Novel immunotherapy against several tumors, such as lung cancer, including nsclc

Abstract

To provide a new efficacious and safe treatment option for cancers such as lung cancer, in particular non-small-cell lung cancer (NSCLC), gastric cancers and brain tumors of different phenotypes.SOLUTION: The present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated cytotoxic T cell (CTL) peptide epitopes, alone or in combination with other tumor-associated peptides, which serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses. The present invention relates to more than 70 novel peptide sequences and their variants derived from HLA class I and HLA class II molecules of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptides, 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 cytotoxic T cell (CTL) peptide epitopes that, alone or in combination with other tumor-associated peptides, serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses. The present invention relates to 67 novel peptide sequences and their variants derived from HLA class I and HLA class II molecules of human tumor cells that can be used in vaccine compositions to elicit anti-tumor immune responses. [Background technology]

[0002] Lung cancer is the leading cause of cancer-related deaths in both men and women. Worldwide, lung cancer is the most common cancer in terms of both incidence and mortality. In 2008, there were 1.61 million new cases and 1.38 million deaths due to lung cancer. Incidence rates are highest in Europe and North America.

[0003] Since 1987, more women have died each year from lung cancer than from breast cancer. Mortality rates have continued to decline significantly for men, by approximately 1.9% per year from 1991 to 2003. Lung cancer mortality rates for women are approaching a plateau after several decades of continuous increases. These trends in lung cancer mortality reflect declining smoking rates over the past 30 years.

[0004] According to the National Cancer Institute (NCI), an estimated 230,000 new cases of lung cancer and 160,000 deaths due to lung cancer are predicted in the United States in 2013.

[0005] Lung cancer is clinically classified for treatment purposes as small cell (13%; SCLC) or non-small cell (87%; NSCLC). Prognosis is usually poor. Of all people with lung cancer, 15% survive 5 years after diagnosis. The disease is often advanced at the time of diagnosis. At presentation, 30-40% of NSCLC cases are stage IV, and 60% of SCLC cases are stage IV.

[0006] Treatment options are determined by the type of cancer (small cell or non-small cell) and stage and include surgery, radiation therapy, chemotherapy, and targeted biologic therapies such as bevacizumab (Avastin®) and erlotinib (Tarceva®). For localized cancer, surgery is usually the treatment of choice. Recent studies suggest that survival in early-stage non-small cell lung cancer is improved by chemotherapy following surgery. Because the disease is usually widespread by the time of detection, radiation therapy and chemotherapy are often used, sometimes in combination with surgery. Chemotherapy, alone or in combination with radiation, is the usual small cell lung cancer treatment of choice; with this regimen, the majority of patients experience remission, which can be long-lasting in some cases.

[0007] Largely due to advances in surgical techniques and combination therapies, the 1-year relative survival rate for lung cancer increased slightly from 37% in 1975-1979 to 42% in 2002. However, the 5-year survival rate for all stages combined is only 16%. In cases detected when the disease is still localized, the survival rate is 49%; however, only 16% of lung cancers are diagnosed at this early stage. Summary of the Invention [Problem to be solved by the invention]

[0008] Despite the above, there remains a need for new effective and safe treatment options for cancers such as lung cancer, particularly non-small cell lung cancer (NSCLC), gastric cancer, and brain tumors of different phenotypes, that improve patient well-being without the use of excessive chemotherapeutic or other drugs that can result in severe side effects.

[0009] The present invention utilizes peptides that stimulate a patient's immune system in a non-invasive manner to act as anti-tumor agents. [Means for solving the problem]

[0010] 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:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92, 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:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92, wherein said variant induces T cells that cross-react with said peptide or a pharmaceutically acceptable salt thereof, and wherein said peptide is not a full-length polypeptide.

[0011] The present invention further relates to peptides of the present invention comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 65, 76 to 84, and 92, or variants thereof that are at least 80%, preferably at least 90%, homologous (preferably at least 80% or at least 90% identical) to SEQ ID NOs: 1 to 65, 76 to 84, and 92, wherein the peptides or variants thereof have a total length of 8 to 100, preferably 8 to 30, and most preferably 8 to 14 amino acids for SEQ ID NOs: 1 to 65, 78 to 84, and 92, and 12 to 100, preferably 12 to 30, and most preferably 12 to 18 amino acids for SEQ ID NOs: 76 and 77.

[0012] The following table shows peptides according to the invention, their respective SEQ ID NOs, and the expected source proteins of these peptides. All peptides in Tables 1a, 1b, and 1c bind to the HLA-AA*02 allele, and peptides in Table 1d bind to the HLA-DR allele. The peptides in Table 1c are further useful in the diagnosis and / or treatment of gastric cancer and / or glioblastoma.

[0013] The Class II peptides of Table 1d are further useful in the diagnosis and / or treatment of gastric and other cancers that overexpress or overpresent MMP12 or POSTN.

[0014] The present invention therefore particularly relates to a peptide of the invention comprising the sequence set forth in SEQ ID NO: 76, or a variant thereof which is at least 80%, preferably at least 90% homologous (preferably at least 80% or at least 90% identical) to SEQ ID NO: 76, said peptide or variant thereof having a total length of 12 to 100, preferably 12 to 30, most preferably 12 to 18 amino acids. The present invention particularly relates to a peptide of the invention consisting of the sequence set forth in SEQ ID NO: 76.

[0015] The present invention also relates in particular to a peptide of the present invention comprising the sequence set forth in SEQ ID NO: 77, 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: 77, wherein said peptide or variant thereof has a total length of 12 to 100, preferably 12 to 30, most preferably 12 to 18 amino acids. The present invention particularly relates to a peptide of the present invention consisting of the sequence set forth in SEQ ID NO: 77.

[0016] [Table 1]

[0017] [Table 2]

[0018] [Table 3]

[0019] [Table 4]

[0020] [Table 5]

[0021] [Table 6]

[0022] 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.

[0023] 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: 65, SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92.

[0024] The present invention further relates to a peptide according to the invention, said peptide being modified and / or comprising a non-peptide bond.

[0025] 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 into the sequence of) an antibody, e.g. an antibody specific for dendritic cells.

[0026] The present invention further relates to a nucleic acid encoding a peptide according to the invention.

[0027] The present invention further relates to a nucleic acid according to the invention which is DNA, cDNA, PNA, RNA or a combination thereof.

[0028] The present invention further relates to an expression vector capable of expressing a nucleic acid according to the invention.

[0029] 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.

[0030] The present invention further relates to antibodies according to the invention and to methods for producing them.

[0031] The present invention further relates to T cell receptors (TCRs) according to the invention, in particular soluble TCRs (sTCRs), and methods for producing them.

[0032] The present invention further relates to a host cell comprising a nucleic acid or an expression vector according to the invention as described above.

[0033] The present invention further relates to a host cell according to the invention which is an antigen-presenting cell.

[0034] The present invention further relates to a host cell according to the invention, wherein the antigen-presenting cell is a dendritic cell.

[0035] The present invention further relates to a method for producing a peptide according to the invention, the method comprising the steps of culturing a host cell according to the invention and isolating the peptide from the host cell or its culture medium.

[0036] The present invention further relates to an in vitro method for generating activated cytotoxic T lymphocytes (CTLs), the method comprising contacting ex vivo CTLs 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 CTLs in an antigen-specific manner, wherein the antigen is any peptide according to the present invention.

[0037] The present invention further relates to a method according to the invention, wherein a sufficient amount of antigen is contacted with antigen-presenting cells, thereby loading the antigen onto class I or II MHC molecules expressed on the surface of appropriate antigen-presenting cells.

[0038] The present invention further relates to a method according to the present invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing the peptides containing SEQ ID NO: 1 to SEQ ID NO: 92, preferably SEQ ID NO: 1 to SEQ ID NO: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or the mutant amino acid sequences.

[0039] The present invention further relates to activated cytotoxic T lymphocytes (CTLs) generated 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.

[0040] The present invention further relates to a method of killing target cells in a patient, which target cells aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, the method comprising the step of administering to the patient an effective number of cytotoxic T lymphocytes (CTLs) according to the present invention.

[0041] The present invention further relates to the use of any of the described peptides, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention or activated cytotoxic T lymphocytes according to the invention as a medicament or in the manufacture of a medicament.

[0042] The present invention further relates to the use according to the invention, wherein said medicament is a vaccine.

[0043] The present invention further relates to a use according to the invention, wherein the medicament is effective against cancer.

[0044] The present invention further relates to the use according to the invention, wherein said cancer cells are lung cancer cells, gastric, gastrointestinal, colorectal, pancreatic or renal cancer cells, and glioblastoma cells.

[0045] The present invention further relates to specific peptide-based marker proteins and biomarkers according to the invention which may be used in the diagnosis and / or prognosis of lung, gastric, gastrointestinal, colorectal, pancreatic or renal cancer, and glioblastoma.

[0046] Furthermore, the present invention relates to the use of these novel targets for cancer therapy.

[0047] Stimulation of an immune response depends on the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor-associated antigens has raised the possibility of using the host's immune system to intervene in tumor growth. Various mechanisms that utilize both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

[0048] Certain elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. Isolation of cytotoxic T cells (CTLs) from tumor-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in innate immune defense against cancer. In particular, CD8+ T cells, which possess major histocompatibility complex (MHC) receptors and recognize class I peptides, usually 8–10 amino acid residues long, derived from proteins or defective ribosomal products (DRIPS) located in the cytosol, play a key role in this response. In humans, MHC molecules are also referred to as human leukocyte antigens (HLA).

[0049] There are two classes of MHC molecules: MHC class I molecules, which are found in most cells with a nucleus. Each MHC molecule is 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 that is used for noncovalent interactions with peptides. MHC class I presents peptides derived from proteolytic cleavage of DRIPs and larger peptides, which are mostly endogenous proteins. MHC class II molecules are found primarily on professional antigen-presenting cells (APCs) and primarily present peptides from exogenous or transmembrane proteins that are internalized by APCs during endocytosis and subsequently processed. Peptide-MHC class I molecule complexes are recognized by CD8-positive cytotoxic T lymphocytes bearing the appropriate T cell receptor (TCR), while peptide-MHC class II molecule complexes are recognized by CD4-positive helper T cells bearing the appropriate TCR. As a result, it is well known that TCR, peptide, and MHC are present in a 1:1:1 stoichiometric ratio.

[0050] CD4+ helper T cells play a key role in inducing and maintaining effective responses by CD8+ cytotoxic T cells. Identification of CD4+ T cell epitopes derived from tumor-associated antigens (TAAs) is crucial for drug development to trigger antitumor immune responses (Kobayashi et al., 2002; Qin et al., 2003; Gnjatic et al., 2003). At tumor sites, T helper cells maintain a CTL-compatible cytokine environment (Mortara et al., 2006) and attract effector cells, such as CTLs, NK cells, macrophages, and granulocytes (Hwang et al., 2007).

[0051] In the absence of inflammation, expression of MHC class II molecules is primarily restricted to cells of the immune system, particularly professional antigen-presenting cells (APCs), such as monocytes, monocyte-derived cells, macrophages, and dendritic cells. In cancer patients, tumor cells have surprisingly been found to express MHC class II molecules (Dengjel et al., 2006).

[0052] 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 CTL effector cells (i.e., CD8+ T lymphocytes).

[0053] Furthermore, it has been shown that CD4-positive T cells that recognize peptides from tumor-associated antigens presented by HLA class II molecules can suppress tumor progression through the induction of antibody (Ab) responses.

[0054] In contrast to tumor-associated peptides that bind to HLA class I molecules, only class II ligands for a few tumor-associated antigens (TAA) have been described so far.

[0055] Because constitutive expression of HLA class II molecules is usually restricted to immune system cells, the possibility of isolating class II peptides directly from primary tumors was not thought to be possible, but Dengjel et al. succeeded in identifying several MHC class II epitopes directly from tumors (WO 2007 / 028574, EP 1760088 B1; (Dengjel et al., 2006)).

[0056] 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, and transcription factors, which are expressed in each tumor cell and are upregulated compared to unaltered cells of the same origin.

[0057] Because both CD8- and CD4-dependent responses synergistically contribute to antitumor effects, the identification and characterization of tumor-associated antigens recognized by either CD8+ CTLs (ligand: MHC class I molecule + peptide epitope) or CD4+ T helper cells (ligand: MHC class II molecule + peptide epitope) is important for the development of tumor vaccines.

[0058] The present invention also relates to two new highly useful MHC class II peptides (set forth in SEQ ID NOs: 76 and 77), which are particularly useful in the diagnosis and / or treatment of gastric cancer, NSCLC, and other cancers that overexpress and / or overpresent MMP12 and POSTN, respectively.

[0059] The present invention also relates to so-called length variants of the MHC class II peptides of the present invention set forth in SEQ ID NO: 76 or 77. As described above, the peptide set forth in SEQ ID NO: 76 consists of the amino acid sequence INNYTPDMNREDVDYAIR (MMP12-peptide), and the peptide set forth in SEQ ID NO: 77 consists of the amino acid sequence TNGVIHVVDKLLYPADT (POSTN-002-peptide). Length variants are typically N- and / or C-terminally extended (1 to 5, preferably 1 to 10 amino acids) or N- and / or C-terminally truncated (1 to 5 amino acids) peptides that can still bind to MHC and elicit a cellular immune response as described herein. As known in the art, peptide binding to class II proteins is not size-constrained and can vary in length from 11 to 30 amino acids. The peptide-binding groove in MHC class II molecules is open at both ends, allowing for the binding of relatively longer peptides. While the "core" 9-residue long fragment contributes most to peptide recognition, flanking regions are also important for peptide specificity for class II alleles (see, e.g., Meydan C, et al., Prediction of peptides binding to MHC class I and II alleles by temporal motif mining. BMC Bioinformatics. 2013;14 Suppl 2:S13. Epub 2013 Jan 21). Those skilled in the art will be able to use the many software tools available (e.g., those described above) to identify binding motifs and thus identify opportunities to extend and / or delete the MHC class II peptide set forth in SEQ ID NO: 76 or 77 to generate length variants.

[0060] For a peptide to initiate (trigger) a cellular immune response, it must 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.

[0061] 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 recognized by T cells bearing specific T cell receptors (TCRs).

[0062] 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, and transcription factors, which are expressed in each tumor cell and are upregulated compared to unmodified cells of the same origin.

[0063] The current classification of tumor-associated antigens comprises the following major 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 named cancer-testis (CT) antigens because their members are expressed in histologically distinct human tumors and are present in normal tissues only on spermatocytes / spermatogonia of the testis and occasionally on the placenta. Because testicular cells do not express class I and II HLA molecules, these antigens cannot be recognized by T cells of normal tissues and are therefore considered immunologically tumor-specific. Well-known examples of CT antigens are MAGE family members or NY-ESO-1. b) Differentiation antigens: These TAAs are shared between tumors and the normal tissue 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. 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. 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. e) TAAs resulting from aberrant post-translational modifications: Such TAAs may arise from proteins that are neither specific nor overexpressed in tumors, but that nevertheless become tumor-associated through post-translational processes that are primarily active in tumors. Examples of this class arise from altered glycosylation patterns that result in novel epitopes in tumors, such as MUC1, or from protein splicing events during degradation that may or may not be tumor-specific. 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 induce T cell responses. Examples of such proteins are the human papillomavirus type 16 proteins E6 and E7, which are expressed in cervical cancer.

[0064] 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 relatively less in healthy tissues. Alternatively, in another 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 certain tumors but also be present at high concentrations (i.e., the number of copies of each peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins directly involved in the transformation of normal cells into tumor cells, for example, due to their function in cell cycle 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). In both cases, such peptides ("immunogenic peptides") derived from tumor-associated antigens should elicit a T-cell response in vitro or in vivo, so the presence of an epitope in the amino acid sequence of the antigen is essential.

[0065] 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.

[0066] Therefore, TAAs are the starting point for tumor vaccine development. Methods for identifying and characterizing TAAs are based on the use of CTLs that can be isolated from patients or healthy individuals, or they are based on the generation of differential transcriptional profiles or differential peptide expression patterns between tumor and normal tissues.

[0067] 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 for 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 perform effector functions ("effector T cells").

[0068] 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.

[0069] T helper cells 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.

[0070] Further uses against cancer are disclosed below in the description of the peptide protein according to the invention.

[0071] ATP-binding cassette, subfamily A (ABC1), member 13 (ABCA13) In humans, the ATP-binding cassette (ABC) family of transmembrane transporters contains at least 48 genes and seven gene subfamilies. The predicted ABCA13 protein consists of 5,058 amino acid residues, making it the largest ABC protein described to date (Prades et al., 2002). Knight et al. determined that ABCA13 protein is expressed in the hippocampus and cortex of mice and humans, both of which are associated with schizophrenia and bipolar disorder (Knight et al., 2009). The ABCA13 gene is located on chromosome 7p12.3, a region that contains loci involved in a genetic disorder affecting the pancreas (Schwachman-Diamond syndrome) and T-cell tumor invasion and metastasis (INM7), and is therefore a positional candidate for these conditions (Prades et al., 2002).

[0072] Matrix metallopeptidase 12 (macrophage elastase) (MMP12) MMP12, also known as human metalloelastase (HME) or macrophage metalloelastase (MME), is a zinc endopeptidase recognized for its ability to degrade elastin. It has a broad substrate range, encompassing other matrix proteins such as collagen, fibronectin, laminin, and proteoglycans, as well as non-matrix proteins such as α-1-antitrypsin. In asthma, emphysema, and chronic obstructive pulmonary disease (COPD), MMP12 may contribute to alveolar destruction and airway remodeling (Cataldo et al., 2003; Wallace et al., 2008). MMP12 has been implicated in macrophage migration and its ability to generate angiostatin from plasminogen contributes to angiogenesis inhibition (Chakraborti et al., 2003; Chandler et al., 1996; Sang, 1998). Like other metalloproteinases, MMP12 is involved in physiological processes such as embryogenesis, wound healing, and the menstrual cycle (Chakraborti et al., 2003; Labied et al., 2009), but also in pathological processes of tissue destruction.

[0073] Although data are based on small numbers of patients in some instances, there is ample evidence in the literature that MMP12 is frequently overexpressed in cancer (Denys et al., 2004; Hagemann et al., 2001; Ma et al., 2009; Vazquez-Ortiz et al., 2005; Ye et al., 2008). However, data are controversial regarding the impact of MMP12 overexpression on clinical parameters and prognosis. While it may be involved in matrix elution and therefore metastasis, it may also inhibit tumor growth through the production of angiostatin, which negatively affects angiogenesis (Gorrin-Rivas et al., 2000; Gorrin-Rivas et al., 1998; Kim et al., 2004).

[0074] In lung cancer, the consequences of MMP12 expression remain controversial. Overexpression of MMP12 in epithelial cells has been reported in inflammation-induced lung remodeling. Upregulation of MMP12 may play a role in the transition from emphysema to lung cancer (Qu et al., 2009). Animal studies suggest that MMP12 expression by stroma or macrophages suppresses lung tumor growth (Acuff et al., 2006; Houghton et al., 2006). However, some studies have shown that overexpression of MMP12 in lung tumors correlates with recurrence, metastatic disease, and shorter recurrence-free survival after resection (Cho et al., 2004; Hofmann et al., 2005).

[0075] Dystonin (DST) DST (BPAG1-e) encodes a member of the plakin protein family of adherens junction plaque proteins. BPAG1-e is expressed in epithelial tissues and anchors keratin-containing intermediate filaments to hemidesmosomals (HDs). HDs are multiprotein adhesion complexes that promote epithelial-stromal adhesion in stratified and complex epithelia. Regulation of their function is crucial in diverse biological processes, such as keratinocyte differentiation and migration in wound healing and cancer invasion, in which cells detach from the substrate and acquire a motile phenotype (Litjens et al., 2006).

[0076] Malignant melanoma is one of the most aggressive tumor types. BPAG1 is expressed in human melanoma cell lines (A375 and G361) and normal human melanocytes. Anti-BPAG1 autoantibody levels in the serum of melanoma patients were significantly higher than those in the serum of healthy volunteers (p<0.01). Anti-BPAG1 autoantibodies may be a promising marker for melanoma diagnosis (Shimbo et al., 2010). DST was associated with breast cancer invasion (Schuetz et al., 2006). The BPAG1 gene is likely involved in the proliferation, apoptosis, invasion, and metastasis of nasopharyngeal carcinoma (NPC) (Fang et al., 2005).

