Novel peptides and combination of peptides for use in immunotherapy against hepatocellular carcinoma (HCC) and other cancers

Abstract

To provide novel peptides, proteins, nucleic acids, and cells for use in immunotherapy against hepatocellular carcinoma (HCC).SOLUTION: The present invention relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides, that 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 them into patients. Peptides bound to molecules of major histocompatibility complex (MHC), 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, that can be used in vaccine compositions for eliciting anti-tumor immune responses or as targets for 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, alone or in combination with other tumor-associated peptides, which can serve as active pharmaceutical ingredients in vaccine compositions, for example, to stimulate anti-tumor immune responses or to stimulate T cells ex vivo for transfer into patients. The peptides bind to molecules of the major histocompatibility complex (MHC), or the peptides themselves can also be targets 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 vaccine compositions to elicit anti-tumor immune responses or as targets for the development of pharmacologically / immunologically active compounds and cells. [Background technology]

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

[0003] The distribution of HCC varies geographically, and incidence rates are gender-dependent. The age-standardized incidence rate (ASR) for HCC in men is highest in East Asia (31.9) and Southeast Asia (22.2), intermediate in Southern Europe (9.5) and North America (9.3), and lowest in Northern Europe and South 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 in Northern Europe (1.9) and Micronesia (1.6).

[0004] The overall prognosis for patients with HCC is poor. The 5-year relative survival rate (5Y-RSR) for HCC is approximately 15% and depends on the stage at diagnosis. For localized HCC, where the cancer is still confined to the liver, the 5Y-RSR is approximately 28%. For regional and distant HCC, where the cancer has grown 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 conjunction with alcohol abuse or HBV or HCV infection, but can also be caused by metabolic diseases such as type II diabetes. As a result, healthy liver tissue is replaced by scar tissue, which increases the risk of cancer development.

[0006] Disease management depends on the stage of the tumor at the time of diagnosis and the overall condition of the liver. When possible, part of the liver (partial hepatectomy) or the entire organ (hepatectomy) is removed surgically. Patients with small or completely resectable tumors, in particular, qualify as candidates for liver transplantation.

[0007] If surgery is not a treatment option, different other treatments are currently available: In tumor ablation, a probe is injected into the liver and the tumor is destroyed by radio waves, microwaves, or cryotherapy; in embolization, the tumor's blood supply is blocked by mechanical or chemical means; and in radiation therapy, high-energy radio waves can be used to destroy tumors.

[0008] Chemotherapy for HCC includes a combination of doxorubicin, 5-fluorouracil, and cisplatin for systemic therapy, and a combination of doxorubicin, floxuridine, and mitomycin C for hepatic arterial infusion. However, most HCCs are highly resistant to chemotherapy drugs (Enguita-German and Fortes, 2014).

[0009] Treatment options for advanced, unresectable HCC are limited to the multi-tyrosine kinase inhibitor sorafenib (Chang et al., 2007; Wilhelm et al., 2004). Sorafenib is the only systemic drug identified to increase survival by approximately 3 months and currently represents the only experimental treatment option for these patients (Chapiro et al., 2014; Llovet et al., 2008).

[0010] Recently, a limited number of immunotherapy trials for HCC have been conducted. Cytokines have been used to activate immune cell subsets 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 few therapeutic vaccination trials have been performed. Butterfield et al. conducted two trials using peptides derived from alpha-fetoprotein (AFP) as a vaccine or DCs loaded with AFP peptides ex vivo (Butterfield et al., 2003; Butterfield et al., 2006). In two separate studies, autologous dendritic cells (DCs) were pulsed ex vivo with 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 problem]

[0012] In a first aspect, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300, or a variant 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 said variant binds to MHC and / or induces cross-reactivity of T cells with said peptide or a pharmaceutically acceptable salt thereof, and wherein said peptide is not the underlying full-length polypeptide.

[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 said peptide or variant thereof has an overall 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 expected genes of origin (base) of those peptides. All peptides in Table 1 are HLA-A * 02, and the peptides in Table 2 bind to HLA-A * The peptides in Table 3 have been previously disclosed in extensive lists as a result of high-throughput screening with high error rates or calculated using algorithms, but have not previously been associated with cancer. * 02. The peptides in Table 4 are additional peptides that may be useful in combination with other peptides of the present invention. * 02, or if displayed, A * 24. The peptides of Table 5 are further useful in the diagnosis and / or treatment of various malignancies associated with overexpression or over-representation of the respective underlying polypeptide.

[0015] Table 1: HLA-A * 02 Peptide according to the present invention; S * = phosphoserine [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0016] Table 2: HLA-A according to the present invention * 24 peptides and their sequence numbers; * = phosphoserine [Table 2]

[0017] Table 3: Additional peptides according to the invention with no previously known cancer association; * = phosphoserine [Table 3-1] [Table 3-2]

[0018] Table 4: Peptides useful for, e.g., personalized cancer therapy; * = phosphoserine [Table 4-1] [Table 4-2]

[0019] The present invention further relates to peptides according to the invention for use in the treatment of proliferative diseases, such as pancreatic cancer, colon or rectal cancer, kidney cancer, brain tumors, and / or leukemia in general.

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

[0021] As shown in Tables 5A and B below, many of the peptides of the present invention can also be used in immunotherapy for other indications. The tables show selected peptides discovered in additional tumor types that showed over-representation (including differential presentation) in 5% or more of the tumor samples measured, or that were presented in 5% or more of the tumor samples measured with a tumor-to-normal tissue geometric mean ratio of greater than 3. Over-representation is defined as higher presentation in tumor samples compared to the normal sample with maximal presentation. Normal tissues tested for over-representation relative to the following were adipose tissue, adrenal gland, blood cells, blood vessels, bone marrow, brain, cartilage, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung, lymph node, nerve, pancreas, thyroid, peritoneum, pituitary gland, pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid, trachea, ureter, and bladder.

[0022] Table 5A: Peptides according to the invention and their specific uses in other cancerous diseases, especially other proliferative diseases; * = phosphoserine [Table 5A-1] [Table 5A-2] [Table 5A-3] [Table 5A-4] [Table 5A-5] Table 5B: Peptides according to the invention and their specific uses in other cancerous diseases, especially other proliferative diseases; * = phosphoserine [Table 5B-1] [Table 5B-2] [Table 5B-3] [Table 5B-4] [Table 5B-5] [Table 5B-6] NSCLC=non-small cell lung cancer, SCLC=small cell lung cancer, RCC=renal cancer, CRC=colon or rectal cancer, GC=gastric cancer, HCC=liver cancer, PC=pancreatic cancer, PrC=prostate cancer, leukemia, BRCA=breast cancer, MCC=Merkel cell carcinoma, OC=ovarian cancer, NHL=non-Hodgkin's lymphoma, AML=acute myeloid leukemia, CLL=chronic lymphocytic leukemia.

[0023] Therefore, another aspect of the present invention relates to the use of at least one peptide according to the invention according to any one of SEQ ID NOs: 1, 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, in a preferred embodiment in the combined treatment of pancreatic cancer.

[0024] Thus, another aspect of the present invention is directed to the sequences of SEQ ID NOs: 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, 256, 257, 258, 259, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421 60, 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 in the combined treatment of colon cancer or kidney cancer.

[0025] Thus, another aspect of the present invention is the preparation of SEQ ID NOs: 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, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 3 2, 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 in the combined treatment of renal carcinoma.

[0026] Thus, another aspect of the present invention is directed to the sequences of 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, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 3 332, 333, 334, 336, 337, 338, 339, 340, 342, 343, 344, 345 and 347, in a preferred embodiment in the combined treatment of brain tumors.

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

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

[0029] Therefore, another aspect of the present invention relates to the use of a peptide according to the invention for the combined treatment of proliferative diseases, preferably selected from the group of HCC, brain tumors, renal cancer, pancreatic cancer, colon or rectal cancer, and leukemia.

[0030] The present invention further relates to peptides according to the invention which have 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 peptides according to the invention, said peptides consisting of or consisting essentially of the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 300 (respectively).

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

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

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

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

[0036] 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 the treatment of diseases and in medicine, in particular in the treatment of diseases including cancer and autoimmune / inflammatory / immunopathological diseases.

[0037] The present invention further relates to antibodies against the peptides according to the present invention, or complexes of said peptides according to the present invention with MHC, and methods for producing them.

[0038] The present invention further relates to T cell receptors (TCRs), in particular soluble TCRs (sTCRs), and cloned TCRs, which are incorporated into autologous or allogeneic T cells, and methods for producing these, as well as methods for producing NK cells or other cells that carry or cross-react with said TCRs.

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

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

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

[0042] The present invention further relates to a method according to the present invention, in which a sufficient amount of antigen is contacted with an antigen-presenting cell, thereby loading the antigen onto a class I or II MHC molecule expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell.

[0043] The present invention further relates to a method according to the invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing or expressing said peptides containing SEQ ID NO: 1 to SEQ ID NO: 300, preferably SEQ ID NO: 1 to SEQ ID NO: 124, and SEQ ID NO: 187 to SEQ ID NO: 218 or variant amino acid sequences.

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

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

[0046] The present invention further relates to the use of any of the described peptides, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention, activated T lymphocytes, T cell receptors or antibodies or other peptides and / or peptide-MHC binding molecules according to the invention as a medicament or in the manufacture of a medicament. Preferably, the medicament is effective against cancer.

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

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

[0049] The present invention further relates to specific peptide-based labeled proteins and biomarkers according to the present invention, referred to herein as "targets," which can be used in the diagnosis and / or prognosis of 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 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 structure provides a binding groove, which is used for noncovalent interactions with peptides. MHC class I molecules are found on most nucleated cells. They present peptides derived primarily from endogenous proteins, defective ribosomal products (DRIPs), and proteolytic cleavage of larger peptides. MHC class II molecules are found primarily on professional antigen-presenting cells (APCs) and present peptides from exogenous or transmembrane proteins that are incorporated into APCs during endocytosis and subsequently processed. Peptide-MHC class I complexes are recognized by CD8+ T cells bearing the appropriate T cell receptor (TCR), while peptide-MHC class II molecule complexes are recognized by CD4+ helper T cells bearing the appropriate TCR. As a result, it is well known that TCR, peptide, and MHC exist in a 1:1:1 stoichiometry.

[0051] CD4+ helper T cells play an important role in inducing and maintaining effective responses by CD8+ cytotoxic T cells. Identification of CD4+ T cell epitopes derived from tumor-associated antigens (TAAs) is crucial for the development of therapeutic agents to stimulate antitumor 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). At the tumor site, T helper cells maintain a cytokine environment favorable for cytotoxic T cells (CTLs) (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), which attracts effector cells such as CTLs, NK cells, macrophages, and granulocytes (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, expression of MHC class II molecules is primarily restricted to immune system cells, particularly professional antigen-presenting cells (APCs), such as monocytes, monocyte-derived cells, macrophages, and dendritic cells. In cancer patients, tumor cells have been found to express MHC class II molecules (Dengjel 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 function of CTLs in anti-tumor immunity. T helper cell epitopes that initiate TH1-type T helper cell responses support the effector function of CD8-positive killer T cells, which includes cytotoxicity directed against tumor cells that display tumor-associated peptide / MHC complexes on their cell surface. In this way, tumor-associated T helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses.

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

[0055] There is evidence that CD4 T cells are direct antitumor effectors ( Braumüller et al., 2013 ; Tran et al., 2014 ).

[0056] Because constitutive expression of HLA class II molecules is usually restricted to immune cells, it was not thought possible to isolate class II peptides directly from primary tumors, but Dengjel et al. succeeded in identifying several MHC class II epitopes directly from tumors (WO 2007 / 028574, EP 1760088 B1).

[0057] The 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 the respective tumor cells and are usually upregulated compared to unmodified cells of the same origin.

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

[0059] For an MHC class I peptide to initiate (elicit) a cellular immune response, it must also bind to an MHC molecule. This process depends on the allele of the MHC molecule and specific polymorphisms in the peptide's amino acid sequence. MHC class I-binding peptides are usually 8-12 amino acid residues long and typically contain two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele possesses a "binding motif" that determines which peptides can specifically bind to the binding groove.

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

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

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

[0063] c) Overexpressed TAAs: Genes encoding ubiquitously expressed TAAs have been detected in histologically distinct tumor types as well as in numerous normal tissues, generally at lower expression levels. While many of the epitopes processed and potentially presented by normal tissues may not reach the T cell recognition threshold, their overexpression in tumor cells can trigger anti-cancer responses by breaking previously established immune tolerance. Prominent examples of this class of TAAs are Her-2 / neu, survivin, telomerase, or WT1.

[0064] d) Tumor-specific antigens: These unique TAAs arise from mutations in normal genes (e.g., β-catenin, CDK4). Some of these molecular alterations are associated with neoplastic transformation and / or progression. Tumor-specific antigens can usually induce a strong immune response without the risk of an autoimmune reaction against normal tissue. On the other hand, these TAAs are most often associated only with the very tumor on which they were identified and are usually not shared among many individual tumors. For proteins with tumor-specific (associated) isoforms, tumor specificity (or association) of a peptide may also occur if the peptide is derived from a tumor (associated) exon.

[0065] e) TAAs resulting from aberrant post-translational modifications: Such TAAs may arise from proteins that are neither specific nor overexpressed in tumors, but nevertheless become tumor-associated antigens through post-translational processes active primarily in tumors. Examples of this class arise from altered glycosylation patterns, resulting in novel epitopes in tumors, such as MUC1, or events such as protein splicing during degradation, which may or may not be tumor-specific.

[0066] f) Oncoviral proteins: These TAAs are viral proteins that may play an important role in the carcinogenesis process and, because they are foreign (not of human origin), can elicit T cell responses. Examples of such proteins are the human papillomavirus type 16 proteins E6 and E7, which are expressed in cervical cancer.

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

[0068] Essentially, any peptide capable of binding to an MHC molecule may serve as a T cell epitope. A prerequisite for the induction of a T cell response in vitro or in vivo is the presence of T cells bearing the corresponding TCR and the absence of immune tolerance to this particular epitope.

[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 generating differential transcriptional profiles or differential peptide expression patterns between tumor and normal tissues.

[0070] However, the identification of genes that are overexpressed in tumor tissues or human tumor cell lines, or that are selectively expressed in such tissues or cell lines, does not provide precise information regarding the use of antigens transcribed from these genes in immunotherapy. This is because only individual subsets of epitopes of these antigens are suitable for such use, because T cells with the corresponding TCR must be present and immune tolerance to this particular epitope must be absent or minimal. Therefore, in a highly preferred embodiment of the present invention, it is important to select only those peptides that are excessively or selectively presented against which functional and / or proliferative T cells exist. Such functional T cells are defined as T cells that can undergo clonally proliferation 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. For the TCRs and antibodies according to the invention, presentation is the determining factor.

[0072] Additional uses, both therapeutic and diagnostic, for cancerous diseases are disclosed below in more detail regarding the underlying proteins (polypeptides) of the peptides of the present 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] Alterations 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 mesothelioma cells ( Sudo et al., 2010 ; Qi et al., 2014 ; Wong et al., 2003 ).

[0075] CPB2 activity has been shown to be significantly reduced 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 ). The protein it encodes, ζ-crystallin, has been shown to directly interact with the mRNA of the anti-apoptotic molecule bcl-2, stabilizing bcl-2 overexpression in T-cell acute lymphocytic leukemia ( Lapucci et al., 2010 ).

