptides or polypeptides, or infected with recombinant viruses, may be used in the preparation of autologous CTLs. S. Walter et al. 2003 (Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC / anti-CD28-coated microspheres. J Immunol. 2003 Nov 15;171(10):4974-8) describe the in vitro priming of T cells using artificial antigen-presenting cells (aAPCs), which is also a suitable method for producing T cells specific for selected peptides. In the present invention, aAPCs were produced by conjugating pre-formed MHC:peptide complexes to surface polystyrene particles (microbeads) by biotin:streptavidin biochemistry. This system allows for precise regulation of MHC density on the aAPC, which enables selective induction of high or low avidity antigen-specific T cell responses with high efficiency from blood samples. In addition to the MHC:peptide complex, aAPCs should carry other proteins with co-stimulatory activity, such as anti-CD28 antibodies, conjugated to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, such as cytokine-like interleukin 12.

[0489] In the preparation of T cells, allogeneic cells may also be used, and the method is detailed in WO 97 / 26328, which is incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used, and antigens such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells may be presented. Furthermore, plant viruses may be used (see, for example, Porta et al. (1994) Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides. Virology. 1994 Aug 1;202(2):949-55), which describes the development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides).

[0490] The activated T cells directed against the peptides of the present invention are useful in therapy. Accordingly, a further aspect of the present invention provides activated T cells obtainable by the method of the present invention described above.

[0491] The activated T cells produced by the above method selectively recognize cells that abnormally express a polypeptide comprising the amino acid sequence of SEQ ID NOs: 1 to 300.

[0492] Preferably, T cells recognize cells by interacting (e.g., binding) with HLA / peptide complexes via their TCRs. T cells are useful in methods of killing target cells in a patient whose target cells abnormally express a polypeptide comprising the amino acid sequence of the present invention, and an effective number of activated T cells are administered to the patient. 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). As an alternative, the T cells are derived from a person other than the patient. Of course, it is preferred if the person is a healthy individual. By "healthy individual", the inventors mean that the individual is generally in good health, preferably has a competent immune system, and more preferably does not suffer from any disease that can be easily examined and detected.

[0493] In vivo, the target cells of the CD8-positive T cells according to the present invention can be tumor cells (which sometimes express MHC class II) and / or stromal cells around the tumor (tumor cells) (which also sometimes express MHC class II; (Dengjel et al., 2006)).

[0494] The T cells of the present invention may be used as an active ingredient of a therapeutic composition. Accordingly, the present invention also provides a method of killing target cells that abnormally express a polypeptide comprising the amino acid sequence of the present invention in a patient, the method comprising the step of administering to the patient an effective number of T cells as defined above.

[0495] By "abnormal expression", the inventors also mean that the polypeptide is overexpressed compared to normal expression levels, or that the gene is silent in the tissue from which the tumor is derived but is expressed in the tumor. By "overexpression", the inventors mean that the polypeptide is present at a level of at least 1.2 times the level present in normal tissue; preferably at least 2 times the level present in normal tissue, more preferably at least 5 times or 10 times the level.

[0496] T cells may be obtained by methods known in the art, such as those described above.

[0497] The protocol for this so-called adoptive immunotransfer of T cells is well known in the art. A review is provided in Gattinoni L, et al. Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol. 2006 May;6(5):383-93. Review. and Morgan RA, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006 Oct 6;314(5796):126-9).

[0498] Any molecule of the invention, i.e., a peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor or nucleic acid encoding the same, is useful for the treatment of disorders characterized by cells that have escaped the immune response. Accordingly, any molecule of the invention may be used as a medicament or in the manufacture of a medicament. The molecule may be used alone or in combination with other molecules of the invention or known molecules.

[0499] Preferably, the agent of the present invention is a vaccine. It is administered directly to the patient, to the affected organ, or systemically, by i.d., i.m., s.c., i.p., and i.v., or applied in vitro to cells derived from the patient or a human cell line, which is subsequently administered to the patient, or used in vitro to select a subpopulation of immune cells derived from the patient, which is then readministered to the patient. When the nucleic acid is administered to cells in vitro, it may be useful to transfect the cells to co-express an immune-stimulating cytokine such as interleukin 2. The peptide may be substantially pure, or combined with an immune-stimulating adjuvant (see below), or used in combination with an immunopotentiating cytokine, or administered by a suitable delivery system such as, for example, liposomes. The peptide may also be conjugated to a suitable carrier such as keyhole limpet hemocyanin (KLH) or mannan (see, for example, WO 95 / 18145). The peptide may also be labeled, may be a fusion protein, or may be a hybrid molecule. The peptide whose sequence is described in the present invention is predicted to stimulate CD4 or CD8 T cells. However, the stimulation of CD8 T cells is more efficient in the presence of assistance provided by CD4 T helper cells. Thus, for MHC class I epitopes that stimulate CD8 T cells, the fusion partner or section of the hybrid molecule preferably provides an epitope that stimulates CD4-positive T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those identified in the present invention.

[0500] In one aspect, the vaccine comprises at least one peptide having the amino acid sequence set forth in SEQ ID NOs: 1 to 300 and at least one additional peptide, preferably 2 to 50, more preferably 2 to 25, even more preferably 2 to 20, and most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. The peptide may be derived from one or more specific TAAs and may bind to MHC class I molecules.

[0501] In another aspect, the vaccine comprises at least one peptide having the amino acid sequences set forth in SEQ ID NOs: 1 to 300, and at least one additional peptide, preferably from 2 to 50, more preferably from 2 to 25, even more preferably from 2 to 20, and most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. The peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

[0502] The polynucleotide is substantially pure or may be contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods for designing and introducing such nucleic acids are well known in the art. An overview is provided, for example, by (Pascolo et al., Human peripheral blood mononuclear cells transfected with messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes in vitro. Cell Mol Life Sci. 2005 Aug;62(15):1755-62). Polynucleotide vaccines are easy to prepare, but the mechanism of action of these vectors in inducing an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and / or RNA such as adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus, or systems based on hybrids containing two or more viral elements. Non-viral delivery systems include cationic lipids and cationic polymers, which are well known in the field of DNA delivery technology. Physical delivery through a "gene gun" or the like may also be used. The peptide or group of peptides encoded by the nucleic acid may, for example, as described above, be a fusion protein with an epitope that stimulates T cells of the respective reverse CDR.