[0077] Matrix Remodeling Association 5 (MXRA5) MXRA5, also known as adlican, encodes an adhesive proteoglycan and belongs to a group of genes involved in ECM remodeling and cell-cell adhesion (Rodningen et al., 2008). The function of MXRA5 in cancer is unknown, but somatic mutations in MXRA5 have been identified in tumors from diverse tissues, including skin, brain, lung, and ovary. RT-PCR for adlican (MXRA5) confirmed microarray findings of overexpression in colon cancer compared with normal colon tissue (13 colorectal tumors and 13 normal tissues) (Zou et al., 2002). A recent study identified matrix-remodeling-associated 5 as the second most frequently mutated gene in NSCLC (after TP53) (Xiong et al., 2012).

[0078] Cyclin-dependent kinase 4 (CDK4) / cyclin-dependent kinase 6 (CDK6) CDK4 is a member of the Ser / Thr protein kinase family. It is a catalytic subunit of a protein kinase complex that is important for progression through the G1 phase of the cell cycle. The activity of this kinase is restricted to the G1-to-S phase transition during the cell cycle, and its expression is primarily regulated at the transcriptional level (Xiao et al., 2007). CDK4 and CDK6 enzymes and their regulators, such as cyclins, play important roles in embryogenesis, homeostasis, and carcinogenesis (Graf et al., 2010).

[0079] CDK4 protein expression levels were significantly increased in lung cancer tissues compared with normal tissues (P<0.001). Patients with higher CDK4 expression had significantly shorter overall survival than those with lower CDK4 expression. Multivariate analysis suggested that CDK4 expression levels were an independent prognostic indicator for survival in lung cancer patients (P<0.001). Furthermore, suppression of CDK4 expression significantly increased the expression of the cell cycle regulator p21 (Wu et al., 2011a). In lung cells expressing the endogenous K-Ras oncogene, ablation of Cdk4, but not Cdk2 or Cdk6, induced an immediate senescence response. This response did not occur in lungs expressing a single Cdk4 allele or in other K-Ras-expressing tissues. Targeting Cdk4 alleles in advanced tumors, detectable by computed tomography scanning, also induces senescence and prevents tumor progression ( Puyol et al., 2010 ).

[0080] Heterogeneous nuclear ribonucleoprotein H1(H)(HNRNPH1) / heterogeneous nuclear ribonucleoprotein H2(H')(HNRNPH2) These genes belong to the ubiquitously expressed subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs). hnRNPs are RNA-binding proteins that complex with heterogeneous nuclear RNAs (hnRNAs). These proteins appear to associate with pre-mRNAs in the nucleus and influence pre-mRNA processing and other aspects of mRNA metabolism and transport.

[0081] hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as a central splicing oncogenic switch that may mediate multiple important aspects of aggressive tumor behavior, including apoptosis evasion and invasiveness, reflecting the reactivation of stem cell patterns (Lefave et al., 2011). Small interfering RNA-mediated knockdown of hnRNPH or A-Raf resulted in MST2-dependent apoptosis. In contrast, forced expression of either hnRNPH or A-Raf partially counteracted etoposide-induced apoptosis (Rauch et al., 2010). Upregulation of hnRNPH / H′ is observed in a few tissues that normally express low levels of cytoplasmic hnRNPH / H′, such as pancreatic adenocarcinoma, hepatocellular carcinoma, and gastric cancer (Honore et al., 2004).

[0082] Tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (TANC2) The TANC family, comprised of TANC1 and TANC2, was identified in 2005 (Han et al., 2010). TANC1 deficiency in mice reduced hippocampal spine density and impaired spatial learning, whereas TANC2 deficiency resulted in embryonic lethality, suggesting that TANC family proteins are involved in regulating dendritic spines, spatial learning, and embryonic development. In contrast, overexpression of TANC1 and TANC2 in cultured neurons increases the density of dendritic spines and excitatory synapses. TANC1 and 2 proteins are primarily expressed in the brain, where a significant proportion of the protein is located within vesicle membranes (Han et al., 2010).

[0083] Ring finger protein 213 (RNF213) RNF213 encodes a protein containing a C3HC4-type RING finger domain, a specialized type of Zn finger that binds two zinc atoms and is thought to be involved in mediating protein-protein interactions.

[0084] The research group provided the first evidence suggesting the involvement of RNF213 in genetic susceptibility to moyamoya disease (Liu et al., 2011b). Another study showed that the RNF213 gene is associated with moyamoya disease susceptibility in a Han Chinese population (Wu et al., 2012).

[0085] Solute Carrier Family 34 (Sodium Phosphate), Member 2 (SLC34A2) SLC34A2 is a pH-sensitive sodium-dependent phosphate transporter. Upregulation of SLC34A2 gene expression in well-differentiated tumors may reflect the cell differentiation process in ovarian carcinogenesis and may serve as a potential marker for the diagnosis and prognosis of ovarian cancer (Shyian et al., 2011). RT-PCR confirmed increased expression of SLC34A2 in papillary thyroid carcinoma (Kim et al., 2010b). Breast cancer tissues also had significantly increased SLC34A2 gene expression compared with normal tissues (Chen et al., 2010a).

[0086] SET and MYND domain-containing 3 (SMYD3) Upregulation of SMYD3, a histone H3 lysine 4-specific methyltransferase, has previously been reported to play an important role in the proliferation of colorectal cancer (CRC) and hepatocellular carcinoma (HCC). Another study revealed that SMYD3 expression is also elevated in most breast cancer tissues. Similar to CRC and HCC, silencing SMYD3 with small interfering RNA (SMI) against this gene resulted in growth inhibition of breast cancer cells, suggesting that increased SMYD3 expression is also essential for breast cancer cell proliferation (Hamamoto et al., 2006). Knockdown of SMYD3 by RNA interference downregulated c-Met expression and inhibited HGF-induced cell migration and invasion (Zou et al., 2009). SMYD3 plays an important role in HeLa cell proliferation and migration / invasion, and may be a useful therapeutic target in human cervical cancer (Wang et al., 2008b).

[0087] Aldo-keto reductase family 1, member C1 (AKR1C1) AKR1C1 and AKR1C2 differ by only seven amino acid residues (Le et al., 2010). AKR1C1 and AKR1C2 regulate the activity of androgens, estrogens, and progesterone, as well as the occupancy and transactivation of their corresponding receptors (Penning et al., 2000; Steckelbroeck et al., 2004). AKR1C enzymes, except for AKR1C4, which is liver-specific, are expressed in different normal and pathological tissues and are therefore associated with several diseases, including lung, breast, prostate, and endometrial cancers, and myeloid leukemia (Brozic et al., 2011; Byrns et al., 2011). Sensitivity to cisplatin appears to be related to AKR1C levels in epithelial lung cancer cell lines (Chen et al., 2010b) and NSCLC patients (Kuang et al., 2012; Stewart, 2010). Thus, overexpression of AKR1C is an indicator of poor prognosis and chemotherapy resistance in human non-small cell lung cancer (NSCLC) (Wang et al., 2007). Overexpression of AKR1C2 is also associated with disease progression in prostate cancer (Huang et al., 2010). Depletion of AKR1C2 expression by RNAi inhibits tumor formation in vivo and in vitro, strongly suggesting that AKR1C2 siRNA may play an important role in blocking liver cancer development (Dong-Dong, 2007).

[0088] Reticulocalvin 1, EF-hand calcium-binding domain (RCN1) / Reticulocalvin 3, EF-hand calcium-binding domain (RCN3) Reticulocalbin 1 (RCN1) is a calcium-binding protein located within the lumen of the ER. Immunohistochemical studies have demonstrated widespread distribution of RCN in various fetal and adult organs, mostly in endocrine and exocrine organs. Overexpression of RCN may play a role in tumorigenesis, tumor invasion, and drug resistance (Fukuda et al., 2007). Reticulocalbin 1 (RCN1) is a cell surface-associated protein present on both endothelial (EC) and prostate cancer (PCa) cell lines. Cell surface RCN1 expression was upregulated by tumor necrosis factor-α treatment of bone marrow endothelial cells (Cooper et al., 2008). RCN1 is upregulated in colorectal cancer (CRC) and localizes within cancer cells or stromal cells adjacent to cancer cells. It may be a novel candidate CRC marker (Watanabe et al., 2008). RCN3 is a member of the CREC (Cab45 / reticulocalvin / ERC45 / calumenin) family of multiple EF-hand Ca2+-binding proteins localized in the secretory pathway (Tsuji et al., 2006). In oligodendroglioma, RCN3 has been suggested as a potentially important candidate gene, but little is known about its function (Drucker et al., 2009).

[0089] Interleukin 8 (IL8) IL-8 is a CXC family chemokine, one of the major mediators of the inflammatory response. This chemokine is secreted by several cell types. It functions as a chemoattractant and is also a potent angiogenic factor. CXC (ELR+) chemokine-like IL-8 induces angiogenesis and may be important in cancers with an angiogenic phenotype, such as NSCLC (Arenberg et al., 1997). Recently, tumor-derived IL-8 has been shown to function as an attractant for circulating tumor cells homing back to the primary tumor (breast cancer, colon cancer, and melanoma tumors), resulting in a more aggressive tumor phenotype (Kim et al., 2009). IL-8 levels are associated with lung cancer risk several years before diagnosis. The combination of IL-8 and CRP is a more robust biomarker for subsequent lung cancer prediction (Pine et al., 2011). Activating mutations in KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLC from elderly male smokers, NSCLC with pleural involvement, and KRAS-mutated adenocarcinoma; IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC (Sunaga et al., 2012).

[0090] Pyrimidine receptor P2Y, G-protein coupled, 6 (P2RY6) P2RY6 belongs to the G protein-coupled receptor family. This family has several receptor subtypes with different pharmacological selectivity, sometimes overlapping with various adenosine and uridine nucleotides. The P2Y6 subtype is expressed at particularly high levels in the placenta, suggesting that P2Y6 plays an important role in placental function. However, the cellular localization of P2Y6 within the placenta is unknown. P2Y6 may play an important role in trophoblast development, differentiation, and neoplasia (Somers et al., 1999). An important role for pyrimidine-activated P2Y receptors in the inflammatory response of lung epithelia has been suggested (Schafer et al., 2003).

[0091] HECT, UBA and WWE domain-containing 1, E3 ubiquitin protein ligase (HUWE1) HUWE1 encodes a member of the HECTE3 ubiquitin ligase family. The HECT domain is at the C-terminus and contains the active site cysteine ​​that forms the intermediate ubiquitin-thioester bond.

[0092] ARF-BP1 (HUWE1) is a critical mediator of both p53-independent and p53-dependent tumor suppressor functions of ARF. Therefore, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors, regardless of p53 status (Chen et al., 2005a). Inactivation of ARF-BP1 stabilized p53 and induced apoptosis (Chen et al., 2006). HUWE1 (HectH9) is overexpressed in multiple human tumors and is essential for the proliferation of a subset of tumor cells (Adhikary et al., 2005; Zhang et al., 2011a). In breast cancer, HUWE1 significantly correlated with relevant prognostic factors and clinical outcome (Confalonieri et al., 2009).

[0093] Versican (VCAN) VCAN is a member of the aggrecan / versican proteoglycan family. VCAN is known to associate with several molecules in the extracellular matrix, including hyaluronan, tenascin, fibulin-1, fibronectin, CD44 and L-selectin, fibrillin, integrins, and link protein (Zheng et al., 2004). VCAN is expressed in a variety of tissues. It is highly expressed during the early stages of tissue development and its expression decreases after tissue maturation. Its expression is also elevated during wound repair and tumor growth (Ghosh et al., 2010). Knockdown of VCAN in human lung adenocarcinoma (A549) cells by RNA interference significantly inhibited tumor growth in vivo but not in vitro (Creighton et al., 2005). VCAN is a direct target of p53. High expression of VCAN has also been found in the peritumoral stromal tissue of early-stage prostate cancer and of breast cancer, where it is associated with aggressive tumor behavior ( Yoon et al., 2002 ).

[0094] Drosha, ribonuclease type III (DROSHA) Drosha is a class 2 RNase III enzyme involved in initiating the processing of microRNAs (miRNAs), or short RNA molecules naturally expressed by cells, which interact with the RNA-induced silencing complex (RISC) to induce cleavage of complementary messenger RNA (mRNA) as part of the RNAi pathway, thereby regulating a wide variety of other genes. MicroRNA molecules are synthesized as long RNA primary transcripts known as pri-miRNAs, which are cleaved by Drosha to generate characteristic stem-loop structures approximately 70 base pairs long known as pre-miRNAs (Lee et al., 2003). Drosha exists as part of a protein complex called the microprocessor complex, which also contains the double-stranded RNA-binding protein Pasha (also known as DGCR8) (Denli et al., 2004), which is essential for Drosha activity and can bind single-stranded fragments of pri-miRNAs required for proper processing (Han et al., 2006). Human Drosha was cloned in 2000, when it was identified as a nuclear dsRNA ribonuclease involved in ribosomal RNA precursor processing (Wu et al., 2000). Drosha was the first human RNase III enzyme to be identified and cloned. Two other human enzymes involved in miRNA processing and activity are Dicer and Argonaute proteins. Both Drosha and Pasha are localized in the cell nucleus, where processing of pri-miRNAs to pre-miRNAs occurs. This latter molecule is then further processed by the RNase Dicer in the cytoplasm to become mature miRNAs (Lee et al., 2003). Drosha and other miRNA-processing enzymes may be important for cancer prognosis (Slack and Weidhaas, 2008).

[0095] Pleckstrin homology domain-containing, family A (phosphoinositide binding specific) member 8 (PLEKHA8) The phosphatidylinositol-4-phosphate adaptor 2 (FAPP2 = PLEKHA8) gene encodes a cytoplasmic lipid transferase containing a pleckstrin homology domain, which has been implicated in vesicle maturation and transport from the trans-Golgi apparatus to the plasma membrane (Cao et al., 2009). Introduction of a ribozyme targeting the FAPP2 gene into colon cancer cells induced their apoptosis in the presence of a Fas agonist antibody. Furthermore, FAPP2 siRNA-transfected glioma and breast tumor cells showed a significant increase in apoptosis (Tritz et al., 2009). Subsequent studies highlighted the role of FAPP2 as a lipid transport protein involved in glycosphingolipid metabolism in the Golgi apparatus (D'Angelo et al., 2012). Phosphoinositol 4-phosphate adaptor protein 2 (FAPP2) plays a key role in glycosphingolipid (GSL) biogenesis, using its C-terminal region to export newly synthesized glucosylceramide from the cytosol-facing glucosylceramide synthase in the cis-Golgi apparatus for further anabolic processing ( Kamlekar et al., 2013 ).

[0096] Acetyl-CoA carboxylase α (ACACA) ACACA is a biotin-containing enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis (Tong and Harwood, Jr., 2006). Upregulation of ACACA has been recognized in several human cancers, where it promotes lipogenesis to meet the demands of cancer cells for rapid growth and proliferation. Therefore, ACACA may be a potent target for cancer intervention, and inhibitors developed for the treatment of metabolic diseases may be potential therapeutic agents for cancer therapy (Wang et al., 2010a). Two studies have shown that silencing ACACA by RNA interference causes growth inhibition and induces cell death to a degree similar to that observed after silencing FASN gene expression (Brusselmans et al., 2005; Chajes et al., 2006). The allosteric inhibitor of ACACA, TOFA (5-tetradecyloxy-2-furoic acid), is cytotoxic and induces apoptosis in lung cancer cells NCI-H460 and colon cancer cells HCT-8 and HCT-15 (Wang et al., 2009a). Another highly potent ACACA inhibitor, soraphen A, blocks lipogenesis and promotes fatty acid oxidation in prostate cancer cells. The cancer cells cease proliferation and ultimately die (Beckers et al., 2007). These findings suggest that inhibition of lipogenesis itself, rather than malonyl-CoA accumulation, may also lead to cancer cell death, ultimately suggesting that ACACA may be a target for antitumor therapy (Brusselmans et al., 2005).

[0097] Integrin, alpha 11 (ITGA11) Integrins play important roles in diverse cellular and developmental processes, including cell proliferation, differentiation, and survival, as well as in carcinogenesis, cancer cell invasion, and metastasis. Integrin α11 (ITGA11 / α11) is localized in stromal fibroblasts and is commonly overexpressed in non-small cell lung cancer (NSCLC). α11 mRNA was overexpressed in both lung adenocarcinoma and squamous cell carcinoma (Wang et al., 2002). α11 plays an important role in the ability of fibroblasts to promote NSCLC cell growth in vivo, and such activity has been reported to be mediated, in part, by their regulation of IGF2 expression (Zhu et al., 2007). Regarding the clinicopathological characteristics of NSCLC patients, overexpression of hMTH1, SPD, HABP2, ITGA11, COL11A1, and CK-19 was significantly correlated with pathological stage (p<0.05). Furthermore, overexpression of hMTH1, SPD, ITGA11, and COL11A1 correlated with lymph node metastasis and poor prognosis ( Chong et al., 2006 ).

[0098] Collagen, type XII, alpha 1 (COL12A1) The COL12A1 gene encodes the α chain of type XII collagen, a member of the FACIT (fibril-associated collagen with interrupted triple helices) collagen family. Type XII collagen is a homotrimer found bound to type I collagen, and this binding is thought to modify the interaction between collagen I fibrils and the surrounding matrix (Oh et al., 1992). COL12A1 may be involved in basement membrane regulation, providing specific molecular bridges between fibrils and other matrix elements (Thierry et al., 2004). COL12A1 is expressed in various connective tissues, including the heart, placenta, lung, skeletal muscle, and pancreas (Dharmavaram et al., 1998), as well as in articular and epiphyseal cartilage (Gregory et al., 2001; Walchli et al., 1994; Watt et al., 1992). COL12A1 was downregulated in tumors with high microsatellite instability compared to the stable group with low or no microsatellite instability ( Ortega et al., 2010 ).

[0099] Elastase, neutrophil expressed (ELANE) Neutrophil elastase (or leukocyte elastase), also known as ELA2 (elastase 2, neutrophil), is a serine proteinase in the same family as chymotrypsin and has broad substrate specificity. It is secreted by neutrophils during inflammation to destroy bacteria and host tissues (Belaaouaj et al., 2000). Human neutrophil elastase (ELANE), a key player in the development of chronic obstructive pulmonary disease, has recently been implicated in the progression of non-small cell lung cancer (NSCLC). It can act at several levels: (i) intracellularly, by eliminating, for example, the adaptor molecule insulin receptor substrate-1 (IRS-1); (ii) on the cell surface, by hydrolyzing receptors such as CD40; and (iii) within the extracellular space, generating elastin fragments, i.e., morphoelastokines, which potently stimulate cancer cell invasiveness and angiogenesis (Moroy et al., 2012). Neutrophil elastase directly induced tumor cell proliferation in both human and mouse lung adenocarcinoma by accessing endosomal compartments within tumor cells and degrading insulin receptor substrate-1 (IRS-1) ( Houghton et al., 2010 ).

[0100] Serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3) Squamous cell carcinoma antigen (SCCA), also known as SERPINB3, is a member of the high-molecular-weight family of serine protease inhibitors (serpins) (Suminami et al., 1991). High levels have been reported in head and neck tissue cancers and other epithelial cancers (Torre, 1998). SCCA has been reported to be overexpressed in tumors compared with peritumoral tissue, suggesting its role as a potential marker for the histological detection of HCC (Pontisso et al., 2004). Serpin B3 / B4, particularly serpin B4, appears to play an important role in abnormal epithelial proliferation. Evaluation of serpin B3 / B4 may have prognostic value in predicting disease progression, especially in patients with increased susceptibility to lung cancer (Calabrese et al., 2012). SCCA1 (SERPINB3) inhibits cell death induced by lysosomal damage while activating caspase-8 independently of the death receptor apoptotic pathway, sensitizing cells to ER stress (Ullman et al., 2011). Several findings suggest that SERPINB3 plays an important role in inducing epidermal barrier disruption. SERPINB3 may be a key determinant of intraepidermal barrier function (Katagiri et al., 2010).

[0101] Kinesin family member 26B (KIF26B) Kinesins are a class of motor proteins found in eukaryotic cells. They move along microtubule filaments and are energized by ATP hydrolysis (hence, kinesins are ATPases). The kinesin family gene Kif26b is a downstream target of Sall1 (Nishinakamura et al., 2011). Kif26b is essential for kidney development by regulating the attachment of mesenchymal cells contacting the ureteric bud. In vitro overexpression of Kif26b resulted in increased cell adhesion through interaction with nonmuscle myosin (Terabayashi et al., 2012; Uchiyama et al., 2010).