[0078] Overexpression of CSRP2 is associated with 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, breast, 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 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 ), while both CYP4F2 and CYP4F3 are overexpressed in pancreatic ductal adenocarcinoma, and only CYP4F2 is overexpressed in ovarian cancer ( Gandhi et al., 2013 ; Alexanian et al., 2012 ).

[0084] CYP4F11 expression 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 variants 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 better clinical outcomes in breast cancer, while on the other hand, overexpression of DAP3 has been reported in thyroid oncocytic tumors and invasive glioblastomas ( 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 directly interact with 19ARF, ultimately leading to the retention of this factor in the cytoplasm and inactivation of 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 cancer and myelodysplastic syndromes, the latter diseases being associated with 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 has been shown to be overexpressed in glioblastoma and to increase glioblastoma cell invasion in response to HGF via activated Rac-1 ( Murray et al., 2014 ).

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

[0094] DROSHA, one of two key enzymes in microRNA biogenesis, is overexpressed in several cancers, including gastrointestinal tumors, breast cancer, and cervical cancer, and appears to promote tumor cell proliferation, colonization, 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] A whole-exome sequencing study revealed a somatic mutation in the DYNC1H1 gene in a patient with intraductal papillary mucinous neoplasm of the pancreas ( Furukawa et al., 2011 ).

[0097] EEF2 protein has been shown to be overexpressed in lung, esophageal, pancreatic, breast, and prostate cancer, glioblastoma multiforme, and non-Hodgkin's 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 have been identified in colorectal adenoma samples ( Bojjireddy et al., 2014 ; Zhou et al., 2013a ).

[0099] EIF2B5 encodes one subunit of translation initiation factor B. A single nucleotide polymorphism in this gene was described to be associated with survival in ovarian cancer (Goode et al., 2010).

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

[0101] EIF4E is a potent oncogene that is elevated in up to 30% of human malignancies, including breast, prostate, lung, and head and neck carcinomas, as well as in many 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 colon cancer ( Line et al., 2002 ).

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

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

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

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

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

[0109] GLUL is overexpressed in human breast cancer cells and astrocytomas ( 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 in-frame mRNA fusions of GOLGA4 and PDGFRB have been reported in myeloproliferative neoplasms ( Senchenko et al., 2003 ; Hidalgo-Curtis et al., 2010 ).

[0112] GPAM is expressed in human breast cancer, where it is associated with altered 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 to be 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] Different studies suggest a key role of HSPA2 in the disease progression of cervical cancer, renal cell carcinoma, and bladder cancer, and intragenic polymorphisms are 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 colon 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 a candidate tumor suppressor gene and is mutated in luminal B breast cancer ( Cornen et al., 2014 ).

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

[0120] CPSF6 was identified as one gene within the “transcription-ready gene cassette” associated with significant differences in metastatic and invasive potential of several tumor types, including breast, colon, liver, lung, esophageal, and thyroid cancer ( Yu et al., 2008 ).

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

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

[0123] NAV2 was shown to be specifically expressed in a group of colon cancers, and treatment of colon cancer cells with antisense oligonucleotides against 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, and 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, NKD1 protein is decreased, but NKD1 mRNA is increased, the former being correlated with increased invasiveness and poor prognosis (Zhang et al., 2011). NKD1 mRNA was also found to be elevated 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 reported a higher frequency of RFNG expression in adenomas compared to carcinomas ( Gu et al., 2012 ; Hopfer et al., 2005 ).

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

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

[0130] RPL17 was 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 cancer ( Takemasa et al., 2012 ; Sun et al., 2005 ).

[0132] SAMM50 encodes a component of the mitochondrial outer membrane sorting and integration apparatus (SAM), which functions in the integration of β-barrel proteins into the outer mitochondrial membrane. A proliferation-promoting chimeric mRNA (SAMM50-PARVB) was detected in breast and ovarian cancer cells and in several samples from breast, stomach, 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 plasmin-α2-plasmin inhibitor complexes have been shown to be a predictor of survival in non-small cell lung cancer, and low α2-antiplasmin activity 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] SHC1 protein levels are elevated in prostate, metastatic breast, ovarian, and thyroid cancers, and in different isoforms, which appear to function as a major adaptor protein mediating steroid mitogenic signals at the non-genomic level ( Alam et al., 2009 ; Rajendran et al., 2010 ).

[0136] AMACR is highly overexpressed in prostate cancer and is therefore 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 concomitant 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 targeting GPC3 for HCC are currently being tested in phase II clinical trials: a humanized GPC3 monoclonal antibody and a vaccine consisting of two GPC3-derived peptides. The peptides used in the latter study differ from those reported in this paper. GPC3 expression has also been identified in all yolk sac tumors, some lung squamous cell carcinomas, and ovarian clear cell carcinomas (Filmus and Capurro, 2013; Kandil and Cooper, 2009).

[0139] MAGEB2 is expressed in the testis and placenta, and in a significant proportion of tumors of various histological types, particularly multiple myeloma and head and neck squamous cell carcinoma, so it is classified as a cancer-testis antigen ( 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 tumor formation by suppressing oncogenic ras activity in mouse models 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 identified 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 was reported to be overexpressed in human papillomavirus 18-positive oral squamous cell carcinoma and was identified as a prostate cancer susceptibility locus ( Lo et al., 2007 ; Nwosu et al., 2001 ).

[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 links endocytic vesicles to microtubules. This gene is highly expressed in Hodgkin's disease and breast cancer Reed-Sternberg cells 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] While CLU may inhibit tumor progression in advanced neoplasms, it may also provide tumors with a significant survival advantage by suppressing multiple therapeutic stressors and promoting metastasis. CLU plays an important role in prostate cancer pathogenesis, and has been shown to control the aggressive behavior of human renal cell carcinoma clear cell tumors through ERK1 / 2 signaling and MMP-9 expression regulation, conferring therapy resistance in 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 cancer, implying a tumor-suppressor 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 in the SLC16A1 gene may affect clinical outcome and can be used to predict response to adjuvant chemotherapy (Kim et al., 2015; Fei et al., 2014a; Fei et al., 2014a).

[0150] Glioblastomas have been shown to release high levels of glutamate, which stimulates tumor cell proliferation and promotes tumor invasion, and may downregulate SLC1A2, which correlates with higher tumor grade, implicating its potential role in glial tumor progression. Furthermore, SLC1A2-CD44 fusion genes have been detected in gastric cancer and may represent a class of gene fusions that establish a pro-tumorigenic metabolic environment favoring tumor growth and survival (Tao et al., 2011; deGroot et al., 2005).

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

[0152] Evidence for the involvement of SLC9A3R1 in cancer development exists in hepatocellular carcinoma, schwannoma, glioblastoma, colorectal cancer, and especially breast cancer ( Saponaro et al., 2014 ).

[0153] NFYC has been reported to promote the expression of oncogenes 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 tissue 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 wide variety of human cancers. The deregulated kinase is oncogenic, and its activation is associated with progression to metastatic stages. ACK1 inhibitors have shown promise in preclinical studies (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 signaling from the sarcomere to the nucleus (Pizon et al., 2002).

[0159] RNA interference of 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 therefore considered a target point for studying the regulatory mechanisms for irinotecan (CPT-11) drug resistance and drug resistance reversal ( Xie et al., 2014 ).

[0161] UGT1A10 is expressed in gastric and biliary 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, thus acting as an indirect antioxidant. Xenobiotic (XRE) and antioxidant (ARE) response elements have been detected in the UGT1A8, UGT1A9, and UGT1A10 promoters (Kalthoff et al., 2010).

[0162] UGT1A8 is primarily expressed in the gastrointestinal tract ( Gregory et al., 2003 ), and mRNA expression is upregulated upon treatment with the chemo-anticancer 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 induced by the putative chemotherapeutic drug β-naphthoflavone ( Hanioca et al., 2012 ).

[0165] UGT1A9 is primarily expressed in the liver and kidney ( Gregory et al., 2003 ). UGT1A9 germline polymorphisms are a possible predictor 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 adenosquamous carcinoma (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 potential prognostic markers for colorectal cancer (Lascorz et al., 2012).

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

[0170] A significant decrease in both USP10 and SIRT6 protein expression was detected in human colon cancer ( Lin et al., 2013 ).

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

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

[0173] VMP1, a stress-induced autophagy-related protein, is also induced by the oncogene KRAS (Lo Re et al., 2012). VMP1 is overexpressed in poorly differentiated human pancreatic cancer in response to chemotherapeutic agents (Gilabert et al., 2013). Marked downregulation of VMP1 is observed in human HCC tissues and correlates closely with multiple tumor nodules, absence of capsule formation, venous invasion, and poor prognosis in 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 to be 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 a fatty acid synthase that is involved in promoting lipid synthesis in different cancer types, including breast, pancreatic, prostate, liver, ovarian, colon, and endometrial cancers ( Wu et al., 2014 ; Zhao et al., 2013 ).

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

[0178] FMO5 is a dominant liver-specific FMO monooxygenase that 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 dedifferentiation in HCC ( Tanaka et al., 2013 ) and in estrogen receptor α-negative breast adenomas ( Mamtani and Kulkarni, 2012 ).

[0180] Genetic variation in 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 activity and has been found to be inactivated by hypermethylation in colon, stomach, uterine, bladder, and lung tumors ( Debauve et al., 2008 ; Castro et al., 2010 ; Garcia-Baquero et al., 2014 ).

[0182] HDAC10 is a histone deacetylase and transcription factor. HDAC10 expression was significantly reduced in gastric cancer tissue compared with adjacent tissue (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 adrenocortical tumors (Fonseca et al., 2012). HDAC10 levels are elevated in chronic lymphocytic leukemia (Wang et al., 2011). The HDAC10-589C>T promoter polymorphism was significantly associated with HCC development and accelerated HCC in patients with chronic HBV infection (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 patients with diffuse large cell lymphoma ( Wong et al., 2014 ).

[0184] HM13 is a signal peptide peptidase that affected cell viability in colorectal adenomas ( Sillars-Hardebol et al., 2012 ).

[0185] Serum HPR levels in patients with malignant lymphoma were significantly higher than in non-diseased controls, and HPR expression increased with disease progression (Epelbaum et al., 1998). HPR expression paralleled increasing breast cancer aggressiveness, and HPR-positive breast cancers were more likely to recur after primary resection and were associated with shorter disease-free intervals (Shurbaji et al., 1991).

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

[0187] HSD17B6 expression in tissues from prostate cancer patients treated with 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, cervix-vaginal cancer, prostate cancer, mantle cell lymphoma, and serous ovarian cancer. Decreased levels of Hsp10 have been reported in bronchial carcinogenesis (David et al., 2013).

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

[0190] IGFBPL1 is a regulator of insulin growth factor receptor 1 (IGFBPL1) and is downregulated by aberrant hypermethylation in breast cancer cell lines. IGFBPL1 methylation is significantly associated with poorer overall and disease-free survival (Smith et al., 2007).

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

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

[0193] The IRS2-derived peptide pIRS-21097-1105 was reported in HLA-A2(+) melanoma and breast, ovarian, and colorectal carcinoma (Zarling et al., 2014). The IRS-21057DD genotype and 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 suppressing the growth of malignant tumors. Mutational analysis revealed ITGA7 mutations in prostate cancer, hepatocellular carcinoma, soft tissue leiomyosarcoma, and glioblastoma multiforme. ITGA7 was downregulated in nonmetastatic prostate cancer and leiomyosarcoma (Tan et al., 2013).

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

[0196] Missense mutations have been identified in SHKBP1, a receptor tyrosine kinase that acts downstream of FLT3, 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-NETs) at different stages (Darmanis et al., 2013).

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

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

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

[0200] LEPR dysregulation has been reported in a variety of malignant cells, including colon, hepatocellular, endometrial, thyroid, breast, and lung cancers ( 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 tissues is significantly higher than in matched control tissues (Li et al., 2014b). LRPPRC levels serve as a prognostic marker for patients with prostate adenocarcinoma (PCA), with patients with high LRPPRC levels surviving for shorter periods after surgery than patients with low LRPPRC levels (Jiang et al., 2014). LRPPRC is abundantly expressed in various tumor types, including lung adenocarcinoma, esophageal squamous cell carcinoma, gastric, colon, breast, and endometrial adenocarcinoma, and lymphoma (Tian et al., 2012).

[0203] MANEA expression is regulated 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 from individual patients than in tumors (Srivenugopal and Ali-Osman, 1997).

[0205] ORM2 glycoforms provide valuable information for distinguishing primary and secondary liver cancer (Mackiewicz and Mackiewicz, 1995). Plasma ORM2 levels were found 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 levels of tetrahydrobiopterin result in increased PAH activity and PAH protein in human liver tumor cells (McGuire, 1991).

[0207] PARP14 is highly expressed in myeloma plasma cells and is associated with disease progression and poor survival. PARP14 is critically involved in JNK2-dependent survival. PARP14 has been shown to promote myeloma cell survival 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 centrosomal 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 undergoes frequent copy number loss in CIN(+) colorectal cancer ( Burrell et al., 2013 ).

[0211] PIPOX expression varies depending on breast cancer subtype, with HER-2-positive 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 reduced in prostate tumors and reduced the carcinogenic potential of prostate cells by metabolizing sarcosine (Khan et al., 2013).

[0212] Increased levels of PSMD4 have been detected in colon 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 with controls. Preoperative urinary concentrations of PLIN2 reflect 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 undergoes frequent rearrangements or deletions in human cancers, particularly in hepatocellular carcinoma, but also in colorectal, head and neck cancers (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) family that charges tRNA with glutamine. ARS expression and polymorphisms are associated with breast cancer and glioblastoma (He et al., 2014b; Kim et al., 2012).

[0216] 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 upregulated PON2, including thyroid, prostate, pancreatic, testicular, endometrial / uterine, liver and kidney cancers, lymphoid tissue, bladder tumors, ALL, and CML, and such overexpression conferred 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 taxotere (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, which 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 with 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 radiochemotherapy 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 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 with tumor suppressor function in estrogen receptor-positive breast, ovarian, and lung cancers (Li and Li, 2014; Huang et al., 2014). In contrast, RASAL2 is oncogenic in triple-negative breast cancer, driving mesenchymal invasion and metastasis (Feng et al., 2014a).

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

[0227] Overexpression of ROCK1 or mutations in the ROCK1 gene, leading to increased kinase activity, have 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, Inv(3) and t(3;3) breakpoints associated with poor prognosis in myeloid leukemia or myelodysplasia cluster within regions located centromeric and downstream of the RPN1 gene (Wieser, 2002).

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

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

[0232] ABCB1 encodes P-glycoprotein (P-gp), which is expressed in normal cells of various organs, including the intestine, liver, kidney, brain, and placenta. Overexpression and genetic polymorphisms of P-gp have been detected in colorectal cancer, tumors originating from the adrenal gland, 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 (MDR / TAP) of subfamily B. ABCB10 has been shown to be involved in cisplatin resistance in KCP-4 human epidermoid carcinoma cells ( Oiso et al., 2014 ).

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

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

[0236] ABCC6 was downregulated in colorectal cancer non-responders to palliative chemotherapy (Hlavata et al., 2012). In contrast, it was upregulated in gemcitabine-resistant human NSCLCA549 cells (Ikeda et al., 2011).