[0503] The agent of the present invention may also contain one or more adjuvants. An adjuvant is a substance that nonspecifically promotes or enhances an immune response (e.g., an immune response against an antigen mediated by CD8-positive T cells and helper T (TH) cells), and is thus considered useful in the agent of the present invention. Suitable adjuvants include 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or a TLR5 ligand derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), resiquimod, ImuFact IMP321, interleukins such as IL-2, IL-13, and IL-21, interferon α or β or their pegylated derivatives, IS patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactide-co-glycolide) [PLG] based and dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila’s QS21 stimulon derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimetics, and other licensed adjuvants such as Ribi’s Detox or Quil or Superfos, but are not limited thereto. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparations have been previously described (Allison and Krummel, 1995 The Yin and Yang of T cell costimulation. Science. 1995 Nov 10;270(5238):932-3). Cytokines may also be used. Several cytokines have been directly associated with influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, which is specifically incorporated herein by reference in its entirety), and acting as immunopotentiators (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich, 1996 Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells Nat Med. 1996 Oct;2(10):1096-103).

[0504] CpG immunostimulatory oligonucleotides have also been reported to enhance the adjuvant effect in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activation of the innate (non-adaptive) immune system through Toll-like receptors (TLRs), mainly TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it promotes the maturation and differentiation of dendritic cells and results in the promotion of TH1 cell activation and potent cytotoxic T lymphocyte (CTL) generation even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a TH2 bias. CpG oligonucleotides exhibit even higher adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which is particularly necessary to induce a strong response when the antigen is relatively weak. They also accelerate the immune response and in some experiments allow a nearly two-fold reduction in antigen dose with an antibody response equivalent to the total vaccine dose without CpG (Krieg, 2006). U.S. Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens for inducing an antigen-specific immune response. A CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical compositions of the invention. Other TLR-binding molecules such as RNA-binding TLR7, TLR8, and / or TLR9 may also be used.

[0505] Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera); dsRNA analogs such as poly(I:C) and their derivatives (e.g., AmpliGen®, Hiltonol®, poly(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA; as well as cyclophosphamide, sunitinib, bevacizumab®, celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, immune-active small molecules and antibodies such as anti-CTLA4; other antibodies that target important structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor); SC58175, but are not limited thereto, and these may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by those skilled in the art without undue experimentation.

[0506] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon α, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, and PLG or virosome microparticle formulations.

[0507] In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon α.

[0508] In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly ICLC (Hiltonol®) and anti-CD40 mAB or combinations thereof.

[0509] This composition is used for parenteral administration such as subcutaneous, intradermal, intramuscular, or oral administration. For this purpose, the peptide and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. Additionally, the composition may contain excipients such as buffers, binders, bulking agents, diluents, flavors, lubricants, etc. The peptide can also be administered together with immunostimulatory substances such as cytokines. A detailed list of excipients that can be used in such compositions can be obtained, for example, from A. Kibbe, Handbook of Pharmaceutical Excipients, 3rd Ed., 2000, American Pharmaceutical Association and pharmaceutical press. The composition can be used for the prevention, prophylaxis and / or treatment of adenomatous or cancerous diseases. Representative formulations are, for example, in European Patent No. 2112253.

[0510] The present invention provides a medicament useful for treating cancer, particularly HCC and other malignancies.

[0511] The present invention (a) a container containing the above-mentioned pharmaceutical composition in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized preparation; and (c) Optionally, (i) instructions for use of the solution, or (ii) reconstitution and / or use of the lyophilized formulation A kit comprising the same is further targeted.

[0512] The kit may further comprise one or more of (iii) buffer, (iv) diluent, (V) filtration, (vi) needle, or (V) syringe. The container is preferably a bottle, vial, syringe or test tube; it may be a multi-use container. The pharmaceutical composition is preferably lyophilized.

[0513] The kit of the present invention preferably comprises a lyophilized formulation of the present invention in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (e.g., double-chamber vials), syringes (such as double-chamber syringes), and test tubes. The container may be formed from a variety of materials such as glass or plastic. Preferably, the kit and / or container includes handling instructions on or associated with the container, which indicate instructions for reconstitution and / or use. For example, the label may indicate that the lyophilized formulation is reconstituted to a peptide concentration as described above. The label may further indicate that the formulation is useful for, or is for, subcutaneous administration.

[0514] The container containing the formulation may be a multi-use vial, which allows for repeated administration (e.g., 2 to 6 administrations) of the reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (e.g., sodium bicarbonate solution).

[0515] When mixing the diluent and the lyophilized preparation, the final peptide concentration in the reconstituted preparation is preferably at least 0.15 mg / mL / peptide (= 75 μg), and preferably 3 mg / mL / peptide (= 1500 μg) or less. The kit may further contain other substances desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and package inserts on which handling instructions are printed.

[0516] The kit of the present invention may have a single container containing the pharmaceutical composition preparation according to the present invention, with or without other components (for example, other compounds or pharmaceutical compositions of these other compounds), or may have separate containers for each component.

[0517] Preferably, the kit of the present invention contains the preparation of the present invention packaged for use in combination with the co-administration of a second compound (such as an adjuvant (e.g., GM-CSF), chemotherapeutic agent, natural product, hormone or antagonist, anti-angiogenic factor or inhibitor, apoptosis inducer or chelating agent, etc.) or a pharmaceutical composition thereof. The components of the kit may be pre-mixed, or each component may be in separate different containers prior to administration to the patient. The components of the kit may be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. Also, the components of the kit may be provided as solids, which may be converted to liquids by the addition of a suitable solvent, preferably provided in a separate different container.

[0518] The container of the therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means for enclosing a solid or liquid. Usually, when there are two or more components, the kit contains a second vial or another container to allow separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit contains a device (for example, one or more needles, syringes, eye droppers, pipettes, etc.) to enable the administration of the active substance of the present invention, which is a component of the present kit.

[0519] This formulation is suitable for peptide administration by any acceptable means such as oral (enteral), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, the administration is s.c., most preferably i.d. administration, and may also be by infusion pump.

[0520] Since the peptide of the present invention is isolated from HCC, the agent of the present invention is preferably used for treating HCC.

[0521] The present invention further includes a method for manufacturing an individualized pharmaceutical for an individual patient, comprising the step of manufacturing a pharmaceutical composition comprising at least one peptide selected from a library of pre-screening TUMAP, wherein at least one peptide used in the pharmaceutical composition is selected for suitability in an individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method may also be adapted for downstream applications such as TCR isolation, or for the production of T cell clones for soluble antibodies and other therapeutic options.

[0522] "Individualized pharmaceutical" shall mean a treatment method specifically adjusted for an individual patient, which is used only for the treatment of such individual patients, including active individualized cancer vaccines and adoptive cell therapy using autologous patient tissues.