[0102] Ankylosis progressive homolog (mouse) (ANKH) ANKH (human homolog of ankylosis progressiva) regulates inorganic pyrophosphate (PPi) transport across cell membranes (Wang et al., 2008a). Some data suggest that ANKH expression and function in vitro and in vivo are suppressed in hypoxic environments and that its effects are regulated by HIF-1 (Zaka et al., 2009). The human ANKH gene is expressed in vivo in a tissue-specific manner, with the highest levels of mRNA expression found in the brain, heart, and skeletal muscle (Guo et al., 2001). Mutations in the ANKH gene have been associated with autosomal dominant craniometaphyseal dysplasia (Kornak et al., 2010). ANKH was significantly upregulated in cervical cancer cell lines with amplification compared with those without amplification (Kloth et al., 2007). Genomic amplification of a region on chromosome arm 5p has been frequently observed in small cell lung cancer (SCLC), implying the presence of multiple oncogenes on this arm. Coe et al. described the identification of a microdeletion that evaded detection by conventional screening and the identification of TRIO and ANKH as novel putative oncogenes (Coe et al., 2005).

[0103] Nuclear RNA transport factor 1 (NXF1) In human cells, the mRNA export factor NXF1 is located in the nucleoplasm and nuclear pore complexes (Zhang et al., 2011b). Transport of mRNA from nuclear transcription sites to cytoplasmic translation sites is an essential process in eukaryotic gene expression. In human cells, the mRNA export factor NXF1 (also known as TAP) accompanies mRNA transcripts out of the nucleus by simultaneously binding to mRNA, mRNA adaptor proteins, and phenylalanine-glycine (FG) repeats of the nuclear pore complex (Kelly and Corbett, 2009). NXF1 is unique among nuclear export factors because it is a multidomain protein that shares no structural or mechanistic similarities with karyopherin proteins, which transport protein cargo, tRNA, and microRNAs through the NPC. NXF1-mediated mRNA export is a process independent of the GTPase Ran (Gruter et al., 1998). Nuclear export of mRNPs is mediated by transport factors such as NXF1, which bind to mRNPs and mediate their translocation through the nuclear pore (NPC) central channel by transient interactions with FG-nucleoporins (Wickramasinghe et al., 2010). mRNAs can be transported either by a bulk transport pathway involving NXF1 / TAP or by a more specialized pathway involving chromosome region maintenance 1 (CRM1) (Siddiqui and Borden, 2012).

[0104] Regulator of G protein signaling 4 (RGS4) RGS4 functions as a GTPase-accelerating protein and regulates μ and δ opioid receptor (MOR and DOR) signaling. Opioid agonist-induced downregulation of RGS4 occurs through the ubiquitin-proteasome pathway and may contribute to maintaining cellular homeostasis in morphine-dependent states (Wang and Traynor, 2011). RGS4 plays an important role in regulating β-cell function (Ruiz, I et al., 2010). Xie et al. proposed that RGS4 is a novel suppressor of breast cancer migration and invasion, a key step in the metastatic cascade (Xie et al., 2009). RGS4 is overexpressed in thyroid cancer. Effective downregulation of its expression level in thyroid cancer cells significantly attenuates their viability, suggesting an important role for RGS4 in thyroid carcinogenesis (Nikolova et al., 2008). RGS4 was found to be differentially expressed in human pancreatic tumor cell lines and to be a possible marker gene for local tumor invasion and liver metastasis of pancreatic cancer (Niedergethmann et al., 2007). Overexpression of RGS4 selectively inhibited G protein-mediated p38 MAPK activation, resulting in delayed and altered lung epithelial cell duct formation by reducing epithelial cell proliferation, migration, and vascular endothelial growth factor (VEGF) expression (Albig and Schiemann, 2005).

[0105] Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) GFPT2 is involved in neurite outgrowth, early neuronal development, neuropeptide signaling / synthesis, and neuronal receptors (Tondreau et al., 2008). Genetic variants in GFPT2 are associated with type II diabetes and diabetic nephropathy (Zhang et al., 2004). Furthermore, the association of SNPs in GFPT2 suggests that genes involved in regulating oxidative pathways may contribute significantly to diabetic chronic renal failure (Prasad et al., 2010). DNA methylation of the GFPT2 gene was examined in primary acute lymphoblastic leukemia (ALL) samples. Patients with multiple CpG island methylation had poorer overall survival (Kuang et al., 2008). GFPT2 plays a role in glutamine metabolism and has been observed to be more highly expressed in mesenchymal cell lines. Glutamine metabolism may play an important role in tumor progression, and inhibition of cellular metabolic pathways may be a form of epigenetic therapy (Simpson et al., 2012).

[0106] Brain endothelial cell adhesion molecule (CERCAM) CERCAM is localized to the endothelial cell surface (Starzyk et al., 2000) and maps to chromosome 9q34.11, a candidate 9q region identified as associated with familial idiopathic scoliosis (Miller et al., 2012). The CEECAM1 gene is widely transcribed within the nervous system and in several secretory tissues, including the salivary glands, pancreas, liver, and placenta (Schegg et al., 2009). The CERCAM protein is structurally similar to the ColGalT enzymes GLT25D1 and GLT25D2. However, its function remains unknown; it appears to be functionally distinct from the related GLT25D1 protein, and the protein does not function as a glycosyltransferase like the GLT25D1 and GLT25D2 proteins (Perrin-Tricaud et al., 2011).

[0107] UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) (GALNT2) GALNT2 catalyzes the first step in mucin-type O-glycosylation of peptides in the Golgi apparatus. These enzymes transfer N-acetylgalactosamine (GalNAc) from UDPGalNAc to the hydroxyl group of serine or threonine in target proteins (Peng et al., 2010). GALNT2 was constitutively expressed at low levels in most or all human adenocarcinoma cell lines from the pancreas, colon, stomach, and breast examined (Sutherlin et al., 1997). Studies have demonstrated that O-glycans and the GALNT gene play important roles in diverse biological functions and human disease development. Risk of epithelial ovarian cancer (Terry et al., 2010) and coronary artery disease (Willer et al., 2008) has been associated with single nucleotide polymorphisms in GALNT2. Aberrant glycosylation of cell surface glycoproteins, resulting from specific alterations in glycosyltransferase activity, is commonly associated with cancer invasion and metastasis. GALNT2 is involved in tumor migration and invasion in gastric cancer ( Hua et al., 2012 ), hepatocellular carcinoma (HCC) ( Wu et al., 2011b ), and human malignant glioma ( Liu et al., 2011a ).

[0108] Heterogeneous nuclear ribonucleoprotein M (HNRNPM) The HNRNPM gene belongs to the ubiquitously expressed subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs). HNRNPM is an abundant component of human hnRNP complexes, which can influence pre-mRNA splicing by regulating its own pre-mRNA splicing (Hase et al., 2006) or by affecting the regulation of alternative splicing of fibroblast growth factor receptor 2 (Hovhannisyan and Carstens, 2007). Proteomic analysis of in vitro purified spliceosomes has detected HNRNPM in the pre-spliceosomal H complex and throughout the spliceosomal assembly (Rappsilber et al., 2002; Wahl et al., 2009). HNRNPM participates in the spliceosomal machinery through its interaction with the CDC5L / PLRG1 spliceosomal subcomplex (Lleres et al., 2010). In human cancer cells, several results indicate that cytoplasmic retention of IMP-3 and HNRNPM results in a significant decrease in proliferation. The nuclear IMP-3-HNRNPM complex is important for the efficient synthesis of CCND1, D3, and G1 and for human cancer cell proliferation (Rivera et al., 2013).

[0109] Basonuclin 1 (BNC1) Basonuclin is a zinc finger protein with a very limited tissue distribution (Tseng, 1998). To date, basonuclin has been detected primarily in basal keratinocytes of stratified squamous epithelia (skin, oral epithelium, esophagus, vagina, and cornea) and in gametogenic cells of the testis and ovary (Tseng and Green, 1994; Weiner and Green, 1998). There is now considerable evidence that basonuclin is a cell-type-specific transcription factor for rRNA genes (rDNA). The zinc finger of basonuclin interacts with three evolutionarily conserved sites within the rDNA promoter (Iuchi and Green, 1999; Tseng et al., 1999). Epigenetic regulation by CpG methylation plays an important role in tumorigenesis and response to cancer therapy. BNC1 is hypomethylated in the radioresistant H1299 human non-small cell lung cancer (NSCLC) cell line. Suppression of BNC1 mRNA expression in H1299 cells also reduced the resistance of these cells to ionizing radiation (Kim et al., 2010a). Aberrant DNA methylation of BNC1 was also detected in chronic lymphocytic leukemia (CLL) samples (Tong et al., 2010). In renal cell carcinoma (RCC), BNC1 methylation was associated with a poorer prognosis, independent of tumor size, stage, or grade (Morris et al., 2010).

[0110] FK506-binding protein 10, 65 kDa (FKBP10) FK506-binding protein 10 (FKBP10) belongs to the FKBP-type peptidyl-prolyl cis / trans isomerase family. It is located in the endoplasmic reticulum and functions as a molecular chaperone (Ishikawa et al., 2008; Patterson et al., 2000). It is highly expressed during lung development and can be reactivated after lung injury in a coordinated manner with extracellular matrix proteins (Patterson et al., 2005).

[0111] Frizzled family receptor 1 (FZD1), Frizzled family receptor 2 (FZD2), Frizzled family receptor 7 (FZD7) The genes FZD2, FZD1, and FZD7 are all from the frizzled gene family; members of this gene family encode seven transmembrane domain proteins that are receptors for Wnt signaling proteins.

[0112] FZD2 gene expression appears to be developmentally regulated, being expressed at high levels in fetal kidney and lung, and adult colon and ovary (Sagara et al., 1998; Zhao et al., 1995).

[0113] The FZD1 protein contains a signal peptide, a cysteine-rich domain in the N-terminal extracellular region, seven transmembrane domains, and a C-terminal PDZ domain-binding motif. FZD1 transcripts are expressed in various tissues, including the lung, heart, kidney, pancreas, prostate, and ovary (Sagara et al., 1998). The expression of Frizzled 1 and 2 receptors has been found to be upregulated in breast cancer (Milovanovic et al., 2004).

[0114] The FZD7 protein contains an N-terminal signal sequence, 10 cysteine ​​residues typical of the cysteine-rich extracellular domains of Fz family members, seven putative transmembrane domains, and an intracellular C-terminal tail with a PDZ domain-binding motif. Expression of the FZD7 gene may downregulate APC function and enhance β-catenin-mediated signaling in poorly differentiated human esophageal carcinoma (Sagara et al., 1998; Tanaka et al., 1998).

[0115] ATPase, Ca++ transport, cardiac muscle, fast-twitch 1 (ATP2A1), ATPase, Ca++ transport, cardiac muscle, fast-twitch 2 (ATP2A2) Both genes (ATP2A1 and ATP2A2) encode SERCA Ca(2+)-ATPase. Sarcoplasmic reticulum (SR)1 / ER calcium ATPase (SERCA) is a calcium pump that couples ATP hydrolysis with calcium transport across the SR / ER membrane (MacLennan et al., 1997). SERCA is encoded by three homologous genes: SERCA1 (ATP2A1), SERCA2 (ATP2A2), and SERCA3 (Wu et al., 1995). Several lines of evidence have emerged indicating that SERCA may also have direct effects on the processes of apoptosis, differentiation, and cell proliferation (Chami et al., 2000; Ma et al., 1999; Sakuntabhai et al., 1999).

[0116] Mutations in ATP2A1 , which encodes SERCA1 , cause several autosomal recessive forms of Brody disease, characterized by impaired muscle relaxation during increased exercise ( Odermatt et al., 1996 ).

[0117] ATP2A2 is an ATPase associated with Darier's disease, a rare autosomal dominant hereditary skin disorder characterized by abnormal keratinization and acanthosis (Huo et al., 2010). Germline mutations in ATP2A2 may predispose to lung and colorectal cancer, and defective ATP2A2 genes may be involved in carcinogenesis (Korosec et al., 2006). Small cell lung cancer (H1339) and adenocarcinoma lung cancer (HCC) cell lines exhibited reduced ER Ca2+ content compared with normal human bronchial epithelial cell lines. The reduced Ca2+ content correlated with decreased expression of SERCA2, which transports calcium into the ER (Bergner et al., 2009). ATP2A2 may be a potential prognostic marker for colorectal cancer (CRC) patients. It was detected in circulating tumor cells (CTCs), and postoperative recurrence was significantly correlated with overexpression of the gene ( Huang et al., 2012 ).

[0118] Laminin, gamma 2 (LAMC2) Laminins, a family of extracellular matrix glycoproteins, are major non-collagenous components of basement membranes. They have been implicated in a wide variety of biological processes, including cell adhesion, differentiation, migration, signal transduction, neurite outgrowth, and metastasis. The LAMC2 gene encodes the laminin-5 γ2 chain, part of laminin-5, a major component of basement membrane domains. LAMC2 was frequently upregulated by promoter demethylation in gastric cancer (Kwon et al., 2011). LAMC2 was found to be overexpressed in vascular melanoma compared with avascular melanoma (Lugassy et al., 2009). LAMC2 is a biomarker for bladder cancer metastasis, and its expression level correlated with tumor grade (Smith et al., 2009b). The LAMB3 and LAMC2 genes were co-expressed in 21 (66%) of 32 non-SCLC cell lines but only in 1 (8%) of 13 SCLC cell lines. Co-expression of the LAMB3 and LAMC2 genes was also observed in all four primary non-SCLC cells examined, but not in the corresponding non-cancerous lung cells (Manda et al., 2000).

[0119] Heat shock 70 kDa protein 2 (HSPA2), heat shock 70 kDa protein 8 (HSPA8) HSPA2 has been identified as a potential tumor-promoting protein expressed at abnormal levels in a subset of human cancers, including breast cancer (Mestiri et al., 2001), cervical cancer (Garg et al., 2010a), bladder urothelial carcinoma (Garg et al., 2010b), nasopharyngeal carcinoma (Jalbout et al., 2003), and malignant tumors (Chouchane et al., 1997). Some levels of HSPA2 gene activity have also been observed in cell lines derived from several human cancers (Scieglinska et al., 2008), while silencing the HSPA2 gene in cancer cells resulted in growth arrest and reduced tumorigenic potential (Rohde et al., 2005; Xia et al., 2008). Furthermore, polymorphisms in the HSPA2 gene have been associated with an increased risk of developing lung cancer (Wang et al., 2010b). Overexpression of HSPA2 correlates with increased cell proliferation, poor differentiation, and lymph node metastasis in human breast, cervical, and bladder urothelial carcinomas (Garg et al., 2010a; Garg et al., 2010b; Mestiri et al., 2001).

[0120] The HSPA8 gene encodes a member of the heat shock protein 70 family, Hsc70, which contains both heat-inducible and constitutively expressed members. HSPA8 binds to nascent polypeptides and promotes correct protein folding (Beckmann et al., 1990). Hsc70 functions as a molecular chaperone, assisting in protein synthesis, folding, assembly, transport between cellular compartments, and degradation (Bukau and Horwich, 1998; Hartl and Hayer-Hartl, 2002). Hsc70 is expressed in nonmalignant breast cells as well as breast cancer cells (Kao et al., 2003; Vargas-Roig et al., 1998), and overexpression of Hsp / hsc70 in chemotherapy-resistant cancer cells (Ciocca et al., 1992; Lazaris et al., 1997) has prompted research into the role of these proteins as potential clinical markers (Ciocca and Calderwood, 2005). This secreted hsc70 chaperone has a potential role in cell proliferation, which may explain the higher tumor growth in cancer cells overexpressing cathepsin D (Nirde et al., 2010). Furthermore, Ruisin et al. reported an association between polymorphisms in this gene and lung cancer risk (Rusin et al., 2004).

[0121] Vacuolar protein sorting 13 homolog B (yeast) (VPS13B) VPS13B was identified as a peripheral membrane protein localized to the Golgi apparatus, where it overlaps with the cis-Golgi matrix protein GM130. Consistent with its subcellular localization, depletion of VPS13B using RNAi causes fragmentation of the Golgi ribbon into ministacks (Seifert et al., 2011). Kolehmainen et al. (2003) identified the COH1 gene, also known as VPS13B, within the Cohen syndrome risk region on chromosome 8q22 (Kolehmainen et al., 2003). Loss-of-function mutations in the VPS13B gene cause autosomal recessive Cohen syndrome (Seifert et al., 2011). Mutations in VPS13B and other genes have been described in gastric and colorectal cancers with microsatellite instability (An et al., 2012).

[0122] CSE1 chromosome segregation 1-like (yeast) (CSE1L) The cell apoptosis susceptibility (CSE1L) gene has been demonstrated to regulate multiple cellular mechanisms, including the mitotic spindle checkpoint, proliferation, and apoptosis. CSE1L is located in both the cytoplasm and the cell nucleus. Nuclear CSE1L regulates the transcriptional activity of p53, a major tumor suppressor protein (Rao et al., 2011; Tanaka et al., 2007). Cytoplasmic CSE1L binds to microtubules; this binding has been shown to stimulate invadopodia extension and promote tumor cell migration (Tai et al., 2010). CSE1L is highly expressed in most cancers, including benign and malignant cutaneous melanocytic lesions (Boni et al., 1999), endometrial cancer (Peiro et al., 2001), ovarian cancer (Brustmann, 2004), breast cancer (Behrens et al., 2001), and bladder urothelial carcinoma (Chang et al., 2012), and its expression has been shown to correlate with cancer progression. Silencing CSE1L may be a possible therapeutic approach for colorectal cancer (Zhu et al., 2013).

[0123] Dihydropyrimidinase-like 4 (DPYSL4) Dihydropyrimidinase-related protein 4 (DPYSL4) is known to be a regulator of hippocampal neuronal development. DPYSL4 is involved in the growth regulation, polarization, and differentiation of dental epithelial cells during tooth bud morphogenesis (Yasukawa et al., 2013). Several studies have demonstrated a role for DPYSL4 in reducing neurite outgrowth potential through inhibition of microtubule polymerization and have also revealed its novel association with vimentin during nuclear condensation prior to neuronal death (Aylsworth et al., 2009). The p53 tumor suppressor gene, which is frequently mutated in a wide variety of tumors, plays an important role in maintaining genomic integrity. Both DPYSL4 mRNA and protein expression were specifically induced by anticancer factors in p53-proficient cells. DPYSL4 is an apoptosis inducer regulated by p53 in response to DNA damage (Kimura et al., 2011).

[0124] Sec61γ subunit (SEC61G) SEC61γ, a heterotrimeric protein channel composed of the subunits SEC61α, β, and γ, is a member of the SEC61 translocon (Greenfield and High, 1999). The SEC61 complex forms transmembrane pores for the translocation of nascent polypeptides into the ER lumen and the integration of transmembrane proteins into the ER bilayer (Osborne et al., 2005). SEC61γ is essential for tumor cell survival and the cellular response to endoplasmic reticulum stress. Furthermore, it is highly overexpressed in malignant cells and nearly absent in normal cells (Lu et al., 2009). Knockdown of SEC61γ expression resulted in apoptosis, abrogation of EGFR / AKT survival signaling (Lu et al., 2009), and tumor cell growth inhibition (Neidert et al., 2012).

[0125] ORM1-like 1 (S. cerevisiae) (ORMDL1) Human genes (ORMDL1, ORMDL2, and ORMDL3) are widely expressed in adult and fetal tissues. They encode transmembrane proteins anchored in the endoplasmic reticulum, likely involved in protein folding in the ER. Using genome sequence analysis, Hjelmqvist et al. (2002) mapped the ORMDL1 gene to chromosome 2q32.2 (Hjelmqvist et al., 2002). ORMDL proteins are master regulators of ceramide biosynthesis in mammalian cells (Siow and Wattenberg, 2012). ORMDL1 is specifically downregulated following presenilin 1 (PS1) mutations (Araki et al., 2008).

[0126] Pecanex-like 3 (Drosophila) (PCNXL3) Pecanex-like protein (PCNXL3) is a multi-spanning membrane protein; it belongs to the pecanex family.

[0127] The PCNXL3 gene was mapped to the chromosomal region 11q12.1-q13. Three novel human tumor-associated translocation breakpoints were located in the chromosome 11q13 region between markers D11S4933 and D11S546. Therefore, PCNXL3 may be an 11q13-associated disease gene (van et al., 2000).