[0237] ACACA expression is upregulated in many human cancers, including breast, prostate, and liver cancer, and has been shown to correlate with enhanced adipogenesis in cancer cells. Various ACACA inhibitors have shown therapeutic efficacy in treating cancer cell lines by suppressing cell proliferation and inducing cell death through apoptosis (Zu et al., 2013).

[0238] ACLY is aberrantly expressed in various tumors, including breast, liver, colon, lung, and prostate cancer, and is inversely correlated with tumor stage and differentiation ( Zu et al., 2012 ).

[0239] ACSL3 is overexpressed in lung cancer and, based on preclinical studies, is a promising novel therapeutic target in lung cancer (Pei et al., 2013). Upregulated ACSL3 expression may serve as a possible biomarker of estrogen receptor-specific breast cancer risk (Wang et al., 2013c).

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

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

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

[0243] AGFG2 was identified as one of 14 candidate predictive genes in identifying hormone receptor-negative or triple-negative breast cancer cases that likely remain free of metastatic recurrence ( Yau et al., 2010 ).

[0244] AGT is a highly potent antiangiogenic factor and has been shown to produce antitumor effects in vitro and in vivo (Bouquet et al., 2006). In transgenic mice, overexpression of human AGT was shown to reduce angiogenesis in liver cancer, thereby delaying tumor progression (Vincent et al., 2009).

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

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

[0247] APOA1 encodes apolipoprotein AI, the major protein component of high-density lipoprotein (HDL) in plasma. In multiple animal tumor models, APOA1 has been shown to exhibit 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-associated 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 six differentially expressed proteins that may predict patients' response to neoadjuvant chemotherapy and recurrence-free survival (Hyung et al., 2011).

[0250] In stage III colorectal cancer patients and in 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 sensitive and specific marker for distinguishing HCC from other metastatic tumors in the liver (Sang et al., 2013).ARG1 may contribute to local immunosuppression in NSCLC (Rotondo et al., 2009).

[0252] The phosphorylated, and therefore more active, form of ARSB protein was found to be increased in peripheral leukocytes from patients with chronic myeloid leukemia compared with healthy donors (Uehara et al., 1983).

[0253] In ovarian cancer cells, downregulation of ASNA1 has been shown to increase sensitivity to the chemotherapy 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 resulted in cytotoxicity against cholangiocarcinoma cells in vitro and significantly suppressed intrahepatic tumor growth and metastasis (Noda et al., 2012).

[0255] ATP1A2 was found among 31 proteins significantly upregulated in glioblastoma (Com et al., 2012). In contrast, ATP1A2 was 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 lysosomal V-ATPase activity. ATP6V1C1 knockdown significantly inhibited tumor growth, metastasis, and osteolytic lesions in vivo in 4T1 mammary tumor cell lines (Feng et al., 2013). ATP6V1C1 was overexpressed in oral squamous cell carcinoma and was associated with tumor cell motility (Otero-Rey et al., 2008).

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

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

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

[0261] A strong decrease in BHMT and BHMT2 transcripts 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 in an Atg13-dependent 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 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 involves the breast cancer susceptibility gene products, BRCA1 and BRCA2 (Ling et al., 2007).

[0264] C19orf80 encodes the hepatocellular carcinoma-associated 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 subnetwork that significantly discriminated late-stage human colorectal cancer ( Nibbe et al., 2009 ).

[0266] CDK6 has been shown to regulate the activity of the tumor suppressor protein Rb. CDK6 may exert its tumor-promoting functions 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 poor prognosis (Cui et al., 2011).

[0268] Inactivating mutations in CLPTM1 have been found 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 during cell differentiation. Increased expression of sialic acid sugars on the surface of cancer cells is a well-known tumor characteristic (Bull et al., 2014).

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

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

[0272] THTPA hydrolysis may be involved in the antiproliferative effects of Ndrg-1, which 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 epigenetic upregulation of the metalloproteinase MMP-9 (Medjkane et al., 2012). While SMYD3 expression is undetectable or very weak in many types of normal human tissues, overexpression of SMYD3 has been associated with the development and progression of gastric, colorectal, hepatocellular, prostate, and breast cancer (Hamamoto et al., 2006; Liu et al., 2014; Liu et al., 2013a).

[0274] A link between STAT2 and tumorigenesis was observed in genetically engineered mice lacking STAT2 ( Yue et al., 2015 ) or constitutively 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] SPBP has also been shown to suppress the transcriptional activity of estrogen receptor alpha (ERα). Overexpression of SPBP inhibited the proliferation of ERα-dependent breast cancer cell lines (Gburcik et al., 2005). Within the cell nucleus, SPBP exhibits relatively low mobility and is enriched 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 component 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 leads to the generation of the inflammatory mediators and chemoattractants anaphylatoxins C3a and C3b (Sahu et al., 1998).

[0278] CLN3 is an anti-apoptotic gene in NT2 neuronal progenitor cells and a few cancer types (Zhu et al., 2014b). It is involved in intracellular trafficking and regulation in neuronal and non-neuronal cells (Rakheja et al., 2008; Getty and Pearce, 2011), and it participates 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 lung or pancreatic cancer cell lines (Rylova et al., 2002).

[0279] SLC13A5 is one of seven CIMP marker genes. CIMP (CpG island methylator 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 coordinate transcriptional regulation during the induction of sialylsulfo-Lex glycan biosynthesis in acute inflammation (Huopaniemi et al., 2004) and sulfation of 6-sulfolactosamine epitopes in human colorectal cancer cell lines (Kamiyama et al., 2006). Colorectal cancer cell lines as well as human colorectal tissue 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 many malignant tumors (Urig and Becker, 2006), and its inhibition can 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 a prime target for drug design (Liu et al., 2012).

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

[0284] SNRPD1 is a core spliceosomal protein that is upregulated in malignant tumors.

[0285] Decreased 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 disease of bone and tumorigenesis ( Komatsu et al., 2012 ).

[0287] PCNA expression predicts survival in anorectal melanoma (Ben-Izhak et al., 2002). Within PCNA, a cancer-associated isoform of PCNA (caPCNA) was identified that contains an abnormal pattern of methyl ester groups on multiple glutamine and aspartic acid residues (Hoelz et al., 2006).

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

[0289] At the molecular level, STAT1 inhibits the growth of both IFN-γ-treated mouse and human tumor cells 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). Increased STAT1 mRNA levels have been shown to be part of a molecular signature associated with better prediction of metastatic outcomes in patients with hormone receptor-negative and triple-negative breast cancer (Yau et al., 2010).

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

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

[0292] Peptides consisting of or consisting essentially of amino acid sequences as set forth herein may have one or two non-anchor amino acids (see below for anchor motifs) exchanged without substantially altering or adversely affecting their ability to bind to human major histocompatibility complex (MHC) class I or II molecules when compared to the unmodified peptide. In another embodiment, in peptides consisting essentially of amino acid sequences as set forth herein, one or two amino acids may be exchanged with their conservative exchange partners (see below) without substantially altering or adversely affecting their ability to bind to human major histocompatibility complex (MHC) class I or II molecules when compared to the unmodified peptide.

[0293] The present invention further relates to peptides according to the invention which are modified and / or comprise non-peptide bonds as described below.

[0294] The present invention further relates to a peptide according to the invention, said peptide being part of a fusion protein, in particular fused to the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii) or fused to (or in the sequence of) an antibody, e.g. an antibody specific for dendritic cells, 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 invention. The present invention further relates to a nucleic acid according to the 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 displaying a nucleic acid according to the 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, as described in more detail below, and methods for producing them. Preferred are antibodies specific for the peptides of the present invention and / or for the peptides of the present invention when bound to their MHC. Preferred antibodies may be monoclonal.

[0299] The present invention further relates to T cell receptors (TCRs), in particular soluble TCRs (sTCRs) targeting 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 a host cell comprising a nucleic acid or an expression vector according to the invention as described above. The present invention further relates to a host cell according to the invention which is an antigen-presenting cell. The present invention further relates to a host cell according to the invention wherein the antigen-presenting cell is a dendritic cell.

[0302] The present invention further relates to aptamer.Aptamer (see, for example, International Publication No. 2014 / 191359 and the documents cited therein) is a short single-stranded nucleic acid or peptide molecule, which can fold into a predetermined three-dimensional structure and recognize specific target structures.They have been considered as a suitable alternative for developing targeted therapy.Aptamer has been shown to selectively bind with a variety of complex targets with high affinity and specificity.

[0303] Aptamers that recognize molecules located on cell surfaces have been identified within the past decade, providing a means for developing diagnostic and therapeutic approaches. Aptamers have been shown to be nearly non-toxic and immunogenic, making them promising candidates for biomedical applications. Indeed, aptamers, such as those recognizing prostate-specific membrane antigen (PSMA), have been successfully used for targeted therapy and have been shown to function in xenograft in vivo models. Furthermore, aptamers that recognize specific tumor cell lines have been identified.

[0304] DNA aptamers can be selected to exhibit broad-spectrum recognition properties for various cancer cells, particularly those derived from solid tumors, while not recognizing non-tumorgenic and primary healthy cells. If the identified aptamer not only recognizes a specific tumor subtype but also interacts with a range of tumors, this makes the aptamer applicable as a so-called broad-spectrum diagnostic and therapeutic agent.

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

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

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

[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 is attached (e.g., a (second) antigen-binding moiety) to a target site, such as a specific tumor cell or tumor stroma, bearing the antigenic determinant (e.g., a peptide conjugate described herein). In another embodiment, the scaffold can activate signaling through its target antigen, such as a T-cell receptor complex antigen. Scaffolds include, but are not limited to, antibodies and fragments thereof, antibody antigen-binding domains comprising an antibody heavy chain variable region and an antibody light chain variable region, binding proteins comprising at least one ankyrin repeat motif and a single-domain antigen-binding (SDAB) molecule, aptamers, (soluble) TCRs, and (engineered) cells such as allogeneic or autologous T cells.

[0309] Each scaffold may comprise a label, which allows the binding scaffold to be detected by determining the presence or absence of a signal provided by the label. For example, the scaffold may be labeled with a fluorescent dye or any other applicable cell marker molecule. Such marker molecules are well known in the art. For example, the fluorescent label provided by a fluorescent dye may allow the binding aptamer to be visualized by fluorescence or laser scanning microscopy or flow cytometry.

[0310] Each scaffold can be conjugated to a second active molecule, e.g., IL-21, anti-CD3, anti-CD28, etc. Respective polypeptide scaffolds are described, e.g., in the Background section of WO 2014 / 071978 A1 and the references cited therein.

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

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

[0313] The present invention further relates to a method according to the invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing said peptide containing SEQ ID NO: 1 to SEQ ID NO: 300, or a variant amino acid sequence thereof.

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

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

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

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

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

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

[0320] The present invention further relates to specific peptide-based labeled proteins and biomarkers according to the present invention, which may be used in the diagnosis and / or prognosis of HCC.

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

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

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

[0324] Certain elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. Isolation of T cells from tumor-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in the innate immune defense against cancer. In particular, CD8+ T cells play a key role in this response by recognizing class I molecules of major histocompatibility complex (MHC)-carrying peptides, typically 8–10 amino acid residues long, derived from proteins or defective ribosomal products (DRIPS) located in the cytosol. In humans, MHC molecules are also referred to as human leukocyte antigens (HLA).

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

[0326] Furthermore, the term "peptide" is intended to include salts of a series of amino acid residues, typically linked together 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 chlorides or acetates (trifluoroacetates). It should be noted that peptides are not salts in vivo, and therefore salts of peptides according to the present invention have substantially different in vivo states from peptides.

[0327] The term "peptide" is also intended to include "oligopeptide." The term "oligopeptide" is used herein to designate a series of amino acid residues typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, provided the correct epitope or epitopes are retained therein. Oligopeptides are typically less than about 30 amino acid residues in length and more than about 15 amino acids in length.

[0328] The term "peptides of the invention" is intended to include peptides consisting of or comprising the peptides set forth in SEQ ID NO: 1 to SEQ ID NO: 300, as defined above.

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

[0330] A peptide, oligopeptide, protein, or polynucleotide encoding such a molecule is "immunogenic" (and therefore an "immunogen" within the context of the present invention) if it is capable of inducing an immune response. For the purposes of the present invention, immunogenicity is more specifically defined as the ability to induce a T-cell response. An "immunogen" is therefore a molecule capable of inducing an immune response, and in the present invention, a molecule capable of inducing a T-cell response. In another embodiment, the immunogen can be a peptide, a peptide-MHC complex, an oligopeptide, and / or a protein used to raise specific antibodies or TCRs thereagainst.

[0331] A class I T cell "epitope" requires a short peptide that binds to a class I MHC receptor, forming a ternary complex (MHC class I α chain, β-2-microglobulin, and peptide) that can be recognized by a T cell bearing a compatible T cell receptor that binds the MHC / peptide complex with appropriate affinity. Peptides binding to MHC class I molecules are typically 8-14 amino acids in length, most typically 9 amino acids in length.

[0332] In humans, there are three distinct genetic loci that encode MHC class I molecules (human MHC molecules also called human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. * 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 frequency of 24, F, and the most frequent HLA-DR serotype. The frequency is calculated using the Hardy-Weinberg formula: F = 1-(1-Gf) 2 The haplotype frequency Gf in the US population was estimated using the linkage disequilibrium (D) method, 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). * 02 or A * The combination of 24 with certain HLA-DR alleles may be more abundant or less abundant than would be predicted from their single frequencies. For details, see Chanock et al. (SJ 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 invention, when included in the vaccines of the invention described herein, preferably comprise * 02 or A * 24. The vaccine may also comprise a pan-binding MHC class II peptide. Thus, the vaccine of the present invention can be used to * 02 positive, A * 24 positive or A * 02 and A* In patients who are positive for 24, cancer can be treated while MHC class II allotype selection is not required due to the pan-binding properties of these peptides.

[0335] For example, A * 02 and A * Combining 24 peptides into one vaccine has the advantage that a higher percentage of any patient population can be treated compared to addressing only one of the MHC class I alleles. While in most populations, less than 50% of patients are addressed with only one of the alleles, the vaccine 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: USA 61%, Western Europe 62%, China 75%, South Korea 77%, and Japan 86% (calculated from www.allelefrequencies.net).

[0336] As used herein, reference to a DNA sequence includes both single-stranded and double-stranded DNA. Thus, a specific sequence refers to the single-stranded DNA of such a sequence, the double-stranded DNA of such a sequence and its complement, and the complement of such a sequence, unless the context clearly indicates otherwise. The term "coding region" refers to the portion of a gene that naturally or normally encodes the expression product of the gene in its natural genomic environment, i.e., the region that encodes the natural expression product of the gene in vivo.

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

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

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

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

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

[0342] The term "fragment," when referring to a coding sequence, means a portion of DNA comprising less than the entire coding region, the expression product of which retains essentially the same biological function or activity as the expression product of the entire coding region.

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

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

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

[0346] The term "isolated" means that a material is removed from its original environment (e.g., the natural environment if it is of natural origin). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide 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 "purified" form. The term "purified" does not require complete purity; rather, it is intended as a relative definition and can include highly purified or only partially purified preparations, as these terms are understood by those of skill in the art. For example, individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. Purification of the starting material or natural substance to at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude, is expressly contemplated. Furthermore, claimed polypeptides having a purity of preferably 99.999%, or at least 99.99% or 99.9%, by weight; even more desirably 99% or greater, are expressly contemplated.