[0523] In the usage of this specification, the term "repository" shall refer to a group of peptides that have been pre-screened for immunogenicity and / or over-presentation in a specific tumor type. The term "repository" is not intended to imply that the specific peptides included in the vaccine are pre-manufactured and stored within a physical facility, although that possibility is also considered. It is explicitly considered that the peptides may be newly manufactured for each individualized vaccine to be produced, or may be pre-manufactured and stored. The repository (e.g., in the form of a database) is composed of tumor-associated peptides that are highly overexpressed within the tumor tissue of HCC patients with various HLA-A, HLA-B, and HLA-C alleles. It may contain MHC class I and MHC class II peptides or extended MHC class I peptides. In addition to tumor-associated peptides collected from several HCC tissues, the repository may contain HLA-A * 02 and HLA-A * 24-labeled peptides. These peptides enable the comparison of the magnitude of T cell immunity induced by TUMAPS in a quantitative manner, and thus lead to important conclusions regarding the ability of the vaccine to elicit an anti-tumor response. Second, they function as important positive control peptides derived from "non-self" antigens in cases where no vaccine-induced T cell response against TUMAPs derived from "self" antigens is observed in patients. Third, they may enable conclusions to be drawn regarding the immune competence status of the patient.

[0524] TUMAPs for the repository are identified using an integrated genomic functional analysis approach that combines gene expression analysis, mass spectrometry, and T cell immunology (XPresident (registered trademark)). The approach ensures that only TUMAPs that truly exist on a high proportion of tumors but are either absent or minimally expressed in normal tissues are selected for further analysis. For the initial peptide selection, HCC samples from patients and blood from healthy donors were analyzed in a stepwise approach: 1. HLA ligands from malignancies were identified by mass spectrometry 2. Genome-scale messenger ribonucleic acid (mRNA) expression analysis was used to identify gene overexpression in malignant tissues (HCC) compared to a series of normal organs and tissues. 3. The identified HLA ligands were compared with the gene expression data. Preferably, peptides that are overpresented or selectively presented on tumor tissues and are encoded by genes that are selectively expressed or overexpressed as detected in step 2 were considered suitable TUMAP candidates for multi-peptide vaccines.

[0525] 4. A literature search was conducted to identify additional evidence to support the validity of the identified peptides as TUMAPs. 5. The relevance of overexpression at the mRNA level was confirmed by redetection on tumor tissues of selected TUMAPs from step 3 and the lack (or rare detection) of detection in healthy tissues.

[0526] 6. To evaluate whether in vivo T cell responses can be induced by the selected peptides, in vitro immunogenicity assays were performed using human T cells from healthy donors as well as HCC patients.

[0527] It is important to understand that the immune response elicited by the vaccine according to the present invention attacks cancers at different cell division stages and different developmental stages. Furthermore, different cancer-related signaling pathways are attacked. This is an advantage over vaccines that may address only one or a few targets and cause easy adaptation of the tumor to the attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Therefore, combinations of several tumor-associated peptides ensure that every tumor has at least a part of the targets. The composition is specifically designed such that each HLA-A * 02 and / or HLA-A * 24-positive tumors are expected to express some of the antigens and cover several independent pathways necessary for tumor growth and maintenance. Two HLA class I alleles (A* 02 and A * For each of the peptide subsets specific to * and * , this is independently and reliably determined based on the underlying experimental analysis. Thus, the vaccine can be readily "off-the-shelf" used for a larger patient population. This means that the pre-selection of patients to be treated with the vaccine can be limited to HLA typing and does not require any additional biomarker assessment regarding antigen expression, yet it is still certain that several targets will not be simultaneously attacked by the induced immune response, which is important for efficacy. (Banchereau et al., 2001; Walter et al., 2012).

[0528] In one aspect, the peptides are pre-screened for immunogenicity before inclusion in the library. By way of non-limiting example, the immunogenicity of the peptides included in the library is determined by a method comprising in vitro T cell priming through repeated stimulation of CD8+ T cells from healthy donors by peptide / MHC complexes and artificial antigen-presenting cells loaded with anti-CD28 antibodies.

[0529] This method is preferred for rare cancers and for patients with rare expression profiles. In contrast to a multi-peptide mixture with a fixed composition, the currently developed library allows for a significantly higher matching of the actual antigen expression in the tumor with the vaccine. In a multi-target approach, for each patient, a single or a combined number of selected "off-the-shelf" peptides are utilized. Theoretically, an approach based on the selection of 5 different antigenic peptides from a library of, for example, 50 antigenic peptides would result in approximately 17 million possible pharmaceutical (DP) compositions alone.

[0530] In one aspect, the peptides are selected for inclusion in the vaccine based on their suitability for individual patients as described herein or based on methods according to the invention as follows.

[0531] From the patient's tumor material and blood samples, HLA phenotypes, transcriptomics, and peptidomics data are collected, and the most appropriate peptides for each patient are identified that contain a "repository" and patient-specific (i.e., mutated) TUMAP. Peptides that are selectively or overexpressed in the patient tumor and, if possible, tested with the patient's individual PBMCs and show strong in vitro immunogenicity are selected.

[0532] Preferably, the peptides included in the vaccine are identified by a method comprising: (a) the step of identifying tumor-associated peptides (TUMAP) presented by tumor samples from individual patients; (b) the step of comparing the peptides identified in (a) with the above-mentioned peptide repository; and (c) the step of selecting at least one peptide from the repository (database) that is associated with the tumor-associated peptides identified in the patient. For example, TUMAP presented by tumor samples is identified by: (a1) comparing the expression data from the tumor sample with the expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or abnormally expressed in the tumor sample; and (a2) correlating the expression data with the sequences of MHC ligands that bind to MHC class I and / or class II molecules in the tumor sample to identify MHC ligands derived from proteins that are overexpressed or abnormally expressed by the tumor. Preferably, the sequence of the MHC ligand is identified by eluting the binding peptides from the MHC molecules isolated from the tumor sample and sequencing the eluted ligands. Preferably, the tumor sample and the normal tissue are obtained from the same patient.

[0533] In addition to, or as an alternative to, using a repository (database) model to select peptides, TUMAPs may be newly identified in a patient and then included in a vaccine. As one example, candidate TUMAPs may be identified in a patient by: (a1) comparing expression data from a tumor sample to expression data from a sample of normal tissue corresponding to the histotype of the tumor sample to identify proteins that are overexpressed or abnormally expressed in the tumor sample; (a2) correlating the expression data to the sequences of MHC ligands that bind to MHC class I and / or class II molecules in the tumor sample to identify MHC ligands derived from proteins that are overexpressed or abnormally expressed by the tumor. As another example, proteins containing mutations unique to a tumor sample may be identified by comparing to normal corresponding tissue from an individual patient, and TUMAPs that specifically target the mutations may be identified. For example, the genome of the tumor, and of the corresponding normal tissue, may be sequenced by whole genome sequencing. To discover non-synonymous mutations in the protein-coding regions of genes, genomic DNA and RNA are extracted from the tumor tissue, and normal non-mutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMCs). The NGS approach applied is limited to re-sequencing of the protein-coding regions (exome re-sequencing). For this purpose, target enrichment kits supplied by vendors are used to capture exon DNA from human samples, followed by sequencing, e.g., by HiSeq2000 (Illumina). In addition, tumor mRNA is sequenced for direct quantification of gene expression and for verification that the mutated genes are expressed in the patient's tumor. The resulting millions of sequence reads are processed through software algorithms. The output list includes mutations and gene expression. Tumor-specific somatic mutations are determined and prioritized by comparison to the diversity of PBMC-derived germline. Next, the newly identified peptides may be tested for immunogenicity as described above for the repository, and candidate TUMAPs that retain appropriate immunogenicity are selected for inclusion in the vaccine.