[0128] Small nuclear ribonucleoprotein 200 kDa (U5) (SNRNP200) Pre-mRNA splicing is catalyzed by the spliceosome, a complex of specialized RNA and protein subunits that removes introns from transcribed pre-mRNA fragments. The spliceosome consists of approximately 80 conserved proteins as well as the small nuclear RNA proteins (snRNPs) U1, U2, U4, U5, and U6. SNRNP200 is a gene required for the unwinding of the U4 / U6 duplex, an essential step in the catalytic activation of the spliceosome (Maeder et al., 2009). SNRNP200 expression has been detected in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Zhao et al., 2009). Mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP) (Benaglio et al., 2011; Liu et al., 2012).

[0129] SAM domain, SH3 domain and nuclear localization signal 1 (SAMSN1) SAMSN1 is a member of a novel gene family of putative adaptor and scaffold proteins containing SH3 and SAM (sterile alpha motif) domains. SAMSN1 is expressed in hematopoietic tissues, muscle, heart, brain, lung, pancreas, endothelial cells, and myeloma. Endogenous SAMSN1 expression has been shown to be upregulated in primary B cells upon differentiation and proliferation-inducing stimuli, and transduction experiments suggest a promoting role for SAMSN1 in B cell differentiation into plasma cells (Brandt et al., 2010). Cell lines and primary cells from acute myeloid leukemia and multiple myeloma patients express SAMSN1 (Claudio et al., 2001). SAMSN1 was downregulated in the large cell lung carcinoma cell line Calu-6 (Yamada et al., 2008). SAMSN1 was differentially expressed in ulcerative colitis-associated cancers ( Watanabe et al., 2011 ).

[0130] Signal Transducer and Activator of Transcription 2, 113 kDa (STAT2) STAT2, a novel trigger for colorectal and skin carcinogenesis, may act to increase gene expression and secretion of proinflammatory mediators, which in turn activate the oncogenic STAT3 signaling pathway (Gamero et al., 2010). STAT2 is a key mediator in the activation of type I IFN-induced apoptosis. More importantly, defects in STAT2 expression or nuclear localization could reduce the efficacy of type I IFN immunotherapy (Romero-Weaver et al., 2010). Lower STAT2 expression was detected in low-grade astrocytomas compared with high-grade astrocytomas. Results indicate a correlation between STAT and PPARγ signaling in glial tumors, further supporting the predicted important role of STATs in regulating the growth and differentiation of these tumors (Ehrmann et al., 2008).

[0131] CCR4-NOT transcription complex, subunit 1 (CNOT1) The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. CNOT1 plays a key role in expressing the enzymatic activity of the CCR4-NOT complex and is therefore important in regulating mRNA deadenylation and mRNA decay. CNOT1 depletion structurally and functionally degrades the CCR4-NOT complex, leading to mRNA stabilization, which in turn leads to increased translation and ER stress-mediated apoptosis. Ito et al. conclude that CNOT1 contributes to cell survival by ensuring the activity of the CCR4-NOT deadenylase (Ito et al., 2011). siRNA-mediated depletion of endogenous CNOT1 or other CCR4-Not subunits in breast cancer cells results in deregulation of ERα target genes (increased induction of the ERα target genes TTF1 and c-Myc). These findings define the function of the human Ccr4-Not complex as a transcriptional repressor of nuclear receptor signaling, which is relevant to understanding the molecular pathways involved in cancer ( Winkler et al., 2006 ).

[0132] Serine hydroxymethyltransferase 2 (mitochondrial) (SHMT2) The SHMT2 gene encodes a mitochondrial pyridoxal phosphate-dependent enzyme that catalyzes the reversible reaction of serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. The encoded product is primarily involved in glycine synthesis. In polygenic diseases such as lung cancer, gene-gene interactions are predicted to play an important role in determining the variability of disease phenotypes. The interactions between MTHFR677, MTHFR1298, and SHMT polymorphisms may have a significant impact on genetic instability in lung cancer patients. Regarding cytogenetic changes, lymphocytes from lung cancer patients exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [NNK] were shown to have a significantly increased frequency of cytogenetic damage in the presence of MTHFR677, MTHFR1298, and SHMT allelic variants (Piskac-Collier et al., 2011). A pharmacogenomic study on the role of polymorphisms in the SHMT gene in the efficacy of 5-FU and FOLFIRI protocols in patients with colorectal cancer revealed a significant effect that also resulted in changes in overall survival ( Timar et al., 2006 ).

[0133] Jun B proto-oncogene (JUNB) JunB is a member of the AP-1 (activator protein 1) family of dimeric transcription factors. AP-1 transcription factors are involved in cell proliferation, transformation, and death (Shaulian and Karin, 2002). JunB may be regulated through the NF-eB pathway, and HGF-induced upregulation of JunB may play an important role in regulating cell proliferation and invasion through MMP-9 expression (Lee and Kim, 2012). JunB appears to play an oncogenic role in lymphoma, particularly Hodgkin's lymphoma (Shaulian, 2010). JunB is an essential upstream regulator of p16 and contributes to maintaining cellular senescence, which prevents the malignant transformation of TAC. Thus, JunB clearly plays an important role in regulating prostate carcinogenesis (Konishi et al., 2008). JunB promotes tumor invasiveness and angiogenesis in VHL-deficient ccRCC ( Kanno et al., 2012 ).

[0134] Transforming, acidic coiled-coil-containing protein 3 (TACC3) (TACC3) TACC3 exists in a complex with ch-TOG (a gene overexpressed in colon and liver tumors) and clathrin, which bridges microtubules to kinetochore fibers. TACC3 is expressed in certain proliferative tissues, including testis, lung, spleen, bone marrow, thymus, and peripheral blood leukocytes. TACC3 expression is altered in several human tumor types. Intracellularly, TACC3 localizes to both centrioles and spindle microtubules, but not to astral microtubules (Hood and Royle, 2011). TACC3 expression correlates with p53 expression, and patients whose tumors highly expressed both TACC3 and p53 had a significantly poorer prognosis than patients whose tumors had low levels of expression of both immunostains (P = 0.006). Increased TACC3 expression may confer a growth advantage to NSCLC and contribute to tumor progression, and TACC3 expression has been suggested to be a strong prognostic indicator of clinical outcome in NSCLC (Jung et al., 2006). Tacc3 may also be a negative regulator of the Notch signaling pathway (Bargo et al., 2010).

[0135] RAD54 homolog B (S. cerevisiae) (RAD54B) The DNA repair and recombination protein RAD54B is encoded by the RAD54B gene in humans. RAD54 binds to double-stranded DNA and exhibits ATPase activity in the presence of DNA. Human RAD54B is a paralog of the RAD54 protein, which plays an important role in homologous recombination (HR). Homologous recombination (HR) is essential for the accurate repair of DNA double-strand breaks (DSBs) (Sarai et al., 2008). Knockdown of RAD54B, a gene known to be somatically mutated in cancer, causes chromosomal instability (CIN) in mammalian cells (McManus et al., 2009). Increased RAD54B gene expression is significantly associated with shorter cancer progression and poorer overall survival in GBM patients (Grunda et al., 2010).

[0136] Eukaryotic translation elongation factor 2 (EEF2) EEF2 encodes a member of the GTP-binding translation elongation factor family. This protein is an essential component of protein synthesis. It promotes the GTP-dependent translocation of nascent protein chains from the A site to the P site of the ribosome. EEF2 was highly expressed in lung adenocarcinoma (LADC) but not in adjacent non-tumorous lung tissue. Patients with high eEF2 expression had a significantly higher rate of early tumor recurrence and a significantly poorer prognosis, suggesting that eEF2 is an anti-apoptotic marker in LADC. Silencing eEF2 expression increased mitochondrial elongation, cellular autophagy, and cisplatin sensitivity. Furthermore, eEF2 was sumoylated in LADC cells, and eEF2 sumoylation correlated with drug resistance (Chen et al., 2011a). EEF2 inhibition causes a rapid cessation of protein synthesis, induces apoptosis, and ultimately leads to cell death, making it an attractive target for cancer therapy. siRNA-induced silencing of EEF2 resulted in specific cytotoxicity of tumor cells ( Chen et al., 2011b ; Wullner et al., 2008 ).

[0137] Cyclin A2 (CCNA2) CCNA2 belongs to the highly conserved cyclin family. Cyclins function as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns, which contribute to the temporal coordination of mitotic events (Deshpande et al., 2005). Human cyclin A2 is a key regulator of S-phase progression and entry into mitosis. CCNA2 binds to and activates CDC2 or CDK2 kinases, thus promoting both the G1 / S and G2 / M cell cycle transitions (Honda et al., 2012). Mutations, amplifications, and overexpression of this gene, which alter cell cycle progression, are frequently observed in a variety of tumors and may contribute to tumorigenesis (Cooper et al., 2009; Kars et al., 2011; Kim et al., 2011; Tompkins et al., 2011). Furthermore, it has been described that CCNA2 expression is associated with poor prognosis in several types of cancer (Yasmeen et al., 2003), and elevated expression of cyclin A correlates with shorter survival (Dobashi et al., 1998).

[0138] Neuroepithelial cell transforming 1 (NET1)41 NET1 is a member of the Rho guanine nucleotide exchange factor family. Members of this family activate Rho proteins by catalyzing the exchange of GTP for GDP. The protein encoded by NET1 interacts with RhoA in the cell nucleus and may have a role in DNA damage repair after ionizing radiation.

[0139] Breast adenocarcinoma cells do not express opioid receptors, but they do express the NET1 gene, which may promote cancer cell migration (Ecimovic et al., 2011). NET1 is upregulated in gastric cancer (GC) tissues and drives the invasive phenotype of this disease (Srougi and Burridge, 2011). NET1 plays a key role in GC cell migration and invasion, which are key aspects of GC progression (Bennett et al., 2011). Higher expression of RhoC and NET1 in human prostate cancer after short-term endocrine therapy suggests that RhoC and NET1 may be therapeutic targets for endocrine therapy (Kawata et al., 2012).

[0140] Chromosome 11 open reading frame 24 (C11orf24) C11orf24 was identified by Twells et al. (2001). The C11orf24 gene has no known similarity to other genes, and its function is unknown. Northern blot analysis detected high expression of a 1.9-kb transcript in the heart, placenta, liver, pancreas, and colon. Lower levels were detected in the brain, lung, skeletal muscle, kidney, spleen, prostate, testis, ovary, and small intestine, and very low levels were detected in the thymus and leukocytes (Twells et al., 2001). The 449-amino acid protein C11orf24 is located on chromosome 11q13, a region identified as a susceptibility region for multiple cancers (Gudmundsson et al., 2009; Purdue et al., 2011).

[0141] Regulator of chromosome condensation 1 (RCC1) Regulator of chromosome condensation 1 (RCC1) is a guanine nucleotide exchange factor for Ran GTPase. Localized production of Ran-GTP by RCC1 on chromatin is important for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation (Hitakomate et al., 2010). Several data suggest that chromosomal binding of mitotic regulators, such as RCC1, Mad2, and survivin, is essential for mitotic progression (Ho et al., 2008). Wong et al. found that nuclear RanGTP levels decrease during the early stages of apoptosis, which correlates with the immobilization of RCC1 on chromosomes. Therefore, they propose that RCC1 initiates apoptosis by reading the histone code generated by caspase-activated Mst1 and reducing nuclear RanGTP levels (Wong et al., 2009).

[0142] Melanoma antigen family F, 1 (MAGEF1) Most known members of the MAGE (melanoma-associated antigen) superfamily are expressed in tumors, testes, and fetal tissues, which is described as a cancer / testis expression pattern (MAGE subgroup I). MAGE subgroup I peptides have been successfully used in peptide and DC vaccination (Nestle et al., 1998; Marchand et al., 1999; Marchand et al., 1999; Marchand et al., 1995; Thurner et al., 1999). In contrast, several MAGE genes (MAGE subgroup II), such as MAGEF1, are widely expressed in all adult and fetal tissues tested and in numerous tumor types, including ovarian, breast, cervical, melanoma, and leukemia (Nestle et al., 1998; Marchand et al., 1999; Marchand et al., 1999; Marchand et al., 1995; Thurner et al., 1999). Nevertheless, overexpression of MAGEF1 could be detected in NSCLC (Tsai et al., 2007) and in 79% of a Taiwanese cohort of colorectal cancer patients (Chung et al., 2010).

[0143] Non-SMC condensin I complex, subunit D2 (NCAPD2) Condensin is a heteropentameric complex originally identified as a component of mitotic chromosomes. NCAPD2 is an essential component of the human condensin complex required for mitotic chromosome condensation. NCAPD2 depletion affects chromosome alignment during metaphase and delays the transition to anaphase (Watrin and Legagneux, 2005). Recent linkage and association studies suggest that the chromosome 12p13 locus likely harbors genetic variants that predispose to Alzheimer's disease (AD). Single-marker linkage revealed that two SNPs in NCAPD2 (rs7311174 and rs2072374) exhibited nominally significant p-values ​​(p = 0.0491 and 0.0116, respectively). These genetic analyses provide evidence that the chromosome 12p13 locus is associated with AD in a Chinese population (Li et al., 2009).

[0144] Chromosome 12 open reading frame 44 (C12orf44) By searching databases for Drosophila Atg13-binding protein orthologs, Mercer et al. (2009) identified human ATG101, also known as C12orf44 (Mercer et al., 2009). The ATG101 gene was mapped to chromosome 12q13.13. The putative 218-amino acid protein was predicted to be a cytoplasmic hydrophilic protein (Hosokawa et al., 2009). Macroautophagy is a catabolic process involving lysosome-mediated degradation of cytoplasmic proteins, organelles, and macromolecules. ATG proteins, such as ATG101, are required for the formation of autophagosomes, double-membrane vesicles that surround and sequester cytoplasmic cargo before fusion with lysosomes. ATG101 (C12orf44) is essential for autophagy (Mercer et al., 2009).

[0145] HECT and RLD domain-containing E3 ubiquitin protein ligase 4 (HERC4) HERC4 belongs to the HERC family of ubiquitin ligases, all of which contain a HECT domain and at least one RCC1 (MIM179710)-like domain (RLD). The 350-amino acid HECT domain is predicted to catalyze thioester formation with ubiquitin and transfer it to a substrate, and the RLD is predicted to act as a guanine nucleotide exchange factor for small G proteins (Hochrainer et al., 2005). The E3 ubiquitin ligase Herc4 is widely expressed in all tissues but is most highly expressed in the testis, particularly during spermatozoonogenesis. Herc4 ligase is required for proper sperm maturation and cytoplasmic droplet removal to become fully functional (Rodriguez and Stewart, 2007).

[0146] Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) IGF2BP3 is a member of the insulin-like growth factor II mRNA-binding protein family, which is implicated in mRNA localization, turnover, and translational control. The protein contains several KH (K homology) domains, which are important for RNA binding and are known to be involved in RNA synthesis and metabolism. Expression occurs primarily during embryonic development and has been described in several tumors. IGF2BP3 is therefore considered an oncofetal protein (Liao et al., 2005). IGF2BP3 may promote tumor cell proliferation by enhancing IGF-II protein synthesis and by inducing cell adhesion and invasion through stabilization of CD44 mRNA (Findeis-Hosey and Xu, 2012). Furthermore, IGF2BP3 expression has been examined in numerous human neoplasms, and evidence has emerged that it mediates migration, invasion, cell survival, and tumor metastasis (Jeng et al., 2009; Kabbarah et al., 2010; Li et al., 2011; Liao et al., 2011; Lu et al., 2011; Hwang et al., 2012; Samanta et al., 2012), and it may also be involved in angiogenesis (Suvasini et al., 2011; Chen et al., 2012). In lung adenocarcinoma, higher IGF2BP3 expression can be detected in moderately or poorly differentiated adenocarcinoma, which may be associated with aggressive biological behavior (Findeis-Hosey et al., 2010; Beljan et al., 2012; Findeis-Hosey and Xu, 2012).

[0147] Cell division cycle 6 homolog (S. cerevisiae) (CDC6) The CDC6 protein functions as a regulator of the early stages of DNA replication. It is localized in the cell nucleus during G1 of the cell cycle but translocates to the cytoplasm at the onset of S phase. CDC6 is also thought to regulate replication checkpoint activation in higher eukaryotic cells through its interaction with ATR (Yoshida et al., 2010). CDC6 is essential for DNA replication, and its deregulation is involved in carcinogenesis. Downregulation of CDC6 by RNA interference (RNAi) has been shown to impair cell proliferation and promote apoptosis (Lau et al., 2006). Overexpression of CDC6 has been observed in several types of cancer. Examples of cancer types that overexpress CDC6 are gastric cancer (Tsukamoto et al., 2008), brain tumors (Ohta et al., 2001), oral squamous cell carcinoma (Feng et al., 2008), cervical cancer (Wang et al., 2009b), and malignant mesothelioma (Romagnoli et al., 2009).

[0148] Fibroblast activation protein, alpha (FAP) Fibroblast activation protein (FAP) is a type II integral membrane glycoprotein belonging to the serine protease family. The putative serine protease activity of FAPα and its in vivo induction pattern may suggest a role for this molecule in regulating fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis (Scanlan et al., 1994). Most normal adult tissues and benign epithelial tumors exhibit little or no detectable FAP expression. However, FAP expression has been detected in malignant breast, colorectal, lung, skin, and pancreatic tumors, fibroblasts in healing wounds, over 90% of the stroma of soft tissue sarcomas, and some fetal mesenchymal cells. FAP has a potential role in cancer growth and metastasis through cell adhesion and migration processes and rapid degradation of ECM components. It is therefore present on tumor cells invading the ECM and on endothelial cells involved in angiogenesis, but is not expressed on inactive cells of the same type ( Dolznig et al., 2005 ; Kennedy et al., 2009 ; Rettig et al., 1993 ; Rettig et al., 1994 ; Scanlan et al., 1994 ; Zhang et al., 2010a ).

[0149] Wingless MMTV integration site family, member 5A (WNT5A) In general, Wnt5a regulates diverse cellular functions, such as proliferation, differentiation, migration, adhesion, and polarity (Kikuchi et al., 2012). It is expressed in undifferentiated human embryonic stem cells (Katoh, 2008). WNT5A is classified as a non-transforming WNT family member, whose role in carcinogenesis remains unclear. It exhibits tumor suppressor activity in several cancers (thyroid, brain, breast, and colorectal) but is aberrantly upregulated in lung, gastric, and prostate cancers (Li et al., 2010). Oncogenic WNT5A activates canonical WNT signaling within cancer stem cells for self-renewal and non-canonical WNT signaling at the tumor-stroma interface for invasion and metastasis (Katoh and Katoh, 2007). WNT5A expression has been described in diverse tumor entities. For example, aberrant Wnt5a protein expression was observed in 28% of prostate cancers, where it promoted aggressiveness (Yamamoto et al., 2010). Furthermore, overexpression of WNT5A has been described to be associated with poor prognosis and / or increased tumor aggressiveness in ovarian cancer (Badiglian et al., 2009), melanoma (Da Forno et al., 2008; Weeraratna et al., 2002), GBM (Yu et al., 2007), lung cancer (Huang et al., 2005), and pancreatic cancer (Ripka et al., 2007). In HCC, the canonical Wnt signaling pathway appears to contribute to tumor initiation, while noncanonical signaling contributes to tumor progression (Yuzugullu et al., 2009).

[0150] TPX2, microtubule-associated, homolog (Xenopus laevis) (TPX2) TPX2 is a spindle assembly factor. It is required for normal assembly of the mitotic spindle and microtubules during apoptosis. TPX2 is required for chromatin- and / or kinetochore-dependent microtubule nucleation (Bird and Hyman, 2008; Moss et al., 2009). De novo synthesized TPX2 is required for nearly all Aurora A activation and for full p53 synthesis and in vivo phosphorylation during oocyte maturation (Pascreau et al., 2009). TPX2 is a cell cycle-related protein overexpressed in numerous tumor types, including meningiomas (Stuart et al., 2010), squamous cell carcinomas of the larynx (SCCL) (Cordes et al., 2010), oral squamous cell carcinomas (SCC) (Shigeishi et al., 2009), hepatocellular carcinomas (HCC) (Satow et al., 2010), pancreatic tumors (Warner et al., 2009), ovarian cancer (Ramakrishna et al., 2010), and lung squamous cell carcinomas (Lin et al., 2006; Ma et al., 2006). It is frequently co-overexpressed with Aurora A, creating a novel functional entity with oncogenic properties (Asteriti et al., 2010). TPX2 expression is a prognostic indicator in lung cancer (Kadara et al., 2009).