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

[0349] The term "active fragment" generally refers to a fragment that is a peptide, polypeptide, or nucleic acid sequence that generates an immune response (i.e., has immunogenicity) when administered alone, or optionally with a suitable adjuvant, or in a vector, to an animal, such as a mammal, for example, a rabbit or mouse, and also including humans, where such immune response takes the form of stimulating a T-cell response in the recipient animal, such as a human. Alternatively, an "active fragment" may also be used to induce a T-cell response in vitro.

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

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

[0352] If there is an alignment between a comparison sequence and a reference sequence for which the percent identity is calculated as above that is approximately identical to or greater than the specified minimum percent identity, then the comparison sequence has a specified minimum percent identity with the reference sequence, even if there is an alignment within the alignment whose percent identity, as calculated as above, is less than the specified percent identity.

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

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

[0355] A less conservative substitution might involve the substitution of another amino acid with similar characteristics but somewhat different in size, such as the substitution of an isoleucine residue for an alanine. A highly non-conservative substitution might involve the substitution of a polar amino acid with an acidic amino acid, or even a basic amino acid. However, even such "radical" substitutions cannot be dismissed as potentially ineffective, because chemical effects are not completely predictable and radical substitutions can lead to serendipitous effects that cannot be predicted from simple chemical principles.

[0356] Of course, such substitutions may involve structures other than conventional L-amino acids. Thus, D-amino acids may be substituted for L-amino acids commonly found in the antigenic peptides of the invention and still be encompassed by the disclosure herein. Furthermore, amino acids bearing non-standard R groups (i.e., R groups other than those found in the 20 conventional amino acids of naturally occurring proteins) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the invention.

[0357] If substitutions at two or more positions are found to result in a peptide with substantially equal or greater antigenic activity, as defined below, combinations of these substitutions are tested to determine whether the combination of substitutions results in an additive or synergistic effect on the antigenicity of the peptide. At most, no more than four positions within a peptide are substituted simultaneously.

[0358] The peptides of the invention may be extended by up to four amino acids, ie, by adding 1, 2, 3 or 4 amino acids to either end in any combination between 4:0 and 0:4.

[0359] The combinations of extensions according to the invention can be seen from Table 7. [Table 7]

[0360] The amino acids for extension / elongation can be the peptide of the original protein sequence or any other amino acid. Extension can be used to increase the stability or solubility of the peptide.

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

[0362] Preferably, when T cells specific for a peptide according to the invention are tested with a substituted peptide, the peptide concentration at which the substituted peptide achieves half-maximal enhancement of lysis over background is about 1 mM or less, preferably about 1 μM or less, more preferably about 1 nM or less, even more preferably about 100 pM or less, and most preferably about 10 pM or less. It is also preferred that the substituted peptide be recognized by T cells from more than one individual, at least two, and more preferably three individuals.

[0363] Thus, epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes, or may include epitopes that differ from the reference peptide by no more than four residues, so long as they have substantially the same antigenic activity.

[0364] MHC class I molecules are found on the majority of cells with nuclei, presenting peptides that are primarily derived from endogenous cytoplasmic or nuclear proteins, DRIPS, and proteolytic cleavage of larger peptides.However, peptides derived from endosomal compartments or exogenous sources are also frequently found on MHC class I molecules.This non-classical mode of class I presentation is referred to in the literature as cross-presentation.

[0365] Because both CD8- and CD4-dependent responses synergistically contribute to antitumor effects, 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) is important in the development of tumor vaccines. Therefore, it is an object of the present invention to provide peptide compositions containing peptides 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 desperately needed. Thus, there is a need to identify other factors that represent biomarkers for cancer in general, and HCC in particular. Furthermore, there is a need to identify factors that can be used in the treatment of cancer in general, and HCC in particular.

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

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

[0369] HLA-bound peptides can be recognized by the immune system, specifically by T lymphocytes, which can destroy cells that present the recognized HLA / peptide complex, such as HCC cells that present the derived peptide.

[0370] The peptides of the present invention have been shown to be capable of stimulating T cell responses and / or to be over-presented, and therefore can be used to produce antibodies and / or TCRs, particularly TCRs according to the present invention (see Example 3). Furthermore, when complexed with the respective MHC, the peptides can similarly be utilized to produce antibodies and / or TCRs, particularly TCRs according to the present invention. The respective methods are well known to those skilled in the art and can also be found in the respective references. Thus, the peptides of the present invention are useful for generating an immune response in patients that can destroy tumor cells. An immune response in patients can be induced by directly administering the described peptides, or appropriate precursors (e.g., extended peptides, proteins, or nucleic acids encoding such peptides), ideally in combination with an agent (i.e., an adjuvant) that enhances immunogenicity. Because the target peptides of the present invention are not presented in equivalent copy numbers on normal tissues, the immune response resulting from such therapeutic vaccination can be expected to be highly specific to tumor cells, preventing the risk of unwanted autoimmune reactions against the patient's normal cells.

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

[0372] Pharmaceutical compositions comprise peptides in either free form or in the form of a pharmaceutically acceptable salt. (See also above.) As used herein, "pharmaceutically acceptable salt" refers to derivatives of the disclosed peptides, wherein the peptides are modified by making acid or base salts of the drug. For example, acid salts are prepared from the free base (typically in which the neutral form of the drug has a neutral NH group) by reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid, as well as inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, and nitric acid and phosphoric acid. Conversely, basic salts of acidic moieties which may be present on the peptide are prepared with pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine and the like.

[0373] In one 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 compositions and / or uses of said compositions, for example in the form of a vaccine, comprising scaffolds reactive with peptides having the sequences set forth in SEQ ID NOs: 1, 2, 7, 225, 228, 301, 303 and 312 or peptides 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: because the peptides were generated from HCC, and because these peptides were determined to be absent or present at lower levels in normal tissue, these peptides can be used to diagnose the presence of cancer.

[0376] The presence of the claimed peptides in blood samples or tissue biopsies can assist pathologists in diagnosing cancer. Detection of specific peptides by antibodies, mass spectrometry, or other methods known in the art can tell pathologists that a tissue sample is malignant, inflammatory, or generally diseased, or can be used as a biomarker for HCC. The presence of peptide groups can allow for classification or subclassification of diseased tissue.

[0377] The detection of peptides on diseased tissue samples allows the evaluation of the benefits of therapies involving the immune system, especially when T lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a well-described mechanism by which infected malignant cells escape immune surveillance. The presence of peptides therefore indicates that this mechanism is not being utilized by the analyzed cells.

[0378] The peptides of the present invention may be used to analyze lymphocyte responses to these peptides, such as T cell or antibody responses to peptides or peptides complexed with MHC molecules. These lymphocyte responses can be used as prognostic markers to determine further treatment steps. These responses can also be used as surrogate markers in immunotherapeutic approaches aimed at inducing lymphocyte responses by different means, such as vaccination with proteins, nucleic acids, or autologous materials, or adoptive transfer of lymphocytes. In the setting of gene therapy, lymphocyte responses to peptides can be taken into account in the evaluation of side effects. Monitoring lymphocyte responses may also be a useful tool for follow-up testing of transplantation therapy, for example, for detecting graft-versus-host disease and host-versus-graft disease.

[0379] Using the peptides of the present invention, specific antibodies against MHC / peptide complexes can be generated and developed. These can be used for therapeutic purposes to target toxins or radioactive substances to diseased tissues. Another use of these antibodies can be to target radionuclides to diseased tissues for imaging purposes such as PET. This application can help detect small metastases or determine the size and precise location of diseased tissues.

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

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

[0382] Yet another aspect of the present invention relates to a method for producing an antibody that specifically binds to human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen, the method comprising the steps of: 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 in complex with the HLA-restricted antigen; isolating mRNA molecules from antibody-producing cells of the non-human mammal; generating a phage display library that displays protein molecules encoded by the mRNA molecules; and isolating at least one phage from the phage display library, wherein the at least one phage displays the antibody capable of specifically binding to the human major histocompatibility complex (MHC) class I or II in complex with the HLA-restricted antigen.Respective methods for producing such antibodies and single-chain class I major histocompatibility complex antibodies, as well as other tools for producing these antibodies, are described in 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-Oct; 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] A further aspect of the present invention provides a method for producing soluble T cell receptors (sTCRs) that recognize specific peptide-MHC complexes. Such soluble T cell receptors can be produced from specific T cell clones, and their affinity can be increased by targeted mutagenesis of complementarity-determining regions. Phage display can be used to select T cell receptors (U.S. Patent No. 2010 / 0113300; Liddy N, et al. Monoclonal TCR-redirected tumor cell killing. Nat Med 2012 Jun;18(6):980-987). To stabilize T cell receptors for phage display and pharmaceutical applications, the α and β chains can be linked, for example, by a non-native disulfide bond, other covalent bonds (single-chain T cell receptors), or a dimerization domain (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). T cell receptors can be linked to domains that recruit effector cells, such as toxins, drugs, cytokines (see, e.g., U.S. Patent No. 2013 / 0115191), anti-CD3 domains, etc., to perform specific functions on target cells.Furthermore, it can be expressed in T cells used for adoptive transfer. Further information can be found in WO2004 / 033685A1 and WO2004 / 074322A1. TCR combinations are described in WO2012 / 056407A1. Further manufacturing methods are disclosed in WO2013 / 057586A1.

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

[0386] To select over-represented peptides, a representation profile is calculated, showing the median sample representation and replicate variation. The profile aligns samples from the tumor entity of interest with a baseline of normal samples. Each of these profiles can then be combined into an over-representation score by calculating the p-value of a linear mixed-effects model (J. Pinheiro, et al., The nlme Package: Linear and Nonlinear Mixed Effects Models. 2007) or 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 identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock-frozen samples were purified to isolate HLA-associated peptides. The isolated peptides were separated and sequenced by online nanoelectrospray ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequences were analyzed using a 16-nucleotide polymorphism (N = 16) of the HCC samples (Figure 1). * 02 positive samples, 13 A * 02:01 N=15 A including positive samples * The fragmentation patterns of the native TUMAPs recorded from 24 HCC-positive samples were confirmed by comparison with those of the corresponding synthetic standard peptides of identical sequences. Because the peptides were directly identified as ligands for HLA molecules in primary tumors, these results provide direct evidence of the native processing and presentation of the identified peptides on primary cancer tissues obtained from 31 HCC patients.

[0388] The discovery pipeline XPRESIDENT® v2.1 (see, e.g., U.S. Patent No. 2013-0096016, the entire contents of which are incorporated herein by reference) enables the identification and selection of plausible over-presented peptide vaccine candidates based on direct relative quantification of HLA-restricted peptide levels on cancer tissues compared to several different non-cancerous tissues and organs. This was achieved by processing acquired LC-MS data with a proprietary data analysis pipeline and developing label-free differential quantification using 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 over-presented in tumors, compared to non-cancerous tissues and organs, were identified.

[0390] HLA-peptide complexes from HCC tissue samples were purified to isolate HLA-binding peptides and 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 in multiple HCC tumors and normal tissues were quantified using ion counting of label-free LC-MS data. The method assumes that the LC-MS signal area of ​​a peptide correlates with its abundance in the sample. All quantitative signals of peptides from various LC-MS experiments were normalized based on central tendency, averaged per sample, and merged into a bar graph called a presentation profile. The presentation profile integrates different analytical methods, such as protein database searching, spectral clustering, charge state deconvolution, and retention time alignment and normalization.

[0392] The present invention relates to 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 at least 90% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 300, or a variant thereof that cross-reacts T cells with said peptide, wherein said 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 NO: 1 to SEQ ID NO: 300, or a variant thereof that is at least 90% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 300, said peptide or variant having an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

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

[0395] The present invention further relates to a peptide according to the invention, wherein the peptide consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 300.

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

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

[0398] The present invention further relates to nucleic acids encoding peptides according to the invention, with the proviso that the peptide is not a complete (full-length) human protein.

[0399] The present invention further relates to a nucleic acid according to the 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 invention.

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

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

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

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

[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 said peptide containing SEQ ID NO: 1 to SEQ ID NO: 300, or said heterologous amino acid sequence.

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

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

[0408] The present invention further relates to the use according to the invention, wherein said cancer cells are HCC cells or other solid or hematological tumor cells, such as pancreatic cancer, brain tumor, renal 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 prognosis of HCC. The present invention also relates to the use of these novel targets for cancer therapy.

[0410] The terms "antibody" or "antibodies" are used broadly herein and include both polyclonal and monoclonal antibodies. In addition to intact 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 expressing an elevated level of a cancer marker gene, and / or inhibition of the activity of an HCC marker polypeptide).

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

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

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

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

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

[0416] Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

[0417] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate antibody fragments, particularly Fab fragments, can be achieved using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in International Publication No. 94 / 29348 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment produces an F(ab')2 fragment and a pFc' fragment.

[0418] Antibody fragments, whether attached to other sequences or not, may also contain insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide additional properties, such as removing / adding amino acids capable of disulfide bonding, increasing its biological lifespan, or altering its secretion characteristics. In any case, the antibody fragment must retain biologically active properties, such as binding activity or modulation of binding in the binding region. Functional or active regions of an antibody may be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to skilled practitioners and may include site-directed mutagenesis of nucleic acids encoding the antibody fragment.

[0419] The antibodies of the present invention may further comprise humanized or human antibodies. Humanized forms, such as non-human (e.g., murine) antibodies, are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.

[0420] Methods for humanizing non-human antibodies are well known in the art. Humanized antibodies typically have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which typically come from an "import" variable region. Humanization can essentially be performed by substituting rodent CDRs or CDR sequences for corresponding human antibody sequences. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable region has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues have been substituted by residues from analogous sites in rodent antibodies.

[0421] Transgenic animals (e.g., mice) can be used that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of antibody heavy chain joining region genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. 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 antibody of the present invention is preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmacologically acceptable carriers include saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles such as films, liposomes, or microparticles. Those skilled in the art will recognize that certain carriers may be more preferable 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 to the bloodstream in an effective form. The antibody can also be administered intratumorally or via a peritumoral route to exert a local as well as a systemic therapeutic effect. Local or intravenous injection is preferred.

[0424] Effective dosages and schedules for administering antibodies may be determined empirically, and performing such determinations is within the skill of those in the art. Those skilled in the art will understand that the antibody dose to be administered will vary depending, for example, on the subject receiving the antibody, the route of administration, the particular antibody type used, and other agents administered. A 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 factors described above. Following antibody administration, preferably to treat HCC, the efficacy of the therapeutic antibody can be assessed in a variety of ways well known to skilled practitioners. For example, standard tumor imaging techniques may be used to monitor the size, number, and / or distribution of cancers in the treated subject. A therapeutically administered antibody that halts tumor growth, causes tumor shrinkage, and / or prevents the development of new tumors compared to the disease course occurring in the absence of antibody administration is an effective antibody for cancer treatment.