[0534] In an exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients by the method described above; (b) comparing the peptides identified in (a) with a library of peptides pre-screened for immunogenicity and overexpression in tumors in comparison with the corresponding normal tissues; (c) selecting at least one peptide from the library that is associated with the tumor-associated peptides identified in the patient; and (d) optionally, selecting at least one newly identified peptide in (a) and confirming its immunogen.

[0535] In an exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; and (b) selecting at least one newly identified peptide in (a) and confirming its immunogenicity.

[0536] Once the peptides for the personalized peptide-based vaccine are selected, the vaccine is manufactured. The vaccine is preferably a liquid formulation consisting of individual peptides dissolved in 33% DMSO.

[0537] Each peptide included in the product is dissolved in DMSO. The concentration of the single peptide solution has to be selected according to the number of peptides included in the product. Equal volumes of the single peptide DMSO solutions are mixed to obtain a solution containing all the peptides included in the product at a concentration of about 2.5 mg / ml per peptide. The mixed solution is then diluted 1:3 with water for injection to obtain a concentration of 0.826 mg / ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22 μm sterile filter. The final bulk solution is obtained.

[0538] The final bulk solution is filled into vials and stored at -20 °C until use. One vial contains 700 μL of solution containing 0.578 mg of each peptide. Of this, 500 μL (approximately 400 μg per peptide) is applied for intradermal injection.

[0539] The invention will be described in the following examples, which describe preferred embodiments, but which are not to be taken as limiting. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

Brief Description of the Drawings

[0540]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 1I

Figure 1J

Figure 1K

Figure 1L

Figure 1M

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 3

Figure 4

Example

[0541] Example 1: Identification and Quantification of Tumor-Related Peptides Presented on the Cell Surface Tissue sample Tumor tissues from patients were obtained from Universitatsklinik fur Allgemeine, Viszeral-und Transplantationschirurgie, Tubingen, Germany; Istituto Nazionale Tumori”Pascale”. Molecular Biology and Viral Oncology Unit, Via Mariano, Naples, Italy; Bio-Options Inc., Brea, CA, USA; ProteoGenex Inc., Culver City, CA, USA; Asterand Europe, Royston Herts, United Kingdom. Informed consent forms based on all patients were obtained prior to surgery. The tissues were snap-frozen immediately after surgery and stored at less than -70 °C until isolation of TUMAP.

[0542] Isolation of HLA peptides from tissue samples HLA peptide storage from snap-frozen tissue samples was performed according to a slightly modified protocol (Falk, K., 1991; Seeger, F.H.T., 1999) for HLA-A *It was obtained by immunoprecipitation from solid tissue using the specific antibody BB7.2, the HLA-A, -B, -C specific antibody W6 / 32, CNBr-activated Sepharose, acid treatment, and ultrafiltration.

[0543] Mass spectrometry The resulting HLA peptide pool was separated according to their hydrophobicity by reverse-phase chromatography (nanoAcquity UPLC system, Waters), and the eluted peptides were analyzed in an LTQ-velos and fusion hybrid mass spectrometer (ThermoElectron) equipped with an ESI source. The peptide pool was directly inserted onto an analytical fused-silica microcapillary column (75 μm inner diameter × 250 mm) packed with 1.7 μm C18 reverse-phase material (Waters) applying a flow rate of 400 nL / min. Subsequently, the peptides were separated using a two-step 180-min binary gradient from 10% to 33% B at a flow rate of 300 nL / min. The gradient was composed of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold-coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using the TOP5 strategy. Briefly, the scan cycle was initiated with a high mass accuracy full scan in the Orbitrap (R = 30000), followed by MS / MS scans of the five most abundant precursor ions in the Orbitrap (R = 7500), and the pre-selected ions were dynamically excluded. Tandem mass spectra were interpreted by SEQUEST and additional manual adjustment. The identified peptide sequences were verified by comparison of the resulting native peptide fragmentation pattern with the fragmentation pattern of synthetic reference peptides with identical sequences.

[0544] Label-free relative LC-MS quantification was performed by ion counting, i.e., by extraction and analysis of LC-MS features (Mueller et al., 2007a). The method assumes that the LC-MS signal area of a peptide correlates with its abundance in the sample. The extracted features were further processed by charge state deconvolution and retention time alignment (Mueller et al., 2007b; Sturm et al., 2008). Finally, all LC-MS features were cross-referenced with the sequence identification results, combining the quantitative data of different samples and the presentation profile from tissue to peptide. The quantitative data were normalized in a dual-mode according to the central tendency considering the variation within technical and biological replicates. In this way, each identified peptide can be associated with the quantitative data, enabling relative quantification between samples and tissues. Furthermore, all quantitative data obtained for peptide candidates were manually inspected to ensure data integrity and confirm the accuracy of the automated analysis. For each peptide, a presentation profile was calculated, showing the mean sample presentation as well as the inter-assay variation. The profiles juxtapose CLL samples against the baseline of normal tissue samples.

[0545] The presentation profiles of representative overpresented peptides are shown in Figure 1. The presentation scores of representative peptides are shown in Table 8.

[0546] Table 8B: Presentation scores. The table enumerates peptides that are very highly overpresented (+++) on tumors compared to the normal tissue panel, highly overpresented (++) on tumors compared to the normal tissue panel, overpresented (+) on tumors compared to the normal tissue panel. S * = Phosphoserine

Table 8B-1

Table 8B-2

Table 8B-3

Table 8B-4

Table 8B-5

Table 8B-6

[0547] Example 2: Expression profiling of the gene encoding the peptide of the present invention The over-presentation or specific presentation of peptides on tumor cells compared to normal cells is sufficient for their usefulness in immunotherapy, and some peptides are tumor-specific even though their originating proteins are also present in normal tissues. Nevertheless, mRNA expression profiling can enhance the level of safety in the selection of peptide targets for immunotherapy. In particular, for treatment options with high safety risks such as affinity-matured TCRs, the ideal target peptides are derived from proteins that are specific to tumors and not found on normal tissues.

[0548] RNA source and preparation Surgically removed tissue specimens were provided as described above (see Example 1) after informed consent forms were obtained from each patient. Tumor tissue specimens were snap-frozen immediately after surgery and then homogenized using a mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, Germany), followed by purification with RNeasy (QIAGEN, Hilden, Germany); both methods were carried out according to the manufacturers' protocols.