[0151] Hyaluronan-mediated motility receptor (RHAMM) (HMMR) The receptor for hyaluronan-mediated motility, RHAMM (HMMR), exerts distinct functions both intracellularly and on the cell membrane. RHAMM can be transported to the cell surface, where it binds to hyaluronan (HA) and interacts with the HA receptor CD44. Processes such as cell motility, wound healing, and invasion are regulated by RHAMM (Sohr and Engeland, 2008). RHAMM (the receptor for HYA-mediated motility) is a hyaluronan (HYA) receptor (Gares and Pilarski, 2000). Cancer cells also display binding sites for HYA (e.g., CD44, RHAMM), which protects cancer cells from immune cell attack. Serum HYA levels are often elevated in metastatic patients (Delpech et al., 1997). Furthermore, the interaction of HYA with RHAMM (HMMR) and CD44 on cancer cells has been proposed to be important for promoting tumor progression and metastasis (Li et al., 2000b). Furthermore, RHAMM is overexpressed in several cancer tissues (Tzankov et al., 2011; Kramer et al., 2010; Twarock et al., 2010; Shigeishi et al., 2009; Zlobec et al., 2008; Li et al., 2000a).

[0152] ADAM metallopeptidase domain 8 (ADAM8) ADAM8 is a member of the ADAM (disintegrin and metalloprotease domain) family. Many ADAM species, including ADAM8, are expressed in human malignancies, where they are involved in regulating growth factor and integrin function, promoting cell proliferation and invasion (Mochizuki and Okada, 2007). ADAM8 expression was positively correlated with EGFR, both of which are expressed primarily in the cytoplasm and on the cell membrane (Wu et al., 2008). ADAM8 was abundantly expressed in most lung cancers examined. Exogenous expression of ADAM8 increased the migratory activity of mammalian cells, suggesting that ADAM8 may play an important role in lung cancer progression (Ishikawa et al., 2004). ADAM8 has been associated with poor prognosis in lung cancer (Hernandez et al., 2010). Overexpression of ADAM8 was associated with shorter patient survival and was a good predictor of distant metastasis in RCC (Roemer et al., 2004b; Roemer et al., 2004a). Furthermore, the expression level and protease function of ADAM8 correlated with the invasive activity of glioma cells, suggesting that ADAM8 may play an important role in tumor invasion in brain tumors (Wildeboer et al., 2006).

[0153] Collagen α-3(VI) chain protein (COL6A3) COL6A3 encodes the α-3 chain, one of three α chains of type VI collagen. This protein domain has been shown to bind to extracellular matrix proteins, an interaction that explains the importance of this collagen in organizing matrix elements.

[0154] Extracellular matrix remodeling through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells. The presence of collagen VI correlated with tumor grade, a prognostic factor for ovarian cancer (Sherman-Baust et al., 2003). COL6A3 is overexpressed in colorectal tumors (Smith et al., 2009a) and salivary gland cancers (Leivo et al., 2005), and is differentially expressed in gastric cancer (Yang et al., 2007). COL6A3 was identified as one of seven genes with tumor-specific splice variants. The tumor-specific splicing alterations examined were highly consistent, allowing clear separation between normal and cancer samples, and in some cases even between different tumor stages (Thorsen et al., 2008).

[0155] Thy-1 cell surface antigen (THY1) Thy-1 (CD90) is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored glycolipid protein expressed on numerous cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblasts. Activation of Thy-1 can promote T cell activation. Thy-1 also influences numerous non-immune biological processes, including cell adhesion, neurite outgrowth, tumor growth, tumor suppression, migration, wound healing, and cell death. Thy-1 is a key regulator of cell-cell and cell-matrix interactions and plays important roles in nerve regeneration, metastasis, inflammation, and fibrosis (Rege and Hagood, 2006b; Rege and Hagood, 2006a). Furthermore, Thy-1 appears to be a marker of adult, but not embryonic, angiogenesis. Cytokines, but not growth factors, upregulate Thy-1, suggesting the importance of inflammation in the pathogenesis of adult angiogenesis (Lee et al., 1998). Compared with normal lung tissue or benign tumor cells, there is a significant overexpression of Thy-1 in the nuclei of lung cancer cells, which is one of the factors associated with the prognosis of NSCLC patients. Therefore, Thy-1 may be a novel potential malignant marker in lung cancer pathology (Chen et al., 2005b). Thy-1 may be considered a surrogate marker for various types of stem cells (mesenchymal stem cells, liver stem cells ("oval cells")) (Masson et al., 2006), keratinocyte stem cells (Nakamura et al., 2006), and hematopoietic stem cells (Yamazaki et al., 2009).

[0156] Deiodinase, Iodothyronine, Type II (DIO2) The protein encoded by the DIO2 gene belongs to the iodothyronine deiodinase family. It is highly expressed in the thyroid gland and may contribute significantly to the relative increase in thyroid T3 production in patients with Graves' disease and goiter (Meyer et al., 2008; de Souza Meyer et al., 2005). Gene expression patterns differ significantly between upward and downward progression of nasopharyngeal carcinoma (NPC). DIO2 gene expression is higher in downward progression (downward = distant metastasis) than in upward progression (local growth and skull base invasion), which may be closely related to the metastatic potential of NPC (Liang et al., 2008). DIO2 mRNA and activity are expressed in brain tumors (Murakami et al., 2000). D2 activity in the lung is similar between peripheral lung tissue and lung cancer tissue (Wawrzynska et al., 2003).

[0157] Periostin, osteoblast-specific factor (POSTN) POSTN is a gene encoding a protein similar to the fasciclin family and involved in cell survival and angiogenesis, and has emerged as a promising marker of tumor progression in various types of human cancers ( Ruan et al., 2009 ).

[0158] High expression of periostin protein or mRNA has been detected in most solid tumors, including breast (Zhang et al., 2010b), colon (Kikuchi et al., 2008), head and neck (Kudo et al., 2006), pancreatic (Kanno et al., 2008), papillary thyroid (Puppin et al., 2008), prostate (Tischler et al., 2010), ovarian (Choi et al., 2010), lung (Takanami et al., 2008), and liver (Utispan et al., 2010) cancers, as well as esophageal squamous cell carcinoma (Kwon et al., 2009). Abnormally high periostin expression in lung cancer correlates with angiogenesis, invasion, and metastasis (Takanami et al., 2008). Silencing periostin in A549 non-small cell lung cancer (NSCLC) cells inhibits tumor cell growth and reduces cell invasion ( Wu et al., 2013 ).

[0159] SLIT1 (Slit homolog 1 (Drosophila)), SLIT2 (Slit homolog 2 (Drosophila)) SLITs (SLIT1, SLIT2, and SLIT3) are a family of secreted proteins that mediate positional interactions between cells and their environment during development by signaling through the ROBO receptor (Hinck, 2004). However, SLIT / ROBO signaling is not restricted to development, and loss of these signals likely plays an important role during tumor progression. Slit and Robo are considered candidate tumor suppressor genes because their promoters are frequently hypermethylated in epithelial cancers (Narayan et al., 2006; Schmid et al., 2007; Latil et al., 2003). SLIT2 or SLIT3 gene expression is silenced in approximately 50% of human breast adenomas (Sharma et al., 2007). SLIT2 hypermethylation is frequently detected in NSCLC and correlates with various clinical features (Suzuki et al., 2013).

[0160] TLX3 (T-cell leukemia homeobox 3) TLX3 (also known as RNX or HOX11L2) belongs to a family of orphan homeobox genes encoding DNA-binding nuclear transcription factors. Members of the HOX11 gene family are characterized by a threonine-47 substitution for a cytosine within the highly conserved homeodomain (Dear et al., 1993). TLX3 is uniquely expressed in the developing medulla oblongata and is required for the proper formation of primary relay visceral sensory neurons and most (nor)adrenergic centers in the brainstem, and is particularly involved in the physiological control of the cardiovascular and respiratory systems (Qian et al., 2001). TLX3 expression has also been detected in leukemia samples from 20% of children and 13% of adults with T-cell acute lymphoblastic leukemia (Cave et al., 2004), although this gene is not involved in normal T-cell differentiation (Ferrando et al., 2004).

[0161] CEP192 (centrosomal protein 192kDa) Centrosomes play important roles in various cellular processes, including spindle formation and chromosome segregation. CEP192 is a centrosomal protein that plays a key role in centrosome biogenesis and function in mammals, Drosophila, and C. elegans (Gomez-Ferreria et al., 2012). It stimulates the formation of a scaffold on which the γ-tubulin ring complex and other proteins involved in microtubule nucleation and spindle assembly become functional during mitosis (Gomez-Ferreria et al., 2007).

[0162] ANKS1A (ankyrin repeat and sterile α motif domain containing 1A) Ankyrin repeat and SAM domain-containing protein 1A is a protein encoded by the ANKS1A gene in humans (Nagase et al., 1996). ANKS1A was first described as a target and signal transduction agent for receptor tyrosine kinases like EGFR and PDGFR (Pandey et al., 2002) and more recently as an interaction partner for the receptor tyrosine kinase EphA8 (Shin et al., 2007). In a recent study, single nucleotide polymorphisms (SNPs) were genotyped in 348 patients with advanced NSCLC. Researchers identified 17 top candidate SNPs associated with prognosis. The SNPs were located within the genomic region of the ANKS1A gene (Lee et al., 2013).

[0163] CEP250 (centrosomal protein 250kDa) The CEP250 gene encodes a core centriolar protein required for centriole-centriole adhesion during interphase of the cell cycle (Mayor et al., 2002). By radiation mixture analysis, Fry et al. (1998) mapped the CEP250 gene to the centromeric region of chromosome 20 at approximately 20q11.2 (Fry et al., 1998). Mayor et al. (2002) found that overexpression of CEP250 in a human osteosarcoma cell line resulted in the formation of large centrosome-associated structures. Although overexpression of CEP250 did not prevent centrosome separation or cell division, it suggested that cell cycle-regulatory activity dissociated CEP250 from the centrosome (Mayor et al., 2002).

[0164] MDN1 (MDN1, midasin homolog (yeast)) MDN1, a yeast midasin homolog, is a protein encoded by the MDN1 gene in humans. Midasin exists as a single-copy gene encoding a well-conserved protein of approximately 600 kDa in all eukaryotes for which data are available. In humans, the gene is located on 6q15 and encodes a predicted protein of 5596 residues (632 kDa) (Garbarino and Gibbons, 2002). Recently, MDN1 has been found to be mutated in the luminal B subtype of breast cancer. MDN1 may play a role in the development and hormone resistance of this aggressive subtype (Cornen et al., 2014).

[0165] OLFM1 (olfactomedin 1) OLFM1, also known as Noelin-1, is a secreted glycoprotein that belongs to a family of proteins containing an olfactomedin domain and plays an important role in regulating neural crest cell production by the neural tube (Barembaum et al., 2000). Olfactomedin was originally identified as a major component of the mucus layer surrounding the chemosensory dendrites of olfactory neurons (Kulkarni et al., 2000). Olfactomedin 1 protein expression was significantly higher in lung adenocarcinoma than in other histological types of lung cancer and normal lung tissue (Wu et al., 2010). Furthermore, OLFM1 is deregulated in endometrial carcinoma, Ewing's sarcoma, and neuroblastoma (Wong et al., 2007; Allander et al., 2002; Khan et al., 2001).

[0166] BUB1B (Benzimidazole-inhibitable budding 1 homolog beta (yeast)) BUB1B, also called BubR1, is a core mitotic checkpoint component that binds to and inhibits the Cdc20-activated anaphase-promoting complex (APC / CCdc20), a ubiquitin E3 ligase that initiates anaphase by orchestrating separase-mediated cleavage of the cohesion rings that connect sister chromatids (Baker et al., 2004). BubR1 contributes to proper chromosome segregation not only through mitotic checkpoint activation but also through regulation of chromosome-spindle attachment (Malureanu et al., 2009; Lampson and Kapoor, 2005). Impaired spindle checkpoint function has been found in many forms of cancer. Mutations in BubR1 are associated with mosaic variegated aneuploidy (MVA), a rare human syndrome characterized by aneuploidy, tumor predisposition, and several premature aging traits, including short life span, growth and mental retardation, cataracts, and facial dysmorphism ( Matsuura et al., 2006 ).

[0167] PI4KA (phosphatidylinositol 4-kinase, catalytic, alpha) Four distinct phosphatidylinositol 4-kinases (PI4Ks) are expressed in human cells. These isoenzymes (PI4KA, PI4KB, PI4K2A, and PI4K2B) catalyze the phosphorylation of phosphatidylinositol (PtdIns) on the cytoplasmic face of the plasma membrane, resulting in the generation of phosphatidylinositol 4-phosphate (PtdIns4P) (Minogue and Waugh, 2012). PI4KA is primarily found in the endoplasmic reticulum (ER). Its activity appears to regulate both the formation of ER exit sites (Blumental-Perry et al., 2006) and the concentration of PtdIns4P in the plasma membrane (Balla et al., 2008). Researchers have found that PI4KA mRNA is more abundant in HCC than in healthy tissues. This upregulation significantly correlated with both poor differentiation and active proliferation rates in HCC. Therefore, PI4KA may be used as a novel molecular marker to improve established prognostic models of HCC ( Ilboudo et al., 2014 ).

[0168] AURKB (Aurora kinase B) Aurora B kinase is a protein that functions in the attachment of mitotic spindles to centromeres (Kim et al., 2011). AURKB is localized to microtubules near kinetochores (Kunitoku et al., 2003). Aurora kinases are overexpressed in various tumor cell lines, suggesting that these kinases may play a role in tumorigenesis and have already become potential targets for cancer diagnosis and therapy (Fu et al., 2007). Recently, a gene signature of five genes (TOP2A, AURKB, BRRN1, CDK1, and FUS) was identified that is closely linked to the prognosis of patients with NSCLC. The results suggested that genes involved in chromosome condensation, such as AURKB, are likely associated with stem-like characteristics and may predict survival in lung adenocarcinoma (Perumal et al., 2012).

[0169] SLC3A2 (solute carrier family 3 (dibasic and neutral amino acid transport activator), member 2) SLC3A2 comprises the light subunit of the large neutral amino acid transporter (LAT1), also known as CD98 (cluster of differentiation 98) (Lemaitre et al., 2005). The CD98 heterodimer consists of a type II single-pass transmembrane heavy chain of approximately 80–85 kDa (CD98hc, also known as 4F2 antigen heavy chain or FRP-1; encoded by the genes SLC3A2 and Slc3a2 in humans and mice, respectively) disulfide-linked to a multipass light chain of approximately 40 kDa (Deves and Boyd, 2000). CD98hc functions in integrin signaling amplification and amino acid transport; both of these functions may contribute to cell survival and proliferation (Cantor and Ginsberg, 2012). Many tumors express CD98hc (SLC3A2), and its expression correlates with poor prognosis in B-cell lymphoma. Furthermore, nearly all studies examining CD98hc or CD98 light chain expression in solid tumors show that their expression correlates with advanced or metastatic tumors (Kaira et al., 2009).

[0170] IFT81 (Intraflagellar transport 81 homolog (Chlamydomonas)) Intraflagellar transport (IFT) of ciliary precursors, such as tubulin, from the cytoplasm to the ciliary tip is involved in the assembly of the cilium, a hair-like organelle found on most eukaryotic cells. Knockdown and rescue experiments of IFT81 using point mutants demonstrated that tubulin binding by IFT81 is required for ciliary formation in human cells (Bhogaraju et al., 2013). Together with IFT74 / 72, IFT81 forms a core complex for assembling IFT particles required for ciliogenesis (Lucker et al., 2005).

[0171] COG4 (oligomeric Golgi apparatus 4 component) The COG complex consists of eight subunits, designated COG1-8 (Ungar et al., 2002; Whyte and Munro, 2001), which are further divided into two subcomplexes, COG1-4 (LobeA) and COG5-8 (LobeB) (Ungar et al., 2005). The COG complex functions in tethering vesicles that recycle resident Golgi proteins (e.g., glycosylation enzymes) (Pokrovskaya et al., 2011). The COG4 gene is located on chromosome 16q22.1 (Reynders et al., 2009). Ungar et al. (2002) concluded that COG4 is important for Golgi structure and function and may affect intracellular membrane trafficking (Ungar et al., 2002).

[0172] NCBP1 (nuclear cap binding protein subunit 1, 80 kDa) The nuclear cap-binding protein complex is an RNA-binding protein that binds to the 5' cap of RNA polymerase II. Kataoka et al. (1994) described the cloning of the gene encoding the 80 kD nuclear cap-binding protein (NCBP1) found in HeLa cell nuclear extracts, which may be involved in mRNA splicing and RNA transport (Kataoka et al., 1994). By hybridizing to genomic DNA from a somatic cell hybrid panel, Chadwick et al. (1996) mapped the NCBP1 gene to 9q34.1 (Chadwick et al., 1996).

[0173] NEFH (neurofilament, heavy chain polypeptide) Neurofilament heavy chain, encoded by NEFH, is one of the major components of neuronal cytoskeletal neurofilaments. The neurofilament heavy chain polypeptide (NEFH, 200 kD) gene, located on chromosome band 22q12.2, has been proposed as a DNA marker for presymptomatic diagnosis of neurofibromatosis type 2 (NF2) families. Loss or downregulation of NEFH has been reported mostly in human autonomic or central neurocytomas (Mena et al., 2001; Segal et al., 1994). Furthermore, absent or reduced NEFH expression has been observed in human prostate cancer (Schleicher et al., 1997), clear cell epithelioid tumor (Tanaka et al., 2000), and small cell lung cancer (Bobos et al., 2006). Interestingly, overexpression of NEFH disrupted normal cellular structure and function and induced cell death (Szebenyi et al., 2002). DETAILED DESCRIPTION OF THE INVENTION

[0174] As used herein, unless otherwise specified, all terms are defined as set forth below.

[0175] The term "peptide" 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. Peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids, 10, 11, 12, 13, or 14 amino acids in length, and in the case of MHC class II peptides, they can be 15, 16, 17, 18, 19, or 20 amino acids in length.

[0176] Furthermore, the term "peptide" is intended to include salts of a series of amino acid residues that are typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. Preferably, the salts are pharmaceutically acceptable salts.

[0177] The term "peptide" is 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.

[0178] 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: 92, as defined above.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] In humans, there are three different genetic loci that encode MHC class I molecules (human MHC molecules also called human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different MHC class I alleles that can be expressed from these loci.

[0183] [Table 7]

[0184] Therefore, for therapeutic and diagnostic purposes, peptides that bind with suitable affinity to several different HLA class II receptors are highly desirable. Peptides binding to several different HLA class II molecules are referred to as promiscuous binders.

[0185] 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.

[0186] 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 DNA synthesis techniques well known to those skilled in the art.

[0187] The term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

[0188] 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.

[0189] As used herein, the term "nucleotides encoding a peptide" 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.

[0190] The term "expression product" refers to a polypeptide or protein that is the natural translation product of a gene and of any nucleic acid sequence that, due to the degeneracy of the genetic code, encodes an equivalent and therefore encodes the same amino acid.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] The term "promoter" refers to a region of DNA involved in RNA polymerase binding to initiate transcription.

[0195] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is of natural origin). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide 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.

[0196] 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.

[0197] 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.

[0198] The term "active fragment" means a fragment that generates an immune response (i.e., has immunogenicity) when administered alone, or optionally with a suitable adjuvant, to an animal, such as a mammal, for example, a rabbit or a mouse, and also including a human, 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.

[0199] 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, when a polypeptide is treated with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptide resulting from such treatment corresponds 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.

[0200] According to the present invention, when referring to a sequence, the term "percent identity" or "percent identical" means that the sequence is compared to the claimed or described sequence ("reference sequence") after alignment of the sequence being compared ("comparison sequence") with the described or claimed sequence. The percent identity is then determined according to the following formula: Percent identity=100[1-(C / R)] where C is the number of differences between the reference sequence and the compared sequence over the alignment length between the reference sequence and the compared sequence; (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence; and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence that differs from an aligned base or amino acid in the comparison sequence constitutes a difference; (iiii) the alignment must start at position 1 of the aligned sequence; R is the number of bases or amino acids in the reference sequence over the length of the alignment with the comparison sequence, and any gaps made in the reference sequence are also counted as bases or amino acids.

[0201] 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.

[0202] The original (unmodified) peptides disclosed herein may, unless otherwise specified, be modified by substitution of one or more residues at different, possibly selective, sites within the peptide chain.