[0425] Since the peptides mentioned in the above table of the invention, and thus their underlying polypeptides, are highly expressed in HCC and at fairly low to very low levels in normal cells, inhibition of 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 into therapeutic strategies for treating or preventing HCC, for example: 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, and the like, are preferred for inhibition, and for antibodies and / or TCRs directed thereto. 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, DDX 11, DDX12P, DECR2, NME4, DENND5B, DYM, EDC4, ERI3, FAM20A, FNDC3A, GPR107, GYG2, HEATR2, IFT81, KCTD3, SHKBP1, KIAA13 24L, KLHL24, MARCH6, MBTPS2, MIR1279, CPSF6, NOC4L, NXF1, PANK2, PCNXL3, PIPSL, PSMD4, PSMD14, SLC35B1, TCP11L2, THNS Most preferred for inhibition, and for antibodies and / or TCRs thereto, are L2, THOC2, TOMM5, TRAPPC6B, TRIM54, TRIM55, TRIM63, UGGT2, URB1, VPS54, WIZ, ZNF451, RFTN2, SCFD1, SERINC5, CCT7P2, CMAS, ANKS1A, C17orf70, CCT7, CDK5RAP2, CLPTM1, APOB, FASN, and / or COPA.

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

[0427] Antisense nucleic acid can be delivered by various approaches.For example, antisense oligonucleotide or antisense RNA can be directly administered to the subject (for example, by intravenous injection) in a form that allows it to be taken up by tumor cells.Alternatively, virus or plasmid vectors that code for antisense RNA (or RNA fragments) can be introduced into cells in vivo.Antisense effects can also be induced by sense sequences; however, the degree of phenotypic change is highly variable.The phenotypic change induced by effective antisense therapy can be evaluated, for example, by changes in target mRNA level, target protein level, and / or target protein activity level.

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

[0429] An alternative strategy for inhibiting the function of a protein selected from the group consisting of the above-mentioned proteins, most preferably APOB, FASN, and / or COPA, involves the use of nucleic acids (e.g., siRNAs, or nucleic acids encoding anti-protein antibodies or portions thereof, which can be transferred into cancer or other cells to result in intracellular antibody expression and secretion), proteins, or small molecules, or any other compounds that target the expression, translation, and / or biological function of this protein.

[0430] In the above-described methods involving administration and uptake of exogenous DNA into subject cells (i.e., gene conversion or transfection), the nucleic acids of the invention can be in the form of naked DNA, or the nucleic acid can be within a vector that delivers the nucleic acid to cells to inhibit HCC marker protein expression. The vector may be a commercially available preparation such as an adenoviral vector (Quantum Biotechnologies, Inc., Laval, Quebec, Canada). Delivery of the nucleic acid or vector into cells may be via a variety of mechanisms. As one example, delivery may be via liposomes, using 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. Additionally, the nucleic acid or vector of the invention may be delivered by electroporation, the technology of which is available from Genetronics, Inc., San Diego, US, as well as by using a Sonoporation device (ImaRx Pharmaceuticals, Inc.). The vector may be delivered in vivo by means of a system such as a retroviral vector system capable of packaging a recombinant retroviral genome. The recombinant retrovirus may then be used to infect cells, thereby delivering to the infected cells an antisense nucleic acid that inhibits the expression of a protein selected from the group consisting of the proteins described above. The exact method of introducing the modified nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are commonly available for this procedure, including the use of adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, and pseudotyped retroviral vectors. Physical transduction techniques, such as liposome delivery and receptor-mediated and other endocytosis mechanisms, may also be used. The present invention may be used in conjunction 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 coupled to radionucleotides (e.g., ribonucleotides) so that tumors can be localized using immunoscintigraphy. 111 In, 99 Tc, 14 C. 131 I, 3 H, 32 P or 35 In one embodiment, the antibody or fragment thereof binds to the extracellular domain of two or more targets of a protein selected from the group consisting of the above-mentioned proteins with an affinity (Kd) of less than 1 x 10 μM.

[0432] Diagnostic antibodies may be labeled with probes suitable for detection by various imaging methods. Probe detection methods include, but are not limited to, fluorescence, optical, confocal, and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin, and other fluorophores; radioisotopes; gold, gadolinium, and other lanthanides; paramagnetic iron; fluorine-18, and other positron-emitting radionuclides. Furthermore, probes may be bifunctional or multifunctional, making them detectable by one or more of the listed methods. These antibodies may be directly or indirectly labeled with the probes. Attaching probes to antibodies, particularly those well-recognized in the art, includes covalent coupling of the probe, incorporation of the probe into the antibody, and covalent coupling of a chelating compound for probe binding. For immunohistochemistry, diseased tissue samples may be fresh or frozen, or may be paraffin-embedded and fixed in a preservative such as formalin. Fixed or embedded sections containing the sample are contacted with labeled primary and secondary antibodies, and the antibodies are used to detect in situ protein expression.

[0433] Thus, as mentioned 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 cross-reactivity of T cells with said peptide. The peptide of the present invention has the ability to bind to human major histocompatibility complex (MHC) class I molecules or extended versions of said peptides to class II.

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

[0435] One skilled in the art would be able to assess whether T cells induced by a particular peptide variant 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, for example, the side chains of one or two amino acid residues are altered (e.g., by replacing them with the side chains of another naturally occurring amino acid residue, or with other side chains) so that the peptide can still bind to an HLA molecule substantially similarly to a peptide consisting of the given amino acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 300. For example, a peptide may be modified so that it binds to an HLA-A molecule in a manner substantially similar to a peptide consisting of the given amino acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 300. *It may be modified to at least maintain, if not improve, its ability to interact with and bind to the binding groove of an appropriate MHC molecule, such as -02 or -DR, and thus at least maintain, if not improve, its ability to bind to the TCR of an activated CTL.

[0437] These T cells can subsequently cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptide defined in embodiments 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 databases (Rammensee H. et al. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3-4):213-9), specific positions of HLA-binding peptides are typically anchor residues that form core sequences that fit the binding motif of the HLA receptor, which is defined by the polar, electrophysical, hydrophobic, and spatial properties of the polypeptide chain that constitutes the binding groove. Thus, one skilled in the art could modify the amino acid sequences set forth in SEQ ID NO:1-300 by retaining the known anchor residues and determine whether such variants maintain the ability to bind to MHC class I or II molecules. The variants of the invention maintain the ability to bind to the TCR of activated T cells, which can subsequently cross-react with and kill cells expressing a polypeptide containing the native amino acid sequence of the cognate peptide as defined in the embodiments of the invention.

[0438] An amino acid residue that does not substantially contribute to interaction with the T cell receptor may be modified by substitution with another amino acid whose incorporation does not substantially affect T cell reactivity or eliminate binding to the relevant MHC. Thus, except as provided, a peptide of the invention may be any peptide (we include by that term an oligopeptide or polypeptide) comprising an amino acid sequence as given or a portion or variant thereof.

[0439] An amino acid residue that does not substantially contribute to interaction with the T cell receptor may be modified by substitution with another amino acid whose incorporation does not substantially affect T cell reactivity or eliminate binding to the relevant MHC. Thus, except as provided, a peptide of the invention may be any peptide (we include by that term an oligopeptide or polypeptide) comprising an amino acid sequence as given or a portion or variant thereof.

[0440] Table 8A: Variants and motifs of 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 suitable. 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 containing the actual epitope. Preferably, the residues flanking the actual epitope are those that do not substantially affect the proteolytic cleavage required to expose the actual epitope during processing.

[0442] Thus, the present invention provides peptides and variants of MHC class I epitopes, wherein the peptides or variants have an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14, i.e., 8, 9, 10, 11, 12, 13, 14 amino acids, and 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 invention have the ability to bind to molecules of the human major histocompatibility complex (MHC) class I or II. The binding of peptides or variants to MHC complexes may be tested by methods known in the art.

[0444] In a particularly preferred embodiment of the invention, the peptide consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 300.

[0445] "Consisting essentially of" is intended to mean that the peptide according to the invention contains, in addition to a sequence according to any of SEQ ID NOs: 1 to 300 or a variant thereof, an additional N- and / or C-terminally located sequence of amino acids which do not necessarily constitute part of the peptide that functions as an epitope for an MHC molecule epitope.

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

[0447] Furthermore, the peptides or variants may be further modified to improve stability and / or binding to MHC molecules in order to elicit a stronger immune response. Methods for such optimization of peptide sequences are well known in the art and include, for example, the introduction of reverse peptide or non-peptide bonds.

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

[0449] Examples of non-peptide bonds are -CH-NH, -CHS-, -CHCH-, -CH=CH-, -COCH-, -CH(OH)CH-, and -CHSO-. U.S. Pat. No. 4,897,445 provides a method for the solid phase synthesis of non-peptide bonds (-CH-NH) in polypeptide chains, involving polypeptides synthesized by standard procedures and non-peptide bonds synthesized by reacting amino acids with amino aldehydes in the presence of NaCNBH.

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

[0451] Additionally, the peptides of the present invention may be synthesized to alter their configuration. For example, the D-isomer of one or more of the amino acid residues of the peptide may be used instead of the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the present invention may be substituted with one of the well-known non-naturally occurring amino acid residues. Changes such as these may serve to increase the stability, bioavailability, and / or binding activity of the peptides of the present invention.

[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, incorporated herein by reference. Chemical modifications of amino acids include, but are not limited to, acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide and sulfhydryl modification of carboxyl groups by performic acid oxidation of cysteine ​​to cysteic acid, mercury derivatization, mixed disulfide formation with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanate at alkaline pH. In this regard, those skilled in the art are referred to Chapter 15 of Current Protocols in Protein Science, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) for more detailed procedures regarding the chemical modification of proteins.

[0453] Briefly, for example, modification of arginyl residues in proteins is often based on the reaction of vicinal dicarbonyl compounds, such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione, to form adducts. Another example is the reaction of arginine residues with methylglyoxal. Cysteines can be modified without the simultaneous modification of other nucleophilic sites, such as lysines and histidines. Consequently, numerous reagents are available for cysteine ​​modification. Company websites, such as Sigma-Aldrich (http: / / www.sigma-aldrich.com), provide information on specific reagents.

[0454] Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during heat treatment of biopharmaceuticals. Specific glutamic acid residues can be modified using Woodward's reagent K. N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide can be used to form intramolecular crosslinks between lysine and glutamic acid residues. For example, diethylpyrocarbonate is a reagent for modifying histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. Reactants of lysine residues and other α-amino groups are useful, for example, in binding peptides to surfaces or protein / peptide crosslinking. Lysine is the attachment site for poly(ethylene)glycol and is the primary modification site for protein glycosylation. Methionine residues in proteins can be modified, for example, with iodoacetamide, bromoethylamine, and chloramine T.

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

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

[0457] Successful modification of therapeutic proteins and peptides with PEG is often associated with increased circulatory half-life, while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used to prepare hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

[0458] Peptides or variants in which the peptide is modified or contains non-peptide bonds are preferred embodiments of the present invention. Typically, peptides and variants (containing at least peptide bonds between amino acid residues) can be synthesized by Fmoc polyamide solid-phase peptide synthesis, as disclosed by 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 the 9-fluorenylmethyloxycarbonyl (Fmoc) group. 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 (for serine, threonine, and tyrosine), butyl esters (for glutamic acid and aspartic acid), butyloxycarbonyl derivatives (for lysine and histidine), trityl derivatives (for cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (for arginine). When glutamine or asparagine is the C-terminal residue, a 4,4'-dimethoxybenzhydryl group is utilized to protect the side chain amide functional group. The solid-phase support is based on a polydimethylacrylamide polymer composed of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and acryloylsarcosine methyl ester (functionalizing agent). The peptide-resin cleavable linker used is an acid-labile 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives, except asparagine and glutamine, which are added using the reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure, are added as their preformed symmetrical anhydride derivatives. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isatin test procedures.Upon completion of synthesis, the peptide is cleaved from the resin support and the side chain protecting groups are simultaneously removed by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture. Commonly used scavengers include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the constituent amino acids of the peptide being synthesized. A combination of solid-phase and solution-phase methods for peptide synthesis is also possible (see, e.g., Bruckdorfer et al., 2004 and references cited therein).

[0459] The trifluoroacetic acid is removed by evaporation under vacuum, followed by trituration with diethyl ether to yield the crude peptide. Any scavengers present are removed by a simple extraction procedure, which, upon lyophilization of the aqueous phase, gives the scavenger-free crude peptide. Reagents for peptide synthesis are commonly available, for example, from Calbiochem-Novabiochem (Nottingham, UK).

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

[0461] Analysis of peptides may be performed using thin layer chromatography, electrophoresis, particularly capillary electrophoresis, solid phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.

[0462] A further aspect of the present invention provides nucleic acids (e.g., polynucleotides) encoding the peptides or peptide variants of the present invention. The polynucleotide may be, for example, either single-stranded and / or double-stranded DNA, cDNA, PNA, RNA, or a combination thereof, or may be a native or stabilized form of polynucleotide, such as a polynucleotide with a phosphorothioate backbone, so long as it encodes the peptide, and may or may not contain introns. Of course, only peptides containing natural amino acid residues linked by naturally occurring peptide bonds may be encoded by the polynucleotide. A still further aspect of the present invention provides expression vectors capable of expressing the polypeptides according to the present invention.

[0463] A variety of methods have been developed for linking polynucleotides, particularly DNA, to vectors via complementary cohesive ends. For example, complementary homopolymeric sequences can be added to the DNA fragment to be inserted into the vector DNA. The vector and DNA fragment are then joined by hydrogen bonding between the complementary homopolymeric tails to form a recombinant DNA molecule.

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

[0465] A desirable method for modifying DNA encoding a polypeptide of the invention uses the polymerase chain reaction as disclosed in 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 to introduce the DNA into a suitable vector, for example, by engineering appropriate restriction enzyme recognition sites, or it may be used to modify the DNA in other useful ways known in the art. If a viral vector is used, a poxvirus or adenovirus vector is preferred.

[0466] The DNA (or RNA, in the case of retroviral vectors) may then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the invention. Thus, DNA encoding the peptide or variant of the invention may be used to construct expression vectors, which are then used to transform suitable host cells for the expression and production of the polypeptide of the invention, according to known techniques, appropriately modified in light of the teachings contained herein. Such techniques include, for example, those disclosed in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

[0467] The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend on the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

[0468] Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate direction and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational control regulatory nucleotide sequences recognized by the desired host, and such controls are generally available within the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the host will be transformed by the vector. Therefore, it is necessary to select transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, along with any necessary control elements, that encodes a selectable trait in transformed cells, such as antibiotic resistance.

[0469] Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

[0470] Host cells transformed with recombinant DNA of the present invention may then be cultured under appropriate conditions known to those of skill in the art and for a period of time sufficient to allow expression of the polypeptide, which may then be recovered, in light of the teachings disclosed herein.

[0471] Numerous expression systems are known, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. Preferably, the system will 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 poly(A) tail and a resistance marker such as neomycin. One example is pSVL, available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector, pMSG, is also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, typically available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405, and pRS406 are Yeast Integrating plasmids (YIps) that incorporate the yeast selectable markers HIS3, TRP1, LEU2, and URA3. Plasmid pRS413-416 is a Yeast Centromeric Plasmid (Ycps). CMV promoter-based vectors (e.g., from Sigma-Aldrich) offer transient or stable expression, cytoplasmic or secreted expression, and N- or C-terminal tagging with various combinations of FRAG, 3xFLAG, c-myc, or MAT. These fusion proteins allow recombinant proteins to be detected, purified, and analyzed. Dual-tagged fusions offer versatility in detection.