[0549] Total RNA from healthy human tissues was obtained commercially (Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, Netherlands; BioChain, Hayward, CA, USA). RNA from several individuals (2 - 123) was mixed such that the RNA from each individual was equally weighted.

[0550] The quality and quantity of all RNA samples were evaluated on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip kit (Agilent).

[0551] Microarray experiments Gene expression analysis of all tumor and normal tissue RNA samples was performed using Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA). All steps were carried out according to the Affymetrix manual. Briefly, double-stranded cDNA was synthesized from 5 - 8 μg of total RNA using SuperScript RTII (Invitrogen) and oligo dT-T7 primers (MWG Biotech, Ebersberg, Germany) as described in the manual. In vitro transcription was performed using the BioArray High Yield RNA Transcript Labelling Kit (ENZO Diagnostics, Inc., Farmingdale, NY, USA) for the U133A array or the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0 array, followed by cRNA fragmentation, hybridization, and staining with streptavidin-phycoerythrin and biotinylated anti-streptavidin antibody (Molecular Probes, Leiden, Netherlands). Images were scanned using an Agilent 2500A GeneArray Scanner (U133A) or an Affymetrix GeneChip Scanner 3000 (U133 Plus 2.0), and data were analyzed using GCOS software (Affymetrix) with default settings for all parameters. For normalization, 100 housekeeping genes provided by Affymetrix were used. Relative expression values were calculated from the signal log ratios given by the software, with normal kidney samples set to 1.0 at discretion. Representative expression profiles of the origin genes of the present invention that are highly overexpressed or exclusively expressed in HCC are shown in Figure 2. Expression scores of additional representative genes are shown in Table 9.

[0552] Table 9: Expression scores. The table lists peptides from genes that are very highly overexpressed (+++) in tumors compared to the normal tissue panel, highly overexpressed (++) in tumors compared to the normal tissue panel, or overexpressed (+) in tumors compared to the normal tissue panel.

Table 9-1

Table 9-2

Table 9-3

Table 9-4

[0553] Example 3: HLA-A * 02 and HLA-A * 24 UV ligand exchange / peptide binding Candidate peptides for the T cell-based therapies according to the invention were further tested for their MHC binding ability (affinity). Individual peptide-MHC complexes were generated by UV ligand exchange, and the UV-sensitive peptide was cleaved upon UV irradiation and exchanged with the peptide of interest to be analyzed. Only peptide candidates that can effectively bind and stabilize the peptide-receptive MHC molecule prevent the dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA based on the detection of the light chain (β2m) of the stabilized MHC complex was performed. The assay was generally carried out as described by Rodenko et al. (Rodenko B, Toebes M, Hadrup SR, van Esch WJ, Molenaar AM, Schumacher TN, Ovaa H. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protoc. 2006;1(3):1120-32.).

[0554] A 96-well MAXISorp plate (NUNC) was coated overnight at room temperature with 2 μg / ml streptavidin in PBS, washed four times, and blocked for 1 hour at 37 °C in 2% BSA containing blocking buffer. The refolded HLA-A * 0201 / MLA-001 monomer served as a standard covering the range of 15 - 500 ng / ml. The peptide-MHC monomers for the UV exchange reaction were diluted 100-fold in blocking buffer. Samples were incubated for 1 hour at 37 °C, washed four times, incubated for 1 hour at 37 °C with 2 μg / ml HRP-conjugated anti-β2m, washed again, and detected with TMB solution stopped with NH2SO4. Absorbance was measured at 450 nm. For the production and manufacture of antibodies or their fragments, and / or T cell receptors or their fragments, candidate peptides showing a high exchange yield (preferably higher than 50%, most preferably higher than 75%) are generally preferred since they show sufficient binding activity to MHC molecules to prevent the separation of the MHC complex.

[0555] Table 10A: MHC Class I Binding Score <20% = +; 20% - 49% = ++; 50% - 75% = +++; >= 75% = ++++

Table 10A

[0556] Table 10B: MHC Class I Binding Score HLA-A of HLA-class I restricted peptides depending on the peptide sequence * 02 or HLA-A * Binding to 24 was classified by peptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++. S * = Phosphoserine

Table 10B-1

Table 10B-2

Table 10B-3

Table 10B-4

Table 10B-5

Table 10B-6

Table 10B-7

[0557] Example 4: In vitro immunogenicity of MHC class I-presented peptides To obtain information on the immunogenicity of the TUMAPs of the present invention, the inventors performed an investigation using an in vitro T cell priming assay based on the repeated stimulation of CD8+ T cells by artificial antigen-presenting cells (aAPCs) loaded with peptide / MHC complexes and anti-CD28 antibodies. In this way, the inventors were able to demonstrate the immunogenicity of 22 HLA-A * 0201-restricted TUMAPs of the present invention and to demonstrate that these peptides are T cell epitopes for which CD8+ precursor T cells exist in humans (Table 11).

[0558] In vitro priming of CD8+ T cells To perform in vitro stimulation of artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD28 antibodies, the inventors first performed positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) from healthy donors obtained from the University clinics Mannheim, Germany, after informed consent, to isolate CD8+ T cells from fresh HLA-A * 02 leukapheresis products.

[0559] PBMC and isolated CD8+ lymphocytes or PBMC were cultured in T cell medium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U / ml penicillin / 100 μg / ml streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany), and 20 μg / ml gentamicin (Cambrex) until use. 2.5 ng / ml of IL-7 (PromoCell, Heidelberg, Germany) and 10 U / ml of IL-2 (Novartis Pharma, Nurnberg, Germany) were also added to the TCM at this stage.

[0560] The generation of pMHC / anti-CD28-coated beads, T cell stimulation, and readout were performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and eight different pMHC molecules per readout condition.

[0561] Purified costimulatory murine IgG2a anti-human CD28 Ab9.3 (Jung et al., 1987) was chemically biotinylated using sulfo-N-hydroxysuccinimide biotin as recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were streptavidin-coated polystyrene particles with a diameter of 5.6 μm (Bangs Laboratories, Illinois, USA).

[0562] The pMHCs used for positive and negative control stimulations were A * 0201 / MLA-001 (peptide ELAGIGILTV from modified Melan-A / MART-1), and A * 0201 / DDX5-001 (YLLPAIVHI from DDX5), respectively.