[0203] Preferably, these substitutions are located at the end of the amino acid chain.Such substitutions can be conservative, for example, amino acids are replaced by amino acids with similar structure and characteristics, such as hydrophobic amino acids being replaced by other hydrophobic amino acids.Even more conservative substitutions are the replacement of amino acids with the same or similar size and chemical properties, such as the replacement of leucine with isoleucine.In the study of the sequence diversity of naturally occurring homologous protein families, certain amino acid substitutions are often tolerated better than others, and these often show a correlation with the similarity of size, charge, polarity and hydrophobicity between the original amino acid and its replacement, which is the basis for the definition of "conservative substitution".

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

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

[0206] Of course, such substitutions may involve structures other than conventional L-amino acids. Thus, D-amino acids may be substituted for L-amino acids normally found in the antigenic peptides of the invention and still be encompassed by the disclosure herein. Furthermore, 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 generate immunogens and immunogenic polypeptides according to the invention.

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

[0208] 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.

[0209] The combinations of extensions according to the present invention can be represented from Table 3:

[0210] [Table 8]

[0211] The amino acids for the extension can be the peptide of the original protein sequence or any other amino acid. The extension can be used to increase the stability or solubility of the peptide.

[0212] The term "T cell response" refers to the specific proliferation and activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I-restricted 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 perforin, induced by the peptide; or degranulation.

[0213] Preferably, when CTLs specific for peptides of SEQ ID NO: 1 to SEQ ID NO: 92 are tested with substituted peptides, the peptide concentration at which the substituted peptide achieves half-maximal lysis enhancement 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 CTLs from two or more individuals, at least two, and more preferably three individuals.

[0214] 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.

[0215] Stimulation of an immune response depends on the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor-associated antigens has now raised the possibility of using the host's immune system to intervene in tumor growth. Various mechanisms that utilize both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

[0216] Certain elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. Isolation of cytotoxic T cells (CTLs) from tumor-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in innate immune defense against cancer. In particular, CD8+ T cells, which possess major histocompatibility complex (MHC) receptors and recognize class I molecules, typically peptides of 8–12 residues, derived from proteins or defective ribosomal products (DRIPS) located in the cytosol, play a key role in this response. In humans, MHC molecules are also referred to as human leukocyte antigens (HLA).

[0217] MHC class I molecules are present on the majority of cells with nuclei, mainly from endogenous cytoplasmic or nuclear proteins, DRIPS, and peptides derived from the 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.

[0218] 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 CTLs (MHC class I molecules) or CD4-positive CTLs (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.

[0219] Given the serious side effects and costs associated with cancer treatment, there is a critical need for better prognostic and diagnostic methods. Thus, there is a need to identify other factors that represent biomarkers for cancer in general, and lung cancer in particular. Furthermore, there is a need to identify factors that can be used in the treatment of cancer in general, and lung cancer in particular.

[0220] The present invention provides peptides that are useful in the treatment of cancer / tumor, preferably lung cancer, even more preferably non-small cell lung cancer (NSCLC), which 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 lung cancer samples (see Example 1 and Figure 1).

[0221] The original genes / proteins (also referred to as "full-length proteins" or "basal proteins") from which the peptides are derived were shown to be highly overexpressed in non-small cell lung cancer, gastric cancer, and glioblastoma (see Example 2 and Figure 2 for NSCLC), compared to normal tissue, demonstrating the high tumor association of the original genes. Furthermore, the peptides themselves were also strongly overexpressed on tumor tissue, but not on normal tissue (see Example 3 and Figure 3).

[0222] HLA-bound peptides can be recognized by the immune system, specifically T lymphocytes / T cells, which can destroy cells that present the recognized HLA / peptide complex, such as lung cancer cells that present the derived peptide.

[0223] The peptides of the present invention have been shown to be capable of stimulating T cell responses and / or to be over-presented, and thus can be used to generate antibodies and / or TCRs, particularly TCRs according to the present invention (see Example 4 and Figure 4). Furthermore, when complexed with the respective MHC, the peptides can similarly be used to generate 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 literature. 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 these peptides), to the patient, ideally in combination with an agent (i.e., an adjuvant) that enhances immunogenicity. Because the target peptides of the present invention are not presented in equivalent copy numbers on normal tissues, the immune response resulting from such therapeutic vaccination can be expected to be highly specific to tumor cells, preventing the risk of unwanted autoimmune reactions against the patient's normal cells.

[0224] Pharmaceutical compositions comprise peptides in either free form or in the form of a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salt" refers to derivatives of the disclosed peptides, wherein the peptides are modified by forming acid or base salts of the drug. For example, acid salts are prepared from the free base (where the neutral form of the drug typically 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, 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.

[0225] In one particularly preferred embodiment, the pharmaceutical composition comprises the peptide as a salt of acetic acid (acetate), trifluoroacetic acid or hydrochloric acid (chloride).

[0226] In addition to being useful for treating cancer, the peptides of the present invention are also useful as diagnostics: because the peptides originated from lung cancer cells, and because these peptides were determined to be absent or present at lower levels in normal tissues, these peptides can be used to diagnose the presence of cancer.

[0227] The presence of the claimed peptides on tissue biopsies can aid pathologists in cancer diagnosis. Detection of specific peptides by antibodies, mass spectrometry, or other methods known in the art can tell the pathologist that the tissue is malignant, inflamed, or generally diseased. The presence of peptide groups can allow for classification or subclassification of diseased tissue.

[0228] 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.

[0229] 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 may be used as prognostic markers to determine further treatment steps. These responses may 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 substances, or adoptive transfer of lymphocytes. In the setting of gene therapy, lymphocyte responses to peptides may 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.

[0230] The peptides of the present invention can be used to generate and develop specific antibodies against MHC / peptide complexes. 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.

[0231] 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.

[0232] It is a further aspect of the present invention to provide an antibody that specifically binds to human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen, wherein the antibody is preferably a polyclonal, monoclonal, bispecific and / or chimeric antibody.

[0233] 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 that express 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 generating such antibodies and single-chain class I major histocompatibility complexes, as well as other tools for generating these antibodies, are described in WO 03 / 068201, WO 2004 / 084798, WO 01 / 72768, WO 03 / 070752, and Cohen CJ, Denkberg G, Lev A, Epel M, Reiter Y. 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, Lev A, Eisenbach L, Benhar I, Reiter Y. Selective targeting of melanoma and APCs using a recombinant antibody with TCR-like specificity directed toward a melanoma differentiation antigen.J Immunol.2003 Sep 1;171(5):2197-207; and Cohen CJ,Sarig O,Yamano Y,Tomaru U,Jacobson S,Reiter Y.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 Disclosed in human recombinant antibodies.J Immunol.2003 Apr 15;170(8):4349-61.

[0234] 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.

[0235] A further aspect of the present invention provides a method for generating soluble T cell receptors that recognize specific peptide-MHC complexes. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by targeted mutagenesis of complementarity-determining regions. Phage display can be used to select T cell receptors (US Patent Publication No. 2010 / 0113300; Liddy N, Bossi G, Adams KJ, Lissina A, Mahon TM, Hassan NJ, et al. Monoclonal TCR-redirected tumor cell killing. Nat Med 2012 Jun;18(6):980-987).To stabilize T cell receptors in phage display and for 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, Glick M, Todorov PT, Baston E, Sami M, Rizkallah P, et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng 2003 Sep;16(9):707-711.; Card KF, Price-Schiavi SA, Liu B, Thomson E, Nieves E, Belmont H, 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, Gao (See GF, Wyer JR, O'Callaghan CA, Boulter JM, Jones EY, 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 toxins, drugs, cytokines (see U.S. Patent No. 2013 / 0115191), effector cell recruitment domains such as anti-CD3 domains, and the like to exert specific functions on target cells. Furthermore, they can be expressed in T cells used for adoptive transfer.

[0236] Further information can be found in WO 2004 / 033685 A1 and WO 2004 / 074322 A1. TCR combinations are described in WO 2012 / 056407 A1. Further production methods are disclosed in WO 2013 / 057586 A1.

[0237] Additionally, they may be used to confirm a pathologist's cancer diagnosis based on a biopsy sample.

[0238] To select over-represented peptides, a representation profile is calculated, showing the median sample representation and replicate variation. The profile juxtaposes the samples of the tumor entity of interest to the 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, D. Bates, S. DebRoy, Sarkar D., R Core team. nlme: Linear and Nonlinear Mixed Effects Models. 2008) and correcting for multiple testing by the 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).

[0239] For identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules were purified from shock-frozen samples, and HLA-associated peptides were isolated. The isolated peptides were separated and sequenced using online nanoelectrospray ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequences were confirmed by comparing the fragmentation patterns of native TUMAPs recorded from NSCLC samples with those of corresponding synthetic reference 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 tumor tissue obtained from NSCLC patients.

[0240] The proprietary 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 developing label-free differential quantification using a proprietary data analysis pipeline, processing acquired LC-MS data, and combining algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, charge state deconvolution, and normalization.

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

[0242] HLA-peptide complexes were purified from 50 shock-frozen NSCLC tumor samples, and HLA-associated peptides were isolated and analyzed by LC-MS.

[0243] All TUMAPs included in this application were identified by this approach on primary NSCLC tumor samples and their presentation on primary NSCLC was confirmed.

[0244] TUMAPs identified in multiple NSCLC 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.

[0245] The present invention therefore relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or a variant thereof that is at least 90% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or a variant thereof that induces T cells that cross-react with said peptide, wherein said peptide is not a full-length polypeptide.

[0246] 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: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or a variant thereof that is at least 90% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, said peptide or variant having an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0247] 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.

[0248] The present invention further relates to peptides according to the invention, which peptides consist of or consist essentially of the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92.

[0249] The present invention further relates to a peptide according to the invention, which peptide is modified and / or comprises a non-peptide bond.

[0250] The present invention further relates to a peptide according to the invention, wherein the peptide is 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.

[0251] The present invention further relates to nucleic acids encoding peptides according to the invention, with the proviso that the peptide is not a fully human protein.

[0252] The present invention further relates to a nucleic acid according to the invention which is DNA, cDNA, PNA, RNA or a combination thereof.

[0253] The present invention further relates to an expression vector capable of expressing a nucleic acid according to the invention.

[0254] 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.

[0255] 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.

[0256] The present invention further relates to a host cell according to the invention which is an antigen-presenting cell.

[0257] The present invention further relates to a host cell according to the invention, wherein the antigen-presenting cell is a dendritic cell.

[0258] The present invention further relates to a method for producing a peptide according to the invention, the method comprising the steps of culturing the host cells described and isolating the peptide from the host cells or their culture medium.

[0259] The present invention further relates to an in vitro method for generating activated cytotoxic T lymphocytes (CTLs), the method comprising contacting ex vivo CTLs 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 CTLs in an antigen-specific manner, wherein the antigen is any peptide according to the present invention.

[0260] The invention further relates to the described methods, wherein a sufficient amount of antigen is contacted with antigen-presenting cells such that said antigen is loaded onto class I or II MHC molecules expressed on the surface of appropriate antigen-presenting cells.

[0261] The present invention further relates to a method according to the present invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing the peptides containing SEQ ID NO: 1 to SEQ ID NO: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or the mutant amino acid sequences.

[0262] The present invention further relates to activated cytotoxic T lymphocytes (CTLs) generated by the method according to the invention, which selectively recognize cells that aberrantly express a polypeptide comprising the amino acid sequence described.

[0263] The present invention further relates to a method of killing target cells in a patient, which target cells aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, the method comprising the step of administering to the patient an effective number of cytotoxic T lymphocytes (CTLs) according to the present invention.

[0264] The present invention further relates to the use of any peptide according to the invention, a nucleic acid according to the invention, an expression vector according to the invention, a cell according to the invention or an activated cytotoxic T lymphocyte according to the invention as a medicament or in the manufacture of a medicament.

[0265] The present invention further relates to a use according to the invention, wherein the medicament is a vaccine.

[0266] The present invention further relates to a use according to the invention, wherein the medicament is effective against cancer.

[0267] The present invention further relates to the use according to the invention, wherein said cancer cells are lung cancer cells, gastric, gastrointestinal, colorectal, pancreatic or renal.

[0268] The present invention further relates to specific marker proteins and biomarkers that may be used in the prognosis of lung cancer.

[0269] Furthermore, the present invention relates to the use of the novel targets as described by the present invention for cancer therapy.

[0270] The terms "antibody" or "antibodies" are used broadly herein and include both polyclonal and monoclonal antibodies. According to the present invention, the term "antibody" also includes native or "intact" immunoglobulin molecules, as well as fragments or polymers of these immunoglobulin molecules, and humanized versions of immunoglobulin molecules, so long as they exhibit any of the desired properties (e.g., specific binding of a lung cancer marker polypeptide, delivery of a toxin to lung cancer cells expressing an increased level of a lung cancer marker gene, and / or inhibition of the activity of a lung cancer marker polypeptide).

[0271] Whenever possible, the antibodies of the present invention may be purchased from commercial sources. Alternatively, the antibodies of the present invention may be produced using well-known methods. Those skilled in the art will understand that either full-length lung cancer marker polypeptides or fragments thereof may be used to produce the antibodies of the present invention. The polypeptides used to produce the antibodies of the present invention may be partially or completely purified from natural sources, or may be produced using recombinant DNA technology.

[0272] For example, cDNA encoding ABCA13, MMP12, DST, MXRA5, CDK4, HNRNPH, TANC2, 1RNF213, SMYD3, and SLC34A2, or any other polypeptide of SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92, or a 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 generate monoclonal or polyclonal antibody preparations that specifically bind to the lung cancer marker polypeptides used to generate antibodies according to the present invention.

[0273] Those skilled in the art will understand that generating two or more different sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining antibodies with the specificity and affinity required for their intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). Antibodies are tested for their desired activity by known methods according to the purpose for which they are used (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on antibody generation and testing, see, for example, 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, immunohistochemical staining of formalin-fixed lung cancer or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested by known clinical testing methods.

[0274] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous antibody population; i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. As used herein, "monoclonal antibody" specifically includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. Pat. No. 4,816,567, the entire contents of which are incorporated by reference).

[0275] 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.

[0276] Monoclonal antibodies may also be produced 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).

[0277] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate 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, published December 22, 1994, 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 Fe fragment. Pepsin treatment results in fragments with two antigen-binding sites that are still capable of cross-linking antigens.

[0278] 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.

[0279] 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 a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0280] Methods for humanizing non-human antibodies are well known in the art. Humanized antibodies typically have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which typically come from an "import" variable region. Humanization can essentially be performed by substituting rodent CDR(s) or CDR(s) sequences for the corresponding human antibody sequences. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable region has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0281] Transgenic animals (e.g., mice) can be used that are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of antibody heavy chain joining region genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge. Human antibodies can also be produced in phage display libraries.

[0282] The antibody of the present invention is preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmacologically acceptable carriers include saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles such as films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the antibody being administered.

[0283] The antibody may 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 may 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.

[0284] Effective dosages and schedules for administering antibodies may be determined empirically, and making such determinations is within the skill of the art. One of ordinary skill in the art will understand that the antibody dose that must be administered will vary depending, for example, on the subject receiving the antibody, the route of administration, the particular antibody type used, and other agents being administered. A typical daily dose of an antibody used alone may range from about 1 μg / kg up to 100 mg / kg body weight per day or more, depending on the factors discussed above. Following antibody administration to treat lung cancer, 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 lung 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 that would occur in the absence of antibody administration, is an effective antibody for treating lung cancer.

[0285] Since the lung tumor markers ABCA13 and MMP12 of the present invention are highly expressed in lung cancer cells and at extremely low levels in normal cells, inhibition of ABCA13 and MMP12 expression or polypeptide activity may be incorporated into any therapeutic strategy for treating or preventing NSCLC.

[0286] The principle of antisense therapy is based on the hypothesis that sequence-specific inhibition 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.

[0287] 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 encoding antisense RNA (or RNA fragments) can be introduced into cells in vivo.Antisense effect can also be induced by sense sequence; 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.

[0288] In a specific example, the inhibition of lung tumor marker function by antisense gene therapy can be achieved by directly administering antisense lung 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 (for example, T7 promoter).Antisense tumor marker RNA can be administered to cells by any of the direct nucleic acid administration methods described below.

[0289] An alternative strategy for inhibiting ABCA13 and MMP12 function using gene therapy involves intracellular expression of an anti-ABCA13, MMP12 antibody, or a portion of an anti-ABCA13, MMP12 antibody. For example, a gene (or gene fragment) encoding a monoclonal antibody that specifically binds to and inhibits the biological activity of an ABCA13, MMP12 polypeptide is placed under the transcriptional control of a specific (e.g., tissue- or tumor-specific) gene regulatory sequence within a nucleic acid expression vector. The vector is then administered to a subject so that it is taken up by lung cancer cells or other cells, which then secrete the anti-ABCA13, MMP12 antibody, thereby inhibiting the biological activity of the ABCA13, MMP12 polypeptide. Preferably, the ABCA13, MMP12 polypeptide is present on the extracellular surface of gastric cancer cells.

[0290] In the above-described methods involving the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transfer 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 gastric tumor 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 to cells may be via a variety of mechanisms. As one example, delivery may be via liposomes, using commercially available liposomal preparations such as Lipofectin, Lipofectamine (GIBCO-25 BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc., Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to standard procedures in the art. Additionally, the nucleic acids or vectors of the invention may be delivered in vivo by electroporation, the technology of which is available from Genetronics, Inc., San Diego, Calif., as well as by means of a SONOPORATION device (ImaRx Pharmaceutical Corp., Tucson, Arizona).

[0291] As one example, vector delivery can be via a viral system, such as a retroviral vector system, which can package a recombinant retroviral genome. The recombinant retrovirus can then be used to infect and thereby deliver an antisense nucleic acid that inhibits the expression of ABCA13 and MMP12 to the infected cells. 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, can also be used. The present invention can be used in conjunction with any of these or other commonly used gene transfer methods.

[0292] The antibody may also be used for in vivo diagnostic assay. Usually, the antibody is labeled with a radionucleotide (such as In, Tc, C, I, H, P or S) so that the antibody can be used to localize tumors by immunoscintigraphy. In one embodiment, the antibody or its fragment binds to the extracellular domain of two or more ABCA13, MMP12 targets, with an affinity value (Kd) of less than 1 x 10 μM.

[0293] Diagnostic antibodies may be labeled with probes suitable for detection by various imaging techniques. Probe detection methods include, but are not limited to, fluorescence, optical, confocal, and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin, and other fluorophores, radioisotopes, gold, gadolinium, and other lanthanides, paramagnetic iron, fluorine-18, and other positron-emitting radionuclides. Furthermore, probes may be bifunctional or multifunctional and detectable by one or more of the listed methods. These antibodies may be directly or indirectly labeled with the probes. Particularly well-recognized techniques for attaching probes to antibodies include covalent coupling of the probe, incorporation of the probe into the antibody, and covalent coupling of a chelating compound for probe binding. For immunohistochemistry, diseased tissue samples may be fresh or frozen, or may be 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 the in situ expressed ABCA13, MMP12 proteins.

[0294] Thus, the present invention provides peptides comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 65, SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or variants thereof that are 90% homologous to SEQ ID NO: 1 to SEQ ID NO: 65, SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or variants thereof that induce T cells that cross-react with said peptides.

[0295] The peptides of the present invention have the ability to bind to molecules of the human major histocompatibility complex (MHC) class I and / or class II.

[0296] 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.

[0297] 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 et al., 2001); (Zaremba et al., 1997; Colombetti et al., 2006; Appay et al., 2006).

[0298] By "variant" of a given amino acid sequence, we mean that, for example, the side chains of one or two amino acid residues are altered (e.g., by replacing them with the side chain of another naturally occurring amino acid residue, or with other side chains) so that the peptide can still bind to an HLA molecule substantially similarly to a peptide consisting of the given amino acid sequence in SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92. For example, the peptide may be modified to at least maintain, if not improve, its ability to interact with and bind to the binding groove of an appropriate MHC molecule, such as HLA-A*02 or -DR, and thus at least maintain, if not improve, its ability to bind to the TCR of an activated CTL.