[0473] The strong human cytomegalovirus (CMV) promoter regulatory region elevates constitutive protein expression levels as high as 1 mg / L in COS cells. In less potent cell lines, protein levels are typically around 0.1 mg / L. The presence of the SV40 origin of replication results in high levels of DNA replication in SV40-permissive COS cells. For example, a CMV vector can contain the pMB1 (a derivative of pBR322) origin of replication in bacterial cells, a b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and an f1 origin. Vectors containing a preprotrypsin leader (PPT) sequence can direct secretion of FRAG fusion proteins into the culture medium for purification using anti-FRAG antibodies, resins, and plates. Other vectors and expression systems for use with 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 expressed sequentially (similar to a "beaded-beads" construct), whereby the peptides or peptide variants may be linked or fused together by a stretch of linker amino acids, e.g., LLLLLL, or may be linked without any additional peptide between them. These constructs may also be used for cancer therapy and may induce immune responses involving both MHC1 and MHCII.

[0475] The present invention also relates to host cells transformed with the polynucleotide vector constructs of the present invention. Host cells can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some circumstances, typically E. coli strains such as E. coli DH5, available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 (ATCC No. 31343), available from the American Type Culture Collection (ATCC), Rockville, MD, USA. Preferred eukaryotic host cells include yeast, insect, and mammalian cells, preferably vertebrate cells such as those derived from mouse, rat, monkey, or human fibroblastic and colonic cell lines. Yeast host cells include YPH499, YPH500, and YPH501, publicly available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryonic cells NIH / 3T3 available from the ATCC as CRL1658, monkey kidney-derived COS-1 cells available from the ATCC as CRL1650, and human embryonic kidney cells 293. Preferred insect cells are Sf9 cells, which can be transfected with baculovirus expression vectors. Reviews regarding the selection of appropriate host cells for expression can be found, for example, in the textbook "Methods in Molecular Biology: Recombinant Gene Expression, Reviews and Protocols," Part One, Second Edition, ISBN 978-1-58829-262-9 by Paulina Balbas and Argelia Lorence, and other references known to those skilled in the art.

[0476] Transformation of suitable cell hosts with the DNA constructs of the present invention can be achieved by well-known methods, which typically depend on the type of vector used. For the 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 useful for transfecting such cells, such as calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation is also useful for transforming and / or transfecting cells and is well known in the art for transforming yeast, bacterial, insect, and vertebrate cells.

[0477] Successfully transformed cells, i.e., cells containing a DNA construct of the present invention, can be identified by well-known techniques such as PCR. Alternatively, the presence of supernatant proteins can be detected using antibodies.

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

[0479] In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell or an antigen-presenting cell. APC loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) was approved by the U.S. Food and Drug Administration (FDA) on April 20, 2010 (sipuleucel-T) for the treatment of asymptomatic or minimally symptomatic metastatic HRPC (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 invention provides a method for producing a peptide or variant thereof, comprising culturing a host cell and isolating the peptide from the host cell or its culture medium.

[0481] In another embodiment, the peptides, nucleic acids, or expression vectors of the present invention are used in medical treatment. For example, the peptides or their variants may be formulated for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Preferred methods of peptide injection include sc, id, ip, im, and iv. Preferred methods of DNA injection include id, im, sc, ip, and iv. For example, a dose of 50 μg to 1.5 mg, preferably 125 μg to 500 μg, of peptide or DNA may be administered, depending on the peptide or DNA. Doses in this range have been successfully used in previous clinical trials (Walter et al., Nature Medicine 18, 1254-1261 (2012)).

[0482] Another aspect of the invention includes an in vitro method for producing activated T cells, comprising contacting ex vivo T cells with antigen-loaded human MHC molecules expressed on the surface of suitable antigen-presenting cells for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigen is used in conjunction with the antigen-presenting cells.

[0483] Preferably, the mammalian cells have no or reduced levels or function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter involved in antigen processing.

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

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

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

[0487] When antigen-presenting cells are transfected to express such epitopes, the cells preferably comprise an expression vector capable of expressing a peptide containing SEQ ID NO: 1 to SEQ ID NO: 300, or a variant amino acid sequence thereof.

[0488] Several other methods may be used to produce T cells ex vivo. For example, autologous tumor-infiltrating lymphocytes may 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) utilized autologous peripheral blood lymphocytes (PLBs) in the preparation of T cells. Furthermore, autologous T cells can also be produced by pulsing dendritic cells with peptides or polypeptides or infecting them with recombinant viruses. B cells may also be used to produce autologous T cells. Furthermore, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses may be used to prepare 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) described ex vivo priming of T cells using artificial antigen-presenting cells (aAPCs), which is also a suitable method for producing T cells against selected peptides. In this study, aAPCs were produced by conjugating preformed MHC:peptide complexes to the surface of polystyrene particles (microbeads) using biotin:streptavidin biochemistry. This system allows for precise control of MHC density on aAPCs, which allows for selective elicitation of high- or low-avidity antigen-specific T cell responses from blood samples with high efficiency. In addition to MHC:peptide complexes, aAPCs should have other proteins with costimulatory activity, such as anti-CD28 antibodies, conjugated to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, such as cytokines like interleukin-12.

[0489] Allogeneic cells may also be used in preparing T cells, and methods are detailed in WO 97 / 26328, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present antigens, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and vaccinia-infected target cells. Plant viruses may also be used (see, e.g., 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] Activated T cells directed against the peptides of the invention are useful in therapy. Thus, a further aspect of the invention provides activated T cells obtainable by the methods of the invention described above.

[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 NO:1 to SEQ ID NO:300.

[0492] Preferably, the T cells recognize cells by interacting with (e.g., binding to) an HLA / peptide complex via their TCR. The T cells are useful in a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, to which an effective number of activated T cells are administered. The T cells administered to the patient may be derived from the patient and activated as described above (i.e., they are autologous T cells). Alternatively, the T cells are derived from another individual rather than the patient. Of course, it is preferred if the individual is a healthy individual. By "healthy individual," we mean that the individual is generally in good health, preferably has a competent immune system, and more preferably is not suffering from any disease that can be easily tested for and detected.

[0493] In vivo, target cells for CD8+ T cells according to the present invention may be tumor cells (which sometimes express MHC class II) and / or stromal cells surrounding the tumor (tumor cells) (which 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 in a therapeutic composition. Accordingly, the present invention also provides a method for killing target cells in a patient that abnormally express a polypeptide comprising an amino acid sequence of the present invention, comprising the step of administering to the patient an effective number of T cells as defined above.

[0495] By "aberrant expression," we 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 originates, but is expressed in the tumor. By "overexpression," we mean that the polypeptide is present at a level at least 1.2 times that present in normal tissue; preferably at least 2 times, more preferably at least 5 or 10 times that present in normal tissue.

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

[0497] Protocols for this so-called adoptive transfer of T cells are well known in the art and are reviewed 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 present invention, i.e., peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor, or nucleic acid encoding same, is useful for treating disorders characterized by cells that escape the immune response. Thus, any molecule of the present 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 present invention or known molecules.

[0499] Preferably, the medicament of the present invention is a vaccine. It can be administered to a patient directly, to an affected organ, or systemically, via id, im, sc, ip, and iv routes; or it can be applied ex vivo to cells derived from the patient or a human cell line, which are subsequently administered to the patient; or it can be used ex vivo to select a subpopulation of immune cells derived from the patient, which are then re-administered to the patient. When nucleic acids are administered ex vivo to cells, it may be useful to transfect the cells to co-express an immunostimulatory cytokine, such as interleukin-2. The peptides may be substantially pure, combined with an immunostimulatory adjuvant (see below), or used in conjunction with an immunostimulatory cytokine, or administered via a suitable delivery system, such as liposomes. The peptides may also be conjugated to a suitable carrier, such as keyhole limpet hemocyanin (KLH) or mannan (see, e.g., WO 95 / 18145). The peptides may also be labeled, be fusion proteins, or be hybrid molecules. The peptides whose sequences are described in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of help provided by CD4 T helper cells. Thus, in addition to MHC class I epitopes that stimulate CD8 T cells, the fusion partner or section of the hybrid molecule suitably provides an epitope that stimulates CD4+ T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those identified in the present invention.

[0500] In one embodiment, the vaccine comprises at least one peptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 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 peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

[0501] In another embodiment, the vaccine comprises at least one peptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 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 peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

[0502] The polynucleotide may be substantially pure or contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for designing and introducing such nucleic acids are well known in the art. A review is provided, for example, by (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 immune responses is not fully understood. Suitable vectors and delivery systems include viral DNA and / or RNA, such as systems based on adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus, or hybrids containing two or more viral elements. Non-viral delivery systems, including cationic lipids and cationic polymers, are well known in the DNA delivery art. Physical delivery, such as via a "gene gun," may also be used. The peptide or peptides encoded by the nucleic acid may be, for example, a fusion protein with an epitope that stimulates T cells of the respective reverse CDRs, as described above.

[0503] The medicaments of the invention may also include one or more adjuvants. Adjuvants are substances that nonspecifically promote or enhance immune responses (e.g., immune responses to antigens mediated by CD8-positive T cells and helper T (TH) cells) and are therefore considered useful in the medicaments of the invention. Suitable adjuvants include 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligands, 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 pegylated derivatives thereof, IS patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide Adjuvants include, but are not limited to, 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 saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants specific for dendritic cells and their preparations (e.g., MF59) have been previously described (Allison and Krummel, 1995 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 implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, the entire contents of which are specifically incorporated herein by reference), and acting as immune enhancers (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich, 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 promote adjuvant effects in vaccine settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it promotes dendritic cell maturation and differentiation, leading to enhanced TH1 cell activation and potent cytotoxic T lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which is particularly necessary for inducing a strong response when the antigen is relatively weak. They also accelerate immune responses, allowing for a nearly two-order reduction in antigen dose in some experiments with antibody responses equivalent to those of the total vaccine dose without CpG (Krieg, 2006). U.S. Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) manufactured by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, may also be used.

[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; and cyclophosphamide, sunitinib, bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Immunoactive small molecules and antibodies, such as Trap, ZD2171, AZD2171, anti-CTLA4, and other antibodies that target critical structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor), and SC58175, may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation.

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

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

[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 a combination thereof.

[0509] The composition may be used for parenteral administration, such as subcutaneous, intradermal, or intramuscular administration, or for oral administration. For this purpose, the peptide and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. The composition may further contain excipients such as buffers, binders, blasting agents, diluents, flavors, lubricants, etc. The peptide may also be administered together with immune stimulants, such as cytokines. A comprehensive list of excipients that may be used in such compositions can be found, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients, 3rd Ed., 2000, American Pharmaceutical Association and Pharmaceutical Press. The composition may be used for the prevention, prophylaxis, and / or treatment of adenomatous or cancerous diseases. Representative formulations are found, for example, in EP 2112253.

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

[0511] The present invention provides (a) a container containing the above-described pharmaceutical composition in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; and (c) optionally, (i) instructions for use of the solution, or (ii) instructions for reconstitution and / or use of the lyophilized formulation.

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

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

[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., dual-chamber vials), syringes (such as dual-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 instructions on or associated with the container, which indicate instructions for reconstitution and / or use. For example, the label may indicate that the lyophilized formulation is to be reconstituted to a peptide concentration as described above. The label may further indicate that the formulation is useful for or intended for subcutaneous administration.

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

[0515] Upon mixing of the diluent and the lyophilized formulation, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg / mL / peptide (=75 μg) and preferably no more than 3 mg / mL / peptide (=1500 μg). The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions.

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

[0517] Preferably, the kits of the invention comprise a formulation of the invention packaged for use in conjunction with the co-administration of a second compound (such as an adjuvant (e.g., GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic factor or inhibitor, an apoptosis inducer or chelator, or a pharmaceutical composition thereof). The kit components may be premixed, or each component may be in a separate, distinct container prior to administration to a patient. The kit components may be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The kit components may also be provided as solids, which may be converted to a liquid by the addition of a suitable solvent, preferably provided in another, distinct container.

[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 will contain a second vial or another container to allow for separate administration. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit will contain a device (for example, one or more needles, syringes, eyedroppers, pipettes, etc.) to allow for the administration of the active substance of the present invention that is a component of the kit.

[0519] The formulation is suitable for administration of the peptide by any acceptable means, such as oral (enteral), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, administration is sc, and most preferably id, and may be by infusion pump.

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

[0521] The present invention further includes a method for producing a personalized medicine for an individual patient, comprising the step of producing a pharmaceutical composition comprising at least one peptide selected from the reservoir of pre-screened TUMAPs, wherein the at least one peptide used in the pharmaceutical composition is selected for suitability in the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be adapted to produce T cell clones for downstream applications such as TCR isolation, or soluble antibodies and other therapeutic options.

[0522] "Personalized medicine" shall mean a therapy specifically tailored for one individual patient, used exclusively for the treatment of such individual patient, including active personalized cancer vaccines and adoptive cell therapy using autologous patient tissue.

[0523] As used herein, the term "reservoir" refers to a group of peptides that have been pre-screened for immunogenicity and / or over-presentation in a particular tumor type. The term "reservoir" is not intended to imply that the specific peptides included in the vaccine are pre-manufactured and stored in a physical facility, although this possibility is also contemplated. It is expressly contemplated that peptides may be produced de novo for each personalized vaccine produced, or may be pre-manufactured and stored. The reservoir (e.g., in the form of a database) is composed of tumor-associated peptides that are highly overexpressed in tumor tissues of 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 reservoir may also contain peptides associated with HLA-A, HLA-B, and HLA-C alleles. * 02 and HLA-A * The vaccines may contain 24 labeled peptides. These peptides allow quantitative comparison of the magnitude of T cell immunity induced by TUMAPs, thus allowing important conclusions to be drawn about the vaccine's ability to induce anti-tumor responses. Secondly, they serve as important positive control peptides derived from "non-self" antigens in cases where no vaccine-induced T cell response to TUMAPs derived from "self" antigens is observed in patients. Thirdly, they may allow conclusions to be drawn about the patient's immunocompetence status.

[0524] TUMAPs for the reservoir are identified using an integrated functional genomics approach that combines gene expression analysis, mass spectrometry, and T-cell immunology (XPresident®). The approach ensures that only TUMAPs that are truly present on a high percentage of tumors but absent or minimally expressed in normal tissues are selected for further analysis. For initial peptide selection, HCC samples from patients and blood from healthy donors were analyzed in a stepwise approach: 1. HLA ligands from malignant agents were identified by mass spectrometry 2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis was used to identify gene overexpression in malignant tissues (HCC) compared with a range of normal organs and tissues. 3. The identified HLA ligands were compared with gene expression data. Peptides that were over- or selectively presented on tumor tissues, preferably encoded by selectively expressed or over-expressed genes 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 supporting the validity of the identified peptides as TUMAPs. 5. The relevance of overexpression at the mRNA level was confirmed by re-detection of selected TUMAPs from step 3 on tumor tissues and their lack of detection (or rare detection) on healthy tissues.