[0563] In the presence of 4 × 12.5 ng of different biotinylated pMHC, 800,000 beads / 200 μl were coated in a 96-well plate, washed, and subsequently 600 ng of biotinylated anti-CD28 was added in a volume of 200 μl. In 200 μl of TCM to which 5 ng / ml of IL-12 (PromoCell) was added, 1 × 10 6 CD8+ T cells were added to 2 × 8 5 washed and coated beads and co-incubated at 37 °C for 3 days to initiate stimulation in a 96-well plate. Next, half of the medium was replaced with fresh TCM to which 80 U / ml of IL-2 was added, and the culture was continued at 37 °C for 4 days. This stimulation cycle was performed a total of 3 times. For pMHC multimer readout using 8 different pMHC molecules per condition, a minor modification involving conjugation to 5 different fluorescent dyes was added, and the two-dimensional combinatorial coding approach as previously described (Andersen et al., 2012) was used. Finally, multimer analysis was performed by staining the cells with Live / dead near-infrared dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany), and fluorescent pMHC multimers. For the analysis, a BD LSRII SORP hematocytometer equipped with appropriate lasers and filters was used. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Evaluation of the multimer analysis was performed using FlowJo software (Tree Star, Oregon, USA). The in vitro primary stimulation of specific multimer+ CD8+ lymphocytes was detected by comparison with negative control stimulation. If at least one evaluable in vitro stimulation well from one healthy donor was found to contain a specific CD8+ T cell line after in vitro stimulation, the immunogenicity of the given antigen was detected (i.e., this well contained at least 1% specific multimer+ in CD8+ T cells and the percentage of specific multimer+ cells was at least 10-fold the median of negative control stimulation).

[0564] In vitro immunogenicity of HCC peptides Among the tested HLA class I peptides, in vitro immunogenicity could be demonstrated by the generation of peptide-specific T cell lines. Representative flow cytometry results after TUMAP-specific multimer staining of the three peptides of the present invention are shown in FIGS. 3 and 4 together with the corresponding negative controls. The results of 22 peptides from the present invention are summarized in Table 11A.

[0565] Table 11A: In Vitro Immunogenicity of HLA Class I Peptides of the Present Invention Representative results of in vitro immunogenicity experiments of the peptides of the present invention conducted by the applicant. <20% = +; 20% - 49% = ++; 50% - 69% = +++; >= 70% = ++++

Table 11A

[0566] Table 11B: In Vitro Immunogenicity of Additional HLA Class I Peptides of the Present Invention Representative results of in vitro immunogenicity experiments for the HLA-A * 24 binding peptides of the present invention. Results of in vitro immunogenicity experiments are shown. Percentages of positive wells and donors (within evaluable) are summarized as shown: 1 - 20% = +; 20% - 49% = ++; 50% - 69% = +++; >= 70% = ++++

Table 11B

[0567] Healthy HLA-A * 02+ Donor Representative Results of Peptide-Specific In Vitro CD8+ T Cell Responses (Figure 3) CD8+ T cells are complexed with anti-CD28 mAb and IMA-APOB-002 (SEQ ID NO: 7) peptide (A, right panel) or IMA-APOB-003 (B, right panel, SEQ ID NO: 1), or IMA-ALDH1L1-001 (C, right panel, SEQ ID NO: 2), respectively, and HLA-A * 02-coated artificial APCs for primary stimulation. After 3 cycles of stimulation, A* 02 / APOB-002(A) or A * 02 / APOB-003(B), or A * Detection of peptide-reactive cells was performed by 2D multimer staining at 02 / ALDH1L1-001. The left panels (A, B, C) show control staining of cells stimulated with irrelevant A * 02 / peptide complex. Live singlet cells were gated on CD8+ lymphocytes. Boolean gates helped exclude false positive events detected by multimers specific for different peptides. The frequency of specific multimer+ cells within CD8+ lymphocytes is shown.

[0568] Healthy HLA-A * Representative results of peptide-specific ex vivo CD8+ T cell responses from 24+ donors (Figure 4) CD8+ T cells were first stimulated using anti-CD28 mAb and HLA-A coated artificial APCs that complex with IMA-KLHL24-001 (SEQ ID NO: 190) peptide (A, right panel) or IMA-APOB-006 (B, right panel, SEQ ID NO: 218), respectively. * After 3 cycles of stimulation, A * 24 / KLHL24-001(A) or A * Detection of peptide-reactive cells was performed by 2D multimer staining at 24 / APOB-006(B). The left panels (A and B) show control staining of cells stimulated with irrelevant A * 24 / peptide complex. Live singlet cells were gated on CD8+ lymphocytes. Boolean gates helped exclude false positive events detected by multimers specific for different peptides. The frequency of specific multimer+ cells within CD8+ lymphocytes is shown.

[0569] Example 5: Peptide Synthesis All peptides were synthesized using standard well-established solid-phase peptide synthesis using the Fmoc strategy. The identity and purity of the individual peptides were determined by mass spectrometry and analytical RP-HPLC. The peptides were obtained as white to off-white lyophilized powders (trifluoroacetate salts) with a purity >50%. All TUMAPs are preferably administered as trifluoroacetate or acetate salts, and other salt forms are also possible.