[0299] These CTLs can subsequently cross-react with cells and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptides defined in embodiments of the present invention. As can be deduced from the scientific literature (Rammensee et al., 1997) and databases (Rammensee et al., 1999), certain positions of HLA-binding peptides typically anchor residues to form core sequences that fit the binding motif of HLA receptors, defined by the polarity, electrophysical properties, hydrophobicity, and spatial properties of the polypeptide chain that constitutes the binding groove. Thus, one skilled in the art can modify the amino acid sequences set forth in SEQ ID NOs: 1 to 65, SEQ ID NOs: 76 to 84, and SEQ ID NO: 92 by maintaining the known anchor residues and determine whether such variants maintain the ability to bind to MHC class I or II molecules. The variants of the present invention maintain the ability to bind to the TCR of activated CTLs, which can subsequently cross-react with cells and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptides defined in embodiments of the present invention.

[0300] An amino acid residue that does not substantially contribute to the interaction with the T cell receptor may be modified by substitution with another amino acid, the incorporation of which does not substantially affect T cell reactivity or eliminate binding to the relevant MHC. Thus, except for the given provisos, the peptide of the invention may be any peptide (we include by that term an oligopeptide or polypeptide) comprising a given amino acid sequence, or a given part or variant thereof.

[0301] [Table 9-1] [Table 9-2] [Table 9-3]

[0302] Longer peptides may also be suitable, usually 8-11 amino acids in length, but MHC class I epitopes 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.

[0303] Thus, the present invention also 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 class II binding peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or 33 amino acids.

[0304] 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. Binding of the peptides or variants to MHC complexes may be tested by methods known in the art.

[0305] In particularly preferred embodiments of the invention, the peptide consists of or consists essentially of the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92.

[0306] "Consisting essentially of" is intended to mean that in addition to the sequence set forth in any of SEQ ID NOs: 1 to 65, 76 to 84, and 92, the peptide according to the invention or a variant thereof contains additional N- and / or C-terminally located stretches of amino acids that are not essential for forming the part of the peptide that functions as an epitope for an MHC molecule epitope.

[0307] Nevertheless, these stretches may be important for providing efficient introduction of the peptides according to the invention into cells. In one embodiment of the invention, the peptide is a fusion protein comprising the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33, hereinafter "Ii"), e.g., from NCBI, GenBank accession number X00497. In other fusions, the peptides of the invention may be fused to antibodies as described herein, or functional parts thereof, in particular within the sequence of the antibody, so as to be specifically targeted by said antibody, or may be fused to or within an antibody specific for, for example, dendritic cells.

[0308] 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.

[0309] In reverse peptide bond, amino acid residues are not linked by peptide (-CO-NH-) bond, and peptide bond is reversed.Such retro-inverso peptidomimetics can be produced by using methods known in the art, for example, as described in Meziere et al. (1997) J.Immunol.159,3230-3237, which is incorporated herein by reference.This approach involves producing pseudopeptides that contain changes that involve the main chain rather than 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-inverso peptides that contain NH-CO bond instead of CO-NH peptide bond are much more resistant to proteolysis.

[0310] 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.

[0311] Peptides comprising the above sequences may be synthesized with additional chemical groups at their amino and / or carboxy termini to enhance the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the amino termini of the peptides. Similarly, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the amino terminus of the peptides. Furthermore, hydrophobic groups, t-butyloxycarbonyl, or amide groups may be added to the carboxy terminus of the peptides.

[0312] Furthermore, the peptides of the present invention may be synthesized to alter their configuration. For example, one or more D-isomers 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.

[0313] 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, formation of mercury derivatives, 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.

[0314] 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. Cysteine ​​can be modified without the simultaneous modification of other nucleophilic sites, such as lysine and histidine. As a result, numerous reagents are available for cysteine ​​modification. Company websites, such as Sigma-Aldrich (http: / / www.sigma-aldrich.com), provide information on specific reagents.

[0315] Selective reduction of disulfide bonds in proteins is also common: disulfide bonds can be formed and oxidized during heat treatment of biopharmaceuticals.

[0316] Specific glutamic acid residues may be modified using Woodward's reagent K. N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide may be utilized to form intramolecular crosslinks between lysine and glutamic acid residues.

[0317] For example, diethylpyrocarbonate is a reagent for modifying histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal.

[0318] Reactivity of lysine residues and other α-amino groups is useful, for example, in attaching peptides to surfaces or in protein / peptide cross-linking. Lysine is the site of attachment of poly(ethylene)glycol and is the primary modification site for protein glycosylation.

[0319] Methionine residues in proteins can be modified, for example, with iodoacetamide, bromoethylamine, and chloramine T. 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.

[0320] 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).

[0321] Successful modification of therapeutic proteins and peptides with PEG is often associated with increased circulatory half-life, while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used for hydrogel preparation. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

[0322] Peptides or variants in which the peptide is modified or contains non-peptide bonds are preferred embodiments of the present invention. Generally, peptides and variants (at least those containing peptide bonds between amino acid residues) may be synthesized in the Fmoc-polyamide format by solid-phase peptide synthesis, as disclosed in Lu et al. (1981) and references therein. Temporary N-amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is carried out using 20% ​​piperidine in N,N-dimethylformamide. Side chain functionalities may be protected as their butyl ethers (for serine, threonine, and tyrosine), butyl esters (for glutamic acid and aspartic acid), butyloxycarbonyl derivatives (for lysine and histidine), trityl derivatives (for cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (for arginine). When glutamine or asparagine is the C-terminal residue, a 4,4'-dimethoxybenzhydryl group is utilized to protect the side-chain amide functionality. The solid-phase support is based on a polydimethyl-acrylamide polymer composed of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin cleavable linker used is an acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives, except for asparagine and glutamine, which are added using a reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isatin test procedures. Upon completion of synthesis, the peptide is cleaved from the resin support and the side chain protecting groups are simultaneously removed by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture.Commonly used scavengers include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the constituent amino acids of the peptide being synthesized. A combination of solid-phase and solution-phase methodologies for the synthesis of peptides is also possible (see, for example, (Bruckdorfer et al., 2004) and references cited therein).

[0323] The trifluoroacetic acid was removed by evaporation under vacuum, followed by trituration with diethyl ether to give the crude peptide. Any scavengers present were removed by a simple extraction procedure, which, upon lyophilization of the aqueous phase, gave the scavenger-free crude peptide. Reagents for peptide synthesis are generally available, for example, from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

[0324] 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.

[0325] Analysis of peptides may be carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, by fast atom bombardment (FAB) mass spectrometry, and by MALDI and ESI-Q-TOF mass spectrometry.

[0326] 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 a polynucleotide in native or stabilized form, such as a polynucleotide with a phosphorothioate backbone, and may or may not contain introns, so long as it encodes a peptide. Of course, only peptides containing natural amino acid residues linked by naturally occurring peptide bonds may be encoded by the polynucleotide. A still further aspect of the present invention provides expression vectors capable of expressing the polypeptides according to the present invention.

[0327] A variety of methods have been developed for linking polynucleotides, particularly DNA, to vectors, for example, via complementary cohesive ends. For example, complementary homopolymer sequences can be added to the DNA fragment to be inserted into the vector DNA. The vector and DNA fragment are then linked by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0328] 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.

[0329] A desirable method for modifying DNA encoding a polypeptide of the invention is to use the polymerase chain reaction as disclosed in (Saiki et al., 1988). This method may be used to introduce the DNA into a suitable vector, for example, by modifying appropriate restriction enzyme recognition sites, or it may be used to modify the DNA in other useful ways known in the art. If a viral vector is used, poxvirus or adenovirus vectors are preferred.

[0330] 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, according to well-known techniques, appropriately modified in light of the teachings contained herein, DNA encoding the peptide or variant of the invention may be used to construct expression vectors which are then used to transform a suitable host cell for the expression and production of the polypeptide of the invention. Such techniques include 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.

[0331] 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.

[0332] Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate orientation and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host; 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.

[0333] Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

[0334] Host cells transformed with recombinant DNA of the present invention can then be cultured under appropriate conditions known to those of skill in the art and for a period of time sufficient to allow for expression of the polypeptide, which can then be recovered, in light of the teachings disclosed herein.

[0335] 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 cell line will be a mammalian cell, such as CHO cells, available from the ATCC Cell Biology Collection.

[0336] 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 expression or secretion, 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.

[0337] The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg / L in COS cells. In less potent cell lines, protein levels are typically about 0.1 mg / L. The presence of the SV40 origin of replication results in high levels of DNA replication in permissive COS cells for SV40 replication. 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.

[0338] In another embodiment, two or more peptides or peptide variants of the invention are encoded and thus expressed sequentially (similar to a "beads and beads" construct), whereby the peptides or peptide variants may be linked or fused together by a stretch of linker amino acids, e.g., LLLLLL, or may be linked without any additional peptide between them.

[0339] 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 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 embryo 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 cells. 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 Paulina Balbas and Argelia Lorence, "Methods in Molecular Biology: Recombinant Gene Expression, Reviews and Protocols," Part One, Second Edition, ISBN 978-1-58829-262-9, and other references known to those skilled in the art.

[0340] Transformation of suitable cell hosts with the DNA construct 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.

[0341] 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.

[0342] 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.

[0343] In a preferred embodiment, the host cell is an antigen-presenting cell, particularly a dendritic cell or an antigen-presenting cell. APC loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) is currently being investigated for the treatment of prostate cancer (sipuleucel-T) (Small et al., 2006; Rini et al., 2006).

[0344] A further aspect of the invention provides a method of producing a peptide or a variant thereof, the method comprising the steps of culturing a host cell and isolating the peptide from the host cell or its culture medium.

[0345] 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)).

[0346] Another aspect of the invention includes an in vitro method of generating activated T cells, the method comprising contacting ex vivo T cells with antigen-loaded human MHC molecules expressed on the surface of suitable antigen-presenting cells for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigen is used in conjunction with the antigen-presenting cells.

[0347] 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.

[0348] The human peptide loading depletion 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 line 2 is available from the ATCC under catalog number CRL19863; the mouse RMA-S cell line is described in Karre et al. 1985.

[0349] Preferably, the host cells do not express substantially any MHC class I molecules prior to transfection. 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.

[0350] When an MHC class I epitope is used as the antigen, the T cells are CD8-positive CTLs.

[0351] When antigen-presenting cells are transfected to express such epitopes, the cells preferably comprise an expression vector capable of expressing peptides containing SEQ ID NO: 1 to SEQ ID NO: 65, and SEQ ID NO: 76 to SEQ ID NO: 84, and SEQ ID NO: 92, or their mutant amino acid sequences.

[0352] Several other methods may be used to generate CTLs in vitro. For example, the methods described by Peoples et al. (1995) and Kawakami et al. (1992) use autologous tumor-infiltrating lymphocytes in generating CTLs. Plebanski et al. (1995) utilize autologous peripheral blood lymphocytes (PLBs) to prepare CTLs. Jochmus et al. (1997) describe generating autologous CTLs by pulsing dendritic cells with peptides or polypeptides or through infection with recombinant viruses. Hill et al. (1995) and Jerome et al. (1993) utilize B cells in generating autologous CTLs. Additionally, pulsing macrophages with peptides or polypeptides or infection with recombinant viruses may be used to prepare autologous CTLs. S. Walter et al. (2003) describe the ex vivo priming of T cells using artificial antigen-presenting cells (aAPCs), which is also a suitable method for generating T cells against selected peptides. In this study, aAPCs were generated 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 selectively eliciting high- or low-avidity antigen-specific T cell responses from blood samples with high efficiency. In addition to MHC:peptide complexes, aAPCs should possess other proteins with costimulatory activity, such as anti-CD28 antibodies, conjugated to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, such as cytokines like interleukin-12.

[0353] Allogeneic cells may also be used in preparing T cells, and methods are described in detail in International Publication No. WO 97 / 26328, which is 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, for example, Porta et al. (1994), which describes the development of cowpea mosaic virus as a high-yield system for the presentation of foreign peptides).

[0354] 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.

[0355] The activated T cells generated by the above method selectively recognize cells that abnormally express polypeptides comprising the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:92, preferably SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92.

[0356] Preferably, the T cells recognize cells by interaction with (e.g., binding to) an HLA / peptide complex through their TCR. The T cells are useful in a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, to which an effective number of activated T cells are administered. The T cells administered to the patient may be derived and activated from the patient 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.

[0357] 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 sometimes also express MHC class II; (Dengjel et al., 2006)).

[0358] The T cells of the present invention may be used as an active ingredient in a therapeutic composition. Accordingly, the present invention also provides a method for killing target cells in a patient whose target cells abnormally express a polypeptide comprising an amino acid sequence of the present invention, the method comprising the step of administering to the patient an effective number of T cells as defined above.

[0359] By "abnormally expressed," 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 "overexpressed," we mean that the polypeptide is present at a level at least 1.2 times the level present in normal tissue; preferably at least 2 times, more preferably at least 5 or 10 times the level present in normal tissue.

[0360] T cells may be obtained by methods known in the art, such as those described above.

[0361] Protocols for this so-called adoptive transfer of T cells are well known in the art and are reviewed in (Gattinoni et al., 2006) and (Morgan et al., 2006).

[0362] Any molecule of the present invention, i.e., peptide, nucleic acid, antibody, expression vector, cell, activated CTL, 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.

[0363] Preferably, the pharmaceutical agent of the present invention is a vaccine. It can be administered directly to a patient, 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 a patient or a human cell line, which are subsequently administered to a patient; or it can be used ex vivo to select a subpopulation of immune cells derived from the patient, which are then re-administered to the patient. When nucleic acids are administered ex vivo to cells, it may be useful to transfect the cells to co-express an immunostimulatory cytokine, such as interleukin-2. The peptides may be substantially pure, combined with an immunostimulatory adjuvant (see below), used in combination with an immunostimulatory cytokine, or administered via a suitable delivery system, such as liposomes. The peptides may also be conjugated to a suitable carrier, such as keyhole limpet hemocyanin (KLH) or mannan (see WO 95 / 18145 and Longenecker, 1993). The peptides may also be labeled, be fusion proteins, or be hybrid molecules. The peptides whose sequences are described in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 CTLs is more efficient in the presence of help provided by CD4 T helper cells. Therefore, for MHC class I epitopes that stimulate CD8 CTLs, the fusion partner or section of the hybrid molecule suitably provides an epitope that stimulates CD4-positive T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those identified in the present invention.

[0364] 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: 92 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.

[0365] 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:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92, 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.

[0366] 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, in (Pascolo et al., 2005). Polynucleotide vaccines are easy to prepare, but the mechanism of action of these vectors in inducing immune responses is not fully understood. Suitable vectors and delivery systems include viral DNA and / or RNA, such as adenovirus, vaccinia virus, retrovirus, herpesvirus, adeno-associated virus, or hybrid-based systems containing components of two or more viruses. Non-viral delivery systems include cationic lipids and cationic polymers and are well known in the DNA delivery art. Physical delivery, such as via a "gene gun," may also be used. The peptide or peptides encoded by the nucleic acid may be a fusion protein with an epitope that stimulates T cells of the respective reverse CDRs, for example, as described above.

[0367] The medicaments of the present invention may also contain one or more adjuvants. Adjuvants are substances that nonspecifically promote or enhance immune responses to antigens (e.g., immune responses mediated by CTLs and helper T (TH) cells) and are therefore considered useful in the medicaments of the present invention. Suitable adjuvants include 1018ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870 and 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 alpha or beta or pegylated derivatives thereof, IS patch, ISS, ISCOMATRIX, ISCOM, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA Adjuvants include, but are not limited to, 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 (e.g., MF59) specific for dendritic cells and their preparations have been previously described (Allison and Krummel, 1995; Allison and Krummel, 1995). Cytokines may also be used.Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, the entire contents of which are specifically incorporated herein by reference), and acting as immune enhancers (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich, 1996).

[0368] 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 compositions, 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.

[0369] 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. Immunoactive small molecules and antibodies, such as cyclohexyl benzoate, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGFTrap, ZD2171, AZD2171, and anti-CTLA4; other antibodies targeting key structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor); and SC58175, may act therapeutically and / or as an adjuvant. 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.

[0370] Preferred adjuvants are 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.

[0371] 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.

[0372] 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, and resiquimod.

[0373] In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod.

[0374] Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly ICLC (Hiltonol®), and anti-CD40 mAB or combinations thereof.

[0375] 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, and lubricants. The peptide may also be administered together with an immunostimulant, such as a cytokine. 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 2113253.

[0376] Nevertheless, further research is needed to provide formulations for specific combinations of peptides, especially combinations with more than 20 peptides, that are stable for more than 12-18 months, depending on the number and physicochemical properties of the peptides of the present invention.

[0377] The present invention provides agents useful in the treatment of cancer, particularly non-small cell lung cancer, gastric cancer, renal cell carcinoma, colon cancer, adenocarcinoma, prostate cancer, benign neoplasms, and malignant melanoma.

[0378] 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, instructions for (i) use of the solution, or (ii) reconstitution and / or use of the lyophilized formulation.

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

[0379] 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.

[0380] 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 the reconstitution and / or use directions. 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.

[0381] 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).

[0382] 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 for use.

[0383] The kits of the present invention may have a single container containing a pharmaceutical composition formulation according to the present invention, with or without added other components (e.g., other compounds or pharmaceutical compositions of these other compounds), or may have separate containers for each component.

[0384] Preferably, the kits of the invention comprise a formulation of the invention packaged for combination with a second compound (such as an adjuvant (e.g., GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic factor or inhibitor, an apoptosis inducer or chelator, or a pharmaceutical composition thereof) or a pharmaceutical composition thereof. The components of the kit may be premixed, or each component may be in a separate, distinct container prior to administration to a patient. The components of the kit may be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit 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.

[0385] 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 other container to allow for separate administration. The kit may also contain a separate container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit will contain a device (e.g., one or more needles, syringes, eyedroppers, pipettes, etc.) to allow for administration of the active substance of the present invention that is a component of the kit.

[0386] 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. Administration may also be by infusion pump.

[0387] Since the peptides of the present invention are isolated from NSCLC, the agents of the present invention are preferably used to treat NSCLC. In a preferred embodiment, the peptides of the present invention derived from ABCA13 and MMP12 are isolated from NSCLC, so the agents of the present invention are preferably used to treat NSCLC.

[0388] The peptides having SEQ ID NOs: 78-92 were isolated from Merkel cell carcinoma and therefore may be used to treat Merkel cell carcinoma.

[0389] The present invention is illustrated, but nevertheless not limited, in the following examples, which now describe preferred embodiments. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0390] [Figure 1-1]A representative mass spectrum from ABCA13-001 demonstrates its presentation on the primary tumor sample NSCLC898. NanoESI-LCMS was performed on a peptide pool eluted from NSCLC sample 898. The mass chromatogram of m / z 543.8318 ± 0.001 Da, z = 2, shows the peptide peak at retention time 86.36 min. B) The peak detected in the mass chromatogram at 86.36 min revealed a signal at m / z 543.8318 in the MS spectrum. C) Collision-induced decay mass spectrum m / z 543.8318 from selected precursors recorded in a nanoESI-LCMS experiment at a given retention time confirmed the presence of ABCA13-001 in the NSCLC898 tumor sample. D) The fragmentation pattern of a synthetic ABCA13-001 reference peptide was recorded and compared to the resulting native TUMAP fragmentation pattern shown in C for sequence verification. [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 1-4] Same as above [Figure 2] mRNA expression profiles of selected proteins in normal tissues and 21 lung cancer samples. a) ABCA13 (Probeset ID: 1553605_a_at) b) MMP12 (Probeset ID: 204580_at) [Figure 3-1] 1. Presentation profiles of selected HLA class I peptides. Presentation profiles were calculated for each peptide showing the average sample presentation as well as replicate variation. The profiles align samples of the tumor entity of interest to a baseline of normal samples. a) ABCA13-001 b) DST-001 c) MXRA5-001 [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 4]Representative results of peptide-specific in vitro immunogenicity of class I TUMAPs. Specific CD8+ T cells were stained with HLA multimers linked to two different fluorochromes. Dot plots show MHC multimer double-positive populations for the stimulatory peptide (left panel) and each negative control stimulus (right panel). [Figure 5] Binding characteristics of POSTN-002 and MMP12-002 to the investigated HLA haplotypes. The diagram shows the binding scores of POSTN-002 and MMP12-002 to five of the seven HLA-DR haplotypes analyzed. [Figure 6] Stability of HLA-POSTN-002 and MMP12-002 complexes after 24 hours at 37°C: Diagram shows the percentage of intact complexes of HLA-POSTN-002 and HLA-MMP12-002 with the corresponding HLA molecules after 24 hours at 37°C. [Figure 7-1] Representative vaccine-induced CD4 T cell responses to CEA-006 in a Class IIICS assay. Following in vitro sensitization, PBMCs from patient 36-031 were analyzed for CD4 T cell responses to CEA-006 (upper panel) and mock (lower panel) at time point pool V8 / EOS. Cells were stimulated with the corresponding peptide and stained for viability, anti-CD3, anti-CD8, anti-CD4, and effector markers (right to left: CD154, TNF-α, IFN-gamma, IL-2, IL-10), respectively. Viable CD4 T cells were analyzed for the proportion of cells positive for one or more effector molecules. [Figure 7-2] Same as above [Figure 8] Immunogenicity of various class II peptides. The diagram shows the immune response assessment of five different class II peptides using ICS, detected in 16 patients for the IMA950 peptide and in 71 patients for the IMA910 peptide. [Example]

[0391] Example 1 Identification and quantification of tumor-associated peptides displayed on the cell surface Tissue samples Patient tumor tissue was provided by the University of Heidelberg, Heidelberg, Germany. Informed consent was obtained from all patients before surgery. Tissues were shock-frozen in liquid nitrogen immediately after surgery and stored at -80°C until TUMAP isolation.