[0526] 6. To assess whether the selected peptides could induce in vivo T cell responses, 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 induced by the vaccine according to the present invention attacks cancers at different cell division stages and different developmental stages. Furthermore, different cancer-associated signaling pathways are attacked. This is an advantage over vaccines that may address only one or a few targets, leading to easy tumor adaptation to attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Therefore, the combination of several tumor-associated peptides ensures that every possible tumor has at least some of the targets. The composition may be composed of peptides that are specific to each HLA-A. * O2 and / or HLA-A * The 24-positive tumors were specifically designed to express several antigens, expected to cover several independent pathways required for tumor growth and maintenance. Two HLA class I alleles (A* 02 and A * For each peptide subset specific to the 24 antigens, this is independently confirmed based on underlying experimental analysis. Thus, the vaccine can be easily used "off the shelf" for larger patient populations. This means that pre-selection of patients to be treated with the vaccine can be limited to HLA typing and does not require any additional biomarker assessment of antigen expression, yet it is still certain that several targets are simultaneously attacked by the induced immune response, which is important for efficacy (Banchereau et al., 2001; Walter et al., 2012).

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

[0529] This approach is preferable for rare cancers and patients with rare expression profiles. In contrast to fixed-composition multipeptide mixtures, currently developed reservoirs allow for significantly higher matching of vaccines with the actual expression of antigens in tumors. Multitargeting approaches utilize several "off-the-shelf" peptides, selected individually or in combination, for each patient. Theoretically, an approach based on the selection of, for example, five different antigenic peptides from a library of 50 antigenic peptides alone would yield approximately 17 million possible drug product (DP) compositions.

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

[0531] HLA phenotype, transcriptomic, and peptidomic data are collected from patient tumor material and blood samples to identify the most relevant peptides for each patient, containing "reservoir" and patient-specific (i.e., mutated) TUMAPs. Peptides are selected that are selectively or overexpressed in patient tumors and, if possible, demonstrate strong in vitro immunogenicity when tested with the patient's individual PBMCs.

[0532] Preferably, peptides to be included in the vaccine are identified by a method comprising the steps of: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; (b) comparing the peptides identified in (a) with the peptide repository described above; and (c) selecting at least one peptide from the repository (database) that is related to the tumor-associated peptides identified in the patient. For example, TUMAPs presented by tumor samples are identified by the following steps: (a1) comparing expression data from the tumor sample with expression data from normal tissue samples corresponding to the tissue type of the tumor sample to identify proteins overexpressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with 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 aberrantly expressed by the tumor. Preferably, the sequences of the MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor sample and sequencing the eluted ligands. Preferably, the tumor sample and normal tissue are obtained from the same patient.

[0533] In addition to or as an alternative to using a reservoir (database) model to select peptides, TUMAPs may be identified de novo in patients and then included in vaccines. As one example, candidate TUMAPs may be identified in patients by (a1) comparing expression data from tumor samples with expression data from normal tissue samples corresponding to the histological type of the tumor sample to identify proteins overexpressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with 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 aberrantly expressed by the tumor. As another example, proteins containing mutations unique to tumor samples may be identified by comparison with normal counterpart tissue from individual patients, and TUMAPs specifically targeting the mutations may be identified. For example, the genomes of tumors and corresponding normal tissues may be sequenced by whole-genome sequencing. To discover nonsynonymous mutations in the protein-coding regions of genes, genomic DNA and RNA are extracted from tumor tissue, and normal, non-mutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMCs). The NGS approach applied is limited to resequencing of protein-coding regions (exome resequencing). For this purpose, exonic DNA from human samples is captured using a supplier-provided target enrichment kit, followed by sequencing, for example, by HiSeq2000 (Illumina). Additionally, tumor mRNA is sequenced for direct quantification of gene expression and for verification that 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 identified and prioritized by comparison with PBMC-derived germline diversity. The newly identified peptides can then be tested for immunogenicity as described above for the reservoir, and candidate TUMAPs that retain appropriate immunogenicity are selected for inclusion in a vaccine.

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

[0535] In one exemplary embodiment, peptides to be 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 peptides are selected for the personalized peptide-based vaccine, the vaccine is manufactured, preferably as 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 must be selected depending on the number of peptides included in the product. Equal amounts of the single peptide DMSO solutions are mixed to obtain a solution containing all peptides included in the product at a concentration of approximately 2.5 mg / ml per peptide. The mixed solution is then diluted 1:3 with water for injection to obtain a concentration of 0.826 mg / ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22 μm sterile filter to obtain the final bulk solution.

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

[0539] The present invention will now be illustrated, but nevertheless not limited, in the following examples which describe preferred embodiments. For purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0540] [Figure 1A]Over-representation of various peptides in normal tissues (dark gray) and HCC (light gray) is shown in Figure 1A. APOB, peptide: ALVDTLKFV(A*02) (SEQ ID NO: 7), from left to right: 1 adipose tissue, 3 adrenal glands, 2 arteries, 2 bone marrow, 7 brains, 3 breasts, 13 colons, 4 esophagi, 2 gallbladders, 3 gastrointestinal tracts, 3 hearts, 16 kidneys, 4 white blood cell samples, 45 lungs, 1 lymph node, 1 ovary, 7 pancreas, 1 peripheral nerve, 1 pituitary gland, 3 pleura, 1 prostate, 6 rectum, 3 skeletal muscles. 1B) ALDH1L1, peptide: KLQAGTVFV(A*02) (SEQ ID NO: 2), from left to right: 1 adipose tissue, 3 adrenal glands, 2 arteries, 2 bone marrow, 7 brains, 3 breasts, 13 colons, 4 esophagi, 2 gallbladder, 3 gastrointestinal tract, 3 hearts, 16 kidneys, 4 white blood cell samples, 45 lungs, 1 lymph node, 1 ovary, 7 pancreas, 1 peripheral nerve, 1 pituitary gland, 3 pleura, 1 prostate, 6 rectum, 3 skeletal muscles; Tissues: Figure 1C) C8B, peptide: AYLLQPSQF(A*24) (SEQ ID NO: 200), from left to right: 2 adrenal glands, 1 artery, 4 brain, 1 breast, 5 colon, 1 heart, 13 kidneys, 9 lungs, 3 pancreas, 2 rectum, 3 skin, 1 spleen, 12 stomachs, 1 thymus, 2 uterus, and 9 liver; Tissues: Figure 1D) RAD23B peptide: KIDEKNFVV (SEQ ID NO: 63), from left to right: 1 serosa, 1 adipose tissue, 3 adrenal glands, 2 arteries, 2 bone marrow, 7 brain, 3 breast, 1 3 colons, 2 gallbladders, 3 gastrointestinal tract, 3 hearts, 12 kidneys, 4 leukocytes, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 pancreas, 1 peripheral nerve, 1 pituitary gland, 3 pleura, 1 prostate, 6 rectums, 3 skeletal muscles, 3 skin, 4 spleens, 5 stomachs, 1 testis, 2 thyroid glands, 3 thyroid glands, 2 uterus, 2 veins, 4 esophagus; Figure 1E) RAD23B peptide: KIDEKNFVV (SEQ ID NO: 63), only samples for which the peptide was reported are shown: 5 cell lines, 1 normal tissue (1 adrenal gland), 16 cancer tissues (2 brain cancers, 4 liver cancers, 5 lung cancers, 1 rectal cancer, 1 bladder cancer, 3 uterine cancers) (from left to right);Figure 1F) RFNGRLPPDTLLQQV (SEQ ID NO: 92), only samples on which the peptide was reported are shown: 1 serous membrane, 1 adipose tissue, 3 adrenal gland, 2 artery, 2 bone marrow, 7 brain, 3 breast, 13 colon, 2 gallbladder, 3 gastrointestinal tract, 3 heart, 12 kidney, 4 leukocyte, 19 liver, 43 lung, 1 lymph node, 1 ovary, 6 pancreas, 1 peripheral nerve, 1 pituitary gland, 3 pleura, 1 prostate, 6 rectum, 3 skeletal muscle, 3 skin, 4 spleen, 5 stomach, 1 testis, 2 thyroid gland, 3 thyroid gland, 2 uterus, 2 vein, 4 esophagus; Figure 1G) RFNG Peptide: RLPPDTLLQQV (SEQ ID NO: 92), only samples for which the peptide was reported are shown: 2 cell lines, 2 normal tissues (2 adrenal glands), 17 cancer tissues (1 brain tumor, 1 breast cancer, 1 esophageal cancer, 5 liver cancers, 4 lung cancers, 1 ovarian cancer, 1 prostate cancer, 2 bladder cancer, 1 uterine cancer) (from left to right); Figure 1H). FLVCR1 peptide: SVWFGPKEV (SEQ ID NO: 104), 1 serous membrane, 1 adipose tissue, 3 adrenal glands, 2 arteries, 2 bone marrow, 7 brain, 3 breast, 13 colon, 2 gallbladder, 3 gastrointestinal , 3 heart, 12 kidney, 4 leukocyte, 19 liver, 43 lung, 1 lymph node, 1 ovary, 6 pancreas, 1 peripheral nerve, 1 pituitary gland, 3 pleura, 1 prostate, 6 rectum, 3 skeletal muscle, 3 skin, 4 spleen, 5 stomach, 1 testis, 2 thyroid, 3 thyroid, 2 uterus, 2 veins, 4 esophagus; Figure 1I) FLVCR1 peptide: SVWFGPKEV (SEQ ID NO: 104). Only samples on which the peptide was reported are shown: 9 cell lines, 1 normal tissue (1 small intestine), 16 cancer tissues (1 brain tumor, 1 breast cancer, 5 liver cancer, 5 lung cancer, 1 skin). 1 skin cancer, 1 stomach cancer, 1 bladder cancer, 1 uterine cancer) (from left to right); Figure 1J) IKBKAP peptide: LLFPHPVNQV (SEQ ID NO: 156) 1 serous membrane, 1 adipose tissue, 3 adrenal gland, 2 artery, 2 bone marrow, 7 brain, 3 breast, 13 colon, 2 gallbladder, 3 gastrointestinal tract, 3 heart, 12 kidney, 4 leukocyte, 19 liver, 43 lung, 1 lymph node, 1 ovary, 6 pancreas, 1 peripheral nerve, 1 pituitary gland, 3 pleura, 1 prostate, 6 rectum, 3 skeletal muscle, 3 skin, 4 spleen, 5 stomach, 1 testis, 2 thyroid gland, 3 thyroid gland, 2 uterus, 2 veins, 4 esophagus;Figure 1K) IKBKAP peptide: LLFPHPVNQV (SEQ ID NO: 156), only samples for which the peptide was reported are shown: 7 cell lines, 2 primary cultures, 1 normal tissue (1 colon), 34 cancer tissues (1 bone marrow cancer, 1 breast cancer, 1 colon cancer, 2 esophageal cancer, 2 leukemia cancers, 4 liver cancer, 11 lung cancer, 3 lymph node cancer, 5 ovarian cancer, 4 bladder cancer) (from left to right); Figure 1L) NKD1 peptide: FLDTPIAKV (SEQ ID NO: 47), 1 serous membrane, 1 adipose tissue, 3 adrenal gland, 2 artery, 2 bone marrow, 7 brain, 3 breast, 13 colon, 2 gallbladder, 3 gastrointestinal tract, 3 heart, 12 kidney 1 kidney, 4 leukocytes, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 pancreas, 1 peripheral nerve, 1 pituitary gland, 3 pleura, 1 prostate, 6 rectum, 3 skeletal muscle, 3 skin, 4 spleen, 5 stomach, 1 testis, 2 thyroid gland, 3 thyroid gland, 2 uterus, 2 veins, 4 esophagus; Figure 1M) NKD1 peptide: FLDTPIAKV (SEQ ID NO: 47). Only samples for which the peptide was reported are shown: 1 other disease (encephalocele), 2 normal tissues (1 lung, 1 spleen), 35 cancer tissues (5 brain cancers, 6 colon cancers, 1 esophageal cancer, 6 liver cancers, 9 lung cancers, 1 ovarian cancer, 1 prostate cancer, 4 rectal cancers, 2 stomach cancers) (from left to right); [Figure 1B] Same as above [Figure 1C] Same as above [Figure 1D] Same as above [Figure 1E] Same as above [Figure 1F] Same as above [Figure 1G] Same as above [Figure 1H] Same as above [Figure 1I] Same as above [Figure 1J] Same as above [Figure 1K] Same as above [Figure 1L] Same as above [Figure 1M] Same as above [Figure 2A]Representative expression profiles of the source genes of the present invention that are highly overexpressed or exclusively expressed in HCC are shown (relative expression compared to normal kidney) in normal tissues (dark gray) and a panel of 12 HCC samples (gray).Figure 2A) APOB, from left to right, 1 adrenal gland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1 white blood cell sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thyroid, 1 bladder, 1 uterine cervix, 1 uterus, 1 vein; Figure 2B) AMACR, from left to right, 1 adrenal gland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1 white blood cell sample, 1 liver, 1 lung, 1 lymph node, 1 ovary; Tissues of 1 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thyroid gland, 1 bladder, 1 uterine cervix, 1 uterus, and 1 vein: Figure 2C) ALDH1L1, from left to right: 1 adrenal gland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1 white blood cell sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thyroid gland, 1 bladder, 1 uterine cervix, 1 uterus, and 1 vein: Figure 2D). F GG, from left to right, 1 adrenal gland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1 white blood cell sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thyroid, 1 bladder, 1 uterine cervix, 1 uterus, 1 vein (tissue: Figure 2E). C8B, from left to right, 1 adrenal gland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1 white blood cell sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 pancreas, 1 placenta. Tissues included: 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thyroid gland, 1 bladder, 1 uterine cervix, 1 uterus, and 1 vein; and Figure 2F) HSD17B6, from left to right: 1 adrenal gland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1 white blood cell sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thyroid gland, 1 bladder, 1 uterine cervix, 1 uterus, and 1 vein. [Figure 2B] Same as above [Figure 2C] Same as above [Figure 2D] Same as above [Figure 2E] Same as above [Figure 2F] Same as above [Figure 3] Representative flow cytometry results after peptide-specific multimer staining are shown. For further explanation, see Example 4. [Figure 4] Representative flow cytometry results after peptide-specific multimer staining are shown. For further explanation, see Example 4. [Example]

[0541] Example 1: Identification and quantification of tumor-associated peptides displayed on the cell surface Tissue samples Patient tumor tissues were obtained from the Universitatsklinik fur Allgemeine, Viszeral-und Transplantationschirurgie, Tübingen, 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; and Asterand Europe, Royston Herts, United Kingdom. Informed consent was obtained from all patients before surgery. Tissues were shock-frozen immediately after surgery and stored at <-70°C until TUMAP isolation.

[0542] Isolation of HLA peptides from tissue samples HLA peptide pools from shock-frozen tissue samples were prepared by HLA-A peptide pooling according to a slightly modified protocol (Falk, K., 1991; Seeger, FHT, 1999). *It was obtained by immunoprecipitation from solid tissue using the O2-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 reversed-phase chromatography (nanoAcquity UPL C system, Waters), and the eluted peptides were analyzed in an LTQ-velos and fusion hybrid mass spectrometer (ThermoElectron) equipped with an ESI source. The peptide pool was directly loaded onto an analytical fused silica microcapillary column (75 μm i.d. × 250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) at a flow rate of 400 nL / min. Peptides were subsequently separated using a two-step 180-min binary gradient from 10% to 33% B at a flow rate of 300 nL / min. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). 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, a scan cycle began with a high-mass-accuracy full scan in the Orbitrap (R = 30,000), followed by an MS / MS scan of the five most abundant precursor ions, also in the Orbitrap (R = 7,500), with preselected ions dynamically excluded. Tandem mass spectra were interpreted using SEQUEST and additional manual adjustments. Identified peptide sequences were confirmed by comparison of the resulting native peptide fragmentation patterns with those of sequence-identical synthetic reference peptides.