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A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300 and mutant sequences thereof that are at least 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 300, wherein the mutant binds to MHC and / or induces a cross-reaction with the mutant peptide of T cells and its pharmaceutically acceptable salts, and the peptide is not a full-length polypeptide. 2. The peptide according to item 1, wherein the peptide has the ability to bind to molecules of human major histocompatibility complex (MHC) class I or II, and the peptide has the ability to be recognized by CD4 and / or CD8 T cells when bound to MHC. 3. The peptide or its mutant according to item 1 or 2, the amino acid sequence of which comprises a continuous series of amino acids described in the group of SEQ ID NO: 1 to SEQ ID NO: 300. 4. The peptide or its mutant according to any one of items 1 to 3, preferably having a full length of 8 to 30, more preferably 8 to 16, and most preferably 8 to 100 amino acids, and the peptide consists of or is essentially composed of the amino acid sequence described in SEQ ID NO: 1 to SEQ ID NO: 300. 5. The peptide according to any one of items 1 to 4, or a variant thereof, wherein the peptide is modified and / or contains non-peptide bonds. 6. The peptide according to any one of items 1 to 5, or a variant thereof, wherein the peptide is part of a fusion protein, particularly comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii). 7. A nucleic acid encoding the peptide according to any one of items 1 to 6, or a variant thereof, which optionally binds to a heterologous promoter sequence. 8. An expression vector capable of expressing the nucleic acid according to item 7. 9. The peptide according to any one of items 1 to 6, or a variant thereof, the nucleic acid according to item 7, or the expression vector according to item 8, for use in medicine. 10. A host cell comprising the peptide according to any one of items 1 to 6, the nucleic acid according to item 7, or the expression vector according to item 8, preferably an antigen-presenting cell such as a dendritic cell. 11. A method for producing the peptide according to any one of items 1 to 6, or a variant thereof, comprising culturing the host cell according to item 10 that presents the peptide according to any one of items 1 to 6 or expresses the nucleic acid according to item 7, or the expression vector according to item 8, and isolating the peptide or its variant from the host cell or its culture broth. 12. An in vitro method for producing activated T lymphocytes, comprising contacting T cells in vitro for a time sufficient to activate the T cells in an antigen-specific manner with antigen-loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell, wherein the antigen is the peptide according to any one of items 1 to 9. 13. Activated T cells produced by the method according to item 12, which selectively recognize cells presenting a polypeptide comprising the amino acid sequence according to any one of items 1 to 5. 14. A method of killing target cells in a patient, comprising the step of administering to the patient an effective number of the activated T cells defined in item 13, wherein the target cells of the patient present a polypeptide comprising the amino acid sequence according to any one of items 1 to 5. 15. A soluble or membrane-bound antibody that specifically recognizes a peptide according to any one of items 1 to 5 or a variant thereof, preferably a peptide according to any one of items 1 to 5 that binds to an MHC molecule or a variant thereof. Optionally, the antibody has additional effector functions such as an immunostimulatory domain or a toxin. 16. Use of a peptide according to any one of items 1 to 6, a nucleic acid according to item 7, an expression vector according to item 8, a cell according to item 10, an activated T lymphocyte according to item 13, or an antibody and other binding molecules according to item 15 for treating cancer or in the manufacture of a cancer therapeutic agent. 17. The use according to item 16, wherein the cancer is most preferably selected from the group of HCC, brain tumor, renal cancer, pancreatic cancer, colon or rectal cancer or leukemia and other tumors, which shows overexpression of APOB, FASN, COPA, preferably GLUL, GPAM, PLIN2, SLC16A1, SLC9A3R1, PCBD1, SEC16A, AKR1C4, ABCB11, HAL, CYP2E1, C4A, C4B, ALDH1L1, CRP, ACSL4, EEF2, HLTF, FBXO22, GALK1, TMCO1, TMEM33, ZNF318, IPO9, AMACR, C1QTNF3, CYP4F8, CYP4F3, CYP4F11, CYP4F12, CYP4F2, MOCOS, A1CF, COL18A1, HPR, LBP, C19orf80, CFHR5, ITIH4, TMEM110, LARP4, LMF2, SLC10A5, SLC16A11, still preferably ANKFY1, C12orf44, C16orf58, CPSF1, DCAF8, PEX19, DDX11, DDX12P, DECR2, NME4, DENND5B, DYM, EDC4, ERI3, FAM20A, FNDC3A, GPR107, GYG2, HEATR2, IFT81, KCTD3, SHKBP1, KIAA1324L, KLHL24, MARCH6, MBTPS2, MIR1279, CPSF6, NOC4L, NXF1, PANK2, PCNXL3, PIPSL, PSMD4, PSMD14, SLC35B1, TCP11L2, THNSL2, THOC2, TOMM5, TRAPPC6B, TRIM54, TRIM55, TRIM63, UGGT2, URB1, VPS54, WIZ, ZNF45, RFTN2, SCFD1, SERINC5, CCT7P2, CMAS, ANKS1A, C17orf70, CCT7, CDK5RAP2, CLPTM1, and peptides of SEQ ID NO: 1 to SEQ ID NO: 300 and another protein derived therefrom. 18. (a) A container comprising a pharmaceutical composition containing the peptide variant according to any one of items 1 to 6, the nucleic acid according to item 7, the expression vector according to item 8, the cell according to item 10, the activated T lymphocyte according to item 13, or the antibody according to item 15, in solution or lyophilized form; (b) Optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; (c) Optionally, at least one additional peptide selected from the group consisting of SEQ ID NOs: 1 to 346, and (d) Optionally, a kit comprising (i) instructions for use of the solution, or (ii) instructions for reconstitution and / or use of the lyophilized formulation. 19. The kit according to item 18, further comprising one or more of (iii) buffer, (iv) diluent, (V) filter, (vi) needle, or (V) syringe. 20. The kit according to item 18 or 19, wherein the peptide is selected from the group consisting of SEQ ID NOs: 1 to 300. 21. a) Identifying tumor-associated peptides (TUMAP) presented by tumor samples from individual patients; b) Comparing the peptides identified in a) with a peptide library pre-screened for immunogenicity and / or over-presentation in tumors compared to normal tissues; c) Selecting at least one peptide from the library that matches the tumor-associated peptide identified in the patient; and d) Based on step c), manufacturing an individualized vaccine or compound-based or cell therapy product A method for manufacturing an individualized anti-cancer vaccine for compound-based and / or cell therapy for an individual patient, comprising: 22. The TUMAP is a1) Comparing expression data from the tumor sample with expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins overexpressed or abnormally expressed in the tumor sample; and a2) Correlating the expression data with the sequences of MHC ligands that bind 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. The method according to item 21, identified by 23. The method according to item 21 or 22, wherein the binding peptide is eluted from the MHC molecules isolated from the tumor sample, and the sequence of the MHC ligand is identified by sequencing the eluted ligand. 24. The method according to any one of items 21 to 23, wherein the normal tissue corresponding to the tissue type of the tumor sample is obtained from the same patient. 25. The peptide included in the storage library aa. performing genome-scale messenger ribonucleic acid (mRNA) expression analysis by a high-throughput method such as microarray or sequencing-based expression profiling, including the step of identifying genes that are overexpressed in malignant tissues compared to normal tissues or tissue groups; ab. selecting a peptide encoded by a gene that is selectively expressed or overexpressed, detected in step aa; ac. determining the induction of an in vivo T cell response by the selected peptide, including an in vitro immunogenicity assay using human T cells from healthy donors or the patient; or ba. identifying HLA ligands from the tumor sample using a mass spectrometry method; bb. performing genome-scale messenger ribonucleic acid (mRNA) expression analysis by a high-throughput method such as microarray or sequencing-based expression profiling, including the step of identifying genes that are overexpressed in malignant tissues compared to normal tissues or tissue groups; bc. comparing the identified HLA ligands with the gene expression data; bd. selecting a peptide encoded by a gene that is selectively expressed or overexpressed, detected in step bc; be. redetecting the TUMAP selected from step bd on tumor tissue, and confirming the relevance of overexpression at the mRNA level by the absence or rare detection on healthy tissue. Determining induction of an in vivo T cell response by the selected peptide, comprising an in vitro immunogenicity assay using human T cells from a healthy donor or said patient The method according to any one of items 21 to 24, identified based on . 26. The method according to any one of items 21 to 25, wherein the immunogenicity of the peptide comprised in the reservoir is determined by a method comprising an in vitro immunogenicity assay, patient immune monitoring for individual HLA binding, MHC multimer staining, ELISPOT assay and / or intracellular cytokine staining. 27. The method according to any one of items 21 to 26, wherein the reservoir comprises a plurality of peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 346. 28. The method according to any one of items 21 to 27, further comprising identifying at least one mutation specific to the tumor sample compared to normal corresponding tissue from the individual patient, and selecting a peptide associated with the mutation for inclusion in a vaccine or for generating a cell therapy. 29. The method according to item 28, wherein the at least one mutation is identified by whole genome sequencing. 30. A soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein the ligand has at least 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300. 31. The T cell receptor according to item 30, wherein the amino acid sequence is at least 88% identical to SEQ ID NO: 1 to SEQ ID NO: 300. 32. The T cell receptor according to item 30 or 31, wherein the amino acid sequence consists of any one of SEQ ID NO: 1 to SEQ ID NO: 300. 33. The T cell receptor according to any one of items 30 to 32, wherein the T cell receptor is provided as a soluble molecule and optionally possesses additional effector functions such as an immune-stimulatory domain or a toxin. 34. A nucleic acid encoding a TCR according to any one of items 30 to 33, optionally binding to a heterologous promoter sequence. 35. An expression vector capable of expressing the nucleic acid according to item 34. 36. A host cell comprising the nucleic acid according to item 34, or a nucleic acid encoding the antibody according to item 15, or the expression vector according to item 35, preferably a T cell or an NK cell. 37. A method for producing a T cell receptor according to any one of items 30 to 33, comprising culturing the host cell according to item 36 and isolating the T cell receptor from the host cell and / or its culture supernatant. 38. a) A peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300; b) A T cell receptor reactive with the peptide and / or peptide-MHC complex according to a); c) A fusion protein comprising the peptide according to a) and the N-terminal amino acids 1 to 80 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) A host cell comprising the expression vector according to d); f) A method comprising contacting T cells in vitro with the peptide according to a) expressed on the surface of an appropriate antigen-presenting cell for a time sufficient to activate the T cells in an antigen-specific manner, and a method of transferring these activated T cells into oneself or another patient, and activated T lymphocytes obtained thereby; g) An antibody or soluble T cell receptor reactive with the peptide and / or peptide-MHC complex according to a), and / or a cell presenting the peptide according to a), and potentially modified by fusion with, for example, an immunoreactive activation domain or a toxin; h) An aptamer that recognizes a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300 and / or a complex of a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300 and an MHC molecule; i) a conjugate or labeled peptide or scaffold as described in any one of a) to h), and a pharmaceutically acceptable carrier; and optionally, a pharmaceutically acceptable excipient and / or stabilizer A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of 39. An aptamer that specifically recognizes a peptide or a variant thereof as described in any one of Items 1 to 5, preferably a peptide or a variant thereof as described in any one of Items 1 to 5 that binds to an MHC molecule. 40. The pharmaceutical composition according to Item 38, comprising at least one peptide selected from SEQ ID NOs: 1, 2, 7, 225, 228, 301, 303, and 312, preferably all of the peptides.