[0392] Isolation of HLA peptides from tissue samples HLA peptide pools from shock-frozen tissue samples were obtained by immunoprecipitation from solid tissues using the HLA-A*02-specific antibody BB7.2, the HLA-A, -B, and -C-specific antibody W6 / 32, CNBr-activated Sepharose, acid treatment, and ultrafiltration according to a slightly modified protocol (Falk, K., 1991; Seeger, FHT, 1999).

[0393] method The resulting HLA peptide pool was separated according to their hydrophobicity by reversed-phase chromatography (Acquity UPLC system, Waters), and the eluted peptides were analyzed in an LTQ-Orbitrap hybrid mass spectrometer (ThermoElectron) equipped with an ESI source. The peptide pool was directly loaded onto an analytical fused silica microcapillary column (75 μm i.d. × 250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) at a flow rate of 400 nL / min. Peptides were subsequently separated using a two-step 180-min binary gradient from 10% to 33% B at a flow rate of 300 nL / min. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Gold-coated glass capillaries (PicoTip, New Objective) were used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using the TOP5 strategy. Briefly, a scan cycle began with a high-mass-accuracy full scan in the orbitrap (R = 30,000), followed by an MS / MS scan of the five most abundant precursor ions, also in the orbitrap (R = 7,500), with 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 pattern with that of a sequence-identical synthetic reference peptide. Figure 1 shows a representative spectrum obtained from tumor tissue of the MHC class I-associated peptide ABCA13-001 and its elution profile on the UPLC system.

[0394] 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 tissue-to-peptide presentation profiles. Quantitative data were normalized in a two-fold manner according to central tendency, accounting for technical and intra-biological replicate variability. In this way, each identified peptide was 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 average sample presentation as well as replicate variability. The profile juxtaposes the NSCLC samples to a baseline of normal tissue samples.

[0395] The presentation profile of a representative over-presented peptide is shown in FIG.

[0396] Example 2 Expression profiling of genes encoding the peptides of the present invention Not all peptides identified as being presented on the surface of tumor cells by MHC molecules are suitable for immunotherapy, because the majority of these peptides are derived from normal cellular proteins expressed by many cell types.Only a small number of these peptides are tumor-associated, and are likely to induce T cells that recognize the tumors they originate from with high specificity.In order to identify such peptides and minimize the risk of autoimmunity induced by vaccination, the inventors focused on peptides derived from proteins that are overexpressed on tumor cells compared with the majority of normal tissues.

[0397] An ideal peptide would be derived from a protein that is specific to the tumor and not present in any other tissue. To identify peptides derived from genes with expression profiles similar to the ideal ones, the identified peptides were assigned to the proteins and genes from which they were derived, respectively, and expression profiles of these genes were generated.

[0398] RNA origin and preparation Surgically removed tissue specimens were provided by the University of Heidelberg, Heidelberg, Germany, after informed consent was obtained from each patient (see Example 1). Tumor tissue specimens were snap-frozen in liquid nitrogen 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.

[0399] 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 RNA from each individual was equally weighted.

[0400] 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).

[0401] 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) were used. 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 using an Agilent 2500A GeneArray Scanner (U133A) or an Affymetrix Gene-Chip Scanner 3000 (U133 Plus 2.0), and data were analyzed using 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 given by the software, with normal kidney samples arbitrarily set to 1.0.

[0402] Representative expression profiles of the source genes of the present invention that are highly overexpressed or exclusively expressed in non-small cell lung cancer are shown in FIG.

[0403] Example 4 In vitro immunogenicity of NSCLC MHC class I-presented peptides To obtain information about the immunogenicity of the TUMAPs of the present invention, we performed an investigation using an in vitro T cell priming assay, based on repeated stimulation of CD8+ T cells with artificial antigen-presenting cells (aAPCs) loaded with peptide / MHC complexes and anti-CD28 antibodies. In this way, we have been able to demonstrate the immunogenicity of nine HLA-A*0201-restricted TUMAPs of the present invention and have demonstrated that these peptides represent T cell epitopes against which CD8+ precursor T cells are present in humans (Table 4).

[0404] Ex vivo priming of CD8+ T cells To perform ex vivo stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD28 antibodies, we first isolated CD8+ T cells from fresh HLA-A*02 leukapheresis products through positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) from healthy donors obtained from Transfusion Medicine Tuebingen, Germany, after informed consent.

[0405] 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.

[0406] 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.

[0407] All pMHC complexes used for aAPC loading and cytometric readout were derived from UV-induced MHC-ligand exchange (Rodenko et al., 2006) with minor modifications. To determine the amount of pMHC monomer obtained by exchange, we performed a streptavidin-based sandwich ELISA as described (Rodenko et al., 2006).

[0408] Purified costimulatory mouse IgG2a anti-human CD28 Ab 9.3 (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).

[0409] The pMHC used for positive and negative control stimulation were A*0201 / MLA-001 (peptide ELAGIGILTV from modified Melan-A / MART-1) and A*0201 / DDX5-001 (YLLPAIVHI from DDX5), respectively.

[0410] 800,000 beads / 200 μl were coated in a 96-well plate in the presence of 4 × 12.5 ng of different biotin pMHC and washed, followed by the addition of 600 ng of biotin anti-CD28 in a volume of 200 μl. Stimulation was initiated in 96-well plates by co-incubating 1 × 106 CD8+ T cells with 2 × 105 washed coated beads in 200 μl of TCM supplemented with 5 ng / ml IL-12 (PromoCell) at 37°C for 3–4 days. Half of the medium was then replaced with fresh TCM supplemented with 80 U / ml IL-2, and incubation continued at 37°C for 3–4 days. This stimulation cycle was performed a total of three times. For pMHC multimer readout, using eight different pMHC molecules per condition, we used a two-dimensional combinatorial coding approach as previously described (Andersen et al., 2012) with minor modifications, including conjugation to five different fluorochromes. Finally, multimer analysis was performed by staining cells with Live / Dead near-infrared dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany), and fluorescent pMHC multimers. Analysis was performed using a BD LSRII SORP hemocytometer equipped with the appropriate lasers and filters. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis was evaluated using FlowJo software (Tree Star, Oregon, USA). Ex vivo priming 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 one 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 the median of the negative control stimulation).

[0411] In vitro immunogenicity of NSCLC peptides To test HLA class I peptides, in vitro immunogenicity can be demonstrated by generating peptide-specific T cell lines. Representative flow cytometry results after TUMAP-specific multimer staining of two peptides of the invention, along with the corresponding negative control, are shown in Figure 4. Results for 25 peptides from the invention are summarized in Table 5.

[0412] [Table 10-1] [Table 10-2]

[0413] Example 5 Peptide synthesis All peptides were synthesized using standard, well-established solid-phase peptide synthesis using the Fmoc-strategy. After purification by preparative RP-HPLC, an ion-exchange procedure was performed to incorporate physiologically compatible counterions (e.g., trifluoroacetate, acetate, ammonium, or chloride).

[0414] The identity and purity of individual peptides were determined by mass spectrometry and analytical RP-HPLC. Peptides were obtained after ion exchange as white to off-white lyophilizates with purities ranging from 90% to 99.7%.

[0415] All TUMAPs are preferably administered as trifluoroacetate or acetate salts, although other salt forms are also possible. In the measurements in Example 4, the trifluoroacetate salts of the peptides were used.

[0416] Example 6 UV Ligand Exchange Candidate peptides for vaccines according to the present invention were further tested for immunogenicity by in vitro priming assays. Individual peptide-MHC complexes required for these assays 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 capable of effectively binding and stabilizing peptide-receptor MHC molecules prevented dissociation of the MHC complexes. 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).

[0417] A 96-well MAXISorp plate (NUNC) was coated with 2 μg / ml streptavidin in PBS overnight at room temperature, washed four times, and blocked in 2% BSA containing blocking buffer at 37°C for 30 minutes. Refolded HLA-A*0201 / MLA-001 monomer served as a standard covering a range of 8-500 ng / ml. Peptide-MHC monomers for UV exchange reactions were diluted 100-fold in blocking buffer. Samples were incubated for 1 hour at 37°C, washed for 4 hours, and then incubated with 2 μg / ml HRP-conjugated anti-β2m for 1 hour at 37°C, washed again, and detected with NH2SO4-quenched TMB solution. Absorbance was measured at 450 nm.

[0418] [Table 11-1] [Table 11-2] [Table 11-3]

[0419] For the generation and production of antibodies or fragments thereof, and / or T cell receptors or fragments thereof, candidate peptides that exhibit high exchange yields (i.e., greater than 40%, preferably greater than 50%, more preferably greater than 70%, and most preferably greater than 80%) are generally preferred, as they exhibit sufficient binding activity to MHC molecules to prevent dissociation of the MHC complex.

[0420] Example 7 Binding and immunogenicity of selected MHC class II peptides HLA class II proteins are classified into three major isotypes, HLA-DR, -DP, and -DQ, which are encoded by numerous haplotypes. The various α and β chain combinations increase the diversity of HLA class II proteins found in any given population. Therefore, in a significant proportion of patients, a selected HLA class II TUMAP must bind to several different HLA-DR molecules (i.e., exhibit promiscuous binding capacity) to contribute to an effective T cell response.

[0421] The promiscuous binding of POSTN-002 and MMP12-002 to various HLA-DR haplotypes and the stability of the complexes formed were assessed in in vitro binding assays by an external service provider as follows.

[0422] Materials and Methods [Table 12]

[0423] List of investigated HLA-DR haplotypes The seven investigated HLA-DR haplotypes were selected according to their frequency in HLA-A*02 and HLA-A*24 positive North American populations (Tables 7.1 and 7.2).

[0424] Data were derived from an analysis of 1.35 million HLA-typed volunteers enrolled in the United States Bone Marrow Donor Service (Mori et al., 1997). The analyzed population was subdivided into the following ethnicities: White Americans (N = 997,193), African Americans (N = 110,057), Asian Americans (N = 81,139), Latinos (N = 100,128), and Native Americans (N = 19,203).

[0425] [Table 13]

[0426] [Table 14]

[0427] Test Principle The ProImmune REVEAL® MHC-peptide binding assay determines the ability of each candidate peptide to bind to a selected HLA class II haplotype and stabilize the HLA-peptide complex. The candidate peptide is then assembled in vitro with a specific HLA class II protein. The level of peptide incorporation into the HLA molecule is measured by the presence or absence of the native conformation of the assembled HLA-peptide complex at time 0 after the refolding procedure is completed (the so-called on-rate).

[0428] The binding ability of a candidate peptide to a particular HLA molecule is compared to those with known very strong binding properties (positive controls) to obtain a corresponding REVEAL® MHC-peptide binding score. Positive control peptides are selected and provided by ProImmune based on their individual experience with each HLA haplotype.

[0429] In addition to the affinity of the peptide for a particular HLA molecule, the enduring stability of the formed HLA-peptide complex is important for the generation of an immune response. Therefore, the presence of formed HLA-peptide complexes is measured after 24 hours of incubation at 37°C. The stability of the formed MHC-peptide complex is then calculated as the percentage ratio of the binding score at 24 hours to the binding score obtained immediately after refolding (hence, at time 0).

[0430] result Analysis of POSTN-002 and MMP12-002 in the REVEAL® MHC-peptide binding assay demonstrated that both peptides bind to various HLA haplotypes. POSTN-002 was shown to form complexes with four of the seven HLA haplotypes examined, and MMP12-002 with five (Figure 5). Neither peptide bound to HLA-DR3 or HLA-DR6. The binding scores detected ranged from 0.02 to approximately 2.5% relative to the positive control, clearly exceeding the scores of the non-binding peptides.

[0431] Stability analysis of the formed HLA-POSTN-002 and HLA-MMP12-002 complexes revealed that three and two of the six HLA-peptide complexes investigated, respectively, were stable after 24 h at 37°C (Fig. 6 ).

[0432] A conclusion about the immunogenicity of a peptide based on its ability to bind to HLA molecules can be made by comparing the peptide's binding score with known immunogenicity. Therefore, five well-studied peptides with determined immunogenicity were selected for this comparison. The immunogenicity of these peptides was determined in vitro in blood samples from vaccinated patients using intracellular cytokine staining (ICS) CD4 T cells.

[0433] Essentially, the ICS assay analyzes the quality of specific T cells in terms of effector function. To do so, peripheral mononuclear cells (PBMCs) were cultured in vitro and subsequently restimulated with the peptide of interest, a reference peptide, and a negative control (here, MOCK). Following restimulation, cells were stained for FN-γ, TNF-α, IL-2, and IL-10 production, as well as for the expression of the costimulatory molecule CD154. The count of affected cells was performed on a flow cytometer (Figure 7).

[0434] Immunogenicity analysis revealed a 100% immune response following vaccination with IMA950 peptides (BIR-002 and MET-005) in 16 patients and a 44% to 86% immune response following vaccination with MA910 peptides (CEA-006, TGFBI-004, and MMP-001) in 71 patients.

[0435] To compare the binding scores of the POSTN-002 and MMP12-002 peptides with those of the IMA910 and IMA950 peptides, all peptides were arranged in tables for each HLA-DR haplotype investigated according to the binding scores detected (Tables 8.1–8.5).

[0436] [Table 15]

[0437] [Table 16]

[0438] [Table 17]

[0439] [Table 18]

[0440] [Table 19]

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Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92, or a variant thereof that is at least 80% homologous to SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92, wherein said variant induces antibodies and / or T cells that specifically bind to said peptide, or to a pharma- ceutically acceptable salt of SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:92, and wherein said peptide is not the underlying full-length polypeptide.

2. 2. The peptide according to claim 1, wherein the peptide or variant thereof comprises an overall amino acid length of 8-100, preferably 8-30, and most preferably 8-14 for peptides comprising SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:78 to SEQ ID NO:84, and SEQ ID NO:92, and has an overall amino acid length of 12-100, preferably 12-30, and most preferably 12-18 amino acids for peptides comprising SEQ ID NO:76 or SEQ ID NO:

77.

3. A peptide according to any one of claims 1 to 2, having the ability to bind to class I or class II molecules of the human major histocompatibility complex (MHC).

4. 4. The peptide according to any one of claims 1 to 3, wherein the peptide consists of or essentially consists of the amino acid sequence set forth in SEQ ID NO:1 to SEQ ID NO:65, and SEQ ID NO:76 to SEQ ID NO:84, and SEQ ID NO:

92.

5. The peptide according to any one of claims 1 to 4, wherein the peptide is modified, extended at the N- and / or C-terminus by up to four amino acids and / or comprises non-peptide bonds.

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

7. A nucleic acid encoding the peptide according to any one of claims 1 to 6.

8. 8. The nucleic acid of claim 7, which is DNA, cDNA, PNA, RNA or a combination thereof.

9. An expression vector capable of expressing the nucleic acid according to claim 7 or 8.

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

11. A host cell comprising a nucleic acid according to claim 7 or 8 or an expression vector according to claim 9, which is not a human embryonic stem cell.

12. The host cell of claim 11 , wherein the cell is an antigen-presenting cell, such as a dendritic cell.

13. A pharmaceutical composition comprising a peptide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 6 and at least one other component selected from the group of pharma- ceutically acceptable, preferably aqueous, carriers and / or excipients such as buffers, binding agents, blasting agents, diluents, flavours, lubricants and immunostimulatory or immunomodulatory substances such as cytokines, immunomodulators, adjuvants and therapeutic agents with immunomodulatory properties.

14. A method for producing a peptide according to any one of claims 1 to 6, comprising the steps of culturing a host cell according to claim 11 or 12 and isolating said peptide from the host cell or its culture medium.

15. 10. An in vitro method for generating activated cytotoxic T lymphocytes (CTLs) or T helper cells (Th cells), comprising ex vivo contacting of CTLs or Th cells with antigen-loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell for a time sufficient to activate the CTLs in an antigen-specific manner, wherein the antigen is a peptide according to any one of claims 1 to 6.

16. 16. The method of claim 15, wherein the antigen is loaded onto a class I or II MHC molecule expressed on the surface of an appropriate antigen-presenting cell by contacting the antigen-presenting cell with a sufficient amount of the antigen.

17. The method according to claim 15 or 16, wherein the antigen-presenting cell comprises an expression vector capable of expressing the peptide according to any one of claims 1 to 6.

18. 18. An activated cytotoxic T lymphocyte (CTL) or T helper cell (Th cell) produced by the method according to any one of claims 15 to 17, wherein the CTL or Th cell selectively recognizes a cell that abnormally expresses a polypeptide comprising an amino acid sequence according to any one of claims 1 to 6.

19. An in vitro method for generating a TCR or sTCR or a fragment thereof specific for a peptide according to any one of claims 1 to 6, comprising the steps of cloning a variable region from an activated cytotoxic T lymphocyte (CTL) or T helper cell (Th cell) according to claim 18 and expressing said TCR or sTCR or a fragment thereof in a suitable host and / or expression system.

20. 5. An isolated binding agent, such as an antibody or fragment thereof, a protein, a nucleic acid, a peptide, a TCR or sTCR or fragment thereof, which binds, preferably specifically, to a peptide according to any one of claims 1 to 4 or to a complex of a peptide according to any one of claims 1 to 4 and an MHC molecule.

21. An isolated T cell receptor that is reactive with an HLA ligand that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

92.

22. 22. The isolated T cell receptor of claim 21, wherein the amino acid sequence is at least 90%, preferably at least 95%, and most preferably 100% identical to SEQ ID NO:1 to SEQ ID NO:

92.

23. 19. A method of killing target cells in a patient, said target cells aberrantly expressing and / or presenting a polypeptide comprising an amino acid sequence as set out in any one of claims 1 to 6, said method comprising the step of administering to said patient an effective number of cytotoxic T lymphocytes (CTLs) or Th cells according to claim 18.

24. 23. Use of a peptide according to any one of claims 1 to 6, a nucleic acid according to claim 7 or 8, an expression vector according to claim 9, a cell according to claims 11 to 12, an activated cytotoxic T lymphocyte according to claim 18, or an antibody or a TCR or a sTCR according to any one of claims 20 to 22 as a medicament, wherein the medicament is effective against cancer.

25. 25. The use according to claim 24, wherein the medicament is a vaccine.

26. 26. The use according to claim 24 or 25, wherein the cancer is selected from non-small cell lung cancer (NSCLC), lung cancer, gastric cancer, and / or glioblastoma.

27. The use according to any one of claims 24 to 26 for adoptive cell therapy in humans.

28. 21. An autologous or allogeneic human cytotoxic T cell (CTL) or T helper cell (Th cell) recombinantly transfected with the T cell receptor of claim 20.

29. (a) (a1) an isolated peptide according to any one of claims 1 to 6; (a2) a T cell receptor, sTCR or a fragment thereof according to any one of claims 20 to 22; (a3) the fusion protein according to claim 6; (a4) the nucleic acid according to claim 7 or 8; (a5) the expression vector according to claim 9, (a6) a host cell according to any one of claims 11 to 12, and (a7) The activated cytotoxic T lymphocyte or T helper cell according to claim 18. an entity selected from the group consisting of: (b) a pharma- ceutically acceptable carrier, and, optionally, (c) at least one other component selected from the group of pharma- ceutically acceptable excipients, buffers, binders, blasting agents, diluents, flavors, lubricants, and immunostimulatory or immunomodulatory substances such as cytokines, immunomodulators, adjuvants, and therapeutic agents with immunomodulatory properties.

23. A pharmaceutical composition comprising:

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