[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 sequence identification results to combine quantitative data from different samples and tissues with peptide presentation profiles. Quantitative data were double-normalized according to central tendency, accounting for variations within technical and biological replicates. In this way, each identified peptide could be correlated with quantitative data, enabling relative quantification between samples and tissues. Furthermore, all quantitative data obtained for peptide candidates was manually inspected to ensure data consistency and confirm the accuracy of the automated analysis. For each peptide, a presentation profile was calculated showing the mean sample presentation as well as replicate variation. The profile juxtaposes the CLL samples to a baseline of normal tissue samples.

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

[0546] Table 8B: Presentation Score. The table lists peptides that are very highly over-represented on tumors compared to normal tissue panels (+++), highly over-represented on tumors compared to normal tissue panels (++), and over-represented on tumors compared to normal tissue panels (+). * = 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 genes encoding the peptides of the present invention The over- or specific expression of peptides on tumor cells compared to normal cells is sufficient for their usefulness in immunotherapy, and some peptides are tumor-specific, even though their origin proteins are also present in normal tissues. Nevertheless, mRNA expression profiling can increase the level of safety in selecting peptide targets for immunotherapy. In particular, for therapeutic options with high safety risks, such as affinity-matured TCRs, ideal target peptides are derived from proteins that are specific to tumors and not found on normal tissues.

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

[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 so that the RNA from each individual was equally weighted.

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

[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 performed 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 Labeling Kit (ENZO Diagnostics, Inc., Farmingdale, NY, USA) for the U133A array or the GeneChip IVT Labeling 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 with an Agilent 2500A GeneArray Scanner (U133A) or an Affymetrix Gene-Chip Scanner 3000 (U133 Plus 2.0), and data were analyzed with GCOS software (Affymetrix) using 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 provided by the software, with normal kidney samples arbitrarily set to 1.0. Representative expression profiles of the source 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 a normal tissue panel (+++), highly overexpressed in tumors compared to a normal tissue panel (++), or overexpressed in tumors compared to a 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 T cell-based therapy according to the present invention were further tested for their MHC binding ability (affinity). Individual peptide-MHC complexes were generated by UV-ligand exchange, in which UV-sensitive peptides were cleaved upon UV irradiation and exchanged with the peptide of interest being analyzed. Only peptide candidates that could effectively bind and stabilize peptide-receptor MHC molecules prevented MHC complex dissociation. To determine the yield of the exchange reaction, an ELISA based on detection of the light chain (β2m) of stabilized MHC complexes was performed. The assay was generally performed 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] 96-well MAXISorp plates (NUNC) were coated with 2 μg / ml streptavidin in PBS overnight at room temperature, washed four times, and blocked in 2% BSA containing blocking buffer for 1 hour at 37°C. * 0201 / MLA-001 monomer served as a standard covering a range of 15-500 ng / ml. For UV exchange reactions, peptide-MHC monomers were diluted 100-fold in blocking buffer. Samples were incubated for 1 hour at 37°C, washed four times, incubated with 2 μg / ml HRP-conjugated anti-β2m for 1 hour at 37°C, washed again, and detected in TMB solution quenched with NH2SO4. Absorbance was measured at 450 nm. For the generation and production of antibodies or their fragments, and / or T cell receptors or their fragments, candidate peptides exhibiting high exchange yields (preferably greater than 50%, most preferably greater than 75%) are generally preferred, as they exhibit sufficient binding activity to MHC molecules to prevent dissociation of the MHC complex.

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

[0556] Table 10B: MHC Class I Binding Scores HLA-A of HLA-class I-restricted peptides depending on peptide sequence * 02 or HLA-A * Conjugation to 24 was classified by peptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++. * = 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 carried out investigations using an in vitro T cell priming assay based on repeated stimulation of CD8+ T cells with artificial antigen-presenting cells (aAPCs) loaded with peptide / MHC complexes and anti-CD28 antibodies. In this way, the inventors have so far demonstrated that the 22 HLA-A * The immunogenicity of 0201-restricted TUMAPs could be demonstrated, demonstrating that these peptides are T cell epitopes against which CD8+ precursor T cells are present in humans (Table 11).

[0558] In vitro priming of CD8+ T cells To perform ex vivo stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD28 antibodies, we first isolated fresh HLA-A antigens through positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) from healthy donors obtained from the University Clinics Mannheim, Germany, after informed consent. * CD8+ T cells were isolated from the 02 leukapheresis product.

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

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

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

[0563] 800,000 beads / 200 μl were coated in a 96-well plate in the presence of 4 × 12.5 ng of different biotin pMHC, washed, and subsequently added with 600 ng of biotin anti-CD28 in a volume of 200 μl. 1 × 10 beads were cultured in 200 μl of TCM supplemented with 5 ng / ml IL-12 (PromoCell). 6 2 × 8 CD8+ T cells 5 Stimulation was initiated in 96-well plates by co-incubation with washed, coated beads (100 μg / well, ... Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis was performed using FlowJo software (Tree Star, Oregon, USA). Ex vivo stimulation of specific multimer+ CD8+ lymphocytes was detected by comparison with negative control stimulation. Immunogenicity of a given antigen was detected if at least one evaluable ex vivo stimulation well from a single healthy donor was found to contain specific CD8+ T cell lines after ex vivo stimulation (i.e., this well contained at least 1% specific multimer+ among CD8+ T cells, and the percentage of specific multimer+ cells was at least 10-fold higher than the median of the negative control stimulation).

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

[0565] Table 11A: In vitro immunogenicity of HLA class I peptides of the invention Representative results of in vitro immunogenicity experiments of the peptides of the present invention carried out 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 invention The HLA-A of the present invention has been carried out by the applicant. * Representative results of an in vitro immunogenicity experiment for 24 restricted peptides. Results of the in vitro immunogenicity experiment are shown. The percentage of positive wells and donors (within assessable range) are summarized as shown: 1-20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++ [Table 11B]

[0567] Healthy HLA-A * Representative results of peptide-specific ex vivo CD8+ T cell responses of O2 donors (Figure 3) CD8+ T cells were stimulated with HLA-A complexes containing 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. * After three 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 with 02 / ALDH1L1-001. Left panels (A, B, C) show unrelated A * Control staining of cells stimulated with O2 / peptide complexes is shown. Viable singlet cells were gated on CD8+ lymphocytes. Boolean gating helped exclude false positive events detected by multimers specific to different peptides. The frequency of specific multimer+ cells among CD8+ lymphocytes is shown.

[0568] Healthy HLA-A * Representative results of peptide-specific ex vivo CD8+ T cell responses of 24+ donors (Figure 4) CD8+ T cells were stimulated with anti-CD28 mAb and HLA-A complexed 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 three cycles of stimulation, A * 24 / KLHL24-001(A) or A * Detection of peptide-reactive cells was performed by 2D multimer staining with 24 / APOB-006 (B). Left panels (A and B) show unrelated A * Control staining of cells stimulated with 24 / peptide complexes is shown. Viable singlet cells were gated on CD8+ lymphocytes. Boolean gating helped exclude false positive events detected by multimers specific to different peptides. The frequency of specific multimer+ cells among CD8+ lymphocytes is shown.

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

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Claims

1. A peptide comprising an amino acid sequence selected from the group of SEQ ID NO:1 to SEQ ID NO:300, and variant sequences thereof that are at least 88% homologous to SEQ ID NO:1 to SEQ ID NO:300, wherein said variant binds to MHC and / or induces cross-reactivity of T cells with said variant peptide and pharmaceutically acceptable salts thereof, and wherein said peptide is not a full-length polypeptide.

2. 2. The peptide of claim 1, wherein the peptide has the ability to bind to a molecule 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. 3. The peptide or variant thereof according to claim 1 or 2, whose amino acid sequence comprises a stretch of amino acids according to the group of SEQ ID NO: 1 to SEQ ID NO:

300.

4. 4. The peptide or variant thereof according to any one of claims 1 to 3, wherein the peptide or variant thereof has an overall length of preferably 8 to 30, more preferably 8 to 16, most preferably 8 to 100 amino acids, and wherein the peptide consists of or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 1 to SEQ ID NO:

300.

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

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

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

8. An expression vector capable of expressing the nucleic acid of claim 7.

9. A peptide or variant thereof according to any one of claims 1 to 6, a nucleic acid according to claim 7, or an expression vector according to claim 8 for use in medicine.

10. A host cell comprising a peptide according to claims 1 to 6, a nucleic acid according to claim 7 or an expression vector according to claim 8, which is preferably an antigen-presenting cell such as a dendritic cell.

11. A method for producing the peptide or variant thereof according to any one of claims 1 to 6, comprising the steps of culturing the host cell according to claim 10, which presents the peptide according to claims 1 to 6 or expresses the nucleic acid according to claim 7, or the expression vector according to claim 8, and isolating the peptide or variant thereof from the host cell or a culture medium thereof.

12. 10. An in vitro method of producing activated T lymphocytes, comprising the step of ex vivo contacting T cells with antigen-loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificial construct that mimics an antigen-presenting cell, for a time sufficient to activate said T cells in an antigen-specific manner, wherein said antigen is a peptide according to any one of claims 1 to 9.

13. Activated T cells produced by the method of claim 12, which selectively recognize cells presenting a polypeptide comprising the amino acid sequence of any one of claims 1 to 5.

14. 14. A method of killing target cells in a patient, wherein the target cells present a polypeptide comprising an amino acid sequence according to any one of claims 1 to 5, comprising the step of administering to the patient an effective number of activated T cells as defined in claim 13.

15. A soluble or membrane-bound antibody which specifically recognizes a peptide or variant thereof according to any one of claims 1 to 5, preferably a peptide or variant thereof according to any one of claims 1 to 5 which binds to an MHC molecule. Optionally, the antibody carries a further effector function, such as an immunostimulatory domain or a toxin.

16. Use of a peptide according to any one of claims 1 to 6, a nucleic acid according to claim 7, an expression vector according to claim 8, a cell according to claim 10, an activated T lymphocyte according to claim 13, or an antibody and other binding molecule according to claim 15 for treating cancer or in the manufacture of a cancer therapeutic.

17. The cancer is most preferably 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, CYP4F 3, CYP4F11, CYP4F12, CYP4F2, MOCOS, A1CF, COL18A1, HPR, LBP, C19orf80, CFHR5, ITIH4, TMEM110, LARP4, LMF2, SLC10A5, SLC16A11, and more 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, NOC4 L, 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 another protein from which the peptides of SEQ ID NOs: 1 to 300 are derived are selected from the group of HCC, brain tumors, renal cancer, pancreatic cancer, colon or rectal cancer or leukemia and other tumors that exhibit overexpression of the protein.

18. (a) a container containing a pharmaceutical composition in solution or lyophilized form, the pharmaceutical composition comprising the peptide variant according to any one of claims 1 to 6, the nucleic acid according to claim 7, the expression vector according to claim 8, the cell according to claim 10, the activated T lymphocyte according to claim 13, or the antibody according to claim 15; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; (c) optionally, at least one more peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:346, and (d) Optionally, a kit comprising instructions for (i) use of said solution, or (ii) reconstitution and / or use of said lyophilized formulation.

19. 20. The kit of claim 18, further comprising one or more of: (iii) a buffer, (iv) a diluent, (V) a filter, (vi) a needle, or (V) a syringe.

20. 20. The kit of claim 18 or 19, wherein the peptide is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

300.

21. a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; b) comparing the peptides identified in a) with a peptide reservoir pre-screened for immunogenicity and / or over-representation in tumors compared to normal tissues; c) selecting at least one peptide from said reservoir that matches a tumor-associated peptide identified in said patient; and d) producing a personalized vaccine or compound-based or cell therapy product based on step c); 1. A method for producing a personalized 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 that are overexpressed or aberrantly expressed in the tumor sample; and a2) correlating the expression data with sequences of MHC ligands that bind to MHC class I / II molecules in the tumor sample to identify MHC ligands derived from proteins that are overexpressed or aberrantly expressed by the tumor; The method of claim 21 , wherein the nucleotide sequence is identified by:

23. 23. The method of claim 21 or 22, wherein the sequence of the MHC ligand is identified by eluting bound peptides from the MHC molecules isolated from the tumor sample and sequencing the eluted ligands.

24. The method of any one of claims 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 contained in the reservoir is aa. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods such as microarray or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue or a group of tissues; ab. Selecting peptides encoded by the selectively expressed or overexpressed genes detected in step aa; determining the induction of an in vivo T cell response by the selected peptides, comprising an in vitro immunogenicity assay using human T cells from a healthy donor or the patient; or b. Identifying HLA ligands from the tumor sample using mass spectrometry; bb. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods such as microarray or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue or a group of tissues; comparing the identified HLA ligands with the gene expression data; bd. Selecting peptides encoded by the selectively expressed or overexpressed genes detected in step bc; be. Re-detecting the TUMAPs selected from step bd on tumor tissue, and confirming that lack of detection or rare detection on healthy tissue correlates with overexpression at the mRNA level; bf. Determining the induction of an in vivo T cell response by the selected peptides, comprising an in vitro immunogenicity assay using human T cells from a healthy donor or the patient; The method of any one of claims 21 to 24, wherein the genotype is identified based on

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

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

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

30. A soluble or membrane-bound T cell receptor reactive with an HLA ligand, said ligand having at least 75% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

300.

31. 31. The T cell receptor of claim 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 of claim 30 or 31, wherein the amino acid sequence consists of any one of SEQ ID NOs: 1 to 300.

33. 33. The T cell receptor of any one of claims 30 to 32, wherein the T cell receptor is provided as a soluble molecule, optionally carrying an additional effector function such as an immunostimulatory domain or a toxin.

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

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

36. A host cell, preferably a T cell or an NK cell, comprising a nucleic acid according to claim 34, or a nucleic acid encoding an antibody according to claim 15, or an expression vector according to claim 35.

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

38. a) a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300; b) T cell receptors reactive with the peptides and / or peptide-MHC complexes described in 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 said nucleic acid; e) a host cell comprising the expression vector of d); f) activated T lymphocytes obtained by a method comprising the step of contacting T cells ex vivo with a peptide according to a) expressed on the surface of suitable antigen-presenting cells for a time sufficient to activate the T cells in an antigen-specific manner, and transferring these activated T cells into autologous or other patients; g) antibodies or soluble T-cell receptors reactive with the peptides and / or peptide-MHC complexes according to a) and / or cells presenting the peptides according to a), potentially modified, for example by fusion with immunostimulatory domains or toxins; 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 with an MHC molecule; i) a conjugated or labeled peptide or scaffold according to any 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 the peptide according to any one of claims 1 to 5 or a variant thereof, preferably the peptide according to any one of claims 1 to 5 or a variant thereof that binds to an MHC molecule.

40. 39. The pharmaceutical composition of claim 38, comprising at least one peptide, preferably all peptides, selected from SEQ ID NOs: 1, 2, 7, 225, 228, 301, 303, and 312.

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