Claims

1. A pharmaceutical for the treatment or diagnosis of hepatocellular carcinoma (HCC), comprising a pharmaceutically acceptable salt of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303, wherein the free form of the peptide has the ability to bind to a major histocompatibility complex (MHC) class I molecule and, when bound to the MHC, is recognized by CD8 T cells.

2. The pharmaceutical according to claim 1, provided in the form of a chloride, acetate or trifluoroacetate.

3. Use of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical for the treatment or diagnosis of hepatocellular carcinoma (HCC).

4. An antibody that specifically recognizes a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 that forms a complex with an MHC molecule in ELISA, immunohistochemical examination, and in vivo imaging of hepatocellular carcinoma (HCC), wherein the antibody is a soluble antibody or a membrane-bound antibody, or An antibody that specifically recognizes a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 that forms a complex with an MHC molecule in ELISA, immunohistochemical examination, and in vivo imaging of hepatocellular carcinoma (HCC), wherein the antibody is a soluble antibody or a membrane-bound antibody, and the antibody is a monoclonal antibody, humanized antibody, bispecific antibody and / or chimeric antibody.

5. Use of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 that forms a complex with an MHC molecule or an antibody that specifically recognizes a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 in the manufacture of a pharmaceutical for the treatment or diagnosis of HCC, or Use of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 that forms a complex with an MHC molecule or an antibody that specifically recognizes a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 in the manufacture of a pharmaceutical for the treatment or diagnosis of HCC, wherein the antibody is a monoclonal antibody, humanized antibody, bispecific antibody and / or chimeric antibody.

6. Use of a T cell receptor (TCR) that is reactive with an HLA ligand in the manufacture of a pharmaceutical for the treatment or diagnosis of HCC, wherein the ligand consists of the amino acid sequence shown in SEQ ID NO: 303 and the TCR is soluble or membrane-bound, or Use of a T cell receptor (TCR) reactive with an HLA ligand in the manufacture of a medicament for the treatment or diagnosis of HCC, wherein the ligand consists of the amino acid sequence shown in SEQ ID NO: 303, the ligand is part of a peptide-MHC complex, and the TCR is soluble or membrane-bound.

7. Use of activated T cells in the manufacture of a medicament for the treatment or diagnosis of hepatocellular carcinoma (HCC), wherein the activated T cells selectively recognize cells presenting a polypeptide comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO:

303.

8. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 or a pharmaceutically acceptable salt thereof, A peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 complexed with an MHC molecule or an antibody that specifically recognizes a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303, A TCR reactive with an HLA ligand, wherein the ligand consists of the amino acid sequence shown in SEQ ID NO: 303, A TCR reactive with an HLA ligand, wherein the ligand consists of the amino acid sequence shown in SEQ ID NO: 303 complexed with an MHC molecule, and Activated T cells that selectively recognize cells presenting a polypeptide comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 303 A pharmaceutical composition for the treatment or diagnosis of hepatocellular carcinoma (HCC), comprising at least one active ingredient selected from the group consisting of

9. The pharmaceutical composition according to claim 8, further comprising an adjuvant, or The pharmaceutical composition according to claim 8, further comprising an adjuvant, wherein the adjuvant is an interleukin, or The pharmaceutical composition according to claim 8, further comprising an adjuvant, wherein the adjuvant is an interleukin, and the interleukin is IL-2, IL-15 or a combination thereof.

10. (a) A container comprising the pharmaceutical composition according to claim 8 in solution or lyophilized form; and (b) A second container containing a diluent or reconstitution solution for the lyophilized formulation, comprising A kit for use in the treatment or diagnosis of hepatocellular carcinoma (HCC), or (a) A container comprising the pharmaceutical composition according to claim 8 in solution or lyophilized form; (b) A second container containing a diluent or reconstitution solution for the lyophilized preparation; and, (c) At least one additional peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 346 excluding SEQ ID NO: 303 comprising A kit for use in the treatment or diagnosis of hepatocellular carcinoma (HCC).

11. (d) The kit according to claim 10, further comprising one or more of (i) a buffer, (ii) a diluent, (iii) a filter, (iv) a needle, (v) a syringe, and (vi) an adjuvant.

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